Avanços no tratamento sistémico de doentes com melanoma metastizado entre 2011 e 2019

AVANÇOS NO TRATAMENTO SISTÉMICO DE DOENTES COM MELANOMA METASTIZADO ENTRE 2011 E 2019 TERESA MARIA SANTOS AMARAL Tese para obtenção do grau de Doutor em Medicina na Especialidade Investigação Clínica na Faculdade de Ciências Médicas | NOVA Medical School da Universidade NOVA de Lisboa Setembro, 2020 2 Eberhard Karls University of Tuebingen Center for Dermatooncology Head: Professor Dr. C. Garbe Department of Dermatology Director: Professor Dr. M. Röcken Progress in systemic therapy of advanced melanoma between 2011 and 2019 Thesis developed based on cumulative publications to obtain the degree of Doctor of Medicine – Clinical Investigation Submitted by Teresa Amaral From Viseu, Portugal 2020 3 Dúbia é a vida, inconstante o que a governa. O que esperamos nem sempre acontece Nem nos falece sempre, Nem há com que a alma uma ou outra cousa espere. in “Poemas de Ricardo Reis. Fernando Pessoa Table of contents ACKNOWLEDGMENTS 7 1. SUMMARY 8 1.1. ENGLISH SUMMARY 8 1.2. PORTUGUESE SUMMARY 12 1.3. PUBLICATIONS INCLUDED IN THE THESIS 16 1.4. ABBREVIATION LIST 18 2. INTRODUCTION 20 2.1. INCIDENCE AND PREVALENCE OF MELANOMA 20 2.2. BIOLOGY OF MELANOMA 21 2.2.1. MELANOMA SUBTYPES 21 2.2.1.1. CUTANEOUS MELANOMA 21 2.2.1.2. MUCOSAL MELANOMA 22 2.2.1.3. UVEAL MELANOMA 23 2.3. THE 8TH EDITION OF THE AJCC CLASSIFICATION 23 2.4. SYSTEMIC THERAPY OF ADVANCED MELANOMA 25 2.4.1. IMMUNOTHERAPY WITH CTLA-4 AND PD1/PDL-1 INHIBITORS 26 2.4.1.1. ANTICTLA4 – IPILIMUMAB 29 2.4.1.2. ANTI-PD-1 – NIVOLUMAB AND PEMBROLIZUMAB 30 2.4.1.3. NIVOLUMAB PLUS IPILIMUMAB 34 2.4.2. TARGETED THERAPY WITH BRAF AND MEK INHIBITORS 35 2.5. SYSTEMIC THERAPIES IN MELANOMA BRAIN METASTASIS 38 2.6. ACCESS TO SYSTEMIC THERAPIES APPROVED IN THE ADVANCED SETTING IN PORTUGAL AND GERMANY 39 3. OBJECTIVES 41 AIM 1) TO DETERMINE LONG-TERM OUTCOMES IN PATIENTS WITH STAGE IV MELANOMA TREATED WITH TARGETED AND IMMUNOTHERAPY BETWEEN 2011-2019 41 AIM 2) TO EVALUATE THE IMPACT IN SURVIVAL OUTCOMES OF TARGETED AND IMMUNOTHERAPY, AND THEIR COMBINATION WITH LOCAL THERAPIES, IN PATIENTS WITH MELANOMA BRAIN METASTASES 41 4. PATIENTS AND METHODS 42 4.1. THE GERMAN CMMR (MANUSCRIPT 1, 2, 5) 42 5 4.1.1. STUDY DESIGN AND DATA SOURCE 42 4.1.2. KEY VARIABLES 43 4.1.3. STATISTICAL ANALYSES 43 4.2. THE CO-BRIM, COMBI-V AND COLUMBUS STUDY DATABASE (MANUSCRIPT 3) 44 4.2.1. STUDY DESIGN AND DATA SOURCE 44 4.2.2. KEY VARIABLES 44 4.2.3. STATISTICAL ANALYSES 45 4.3. THE MULTICENTRIC STUDIES – GERMAN CMMR IN COMBINATION WITH LOCAL DATABASES (MANUSCRIPT 4,6) 45 4.3.1. STUDY DESIGN AND DATA SOURCE 45 4.3.2. KEY VARIABLES 46 4.3.3. STATISTICAL ANALYSES 46 5. RESULTS 48 6. MANUSCRIPTS 50 7. DISCUSSION 117 7.1. SYSTEMIC TREATMENT OF STAGE IV CUTANEOUS MELANOMA 117 7.2. PRIMARY RESISTANCE TO PD-1 BASED IMMUNOTHERAPY 119 7.3. COMBINED BRAF/MEK INHIBITORS IN STAGE IV BRAFV600 MUTATED CUTANEOUS MELANOMA 121 7.4. SYSTEMIC TREATMENT OF STAGE IV UVEAL MELANOMA 123 7.5. SYSTEMIC AND LOCAL TREATMENT OF MELANOMA BRAIN METASTASES 124 7.6. SAFETY PROFILE OF IMMUNE CHECKPOINT INHIBITORS AND BRAF/MEK INHIBITORS 128 8. LIMITATIONS AND CONCLUSIONS 132 9. REFERENCES 133 10. CURRICULUM VITAE 146 11. FULL LIST OF PUBLICATIONS 149 12. APPENDIX 154 6 Tables and figures Table 1: Overview of the TNM classification for cutaneous melanoma ........................ 25 Table 2: Summary of results from the trials investigating PD-1 based immunotherapy in advanced melanoma ..................................................................................................... 32 Table 3: Summary of results from the trials investigating targeted therapy in advanced melanoma ..................................................................................................................... 37 Figure 1: Timing of re-imbursement for systemic therapies in stage IV melanoma, in Portugal and Germany .................................................................................................. 26 Figure 3: The priming phase and CTLA-4 blockade .................................................... 28 Figure 4: The effector phase and the PD-1/PD-L1/2 blockade .................................... 29 Figure 5: Mechanism of immune-related adverse events associated with immune checkpoint inhibition .................................................................................................... 130 Figure 6: Proposed therapeutic algorithm for the management of immune related adverse events ............................................................................................................ 131 Acknowledgments 7 Acknowledgments “Those who pass by us, do not go alone, and do not leave us alone; they leave a bit of themselves, and take a little of us.” Antoine de Saint-Exupéry I have been very fortunate all my life. I had a wonderful childhood, my family loved me very much, and they always believed in me. Later on, when I pursued a medical career, and despite all the hurdles, I always had someone who truly believed in me. I am now the result of all the people that passed by me, and left something of them. I treasure all the gifts I received. I am sure they also took part of me with them. It is somehow unfair to only name some here. However, I will do so, since this is also the place to thank them for their presence and inspiration: • My grandmother Maria, my father, my mother, my sister and brother, my nephews. • My Ph.D. mentor and supervisor, Claus Garbe, for his support and commitment with my professional growth. His leadership skills taught me more than I could expect. • My Ph.D. supervisor, Thomas Eigentler, for his positivity, mentorship, support and enthusiasm every day. • My mentors during my time as medical student, and later during my training as medical oncologist; my colleagues and co-authors, my students, and my patients. • My mentors and colleagues from the Portuguese Air Force. • My ESMO family. Finally, I want to thank my friends - you know who you are - for their unconditional support and love during this journey. I am fully committed to honor all the energy and thoughtfulness you dedicated to me, by staying true to myself. Summary 1. Summary 1.1. English Summary Background: Ten years ago, stage IV melanoma carried a dismal prognosis, with very short survival. The therapies available then, namely dacarbazine, temozolomide, and other chemotherapies, alone or in combination with surgery and/or radiotherapy, were unable to provide clinically significant benefit. In the last decade, the medical community testified a revolution in the treatment of advanced melanoma with the introduction of immune checkpoint inhibitors, and targeted therapy with mitogen-activated protein kinase inhibitors (MAPKi). Both therapies were able to provide sustainable overall survival benefits in stage IV melanoma. Here, we aimed to investigate whether the survival advantages seen in phase II, and III clinical trials investigating the afore mentioned therapies, were reproducible or not in a real-world setting. Patients and methods: In this retrospective, multicentric analysis, we included stage IV melanoma patients, diagnosed between 2011 and 2018, treated in the skin cancer center in Tuebingen, or in other German centers, and prospectively documented in the German Central Malignant Melanoma Registry (CMMR). The following patients’ and tumor data were collected from the CMMR: year of birth, gender, date of primary tumor diagnosis, type of melanoma, localization, tumor thickness, Clark level, presence of ulceration, presence of regression, date of sentinel lymph node biopsy (if applicable), presence of lymph node metastases, stage at first diagnosis and date and localization of recurrence. The following variables were further included, based on patients’ clinical chart review: date and type of local and systemic therapy in stage IV, date and type of best overall response, according to the Response Evaluation Summary 9 Criteria in Solid Tumors version 1.1 (RECIST 1.1), date of progressive disease, date of last contact, cause of death, and death date. Patients included had a minimum follow-up of three months. Progression free survival (PFS) was defined as the time between date of stage IV diagnosis, start date of systemic therapy, or date of melanoma brain metastases (MBM) diagnosis, depending on the type of population evaluated, and date of progressive disease, or last contact or death, for the patients that didn’t progress. Overall survival (OS) was defined as the time between stage IV diagnosis, start date of systemic therapy, or date of MBM diagnosis, depending on the type of population evaluated, and date of last contact or death. Kaplan-Meier estimates were used for the calculation of PFS and OS. Differences between groups were assessed using the log-rank test. When applicable, hazard ratios (HR) with 95% confidence intervals (CI) were calculated to quantify the impact on survival. Multivariate logistic regression models were used to examine associations between variables, when appropriate. Results were reported as two-sided p values with 95% CIs. All p-values presented are two-sided tests of statistical significance at 0.05. For the indirect comparison between MAPKi, the Bucher method was used. All analyses were submitted to and approved by the local Ethics Committee. Results: Six publications reporting data from 3143 stage IV melanoma patients were included in this thesis. The 3-years (3-y) OS rate for patients treated with first-line chemotherapy, the most used systemic therapy in 2011-2014 was 15.9% (95% CI: 8.8-23). For patients treated with first-line immunotherapy in the same period, the 3-y OS rate was 37.4% (95% CI: 16.6-58.2). In the period of 2015-2018, the 3-y OS rate for patients treated with first-line immunotherapy almost duplicated compared to the 2011-2014 period, and was 64.6% (95% CI: 53.2–76) for patients achieving a complete response (CR), Publications included in the thesis 16 1.3. Publications included in the thesis 1) Forschner A, Eichner F, Amaral T, Keim U, Garbe C, Eigentler TK. Improvement of overall survival in stage IV melanoma patients during 2011-2014: analysis of real-world data in 441 patients of the German Central Malignant Melanoma Registry (CMMR). Journal of cancer research and clinical oncology. 2017; 143: 533-40. (IF=3.656) 2) Amaral T, Seeber O, Mersi E, Sanchez S, Thomas I, Meiwes A, Forschner A, Leiter U, Eigentler T, Keim U, Garbe C. Primary Resistance to PD-1-Based Immunotherapy-A Study in 319 Patients with Stage IV Melanoma. Cancers. 2020; 12. (IF=6.102) 3) Glutsch V, Amaral T, Garbe C, Thoms KM, Mohr P, Hauschild A, Schilling B. Indirect Comparison of Combined BRAF and MEK Inhibition in Melanoma Patients with Elevated Baseline Lactate Dehydrogenase. Acta dermatovenereologica. 2020; 100: adv00174. (IF=4.016) 4) Heppt MV, Amaral T, Kähler KC, Heinzerling L, Hassel JC, Meissner M, Kreuzberg N, Loquai C, Reinhardt L, Utikal J, Dabrowski E, Gesierich A, Pföhler C, Terheyden P, Thoms K-M, Zimmer L, Eigentler TK, Kirchberger MC, Stege HM, Meier F, Schlaak M, Berking C. Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study. Journal for immunotherapy of cancer. 2019; 7: 299. (IF=9.913) 5) Amaral T, Tampouri I, Eigentler T, Keim U, Klumpp B, Heinrich V, Zips D, Paulsen F, Gepfner-Tuma I, Skardelly M, Tatagiba M, Tabatabai G, Garbe C, Forschner A. Immunotherapy plus surgery/radiosurgery is associated with favorable survival in patients with melanoma brain metastasis. Immunotherapy. 2019; 11: 297-309. (IF=2.964) 6) Amaral T, Kiecker F, Schaefer S, Stege H, Kaehler K, Terheyden P, Gesierich A, Gutzmer R, Haferkamp S, Uttikal J, Berking C, Rafei-Shamsabadi D, Reinhardt L, Meier F, Karoglan A, Posch C, Gambichler T, Pfoehler C, Thoms K, Tietze J, Debus D, Herbst R, Emmert S, Loquai C, Hassel JC, Meiss F, Tueting T, Heinrich V, Eigentler T, Garbe C, Zimmer L. Combined immunotherapy with nivolumab and ipilimumab with and without local therapy in patients with melanoma brain metastasis: a DeCOG* study in 380 patients. Journal for immunotherapy of cancer. 2020; 8. (IF=9.913) Publications included in the thesis 17 Total impact of the publications included in the current thesis, according to the Portuguese current legislation - Diário da República, 2.a série — N.o 153 — 7 de agosto de 2015; Regulamento n.o 519/2015, Capítulo II, artigo 20º, ii - Trabalhos científicos alternativos à Tese: • Impact factor as first author – 37.96 • Impact factor as not first author – 12.63 • Total impact factor – 50.59 Abbreviation list 18 1.4. Abbreviation list AE Adverse Events AJCC American Joint Committee on Cancer ALM Acral Lentigo Melanoma APC Antigen Presenting Cells ASCO American Society of Clinical Oncology BRAF B-Raf Proto-Oncogene v-Raf murine Sarcoma Viral Oncogene Homolog B irAE Immune Related Adverse Events IARC International Agency of Research on Cancer CI Confidence Interval CMMR German Central Malignant Melanoma Registry CTCAE Common Terminology Criteria for Adverse Events CTLA-4 Cytotoxic T-lymphocyte-associated protein 4 CR Complete Response DCR Disease Control Rate EMA European Medicines Agency ECOG PS Eastern Cooperative Oncology Group Performance Status EORTC European Organisation for Research and Treatment of Cancer ESMO European Society of Medical Oncology FDA Food and Drug Administration HLA Human Leukocyte Antigen HR Hazard Ratio ICI Immune Checkpoint Inhibitors LDH Lactate Dehydrogenase LMM Lentigo Melanoma MAPKi Mitogen-activated Protein Kinase Kinase inhibitors MBM Melanoma Brain Metastases MEK Mitogen-activated Protein Kinase Kinase NM Nodular Melanoma ORR Overall Response Rate OS Overall Survival mOS Median Overall Survival PD-1 Programmed Cell Death Protein 1 Abbreviation list 19 PD-L1 Programmed Cell Death Ligand 1 PD Progressive Disease PFS Progression Free Survival PR Partial Response RECIST 1.1 Response Evaluation Criteria in Solid Tumors version 1.1 SD Stable Disease SIRT Selective Internal Radiotherapy SOC Standard of Care SPO Sociedade Portuguesa de Oncologia SSM Superficial Spreading Melanoma TACE Trans-arterial Chemoembolization TCR T-cell receptors ULN Upper Limit Normal UV Ultraviolet WG Working Group y years Introduction 20 2. Introduction 2.1. Incidence and prevalence of melanoma In recent years, there was a worldwide increase of melanoma incidence. 1 By 2020, the number of newly diagnosed melanomas worldwide is expected to reach 279,938, and it is estimated that about 67,809 people will die from this disease. The numbers are from the International Agency for Research on Cancer (IARC). 2 Other national reports provided similar data, showing that the increased incidence rate of melanoma is a global issue. 3-5 The incidence rate of cutaneous melanoma is higher in white populations compared to Spanish, Afro-American, Indian and Asian. 6 The mean annually age-adjusted incidence of melanoma in whites per 100 000 persons is 18.4 and in Spanish, AfroAmericans, Indians and Asians it is 2.3, 0.8, 1.6 and 1.0, respectively. In most European countries, the melanoma incidence rate almost doubled between 1990 and 2005, increasing between + 2% and + 10% annually. 7 In 2012, melanoma was the fifth most frequent solid tumor entity in Germany. The incidence rate according to the age-standardization rose from 12.4/100,000 to 19.2/100,000 between 1999 and 2012, representing an increase of approximately 55%. 3 In this publication we reported an increase in incidence of melanoma from 12.7/100,000 to 19.2/100,000 in men and from 12.1/100,000 to 19.2/100,000 in women, which represents an average annual increase of +3.1% and +3.5% in men and women, respectively. There was a marked annual increase of 10.9% for both sexes between 2006 and 2009, that coincided with the period when the nationwide skin cancer screening program was introduced. 3 An extrapolation of the data until 2030 shows that the age-standardized incidence rates will continue to increase. In Germany, the expected incidence rates are 31/100,000 and 30/100,000 for men and women, corresponding to a relative increase of about 60% for both sexes. 3 Introduction 21 The last IARC report shows that in 2018 in Portugal, the age-standardized incidence rate (World) per 100,000 for both sexes, all ages and for all cancers was 259,5. 8 For melanoma, the rate was 6.4 compared to 21.6 in Germany, in the same report. 2.2. Biology of melanoma 2.2.1. Melanoma subtypes 2.2.1.1. Cutaneous Melanoma The two most common subtypes of cutaneous melanoma concern the 1) superficial spreading melanoma (SSM) in about 57% of cases, and the 2) nodular melanoma (NM) in about 21% of cases. Other less frequent melanoma subtypes are 3) the lentigo maligna melanoma (LMM) in about 9% of cases, occurring mainly in chronically sun-exposed skin of older patients, and the 4) acral-lentiginous melanoma (ALM) in about 4% of cases, which can be located in fingers, toes, palms and soles, and is associated with a poorer prognosis. 9-12 There is robust evidence that cutaneous melanoma is associated with the intermittent exposure to ultraviolet (UV) radiation and history of sunburns early in life, namely throughout childhood and adolescence; however, the risk appears to be present regardless the age group. 13-15 The change in leisure and holiday patterns in the later decades, and also the change on the type of protective clothes used while sunbathing, resulted in significantly increase to UV exposure. This is the main reason for the global increase of this tumor entity. 16,17 Approximately 6% of all diagnosed melanomas occur in body regions that have little exposure to UV radiation. Contrary, the majority of melanomas (94%) are located in body Introduction 22 regions frequently or intermittently exposed to UV radiation, such as the face, chest, back, arms and legs. 9,18 The development of melanomas as its correlation with UV exposure has been shown previously by several groups. In 2010 the whole spectrum of somatic mutations in the entire genome of a melanoma metastasis was catalogued for the first time. 19 It was shown that about 70% of the detected single base substitutions were of the type C-T and also around 70% of the dinucleotide substitutions were of the type CC-TT. It is largely known that these are "signature mutations" for exposure to UV radiation, and therefore, this finding represents an important proof of the connection between the development of melanoma and UV radiation exposure. Due to this relation between UV radiation exposure and cutaneous melanoma, this subtype of melanoma belongs to the human malignancies with the highest tumor mutational burden. 20 The high tumor mutational burden has been associated with response to immunotherapy in melanoma and other solid tumors. 21,22 2.2.1.2. Mucosal Melanoma About 55% of the mucosal melanomas are diagnosed in the head and neck area, 24% the anorectal region and 21% the genital tract. 23-27 This type of melanoma is usually diagnosed in later stages, which might contribute to the less favorable prognosis. The lower expression of the programmed death ligand 1 (PD-L1) and overall lower tumor mutational burden, may also contribute to the lower response rates to immune checkpoint inhibitors. 28-31 Introduction 23 2.2.1.3. Uveal Melanoma Uveal melanomas have a different metastatic pattern from cutaneous melanomas. Since the eye has no lymphatic system, almost all uveal melanoma metastases are found directly in the liver via hematogenic spread. This particular pattern of metastatic spread turns these patients into perfect candidates for liver directed therapeutic approaches including surgical resection, trans-arterial chemoembolization (TACE), 32-35 and selective internal radiotherapy (SIRT). 36-38 Both methods, TACE and SIRT, achieve good response rates of up to 57% and 63%, respectively. Similar to mucosal melanoma, the response rates to immune checkpoint inhibitors are considerably lower than for cutaneous melanoma. 39-42 However, combined immunotherapy has been shown to be a safe and more effective therapeutic option for these patients, when compared to chemotherapy or PD-1 monotherapy. 43 2.3. The 8th edition of the AJCC classification Melanoma, like other solid tumors, is staged according to the TNM classification. The new TNM classification was published in 2017 by the American Joint Committee on Cancer (AJCC). 44 The changes introduced affected mostly the classification of stage III melanoma with a new sub-stage IIID. Considering that adjuvant therapy has become available and approved in stage III as well, a very short discussion on the implications of these new classification is of importance. The 8th edition of the AJCC melanoma classification displays what can be defined as a very favorable outcome for stage III patients. In fact, with these outcomes, the discussion arose whether there was the need to treat all stage III patients. It was particularly striking that stage IIC had worse prognosis than stage IIIA. Several authors addressed this aspect, and our group did that as well. 45-47 By evaluating three different datasets of Introduction 24 untreated stage III patients, i.e., patients from the placebo arm of the European Organisation for Research and Treatment of Cancer (EORTC) 18991 and 18071 trials, and from a Tuebingen cohort, we have discussed and pointed out that, the survival rates presented in the new AJCC classification might be overrated. 48 This has implications in both trials’ design and in indication for systemic treatment. We advocated that all stage IIIB and IIIC patients should receive systemic therapy, and that for stage IIIA patients, the decision should be individualized. There were not many stage IIIA patients included in the clinical trials evaluating systemic therapy, and the prognosis is considerably favorable. As for stage IV, a new substage - M1d - was introduced for patients with brain metastases, recognizing that these patients, when left untreated, have a different and worse prognosis compared with other stage IV patients. The M1d substage includes patients with brain metastases with or without metastases in other organs. The M1c category no longer includes patients with brain metastases, and elevated lactate dehydrogenase (LDH) no longer defines M1c category. LDH level is defined by using a suffix next to the M category: normal LDH is identified as 0 (zero) and elevated LDH is identified as 1 (one). No suffix is used if the LDH value is not recorded or is unspecified. The particular subgroup of M1d patients is one of the focus of this thesis (Manuscripts 5 and 6). Table 1 displays a very simplified summary of the TNM classification for cutaneous melanoma. Introduction 25 Table 1: Overview of the TNM classification for cutaneous melanoma Stage Criteria I Tumor thickness up to 2mm without ulceration no metastases II Tumor thickness > 1mm up to 2mm with ulceration Any tumor with a tumor thickness of more than 2mm no metastases III Any tumor thickness with metastases in nearby skin areas or lymph nodes IV Any tumor thickness with metastases in distant skin areas, lymph nodes or organs (e.g. liver, lung, brain, ...) 2.4. Systemic therapy of advanced melanoma The landscape of the systemic therapy in melanoma has changed considerably in the last 10 years. For decades, different combinations of chemotherapy have been investigated in advanced melanoma, but these therapies produced very marginal benefits. 49 Until the introduction of immune checkpoint inhibitors (ipilimumab, nivolumab and pembrolizumab), and targeted therapies with BRAF and MEK inhibitors (vemurafenib plus cobimetinib, dabrafenib plus trametinib and encorafenib plus binimetinib) in the therapeutic arsenal, there was no effective therapy for patients diagnosed with advanced melanoma. Targeted therapies can be offered to patients whose melanomas carry a BRAFV600 mutation, and immune checkpoint inhibitors can be used to treated both BRAFV600 mutated and BRAF wild-type melanomas. Figure 1 depict a simplified timeline of the systemic therapies currently available and their timing of re-imbursement in the last decade in Portugal and Germany. Introduction 32 Based on the results from these trials, nivolumab and pembrolizumab were approved by the FDA and the EMA for the treatment of inoperable metastatic melanoma. 86,87 Over the years, the dosing regimen of nivolumab and pembrolizumab has been simplified, and instead of a weight-based dose, similar to chemotherapy, a flat dose of 240mg every 2 weeks or 480mg every 4 weeks is now used for nivolumab. Pembrolizumab can be administered every 3 weeks (200 mg) or every 6 weeks (400mg). The results achieved with flat dose and longer intervals were similar to the weight-based dose and shorter intervals. 88,89 Introduction Table 2: Summary of results from the trials investigating PD-1 based immunotherapy in advanced melanoma 1 Study Immune checkpoint inhibitors CHECKMATE KEYNOTE 067 037 066 001 002 006 Agent(s) Nivo + Ipi Nivo Nivo Nivo Pembro Pembro Pembro (all pts) (tx naive) (2mg/kg) (10mg/kg) (Q2W) (Q3W) Patients, n (study arm) 314 316 272 210 655 151 180 181 279 277 BRAF mutant, % 32 31 22 0 24 --- 24.4 22.1 35 35 ECOG ≥ 1, % 26 25 40 28.6 --- --- 44.4 44.8 30 32 M1c, % 58 58 75 61 8 --- 82.2 82.3 64 68 LDH > ULN, % 48 47 52 37.6 38 --- 43.3 39.8 29 35 Follow up, months >60 >60 24 38.4 55 55 28 28 45.9 Median OS, months NR 36.9 15.7 37.5 23.8 38.6 13.4 14.7 32.7 1-yr OS, % 73 74 58.9 --- --- --- 53.7 55.6 --- 2-yr OS, % 64 59 38.7 --- --- --- 35.9 38.2 55.2 3-yr OS, % 58 52 --- 31.2 --- --- --- --- 48.1 4-yr OS, % 53 46 --- --- 38 48 --- --- 42.3 5-yr OS, % 52 44 --- --- 34 41 --- --- 38.7 Introduction 34 Median PFS, months 11.5 6.9 3.1 5.1 8.3 16.9 2.9 3.0 8.4 1-yr PFS, % 50 43 --- --- --- --- 22.1 27.9 --- 2-yr PFS, % 41 37 --- --- --- --- 16 22 32.7 3-yr PFS, % 39 32 --- 32.2 --- --- --- --- 28.8 4-yr PFS, % 37 31 --- --- 25 35 --- --- 23 5-yr PFS, % 36 29 --- --- 21 29 --- --- --- ORR, % 58 44.6 27 42.9 41 52 22 28 42 CR/PR, % 22/36 19/26 27 40/50 16/25 25/27 --- --- 13/29 Median DOR, months NR NR 31.9 NR --- NR 22.8 NR NR Related AEs, % 96 87 77 77.7 86 --- 56.7 59.2 79 Discontinuation due to AE % 31 8 15 8.7 8 --- 13.5 16.2 10 CTCAE grade 3/4 AEs, % 59 23 11 15 17 --- 3.3 6.1 17 17 NR= not reached 2 Introduction 2.4.1.3. Nivolumab plus ipilimumab Combination of nivolumab and ipilimumab is the current SOC for stage IV melanoma. This combination was investigated in a prospective, randomized phase 3 study, the CheckMate 067 study, recently updated with survival data after 5 years of follow-up. 78,90 This study compared nivolumab plus ipilimumab with nivolumab monotherapy and ipilimumab monotherapy in treatment-naïve patients. Although this trial was not powered to detect a difference between the combination therapy and the monotherapies, the consecutive survival updates have shown that the difference in terms of OS rates between nivolumab plus ipilimumab and the monotherapy arms have been consistently increasing. The 2-y, 3-y, 4-y, and 5-y OS rates difference between nivolumab plus ipilimumab and nivolumab alone are 5%, 6%, 7% and 8%, respectively. 78,91,92 In the subgroup of patients with BRAFV600 mutation receiving combined immunotherapy or nivolumab monotherapy, the difference in the 5-y OS rates is even more notorious (60% versus 46%). The authors of the Checkmate 067 also evaluated the subsequent therapies received by the patients included. The time until receiving another therapy is an important aspect, not only in terms of survival but also in terms of quality of life. The median time from trial inclusion to subsequent systemic therapy was more than 60.0 months (median not reached) in the nivolumab plus ipilimumab group, 25.2 months in the nivolumab group, and 8.0 months in the ipilimumab group. Besides the time to subsequent systemic therapy, another aspect evaluated was the median treatment-free interval. Here, patients treated with combined immunotherapy also derived greater benefit compared to those receiving nivolumab or ipilimumab monotherapy; 18.1 months versus 1.8 months and 1.9 months, respectively. This was mirrored in the percentage of patients who were not Introduction 36 receiving subsequent systemic at the time of the 5-y follow-up survival analysis: 74% in the combination arm, 58% in the nivolumab arm, and 45% in the ipilimumab arm. 78 The superior results of the combination therapy were, however, associated with a higher toxicity. Common Terminology Criteria for Adverse Events (CTACE) grade 3-4 treatment-related adverse events were seen in 59% of the patients receiving nivolumab plus ipilimumab, in 22% of the patients receiving nivolumab and in 28% of those treated with ipilimumab. 92 Taking that into consideration, the Checkmate 511 trial investigated whether a lower dose of ipilimumab (1mg/kg) combined with a higher dose of nivolumab (3mg/kg) could derive the same efficacy benefits as the standard dose, while resulting in lower toxicity rates. 93 With a lower dose of ipilimumab, the CTCAE grade 3-4 toxicity was reduced by half with a comparable efficacy. The follow-up in this study is however shorter than in the Checkmate 067 (18 months versus 60 months), and longer follow-up is required to confirm these results. 93 2.4.2. Targeted therapy with BRAF and MEK inhibitors Approximately 40 to 60% of cutaneous melanomas harbor mutations in BRAF that lead to constitutive activation of downstream signaling through the MAPK pathway. 94 Approximately 90% of these mutations result in the substitution of glutamic acid for valine at codon 600 (BRAFV600E), although other activating mutations are known (e.g., BRAFV600K and BRAFV600R). Vemurafenib, was the first selective BRAFV600 inhibitor to be investigated in the treatment of advanced melanoma. 95 The BRIM-3 trial was a phase 3 randomized clinical trial that compared vemurafenib with the SOC dacarbazine in patients with previously untreated, metastatic melanoma with the BRAFV600E Introduction 37 mutation. The results showed that vemurafenib was superior to dacarbazine in terms of prolonging PFS and OS. Dabrafenib is another BRAF inhibitor investigated at the same time as vemurafenib with similar results in terms of efficacy. 96 The difference between the both BRAF inhibitors is mainly related with the toxicity profile. Dabrafenib induces almost no photosensitivity compared to vemurafenib (41%), fewer keratoacanthomas and squamous cell carcinomas (7% versus 20-30%). Arthralgia (56%), fatigue (46%) and rash (41%) were commonly reported with vemurafenib treatment. 95 On the other hand, pyrexia is the most common adverse event associated with dabrafenib treatment - almost 50% of the patients reported pyrexia that led to treatment interruption. Treatment with BRAF inhibitors monotherapy induces high response rates but resistance supersedes shortly after. 95,97,98 Combination with another MAPK inhibitor, in this case a MEK inhibitor, is one of the ways to overcome the resistance and short duration of response of monotherapy with BRAF inhibitors. 99 Three different BRAF/MEK combinations are currently available for patients with advanced melanoma. These combinations were investigated in randomized phase III trials, and compared with BRAF inhibitors monotherapy showing improved survival outcomes in BRAFV600 mutated melanoma. The combination of vemurafenib plus cobimetinib was investigated in the coBRIM trial, dabrafenib plus trametinib was investigated in the COMBI-d and COMBI-v study, and encorafenib plus binimetinib was investigated in the COLUMBUS study. 100-103 Recently, a pooled analysis evaluating the survival of BRAFV600 mutated patients treated with BRAF/MEK inhibitors in the COMBI-d and COMBI-v trials showed that, with a median follow-up of 5-y, the OS rate was 34%. A complete response was observed in Introduction 38 19% of the patients, and in this subgroup the 5-y OS rate was 71% (95% CI, 62 to 79). 104 These results show that if targeted therapy is chosen to treat patients with BRAFV600 mutated melanoma, combined targeted therapy and not monotherapy should be used. Since the efficacy and survival outcomes are very similar in with the three combinations of BRAF/MEK inhibitors, the combination chosen is mostly related with the safety profile that differs between them. Our recently published indirect analysis showed a non-significant risk reduction for progression and death in the subgroup with elevated baseline LDH receiving vemurafenib plus cobimetinib, compared with dabrafenib plus trametinib and encorafenib plus binimetinib. Therefore, in this subgroup of patients, combination of vemurafenib plus cobimetinib might be considered. 105 Table 3 provides a summary of the survival outcomes in trials investigating targeted therapy in BRAFV600 mutated melanoma patients. Table 3: Summary of results from the trials investigating targeted therapy in advanced melanoma Study Combination targeted therapy COMBI-d COMBI-v CoBRIM COLUMBUS Agent(s) D + T D + T V + C E + B Patients, n (study arm) 211 352 247 577 (Part1) 258 (Part2) ECOG ≥ 1, % 27 29 24 29 27 M1c, % 67 63 59 64 67 LDH > ULN, % 36 34 46 29 31 Follow up, months ≥ 36.0 23 21.2 36.8 Median OS, months 25.1 26.1 22.5 33.6 1-yr OS, % 74 73 74.5 76 2-yr OS, % 52 53 49.0 58 Introduction 39 3-yr OS, % 44 45 38.5 47 4-yr OS, % 37 34.7 39 5-yr OS, % 34 --- --- Median PFS, months 11.0 12.1 12.3 14.9 12.9 1-yr PFS, % --- --- --- 56 2-yr PFS, % 30 30 --- 37 3-yr PFS, % 22 24 --- 29 4-yr PFS, % 21 --- 25 5-yr PFS, % 19 --- --- ORR, % 69 67 70 64 66 CR/PR, % 16/53 19/48 16/54 13/51 8/58 Median DOR, months 12 13.8 13.0 18.6 12.7 Related AEs, % 97 99 99 98 98 Discontinuation due to AE % 14 16 13 15 12 CTCAE grade 3/4 AEs, % 48 57 77 64 47 D+T= Dabrafenib + Trametinib; V+C= Vemurafenib + Cobimetinib; E+B= Encorafenib + Binimetinib. 2.5. Systemic therapies in melanoma brain metastasis Patients with MB pose a particular therapeutic challenge, and have a worse prognosis compared to stage other IV patients. This has been acknowledged in the new AJCC classification, that included patients with MBM in a particular subgroup – M1d. 44 This particular subgroup of patients and their outcomes to systemic therapy have been evaluated separately in this thesis (Manuscripts 5 and 6). The studies evaluating systemic therapy in stage IV melanoma patients have systematically excluded patients with brain metastases. In fact, the presence of active MBM is an Introduction 40 exclusion criterion for the great majority of phase III clinical trials, regardless the tumor entity. Trials specifically investigating immunotherapy and targeted therapy in patients with MBM have shown that these therapies are also effective intracranially, and that the intracranial response rate is similar to the extracranial response. 106-109 Currently, there is evidence that PD-1-based immunotherapy, and particularly combined immunotherapy with nivolumab and ipilimumab might be more effective than BRAF/MEK inhibitors. 110,111 For patients with MBM, the combination of local and systemic therapies has long been investigated. Retrospective data show that the patients receiving a combination of local therapy and systemic therapy have better outcomes when compared to patients who do not receive local therapy. 112-119 The timing of the local therapy, i.e., up-front or later in the course of the disease, doesn’t seem to be statistically significant. However, local therapies given up-front seem to derive better outcomes. 120-123 There is still ongoing debate whether some patients might be better served with systemic therapy alone, considering the very positive outcomes seen in clinical trials. Not applying local therapy reduces local complications, potential cognitive impairment, and might be particularly adequate for patients with a low number of asymptomatic MBM. This question along with the best sequence regarding local and systemic therapy will be addressed in ongoing/planed clinical trials. 124,125 2.6. Access to systemic therapies approved in the advanced setting in Portugal and Germany Despite the positive results of systemic therapy in advanced melanoma patients, these therapies are not available for all patients at the same time. In Germany, therapies that Introduction 41 are approved by EMA become available and reimbursed in the clinical practice immediately after their approval. In other countries, namely in Portugal, the process is different (see Figure 1). The asymmetric re-imbursement process precludes access to effective therapy. Particularly in melanoma, this asymmetric access to systemic therapies was investigated in 2017. The authors of this work reported that, at the time, more than 5000 patients with metastatic melanoma per year in Europe did not have access to recommended first-line innovative treatments. This obviously translates into pourer survival outcomes in patients from countries that do not have access to innovative therapies. In Europe, the European Society of Medical Oncology Magnitude of Clinical Benefit Scale working group (ESMO-MCBS WG), of which I am current member, has addressed this topic. The evolving classification has been available for the last five years, and using it one can easily identify and define whether the therapies available in a defined setting should or shouldn’t be reimbursed, based in criteria such as improved survival outcomes, toxicity, and quality of life. 126-128 The progress in systemic therapy of advanced melanoma, which is the topic of this thesis, is only important in the extent that it is available for all patients who could benefit from it. 129 Patients and methods 48 square test and logistic regression, as appropriate. Results were reported as two-sided p values with 95% CIs. All p-values presented are two-sided tests of statistical significance at 0.05. All analyses were carried out with SPSS statistics version 23.0 (IBM) or GraphPad Prism version 5.01 (GraphPad Software). For manuscript 6, estimates of survival rates, OS and follow-up time were calculated considering the date of melanoma brain metastases diagnosis, and last patient contact or death. Kaplan-Meier estimates were used for the calculation of OS. Differences between groups were assessed using the log-rank test. When appropriate, multivariate logistic regression models were used to examine associations. Results were reported as two-sided p values with 95% CIs. All p-values presented are two-sided tests of statistical significance at 0.05. All statistical analyses were performed using SPSS v.25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp). Results 49 5. Results For the elaboration of this thesis, 6 original manuscripts were included, and will be presented in dedicated separate sections. To evaluate the long-term outcomes in patients with stage IV melanoma treated with immunotherapy and targeted therapy between 2011-2019 (Work project 1), the following manuscripts were considered: • Improvement of overall survival in stage IV melanoma patients during 20112014: analysis of real-world data in 441 patients of the German Central Malignant Melanoma Registry (CMMR) • Primary Resistance to PD-1-Based Immunotherapy - A Study in 319 Patients with Stage IV Melanoma • Indirect Comparison of Combined BRAF and MEK Inhibition in Melanoma Patients with Elevated Baseline Lactate Dehydrogenase • Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study Results 50 For evaluating the impact in survival outcomes of immunotherapy and targeted therapy, and their combination with local therapies in patients with melanoma brain metastases (Work project 2), the following manuscripts were considered: • Immunotherapy plus surgery/radiosurgery is associated with favorable survival in patients with melanoma brain metastasis • Combined immunotherapy with nivolumab and ipilimumab with and without local therapy in patients with melanoma brain metastasis: a DeCOG* study in 380 patients For all work projects as first author, Teresa Amaral, led the design of the study, performed the data analyses, interacting with biostatisticians when needed, performing data analysis, interpreting results, and writing all the manuscripts that are presented in this thesis. For all the work projects as co-author, Teresa Amaral was involved in the design of the study and in the data analysis, interpreted and wrote the results, and co-wrote the manuscript. Manuscripts 6. Manuscripts Manuscript 1 52 1. "Improvement of overall survival in stage IV melanoma patients during 2011-2014: analysis of real-world data in 441 patients of the German Central Malignant Melanoma Registry (CMMR)." Forschner, A., F. Eichner, T. Amaral, U. Keim, C. Garbe and T. K. Eigentler (2017). J Cancer Res Clin Oncol 143(3): 533-540. Manuscript 1 53 1 3 J Cancer Res Clin Oncol (2017) 143:533–540 DOI 10.1007/s00432-016-2309-y ORIGINAL ARTICLE – CLINICAL ONCOLOGY Improvement of overall survival in stage IV melanoma patients during 2011–2014: analysis of real-world data in 441 patients of the German Central Malignant Melanoma Registry (CMMR) Andrea Forschner1 · Felizitas Eichner1,2 · Teresa Amaral1,3 · Ulrike Keim1 · Claus Garbe1 · Thomas Kurt Eigentler1 Received: 28 October 2016 / Accepted: 17 November 2016 / Published online: 22 November 2016 © Springer-Verlag Berlin Heidelberg 2016 targeted therapy had a better OS (median 14 months) than patients with ipilimumab treatment (median 7 months). Among all patients with first-line systemic treatment, outcome of patients diagnosed in the years 2013/2014, compared to 2011 and 2012, showed an improved survival. Three-year OS for patients that entered stage IV in 2013/2014 was 37% compared to those that entered stage IV in 2011 (18%) and 2012 (20%). Conclusion The analysis of real-world data of treatment of metastatic melanoma showed an improvement of OS with both immunotherapy and targeted therapy. In case of cerebral metastasis, patients treated with targeted therapy showed a longer median OS than patients treated with ipilimumab. Keywords Melanoma · Survival · Checkpoint inhibitors · Targeted therapy · Chemotherapy · Pembrolizumab · Nivolumab · Ipilimumab · Brain metastasis Introduction Systemic treatment of metastasized melanoma has been disappointing for decades. Chemotherapy with dacarbazine or carboplatin and paclitaxel resulted in median survival times of 7–9 months, and for a long time, no other treatment regime was found to prolong survival in advanced metastatic melanoma patients (Dummer et al. 2012; Eigentler et al. 2003; Pflugfelder et al. 2011; Tsao et al. 2004). At that time, one-year overall survival (OS) of stage IV melanoma patients was about 25–30% (Balch et al. 2009). It is impressive to see that in the last 5 years 1-year OS for stage IV patients reported in clinical trials has risen to over 70%. This dramatic improvement is due to the Abstract Background During 2011 and 2014, new treatment modalities like tyrosine kinase inhibitors and checkpoint inhibitors were introduced into the therapy of metastatic melanoma. This study addresses the question whether overall survival (OS) of metastatic melanoma patients has already been improved in 441 patients diagnosed with metastatic melanoma between 2011 and 2014 in the real-world setting at the University Hospital Tuebingen. Methods All patients were documented with their different therapies by the CMMR and followed up until March 2016. Survival probabilities were calculated by Kaplan–Meier estimators, and log-rank tests were used to evaluate significances. Hazard ratios were estimated by Cox regression analysis for survival probabilities and prognostic factors in stage IV melanoma. Results Best OS was observed in patients (n = 93) treated by metastasectomy as primary treatment with the intention to completely excise all metastases (3-year OS 61%). OS for patients with first-line systemic treatment (n = 258) was unfavorable in general (3-year OS 23%). Of those, the most favorable outcome was observed in patients without brain metastasis and treated with immunotherapy (mostly ipilimumab), as first-line treatment (median OS 35 months, 3-year OS 43%). In case of brain metastases, patients with * Andrea Forschner [email protected] 1 Department of Dermatology, University Medical Center Tübingen, Liebermeisterstr. 25, 72076 Tübingen, Germany 2 Graduate School of Life Sciences, Utrecht University, Utrecht, Netherlands 3 Portuguese Air Force Health Direction, Paço do Lumiar, 1649-020 Lisbon, Portugal Manuscript 1 54 534 J Cancer Res Clin Oncol (2017) 143:533–540 1 3 availability of multiple new drugs. So, it has become possible to inhibit the mitogen-activated protein kinase (MAPK) pathway in BRAF V600-mutant melanoma selectively by using BRAF/MEK inhibitors. Furthermore, checkpoint inhibitors enable an increase in host T cell response against tumor cells. Monotherapy with BRAF inhibitors led to an increase in the median progression-free survival (PFS) of about 5 months and 1-year OS reached 68% (Chapman et al. 2011; Hauschild et al. 2012). Combinations of BRAF and MEK inhibitors prolonged median PFS to even ~10 months and improved 1-year OS to 74% (Larkin et al. 2014; Long et al. 2014). Concerning checkpoint inhibitors, treatment with ipilimumab resulted in a 1-year OS of 46% (Robert et al. 2011), whereas treatment with PD-1 inhibitors showed a one-year OS of ~70% (Robert et al. 2015a, b). These new treatment options have significantly improved the prognosis of metastasized melanoma patients—but mainly in clinical trials with selected cohorts. For patient care, it is even more important to assess their efficacy in the real-world setting. To determine whether these new drugs can provide a survival benefit for patients in a real-world setting also, we performed an OS analysis of our patients suffering from advanced melanoma who entered stage IV between 2011 and 2014 and received treatment at the University Hospital Tuebingen. Methods Our analysis was performed on prospectively collected data of stage IV melanoma patients entered into the Central Malignant Melanoma Registry (CMMR). Routinely, all melanoma patients of our hospital are registered in the CMMR. Informed consent was obtained from all patients included in this study. Captured data include general information like date of birth, sex, origin and date of death, if applicable. In addition, the CMMR provides melanomaspecific variables such as localization, size, histological type, Breslow’s tumor thickness and Clark level. For stage IV patients, 61 additional variables are documented including localization of metastases, BRAF, KIT and NRAS mutation status, treatment lines, period of treatment, treatment regimens, best response, dose modifications and type of outcome. Adverse events or serious adverse events are not captured. Data are entered into the database each time the patient visits the hospital for a treatment cycle, surgery or radiotherapy. Phone contact with the patient, the family or external treating practitioners is included into the patient file if the patient had no contact with hospital for an extended amount of time. Statistical analysis was performed using the statistical program for social sciences SPSS version 23 (IBM, New York, USA). Survival probabilities and median survival with 95% confidence intervals (CI) were estimated according to the Kaplan–Meier method with the time depicted in months. A log-rank test was performed to reveal possible differences between the groups, and p-values < 0.05 were considered statistically significant. Afterward, a Cox regression model was fitted to obtain hazard ratios (HR) and their 95% confidence intervals. Follow-up time was defined from the date entering stage IV to the date of last known contact or death. This study was approved by the local ethics committee of the University of Tuebingen (reference number 676/2016BO2). Results In September 2016, the CMMR comprised a total number of over 13,900 patients with invasive melanomas born between 1884 and 2007, who were treated at the University Hospital Tuebingen. The selected patient cohort for these analyzed consisted of 187 women and 254 men. Followup ranged from 10 days to 61 months with a median follow-up time of 14 months. The median age at time point of advanced disease was 59 years [19Y–96Y, interquartile range 48Y–72Y]. When entering stage IV, 105 patients (23.8%) had cerebral metastases, 256 patients (58%) lung metastases and 138 patients (30%) liver metastases. OS between patients with metastases only to distant skin or lymph nodes (M1a, n = 53), metastases of the lung (M1b, n = 75) and metastases of other organs or increased LDH (M1c, n = 313) showed a significant survival difference (p < 0.0001, Fig. 1). One-year OS was best for M1a patients with 86.6%, followed by M1b patients with 74.3% and M1c patients with 51.6% (Table 1). In the first-line situation, patients treated surgically (n = 93) had a significantly improved OS over patients treated systemically (n = 258) (p < 0.0001, Fig. 2). Furthermore, patients whose metastases could be removed completely by surgery had the most favorable prognosis and a 1-year OS of 76.8% compared to 57.5% of systemically treated patients (Table 1). Of the 258 patients who received first-line systemic treatment, 47% were BRAF wild type and 41% were BRAF mutated. In 12% of the cases, the mutation status was not determined. Of them, 37% suffered from uveal melanoma and the others were diagnosed with stage IV in 2011 when BRAF inhibitors were not available outside of studies in Germany. Figure 3 shows a comparison of OS for the different systemic treatment options. Sixty-eight patients received targeted therapy, 52 patients immunotherapy and 132 patients chemotherapy. Six patients were excluded from this analysis because they were treated in Manuscript 1 55 535J Cancer Res Clin Oncol (2017) 143:533–540 1 3 a blinded clinical trial. Twelve of the 68 patients (17.6%) with targeted therapy were treated by a combination of BRAF and MEK inhibitor, seven (10.3%) received MEK inhibitor only and 49 (72%) received BRAF inhibitor monotherapy. Forty-two of the 52 (80.8%) patients with immunotherapy received ipilimumab and five (9.6%) patients PD-1 antibodies. The five remaining patients were treated with the bispecific antibody L19 IL2 in clinical trials, three of them in combination with dacarbazine. We calculated a significant difference between the treatments options (p = 0.003). Best OS was detected in patients treated with immunotherapy. For these patients, median OS was 33 months, compared to 16 months for patients receiving targeted therapy and 11 months for patients in the chemotherapy group (Fig. 3; Table 1). Fig. 1 Kaplan–Meier survival curves by stage M1a, M1b and M1c, p < 0.0001 Table 1 Overall survival in months of subgroups of stage IV melanoma patients Subgroup Median OS [months] (95% CI) One-year OS [%] (95% CI) Two-year OS [%] (95% CI) Three-year OS [%] (95% CI) HR (95% CI) Stage M1a (n = 53) Not reached 86.6 (77.4–95.8) 72.1 (59.6–84.6) 59 (43.3–74.7) 1 Stage M1b (n = 75) 26 (14.4–37.6) 74.3 (64.3–84.3) 53.6 (41.8–65.4) 41.7 (28.6–54.8) 1.75 (1.02–3.03) Stage M1c (n = 313) 12 (9,8–14.2) 51.6 (46.1–57.1) 31.9 (26.6–37.2) 24 (18.7–29.3) 3.05 (1.91–4.87) First-line surgery (n = 93) Not reached 76.8 (68.3–85.5) 66.8 (56.8–76.8) 61.1 (50.1–72.1) 0.36 (0.25–0.53) First-line systemic therapy (n = 258) 15 (12.1–18) 57.5 (51.4–63.6) 35.8 (29.7–41.9) 23.4 (17.1–29.7) 1 First-line targeted therapy (n = 68) 16 (10.6–21.4) 64.7 (53.3–76.1) 36.3 (24.5–48.1) 27.6 (15.8–39.4) 0.73 (0.52–1.03) First-line immunotherapy (n = 52) 33 (21.7–44.3) 67.1 (54.4–79.8) 60.3 (46.6–74.0) 37.4 (16.6–58.2) 0.5 (0.32–0.77) First-line chemotherapy (n = 132) 11 (7.6–14.4) 49.6 (41–58.2) 26.9 (19.1–34.7) 15.9 (8.8–23) 1 Targeted therapy + brain metastases (n = 24) 14 (5.4–22.6) 58.3 (38.5–78.1) 28.6 (10.2–47) 15.9 (17.2–33.1) 1 Targeted therapy no brain metastases (n = 44) 17 (12.1–21.9) 68.2 (54.5–82) 40.8 (25.7–55.9) 34 (18.7–49.3) 0.75 (0.42–1.35) Immunotherapy + brain metastases (n = 10) 7 (0–17.9) 50 (19–81) 40 (9.6–70.4) 1 Immunotherapy no brain metastases (n = 42) 35 (25.5–44.5) 71.2 (71.1–71.3) 65.4 (65.3–65.6) 43.1 (43.3–42.9) 0.38 (0.15–0.94) Chemotherapy + brain metastases (n = 23) 9 (4.3–13.7) 26.1 (26–26.3) 1 Chemotherapy no brain metastases (n = 109) 14 (10.3–17.7) 54.7 (54.6–54.8) 32.9 (32.8–33) 19.4 (19.3–19.5) 0.45 (0.28–0.72) 2011 stage IV diagnosis (n = 68) 12 (8.3–15.7) 51.5 (51.4–51.6) 30.9 (30.8–31.0) 17.6 (17.5–17.7) 1 2012 stage IV diagnosis (n = 70) 13 (8.6–17.4) 58.2 (58.1–58.3) 32.2 (32.1–32.3) 19.7 (19.6–19.8) 0.82 (0.61–1.11) 2013/2014 stage IV diagnosis (n = 114) 17 (12.8–21.2) 60.2 (60.1–60.3) 41.6 (41.5–41.7) 37.2 (37.1–37.3) 0.79 (0.6–1.04) Manuscript 1 56 536 J Cancer Res Clin Oncol (2017) 143:533–540 1 3 In the group of patients receiving targeted therapy, 24 of 68 (35.3%) had brain metastases, but only ten of 52 patients (19.2%) were in the immunotherapy group and 23 of 132 patients (17.4%) were in the chemotherapy group. For patients treated with targeted therapy, median OS was 14 months for patients with brain metastases compared to 17 months for patients without brain metastases (Fig. 4; Table 1). Patients who received immunotherapy as first-line treatment had a median OS of 7 months in case of brain metastases compared to 35 months for patients without brain metastases (Fig. 5; Table 1). For more details, please refer to Table 1. OS was significantly different between patients with and without brain metastases who were treated with first-line chemotherapy (p = 0.001; median OS 9 months for patients with cerebral metastases vs. 14 months for patients without brain metastases; Table 1). An illustration of the survival curves of patients treated systemically as first option depending on the year of entering stage IV melanoma is provided in Fig. 6. In 2011, 70.1% of the patients were treated with chemotherapy, 11.8% with immunotherapy and 16.2% with targeted therapy. In 2012, 32.9% of the systemically treated patients Fig. 2 Kaplan–Meier survival curves by first-line surgical vs. systemic treatment, p < 0.0001 Fig. 3 Kaplan–Meier survival curves by first-line systemic treatment type, p = 0.003 Manuscript 1 57 537J Cancer Res Clin Oncol (2017) 143:533–540 1 3 received targeted therapy first line, 11.4% immunotherapy and 55.7% chemotherapy, whereas in the years 2013/2014, 32.8% of the patients were treated with targeted therapy, 31.6% with immunotherapy and only 38.6% with chemotherapy. Median OS for patients entering stage IV in 2011 was 12 months, in 2012 13 months and in 2013/2014 17 months. The difference in survival probability between 2012 and 2013/2014 was rather small after 1 year, but increased during longer follow-up. Three-year OS was 19.7% for the 2012 group and 37.2% for the 2013/2014 group (Table 1). Discussion In general, our analyses of real-world treated melanoma patients confirmed findings of clinical trials of novel drugs to improve OS. Not surprisingly, the improvement was less extensive compared to clinical trials, as these normally include selected patients, only. In contrast, in our study all real-life patients were included, who, e.g., suffered from brain metastases, had comorbidities or had a decreased performance status. This approach is important as these are the patients in the daily clinical practice. Fig. 4 Kaplan–Meier survival curves by first-line targeted therapy and cerebral status, p = 0.329 Fig. 5 Kaplan–Meier survival curves by first-line immunotherapy and cerebral status, p = 0.027 Manuscript 2 64 Cancers 2020,12, 1027 3 of 13 time of stage IV diagnosis, localization and number of metastatic organs, type of systemic therapy for stage IV disease and respective start and end dates, response at the first tumor assessment after systemic therapy start, best overall response to systemic therapy, and time of last follow-up or death from any cause. Primary resistance was defined as progressive disease (PD) at the time of first tumor assessment after immunotherapy start. In our center, this is performed after 12 weeks (+/  5 days). This evaluation was performed using RECIST 1.1 [ 31 ]. Patients with CR, PR, and SD were considered to have disease control (DC). Best overall response to first-line immunotherapy was defined as the best response—intracranial and extracranial—that patients achieved during the time they were treated. Taking that into consideration, patients for whom the best overall response was PD were, by definition, patients with primary resistance. These patients did not continue to receive immunotherapy, since the clinical evaluation also determined that they were not deriving benefit from the ongoing therapy. Pseudoprogression was considered for patients who were classified as having PD by RECIST 1.1 [ 31 ] at the time of first assessment after immunotherapy start but, due to clinical benefit, continued receiving immunotherapy and had a response later in the course of their disease. These patients were not considered as primary resistant and were included in the group of disease control. 2.2. Statistical Analysis Statistical analysis was performed using the statistical program for social sciences SPSS Version 25 (IBM, New York, NY, USA). STATA ® v15 (StataCorp LLC, College Station, TX, USA) was used to generate the final version of the Kaplan–Meier survival curves. Descriptive statistical analyses, frequency tables, and chi-square tables were used to characterize the patients’ population. Variables with missing information were excluded from the respective analysis. Follow-up time was defined as the time between the date of stage IV diagnosis and the date of the last follow-up or death from any cause. Survival analyses were performed according to the Kaplan–Meier method. In addition, the 1-, 2-, and 3-year survival rates were calculated with a 95% confidence interval. Factors that were significant in the univariate analysis were included into the multivariate logistic regression analysis. The level of significance was 0.05 (two-sided) in all analyses. The cut-o↵date for data analysis was March 2019. 3. Results 3.1. Univariate and Multivariate Analysis Table 1shows characteristics of the study population: 192 patients (60%) had disease control (SD, PR, CR) and 127 (40%) patients had primary resistance. The median age of the patients at the time of stage IV melanoma diagnosis was 68 years; interquartile range (IQR) (56–77). Age was not associated with primary resistance. Thirty-five patients (11%) had more than 3 organs with metastases at the time of immunotherapy beginning and 292 patients (89%) had 1–3 organs with metastases. Sixty-three (19%) patients had brain metastases and 118 (36%) patients had liver metastases. The number of organs involved, the presence of brain metastases, and the presence of liver metastases were not associated with primary resistance in our analysis. Manuscript 2 65 Cancers 2020,12, 1027 4 of 13 Table 1. Patients characteristics, univariate and multivariate analysis for the whole cohort, according to best overall response to first-line PD-1-based immunotherapy. Characteristics ICI Cohort n=319 n(%) n(%) Univariate Analysis 2Test Cancers 2020, 12, x 4 of 14  Table 1. Patients characteristics, univariate and multivariate analysis for the whole cohort, according to best overall response to first-line PD-1-based immunotherapy. Characteristics ICI Cohort n = 319 n (%) n (%) Univariate Analysis ࣑2 test♣ Multivariate Logistic Regression Analysis Primary Resistance n = 127 (40) DC (CR, PR, SD) n = 192 (60) Age Distribution 0.732 Median (years [IQR]) 68 (56–77) 65 (55–78) 68 (56–77) <60y 101 (32) 37 (29) 64 (33) 60y–75y 114 (36) 47 (37) 67 (35) >75y 104 (32) 43 (34) 61 (32) Sex 0.049 0.822 Male 192 (60) 68 (54) 124 (65) Female 127 (40) 59 (46) 68 (35) Tumor localization * 0.000 0.001 Head and neck 54 (22) 12 (13) 42 (27) Trunk 73 (29) 18 (20) 55 (34) Extremity 109 (43) 54 (59) 55 (34) Other 15 (6) 7 (8) 8 (5) Histological subtype * 0.007 0.452 SSM 76 (32) 31(37) 45 (29) NM 72 (30) 18(21) 54 (35) LMM 13 (6) 0 13 (9) ALM 30 (12) 15 (18) 15 (10) Mucosal 15 (6) 7 (8) 8 (5) Other 32 (14) 14 (16) 18 (12) Stage at initial diagnosis * 0.114 I 48 (17) 19 (18) 29 (17) II 84 (31) 25 (23) 59 (35) III 95 (35) 38 (37) 57 (34) IV 47 (17) 24 (22) 23 (14) Number of organs with metastases 0.098 0.470 1-3 285 (89) 109 (86) 176 (92) >3 34 (11) 18 (14) 16 (8) Brain metastases 0.618 No 258 (81) 101 (79) 157 (82) Yes 61 (19) 26 (21) 35 (18) Liver metastases 0.139 No 204 (64) 75 (59) 129 (67) Yes 115 (36) 52 (41) 63 (33) BRA F mutation * 0.844 BRAFmut 88 (45) 32 (44) 56 (46) BRAFwt 106 (56) 40 (56) 66 (54) LDH level * 0.029 0.532 Normal 190 (68) 67 (60) 123 (73) Elevated 90 (32) 44 (40) 46 (27) S100B level * 0.000 0.008 Normal 157 (56) 44 (40) 113 (65) Elevated 125 (44) 65 (60) 60 (35) * patients with no information available were excluded in the respective analysis; IQR = interquartile range; ♣ Chisquare test performed between the two groups—primary resistance and disease control; ICI = immune-checkpoint inhibitors cohort—145 patients received first-line treatment with nivolumab plus ipilimumab and 174 received antiPD-1 antibodies monotherapy (nivolumab n = 46 and pembrolizumab n = 128).; y = years; SSM = superficial spreading melanoma; NM = nodular melanoma; LMM = lentigo malignant melanoma; ALM = acral lentiginous melanoma; BRAFmut = presence of BRAFV600E/K mutation; BRAFwt = BRAF wild-type; LDH = lactate dehydrogenase; S100B = tumor marker protein S100B. p-values that are statistically significant are noted in bold. In our cohort we had slightly more men (60%) than women (40%). In the univariate analysis, sex was a statistically significant factor associated with primary resistance, with male patients having better outcomes than female patients. In the multivariate logistic regression analysis, sex was not a statistically significant factor. Multivariate Logistic Regression Analysis Primary Resistance n=127 (40) DC (CR, PR, SD) n=192 (60) Age Distribution 0.732 Median (years [IQR]) 68 (56–77) 65 (55–78) 68 (56–77) <60y 101 (32) 37 (29) 64 (33) 60y–75y 114 (36) 47 (37) 67 (35) >75y 104 (32) 43 (34) 61 (32) Sex 0.049 0.822 Male 192 (60) 68 (54) 124 (65) Female 127 (40) 59 (46) 68 (35) Tumor localization * 0.000 0.001 Head and neck 54 (22) 12 (13) 42 (27) Trunk 73 (29) 18 (20) 55 (34) Extremity 109 (43) 54 (59) 55 (34) Other 15 (6) 7 (8) 8 (5) Histological subtype * 0.007 0.452 SSM 76 (32) 31(37) 45 (29) NM 72 (30) 18(21) 54 (35) LMM 13 (6) 0 13 (9) ALM 30 (12) 15 (18) 15 (10) Mucosal 15 (6) 7 (8) 8 (5) Other 32 (14) 14 (16) 18 (12) Stage at initial diagnosis * 0.114 I 48 (17) 19 (18) 29 (17) II 84 (31) 25 (23) 59 (35) III 95 (35) 38 (37) 57 (34) IV 47 (17) 24 (22) 23 (14) Number of organs with metastases 0.098 0.470 1-3 285 (89) 109 (86) 176 (92) >3 34 (11) 18 (14) 16 (8) Brain metastases 0.618 No 258 (81) 101 (79) 157 (82) Yes 61 (19) 26 (21) 35 (18) Liver metastases 0.139 No 204 (64) 75 (59) 129 (67) Yes 115 (36) 52 (41) 63 (33) BRAF mutation * 0.844 BRAFmut 88 (45) 32 (44) 56 (46) BRAFwt 106 (56) 40 (56) 66 (54) LDH level * 0.029 0.532 Normal 190 (68) 67 (60) 123 (73) Elevated 90 (32) 44 (40) 46 (27) S100B level * 0.000 0.008 Normal 157 (56) 44 (40) 113 (65) Elevated 125 (44) 65 (60) 60 (35) * patients with no information available were excluded in the respective analysis; IQR =interquartile range; Cancers 2020, 12, x 4 of 14  Table 1. Patients characteristics, univariate and multivariate analysis for the whole cohort, according to best overall response to first-line PD-1-based immunotherapy. Characteristics ICI Cohort n = 319 n (%) n (%) Univariate Analysis ࣑2 test♣ Multivariate Logistic Regression Analysis Primary Resistance n = 127 (40) DC (CR, PR, SD) n = 192 (60) Age Distribution 0.732 Median (years [IQR]) 68 (56–77) 65 (55–78) 68 (56–77) <60y 101 (32) 37 (29) 64 (33) 60y–75y 114 (36) 47 (37) 67 (35) >75y 104 (32) 43 (34) 61 (32) Sex 0.049 0.822 Male 192 (60) 68 (54) 124 (65) Female 127 (40) 59 (46) 68 (35) Tumor localization * 0.000 0.001 Head and neck 54 (22) 12 (13) 42 (27) Trunk 73 (29) 18 (20) 55 (34) Extremity 109 (43) 54 (59) 55 (34) Other 15 (6) 7 (8) 8 (5) Histological subtype * 0.007 0.452 SSM 76 (32) 31(37) 45 (29) NM 72 (30) 18(21) 54 (35) LMM 13 (6) 0 13 (9) ALM 30 (12) 15 (18) 15 (10) Mucosal 15 (6) 7 (8) 8 (5) Other 32 (14) 14 (16) 18 (12) Stage at initial diagnosis * 0.114 I 48 (17) 19 (18) 29 (17) II 84 (31) 25 (23) 59 (35) III 95 (35) 38 (37) 57 (34) IV 47 (17) 24 (22) 23 (14) Number of organs with metastases 0.098 0.470 1-3 285 (89) 109 (86) 176 (92) >3 34 (11) 18 (14) 16 (8) Brain metastases 0.618 No 258 (81) 101 (79) 157 (82) Yes 61 (19) 26 (21) 35 (18) Liver metastases 0.139 No 204 (64) 75 (59) 129 (67) Yes 115 (36) 52 (41) 63 (33) BRA F mutation * 0.844 BRAFmut 88 (45) 32 (44) 56 (46) BRAFwt 106 (56) 40 (56) 66 (54) LDH level * 0.029 0.532 Normal 190 (68) 67 (60) 123 (73) Elevated 90 (32) 44 (40) 46 (27) S100B level * 0.000 0.008 Normal 157 (56) 44 (40) 113 (65) Elevated 125 (44) 65 (60) 60 (35) * patients with no information available were excluded in the respective analysis; IQR = interquartile range; ♣ Chisquare test performed between the two groups—primary resistance and disease control; ICI = immune-checkpoint inhibitors cohort—145 patients received first-line treatment with nivolumab plus ipilimumab and 174 received antiPD-1 antibodies monotherapy (nivolumab n = 46 and pembrolizumab n = 128).; y = years; SSM = superficial spreading melanoma; NM = nodular melanoma; LMM = lentigo malignant melanoma; ALM = acral lentiginous melanoma; BRAFmut = presence of BRAFV600E/K mutation; BRAFwt = BRAF wild-type; LDH = lactate dehydrogenase; S100B = tumor marker protein S100B. p-values that are statistically significant are noted in bold. In our cohort we had slightly more men (60%) than women (40%). In the univariate analysis, sex was a statistically significant factor associated with primary resistance, with male patients having better outcomes than female patients. In the multivariate logistic regression analysis, sex was not a statistically significant factor. Chi-square test performed between the two groups—primary resistance and disease control; ICI =immune-checkpoint inhibitors cohort—145 patients received first-line treatment with nivolumab plus ipilimumab and 174 received antiPD-1 antibodies monotherapy (nivolumab n=46 and pembrolizumab n=128 ).; y=years ; SSM =superficial spreading melanoma; NM =nodular melanoma; LMM =lentigo malignant melanoma; ALM =acral lentiginous melanoma; BRAFmut =presence of BRAFV600E/Kmutation; BRAFwt =BRAF wild-type; LDH =lactate dehydrogenase; S100B =tumor marker protein S100B. p-values that are statistically significant are noted in bold. In our cohort we had slightly more men (60%) than women (40%). In the univariate analysis, sex was a statistically significant factor associated with primary resistance, with male patients having better outcomes than female patients. In the multivariate logistic regression analysis, sex was not a statistically significant factor. Tumor localization was significantly associated with primary resistance. Tumors of the extremities, including acral melanomas, showed significantly increased primary resistance. Tumor localization remained a significant factor in multivariate logistic regression analysis. The histological subtype was Manuscript 2 66 Cancers 2020,12, 1027 5 of 13 also associated with primary resistance. Primary resistance was found especially in acral lentiginous melanoma, mucosal melanoma, and other melanomas. In the multivariate logistic regression analysis, however, the histological subtype was not a significant factor. Another significant factor in the univariate analysis was an elevated level of the tumor marker protein S-100B, which was associated with a significantly increased primary resistance, both in the univariate and in the multivariate logistic regression analysis. An elevated LDH level was also significantly associated with increased primary resistance in the univariate analysis, but was not significant in the multivariate logistic regression analysis. The following variables were not significant either in univariate analysis or in multivariate analysis: stage at initial diagnosis, number of metastatic organs, presence of brain metastases, presence of liver metastases, and BRAF mutation status. Table S1 shows characteristics of the study population where the primary resistance group includes patients with PD at the first evaluation after starting immunotherapy and patients with SD with a duration of less than 6 months (n=169), and the DC group includes patients with CR, PR, and SD with a duration of more than 6 months (n=190). The results are similar to the ones described above, except that the number of organs with metastases is a statistically significant factor in the univariate analysis and LDH level is no longer statistically significant. 3.2. Survival Analysis The median overall survival (OS) and the 1-year, 2-year, and 3-year OS rates are summarized according to the best response in Table 2. The 2-year OS rates were 96% for patients with CR, 84% for patients with PR, and 64% for patients with SD. Patients with primary PD had a 2-year OS rate of 15%. The corresponding progression-free survival (PFS) rates are summarized in Table 3. Here, 2-year PFS was 81% for CR, 63% for PR, 22% for SD, and 3% for PD. The 2-year OS rate in the disease control (SD +PR +CR) group was 81% versus 15% in patients with primary resistance. As for the 2-year PFS rate it was 56% versus 3%, respectively. Table 2. Median overall survival and overall survival rates for patients receiving first-line PD-1-based immunotherapy according to best overall response and type of immunotherapy. Best Response Median OS (Months; 95% CI) OS (%; 95% CI) 1-Year 2-Year 3-Year CR n=50 (15.7%) not reached 100% 95.7 (87.3–100) 87.7 (70.8–100) PR n=80 (25.1%) not reached 89.5 (82.1–96.9) 84.4 (74.4–94.4) 84.4 (74.4–94.4) SD n=62 (19.4%) 28 (22.9–33.1) 86.3 (77.5–95.1) 63.8 (47.7–79.9) 24.6 (2.6–46.5) PD n=127 (39.8%) 11 (9.0–13.0) 41.3 (31.9–50.7) 14.7 (7.4–22.0) 10.1 (3.4–16.8) DC n=192 (60.2%) not reached 91.3 (87.0–95.6) 81.0 (73.7–88.3) 64.6 (53.2–76) PD-1 monotherapy n=174 (66.2%) 26 (19.7–32.3) 71.1 (64.0–78.2) 53.3 (45.1–61.5) 41.3 (32.1–50.5) PD-1 +CTLA4 n=145 (54.6%) 31 (17.2–44.8) 72.8 (65–80.6) 54.5 (42.9–66.1) 42.5 (24.1–60.9) OS =overall survival; CR =complete response; PR =partial response; SD =stable disease; PD =progressive disease; DC =disease control (CR +PR +SD); PD-1 monotherapy =nivolumab or pembrolizumab; PD-1 +CTLA4 =nivolumab plus ipilimumab. Manuscript 2 67 Cancers 2020,12, 1027 6 of 13 Table 3. Median progression-free survival and progression-free survival rates for patients receiving first-line PD-1-based immunotherapy according to best overall response and type of immunotherapy. Best Response Median PFS (Months; 95% CI) PFS (%; 95% CI) 1-Year 2-Year 3-Year CR n=50 (15.7%) Not reached 87.6 (78.4–96.8) 81.2 (68.9–93.5) 72.2 (52.2–92.2) PR n=80 (25.1%) 37 (14.97–59.03) 74.4 (64.2–85.0) 62.7 (50.0–75.4) 62.7 (50.0–75.4) SD n=62 (19.4%) 12 (8.97–15.03) 43.0 (29.3–56.7) 21.8 (6.3–37.3) - PD n=127 (39.8%) 4 (3.56–4.44) 8.7 (3.8–13.6) 3.2 (0–6.5) 1.1 (0–3.1) DC n=192(60.2%) 33 (20.4–45.6) 68.1 (61.0–75.2) 56.2 (51.8–64.8) 48.7 (37.7–59.7) PD-1 monotherapy n=174 (66.2%) 8 (5.5–10.5) 40.3 (32.7–47.9) 30.5 (23.1–37.9) 24.1 (16.3–31.9) PD-1 +CTLA4 n=145 (54.6%) 9 (1.8–16.2) 48.5 (40.1–56.9) 39 (29.2–78.8) - PFS =progression-free survival; CR =complete response; PR =partial response; SD =stable disease; PD =progressive disease ; DC =disease control (CR +PR +SD); PD-1 monotherapy =nivolumab or pembrolizumab; PD-1 +CTLA4 =nivolumab plus ipilimumab. A statistically significant di↵erence can be seen in OS when patients are classified as having primary resistance or disease control at the time of first tumor assessment after starting immunotherapy (Figure 1A; p<0.0001). The same is true for PFS (Figure 1B; p<0.0001). After three years, a plateau was formed for the group with disease control at a level of 65%, while the PFS rate for primary resistance decreased to 10%. After three years, a certain plateau formation around a 45% PFS rate was also visible in patients with disease control, while in patients with primary resistance, the PFS rate dropped to 1%. The OS curves according to Kaplan and Meier show that in cases of CR and PR, the survival remained largely stable after two years. This was not the case in patients that achieved SD, where there was a relatively steep drop in the survival curve after 18 months, leading to OS rates very close to those in patients with PD (Figure 1C; p<0.0001). In the PFS analysis, there were even clearer di↵erences between CR and PR. After the first year, there was a clear drop in PFS rates for PR compared to CR. After three years, patients with SD had approximately the same survival rates as patients with PD (Figure 1D; p<0.0001). Table S2 shows the patients characteristics for the whole cohort, considering the type of first-line immunotherapy received. One hundred and seventy-four patients received monotherapy with anti-PD-1 (nivolumab or pembrolizumab), while 145 patients were treated with the combination of nivolumab plus ipilimumab. The survival curves for OS overlapped completely, and in our cohort, there was no apparent benefit for the combination treatment (Figure 1E; p=0.993). The survival curves for PFS separated approximately eight months after the start of treatment with a slightly more favorable course for the combined regimen, but this di↵erence was not statistically significant (Figure 1F; p=0.216). In order to evaluate whether primary resistance can be predicted based on pre-existing risk factors, three subgroups were defined, considering the two factors that were significant in the multivariate regression analysis (i.e., primary tumor localization and protein S-100B level) (Figure S1). The subgroups were defined as follows: no risk factor (low-risk), one risk factor (intermediate-risk) and two risk factors (high-risk). The survival analysis showed that OS overlapped for the low and intermediate subgroups, while a significantly less favorable survival was observed for high-risk patients (Figure S1A; p<0.0001). There was no significant di↵erence in terms of PFS (Figure S1B; p=0.230). Finally, the analysis where the primary resistance group included patients with PD and SD for less than six months and the DC group included patients with CR, PR, and SD for more than six months Manuscript 2 68 Cancers 2020,12, 1027 7 of 13 (Figure S2A,B) showed that the di↵erence in terms of OS and PFS remained statistically significant (p<0.0001). Figure 1. ( A ). Overall survival according to response to first-line PD-1-based immunotherapy ( p<0.0001 ); ( B ). progression-free survival according to response to first-line PD-1-based immunotherapy (p<0.0001); ( C ). overall survival according to best overall response to first-line PD-1-based immunotherapy (p<0.0001); ( D ). progression-free survival according to best overall response to first-line PD-1-based immunotherapy (p<0.0001); ( E ). overall survival according to the type of first-line PD-1-based immunotherapy (p=0.993); ( F ). progression-free survival according to the type of first-line PD-1-based immunotherapy (p=0.216). Manuscript 2 69 Cancers 2020,12, 1027 8 of 13 3.3. Second-Line Therapies and Outcomes Tables S3 and S4 show the type of second-line therapy in patients with primary resistance, and also the best overall response achieved, according to the BRAF mutation status. Approximately 50% (n=63) of the patients with primary resistance to immunotherapy received a second-line therapy. Sixty-four patients did not receive further systemic therapies. Twenty-one patients had tumors harboring a BRAFV600E/Kmutation, 20 patients had BRAF wild-type tumors, and in 22 patients, there was no information regarding BRAF mutation status. The majority of the patients with BRAFV600E/Kmutation (17/21) received targeted therapy with BRAF plus MEK inhibitors, three received immunotherapy, and one patient chemotherapy. Patients with BRAF wild-type tumors received in equal number immunotherapy (10/20) and chemotherapy (10/20). Information on the best overall response for the second-line systemic therapy was available for 58 patients. In five patients, this information was not available. Patients with tumors harboring a BRAFV600E/Kmutation received predominantly targeted therapy which resulted in a high response rate (CR or PR) of 63%. In patients with BRAF wild-type tumors treated either with second-line immunotherapy or chemotherapy, the response rate was only 11% (Table S3). 3.4. Pseudoprogression In our cohort (n=319), we identified six patients with pseudoprogression. Of these six patients, five showed initial PD but later achieved SD as best overall response to immunotherapy, and in one patient after initial PD, the best overall response to immunotherapy was CR. 4. Discussion Our study shows that patients with primary resistance and tumor progression at the time of first tumor assessment after starting immunotherapy have a highly significantly unfavorable survival rate as compared to those who achieve disease control. Response at the time of first tumor assessment after starting immunotherapy is a better predictive factor for survival than other pretreatment risk factors for the development of primary resistance. Achieving an objective remission (CR or PR) is decisive for favorable OS. The median OS for patients with SD is significantly better than for patients with primary resistance (28 months versus 11 months). After three years, however, the survival curves converge strongly at an unfavorable level. This convergence is even more pronounced for PFS. In the multivariate logistic regression analysis, only two significant risk factors for primary resistance to immunotherapy were identified. These were primary tumor localization and an elevated level of protein S-100B. In the univariate analysis, an elevated level of LDH was also a significant factor, but this did not remain significant in the multivariate analysis. The decisive factor here may be that the LDH value and the protein S-100B value usually increase in parallel, and that the S-100B value increases earlier and in more patients. In the univariate analysis, the histological subtype was also a significant factor. Here there is an overlap with tumor localization, since ALM is more commonly seen in the extremities, and mucosal melanomas were classified in the other localizations group. The higher discriminatory power was observed for the tumor localization. There is also a relationship between tumor localization and sex, as melanomas in the extremities occur more frequently in females, and females have a less favorable response to immunotherapy than males [ 32 – 34 ]. Accordingly, sex was a significant risk factor in the univariate analysis but not in the multivariate analysis. The definition of risk groups considering the two risk factors that remained significant in the multivariate analysis (i.e., primary tumor localization and protein S-100B) showed a relatively low predictive value. We observed a statistically significant di↵erence in terms of OS between highand intermediateand low-risk groups, whereas this di↵erence was not observed in PFS. The three-year PFS and three-year OS rates reported here for PD-1 monotherapy and the combination of nivolumab plus ipilimumab are lower than those reported in the CheckMate 067 Manuscript 2 70 Cancers 2020,12, 1027 9 of 13 trial [ 35 ]. This might be partially explained by the selection of the patients included in that study compared to the unselected population in our cohort. As an example, patients with (active) brain metastases are typically excluded from clinical trials. In fact, only 3.6% of the patients included in the CheckMate 067 trial had brain metastases compared to 19% in our study. In our cohort, 36% of the patients also had liver metastases, which is associated with worse response to immunotherapy [ 36 , 37 ]. The percentage of patients that had elevated LDH is similar in both reports (32% in our cohort vs. 36% in the CheckMate 067 trial). In the CheckMate 067 trial, the S100B levels, which are a known prognostic factor [ 38 , 39 ], were not reported; in our study, 44% of the patients had elevated S100B. Together, these aspects define a collective of patients that probably had a worse prognosis compared to the patients included in the clinical trial. In our cohort, there was no di↵erence in terms of OS between patients treated with PD-1 monotherapy and those receiving nivolumab plus ipilimumab. This might be related to the median follow-up time of only 22 months, shorter than the last update from the CheckMate 067 trial, where the di↵erence between combined immunotherapy and monotherapy was clearer with a five-year follow-up [ 4 ]. In our cohort, a significantly higher proportion of patients with BRAFV600E/Kmutation received combined immunotherapy (p=0.003). This subgroup seemed to respond better to combined immunotherapy compared to PD-1 monotherapy [ 4 , 40 ], and this might explain why we started to see a separation of the PFS curves. Possibly with a longer follow-up, a di↵erence in OS can be expected. The absence of di↵erence in terms of OS in these two subgroups is probably also linked to the fact that they were not homogenous, with a selection bias regarding the type of immunotherapy. Older patients, who seem to respond better to immunotherapy [ 41 ], received predominantly PD-1 monotherapy (p=0.007), patients with more than three metastatic organs received preferably combined immunotherapy (p=0.043), and a significantly higher proportion of patients with brain metastasis were also treated with nivolumab plus ipilimumab (p=0.018). Only 50% of the patients with primary resistance to immunotherapy received a second systemic therapy, similar to the percentage of patients reported in other series receiving a second-line therapy [ 42 ]. In our cohort, patients with BRAFV600E/Kmutation received predominantly targeted therapy, and in this subgroup, 63% of patients had a response (CR or PR). This was slightly higher than previously published [ 43 ], but in our cohort, only 21 patients with BRAFV600E/Kmutation received second-line therapy, and therefore the outcomes need to be interpreted cautiously. Nevertheless, our group and others have already demonstrated that patients with BRAFV600E/Kfirst-line immunotherapy followed by targeted therapy, similar to what patients in this cohort received, seem to have better outcomes than the inverse sequence [ 44 , 45 ]. The high response rate in our cohort might be explained by this favorable therapy sequencing. On the other hand, for patients with BRAF wild-type tumors, only one patient responded to second-line therapy. Again, the number of patients was low (n=20), but these results show that a second-line therapy in the BRAF wild-type cohort is not possible in a high number of patients and, when possible, still has a small impact on survival. Strengths of this investigation are the fact that the data included was from a German certified skin cancer center with high standards for data quality. Three hundred and nineteen patients were analyzed, which is a large cohort of patients with stage IV melanoma managed with PD-1-based immunotherapy in a routine clinical setting. This high number of patients allowed us to perform subgroup analyses, with results of reasonable sensitivity. Further, this study provides follow-up data covering a period of up to 22 months. The study limitations are related to its retrospective and monocentric design. Patients included were those receiving first-line immunotherapy for stage IV melanoma and for whom a response to therapy was documented. Since no other selection criteria were applied, the heterogeneity of the study population might have contributed to the di↵erences observed in survival. Another limitation is the absence of histological confirmation of progressive disease in all patients with primary resistance. Manuscript 2 71 Cancers 2020,12, 1027 10 of 13 This approach is currently changing and, in the future, it would certainly be of value to include other factors in the definition of primary resistance. 5. Conclusions Patients with progressive disease at the first tumor assessment after starting first-line PD-1-based immunotherapy have a very unfavorable prognosis. Predicting primary resistance based on pre-existing risk characteristics is possible only to a limited extent. Response at time of first tumor assessment after starting immunotherapy is a stronger predictive factor. In future analysis, other factors, namely histological and molecular characterization of the progressive lesions, should be included in the definition of primary resistance. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/4/1027/s1, Figure S1: ( A ) Overall survival according to risk groups (low, intermediate, and high) (p<0.0001). The factors used were primary tumor localization and protein S-100B level. The subgroups were defined as follows: no risk factor (low), one risk factor (intermediate), and two risk factors (high). ( B ) Progression-free survival according to risk groups (low, intermediate, and high) (p=0.230). The factors used were primary tumor localization and protein S-100B level. The subgroups were defined as follows: no risk factor (low), one risk factor (intermediate), and two risk factors (high); Figure S2: ( A ) Overall survival for the disease control group (complete response, partial response, and stable disease for more than 6 months) and primary resistance (progressive disease and stable disease for less than 6 months). ( B ) Progression-free survival for the disease control group (complete response, partial response, and stable disease for more than 6 months) and primary resistance (progressive disease and stable disease for less than 6 months); Table S1: Patients characteristics and univariate analysis for the whole cohort. In this analysis, the primary resistance group includes progressive disease at the time of first tumor response evaluation after immunotherapy plus stable disease for less than 6 months. The disease control group includes complete response, partial response, and stable disease for longer than 6 months; Table S2: Patients characteristics and univariate analysis for the whole cohort according to type of first-line immunotherapy; Table S3: Second-line therapies in patients with primary resistance considering BRAF mutation status; Table S4: Best overall response to second-line therapies in patients with primary resistance considering BRAF mutation status. Author Contributions: Conceptualization: T.A., O.S., C.G.; Methodology: T.A., O.S., C.G.; Software: T.A., O.S., T.E., C.G.; Validation: T.A., O.S., T.E., C.G.; Formal Analysis: T.A., O.S., U.K., E.M., S.S., T.E., C.G.; Data Interpretation: all authors; Writing—Original Draft Preparation: T.A., O.S., E.M., S.S., C.G.; Writing—Review & Editing: all authors; Visualization: all authors; Supervision: T.A., O.S., C.G..; Project Administration: T.A., O.S., C.G.; Final Approval: all authors; Funding Acquisition: C.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the German Central Malignant Melanoma Registry. Conflicts of Interest: T.A.: Amaral reports personal fees and travel grants from BMS, grants, personal fees, and travel grants from Novartis, personal fees from Pierre Fabre, grants from Neracare, grants from Sanofi, outside the submitted work. I.T.: Thomas reports travel grants from Novartis, Abbvie, and Roche, outside the submitted work. A.F.: Forschner served as consultant to Roche, Novartis, MSD, Pierre-Fabre; received travel support from Roche, Novartis, BMS, Pierre-Fabre, received speaker fees from Roche, Novartis, BMS, MSD, and CeGaT, outside the submitted work. U.L.: Leiter reports personal fees, speaker fees, and grants from MSD, speaker fees from Roche, Novartis, Sun Pharma, Sanofi, outside the submitted work. T.E.: Eigentler reports personal fees from Amgen, grants and personal fees from BMS, personal fees from MSD, grants and personal fees from Novartis, personal fees from Pierre Fabre, grants and personal fees from Roche, grants and personal fees from Sanofi, outside the submitted work. C.G.: Garbe reports grants and personal fees from BMS, personal fees from MSD, during the conduct of the study; personal fees from Amgen, grants and personal fees from NeraCare, grants and personal fees from Novartis, personal fees from Philogen, grants and personal fees from Roche, grants and personal fees from Sanofi, outside the submitted work. O.S., E.M., S.S., A.M., and U.K. have nothing to disclose. References 1. Franken, M.G.; Leeneman, B.; Gheorghe, M.; Uyl-de Groot, C.A.; Haanen, J.B.A.G.; van Baal, P.H.M. A systematic literature review and network meta-analysis of e↵ectiveness and safety outcomes in advanced melanoma. Eur. J. Cancer 2019,123, 58–71. [CrossRef][PubMed] 2. Hodi, F.S.; O’Day, S.J.; McDermott, D.F.; Weber, R.W.; Sosman, J.A.; Haanen, J.B.; Gonzalez, R.; Robert, C.; Schadendorf, D.; Hassel, J.C.; et al. Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 2010,363, 711–723. [CrossRef][PubMed] Manuscript 2 72 Cancers 2020,12, 1027 11 of 13 3. Robert, C.; Schachter, J.; Long, G.V.; Arance, A.; Grob, J.J.; Mortier, L.; Daud, A.; Carlino, M.S.; McNeil, C.; Lotem, M.; et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015 ,372, 2521–2532. [CrossRef][PubMed] 4. Larkin, J.; Chiarion-Sileni, V.; Gonzalez, R.; Grob, J.-J.; Rutkowski, P.; Lao, C.D.; Cowey, C.L.; Schadendorf, D.; Wagsta↵, J.; Dummer, R.; et al. Five-year survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med. 2019,381, 1535–1546. [CrossRef] 5. Maio, M.; Grob, J.-J.; Aamdal, S.; Bondarenko, I.; Robert, C.; Thomas, L.; Garbe, C.; Chiarion-Sileni, V.; Testori, A.; Chen, T.-T.; et al. Five-year survival rates for treatment-naive patients with advanced melanoma who received ipilimumab plus dacarbazine in a phase III trial. J. Clin. Oncol. 2015 ,33, 1191–1196. [CrossRef] 6. Schadendorf, D.; Hodi, F.S.; Robert, C.; Weber, J.S.; Margolin, K.; Hamid, O.; Patt, D.; Chen, T.-T.; Berman, D.M.; Wolchok, J.D. Pooled analysis of long-term survival data from phase II and phase III trials of ipilimumab in unresectable or metastatic melanoma. J. Clin. Oncol. O↵. J. Am. Soc. Clin. Oncol. 2015 ,33, 1889–1894. [CrossRef] 7. Fares, C.M.; Van Allen, E.M.; Drake, C.G.; Allison, J.P.; Hu-Lieskovan, S. Mechanisms of resistance to immune checkpoint blockade: Why does checkpoint inhibitor immunotherapy not work for all patients? Am. Soc. Clin. Oncol. Educ. Book 2019,39, 147–164. [CrossRef] 8. Shin, D.S.; Zaretsky, J.M.; Escuin-Ordinas, H.; Garcia-Diaz, A.; Hu-Lieskovan, S.; Kalbasi, A.; Grasso, C.S.; Hugo, W.; Sandoval, S.; Torrejon, D.Y.; et al. Primary Resistance to PD-1 blockade mediated by JAK1/2 mutations. Cancer Discov. 2017,7, 188–201. [CrossRef] 9. Larkin, J.; Chiarion-Sileni, V.; Gonzalez, R.; Grob, J.J.; Cowey, C.L.; Lao, C.D.; Schadendorf, D.; Dummer, R.; Smylie, M.; Rutkowski, P.; et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N. Engl. J. Med. 2015,373, 23–34. [CrossRef] 10. Robert, C.; Long, G.V.; Brady, B.; Dutriaux, C.; Maio, M.; Mortier, L.; Hassel, J.C.; Rutkowski, P.; McNeil, C.; Kalinka-Warzocha, E.; et al. Nivolumab in previously untreated melanoma without BRAF mutation. N. Engl. J. Med. 2015,372, 320–330. [CrossRef] 11. Robert, C.; Thomas, L.; Bondarenko, I.; O’Day, S.; Weber, J.; Garbe, C.; Lebbe, C.; Baurain, J.-F.; Testori, A.; Grob, J.-J.; et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N. Engl. J. Med. 2011,364, 2517–2526. [CrossRef][PubMed] 12. Jenkins, R.W.; Barbie, D.A.; Flaherty, K.T. Mechanisms of resistance to immune checkpoint inhibitors. Br. J. Cancer 2018,118, 9–16. [CrossRef][PubMed] 13. Seto, T.; Sam, D.; Pan, M. Mechanisms of primary and secondary resistance to immune checkpoint inhibitors in cancer. Med. Sci. 2019,7, 14. [CrossRef][PubMed] 14. Fuereder, T. Resistance to immune checkpoint inhibitors. Next steps and combinational approaches. Memo Mag. Eur. Med Oncol. 2019,12, 123–127. [CrossRef] 15. Veldman, J.; Visser, L.; Berg, A.v.d.; Diepstra, A. Primary and acquired resistance mechanisms to immune checkpoint inhibition in Hodgkin lymphoma. Cancer Treat. Rev. 2020,82, 101931. [CrossRef] 16. Sharma, P.; Hu-Lieskovan, S.; Wargo, J.A.; Ribas, A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell 2017,168, 707–723. [CrossRef] 17. Zaretsky, J.M.; Garcia-Diaz, A.; Shin, D.S.; Escuin-Ordinas, H.; Hugo, W.; Hu-Lieskovan, S.; Torrejon, D.Y.; Abril-Rodriguez, G.; Sandoval, S.; Barthly, L.; et al. Mutations associated with acquired resistance to PD-1 Blockade in Melanoma. N. Engl. J. Med. 2016,375, 819–829. [CrossRef] 18. Lad á nyi, A.; Kiss, J.; Somlai, B.; Gilde, K.; Fejos, Z.; Mohos, A.; Gaudi, I.; T í m á r, J. Density of DC-LAMP(+) mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor. Cancer Immunol. Immunother. CII 2007 ,56, 1459–1469. [CrossRef] 19. Wu, W.; Wang, W.; Wang, Y.; Li, W.; Yu, G.; Li, Z.; Fang, C.; Shen, Y.; Sun, Z.; Han, L.; et al. IL-37b suppresses T cell priming by modulating dendritic cell maturation and cytokine production via dampening ERK/NF-B/S6K signalings. Acta Biochim. Et Biophys. Sin. 2015,47, 597–603. [CrossRef] 20. Lindenberg, J.J.; van de Ven, R.; Lougheed, S.M.; Zomer, A.; Santegoets, S.J.A.M.; Griffioen, A.W.; Hooijberg, E.; van den Eertwegh, A.J.M.; Thijssen, V.L.; Scheper, R.J.; et al. Functional characterization of a STAT3-dependent dendritic cell-derived CD14+cell population arising upon IL-10-driven maturation. Oncoimmunology 2013 , 2, e23837. [CrossRef] Manuscript 2 73 Cancers 2020,12, 1027 12 of 13 21. Strauss, L.; Bergmann, C.; Szczepanski, M.; Gooding, W.; Johnson, J.T.; Whiteside, T.L. A Unique Subset of CD4+CD25highFoxp3+T cells secreting interleukin-10 and transforming growth factor-  1 mediates suppression in the tumor microenvironment. Clin. Cancer Res. 2007,13, 4345. [CrossRef] 22. Viguier, M.; Lemaître, F.; Verola, O.; Cho, M.-S.; Gorochov, G.; Dubertret, L.; Bachelez, H.; Kourilsky, P.; Ferradini, L. Foxp3 expressing CD4+CD25high regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J. Immunol. 2004 ,173, 1444. [CrossRef] [PubMed] 23. Togashi, Y.; Shitara, K.; Nishikawa, H. Regulatory T cells in cancer immunosuppression-implications for anticancer therapy. Nat. Rev. Clin. Oncol. 2019,16, 356–371. [CrossRef][PubMed] 24. Pardoll, D.M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 2012 ,12, 252–264. [CrossRef][PubMed] 25. Wu, Y.; Chen, W.; Xu, Z.P.; Gu, W. PD-L1 Distribution and perspective for cancer immunotherapy—Blockade, knockdown, or inhibition. Front. Immunol. 2019,10, 2022. [CrossRef] 26. Snyder, A.; Makarov, V.; Merghoub, T.; Yuan, J.; Zaretsky, J.M.; Desrichard, A.; Walsh, L.A.; Postow, M.A.; Wong, P.; Ho, T.S.; et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N. Engl. J. Med. 2014,371, 2189–2199. [CrossRef][PubMed] 27. McGranahan, N.; Furness, A.J.S.; Rosenthal, R.; Ramskov, S.; Lyngaa, R.; Saini, S.K.; Jamal-Hanjani, M.; Wilson, G.A.; Birkbak, N.J.; Hiley, C.T.; et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science (N. Y.) 2016,351, 1463. [CrossRef][PubMed] 28. del Campo, A.B.; Kyte, J.A.; Carretero, J.; Zinchencko, S.; M é ndez, R.; Gonz á lez-Aseguinolaza, G.; Ruiz-Cabello, F.; Aamdal, S.; Gaudernack, G.; Garrido, F.; et al. Immune escape of cancer cells with beta2-microglobulin loss over the course of metastatic melanoma. Int. J. Cancer 2014 ,134, 102–113. [CrossRef] [PubMed] 29. Forschner, A.; Battke, F.; Hadaschik, D.; Schulze, M.; Weißgraeber, S.; Han, C.-T.; Kopp, M.; Frick, M.; Klumpp, B.; Tietze, N.; et al. Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma–results of a prospective biomarker study. J. Immunother. Cancer 2019,7, 180. [CrossRef][PubMed] 30. Gershenwald, J.E.; Scolyer, R.A.; Hess, K.R.; Sondak, V.K.; Long, G.V.; Ross, M.I.; Lazar, A.J.; Faries, M.B.; Kirkwood, J.M.; McArthur, G.A.; et al. Melanoma staging: Evidence-based changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA A Cancer J. Clin. 2017 ,67, 472–492. [CrossRef] 31. Eisenhauer, E.A.; Therasse, P.; Bogaerts, J.; Schwartz, L.H.; Sargent, D.; Ford, R.; Dancey, J.; Arbuck, S.; Gwyther, S.; Mooney, M.; et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer 2009,45, 228–247. [CrossRef][PubMed] 32. Grassadonia, A.; Sperduti, I.; Vici, P.; Iezzi, L.; Brocco, D.; Gamucci, T.; Pizzuti, L.; Maugeri-Sacc à , M.; Marchetti, P.; Cognetti, G.; et al. E↵ect of Gender on the Outcome of Patients Receiving Immune Checkpoint Inhibitors for Advanced Cancer: A Systematic Review and Meta-Analysis of Phase III Randomized Clinical Trials. J. Clin. Med. 2018,7, 542. [CrossRef] 33. Wu, Y.; Ju, Q.; Jia, K.; Yu, J.; Shi, H.; Wu, H.; Jiang, M. Correlation between sex and efficacy of immune checkpoint inhibitors (PD-1 and CTLA-4 inhibitors). Int. J. Cancer 2018,143, 45–51. [CrossRef][PubMed] 34. Conforti, F.; Pala, L.; Bagnardi, V.; De Pas, T.; Martinetti, M.; Viale, G.; Gelber, R.D.; Goldhirsch, A. Cancer immunotherapy efficacy and patients’ sex: A systematic review and meta-analysis. Lancet Oncol. 2018 ,19, 737–746. [CrossRef] 35. Wolchok, J.D.; Chiarion-Sileni, V.; Gonzalez, R.; Rutkowski, P.; Grob, J.-J.; Cowey, C.L.; Lao, C.D.; Wagsta↵, J.; Schadendorf, D.; Ferrucci, P.F.; et al. Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N. Engl. J. Med. 2017,377, 1345–1356. [CrossRef][PubMed] 36. Bilen, M.A.; Shabto, J.M.; Martini, D.J.; Liu, Y.; Lewis, C.; Collins, H.; Akce, M.; Kissick, H.; Carthon, B.C.; Shaib, W.L.; et al. Sites of metastasis and association with clinical outcome in advanced stage cancer patients treated with immunotherapy. BMC Cancer 2019,19, 857. [CrossRef] 37. Tumeh, P.C.; Hellmann, M.D.; Hamid, O.; Tsai, K.K.; Loo, K.L.; Gubens, M.A.; Rosenblum, M.; Harview, C.L.; Taube, J.M.; Handley, N.; et al. Liver Metastasis and Treatment Outcome with Anti-PD-1 Monoclonal Antibody in Patients with Melanoma and NSCLC. Cancer Immunol. Res. 2017,5, 417–424. [CrossRef] Manuscript 3 80 ActaDV ActaDV Advances in dermatology and venereology Acta Dermato-Venereologica MAPKi in melanoma patients with elevated LDH Acta Derm Venereol 2020 DJLYHQFRPELQDWLRQPLJKWSURYLGHJUHDWHUEHQH¿WLQD particular subgroup of patients. Although indirect comparisons of pivotal trials warrant great caution, the usage of vemurafenib as comparator coBRIM, COMBI-v and COLUMBUS part 1 allows to perform a Bucher analysis (18). Looking at the vemurafenib monotherapy arms, results indicate similar response to treatment and prognosis due to resemblance across multiple endpoints (e.g. PFS and OS) in the respective vemurafenib groups (1, 3, 17). Prognostic factors, such as ECOG PS and degree of organ involvement, were comparable across the trials (Table I) (2, 12, 13). However, the coBRIM trial included the highest percentage of patients with elevated baseline levels of LDH in the combination arm (46%) compared with only 34% in the COMBI-v and 29% in the COLUMBUS trial (1–3). In real-world dataVHWVXSWRRISDWLHQWVUHFHLYLQJGXDO0$3.L¿UVWOLQH showed an elevated LDH resembling the coBRIM cohort (19, 20). Although an elevated baseline LDH accounts for a worse outcome, the HR for progression or death in the total trial populations was comparable across the 3 trials (Table I). This indicated an advantage of vemurafenib plus cobimetinib in the subgroup with elevated baseline LDH. 7KHFR%5,0GDWDFRQ¿UPHGWKLVK\SRWKHVLVUHJDUGLQJ a PFS advantage of combined BRAF and MEK inhibition with vemurafenib plus cobimetinib compared with a vemurafenib monotherapy independent of the baseline LDH level (Fig. 1). Consequently, BRAF-V600 mutated SDWLHQWVZLWKDQHOHYDWHGEDVHOLQH/'+OHYHOPLJKWEHQH¿W from a combined TT with vemurafenib and cobimetinib to a similar extend as patients with normal LDH do when compared with vemurafenib monotherapy. This could not be demonstrated for dabrafenib plus trametinib or encorafenib plus binimetinib (Fig. 1). The Bucher analysis FRQ¿UPHGWKHVH¿QGLQJVVKRZLQJDQRQVLJQL¿FDQWDGvantage for vemurafenib plus cobimetinib in the subgroup with elevated baseline LDH compared with dabrafenib plus trametinib and encorafenib plus binimetinib regarding PFS (Table II). Although this retrospective indirect Bucher analysis does not allow an exclusion of all selection bias, our results indicate that the LDH level should EHFRQVLGHUHGZKHQFKRRVLQJDVSHFL¿F%5$)DQG0(. inhibitor to achieve disease control. ,QFRQWUDVWWRWKH3)6GDWDRXUDQDO\VLVGLGQRWDI¿UP DEHQH¿FLDOHIIHFWRIYHPXUDIHQLE SOXVFRELPHWLQLE compared with dabrafenib and trametinib, and showed only a slight advantage compared with encorafenib and binimetinib regarding OS. Likewise, the coBRIM data do not provide an OS advantage for combined TT compared with vemurafenib monotherapy. Therefore, the choice of a particular BRAF and MEK inhibitor combination seems to have no impact on OS. However, when interSUHWLQJHI¿FDF\UHVXOWVVXFKDV26SULRUDQGVXEVHTXHQW treatment regimens, such as immunotherapies, as well as prognostic factors apart from LDH have to be taken into consideration, creating a potential bias. %HVLGHVHI¿FDF\VDIHW\DQG WROHUDELOLW\ DUHRIKLJK clinical relevance and have an impact on treatment recommendations. Distinct patterns of treatment-related adverse events can be found in melanoma patients receiving dabrafenib + trametinib, vemurafenib + cobimetinib or encorafenib + binimetinib. Pyrexia is most frequently observed in patients receiving dabrafenib + trametinib, while vemurafenib + cobimetinib causes the highest number of cutaneous adverse events (AEs), and encorafenib + binimetinib leads to more nausea and constipation than the other combinations (1–3). In an indirect comparison similar to ours, a lower incidence of treatment-related AEs was found for dabrafenib + trametinib compared with vemurafenib + cobimetinib (18). However, when looking at any AE, serious AEs or AEs leading to treatment discontinuation, no differences were observed. Regarding OS and PFS (dabrafenib + trametinib vs. vemurafenib + cobimetinib), Daud et al. calculated a HR of 0.94 and 1.05, respectively, when applying the Bucher method. In contrast to our indirect comparison, earlier data cuts were used and most importantly, the total patient populations of the combination arms were analysed. We cannot provide any data explaining the differences observed. Lactate accumulating in the tumour microenYLURQPHQWPLJKWFDXVHDFLGL¿FDWLRQGHFUHDVLQJWKHS+ (19). Since it is known that bioavailability of dabrafenib is dependent on pH, while that of vemurafenib is not (20), our hypothesis is that antineoplastic activity of dabrafenib, but not vemurafenib, is pH dependent. Experimental and pharmacokinetic data are needed to test this hypothesis. Elevated LDH is a very important biomarker in advanced melanoma, and has been incorporated into the AJCC Melanoma Staging system since 2009 (5). Three UHFHQWSRROHGDQDO\VHVFRQ¿UPHGDQHOHYDWHG/'+DV predictive factor for shorter PFS and OS in melanoma patients receiving combined TT (6–8). In the real-world setting, melanoma patients receiving palliative MAPKi ¿UVWOLQHKDYHSRRUSURJQRVWLFIHDWXUHVLQFOXGLQJEXWQRW limited to, elevated LDH (21, 22). There might be other subgroups in which a particular treatment regime might tend to be superior to the others. However, taking other biomarkers, such as involvement of particular organs or the sum of lesions diameters, into account was not possible. Patient cohorts are slightly heterogeneous across the 3 trials and more importantly, the way the trials are reported limits the availability of data for comparisons. In conclusion, there is no statistically significant GLIIHUHQFHLQHI¿FDF\EHWZHHQWKH77FRXSOHVXVLQJ the Bucher method. However, our data indicate a trend towards a lower risk for progression or death in melanoma patients with elevated LDH when receiving vemurafenib + cobimetinib in comparison with dabrafenib WUDPHWLQLEDQGHQFRUDIHQLEELQLPHWLQLEDV¿UVWOLQH therapy. In light of the current preference to use dual MAPKi instead of immune-checkpoint blockade in pa- Manuscript 3 81 ActaDV ActaDV Advances in dermatology and venereology Acta Dermato-Venereologica V. Glutsch et al.6/6 www.medicaljournals.se/acta tients with poor prognostic features including elevated LDH, our indirect analysis might provide a rationale to XVHDVSHFL¿FWUHDWPHQWUHJLPH+RZHYHUWKLV¿QGLQJ needs to be validated prospectively. Although a Bucher analysis partially retains the randomization of the individual trials, data provided by an indirect comparison must be interpreted with caution. ACKNOWLEDGEMENTS The authors would like to thank Susanne Schwenke from SchwenNH&RQVXOWLQJIRUSHUIRUPLQJWKHVWDWLVWLFDODQDO\VHV1RVSHFL¿F funding was received to perform this study. This publication was supported by the Open Access Publication Fund of the University of Wuerzburg. &RQÀLFWVRILQWHUHVWVG has received honoraria from Bristol-Myers Squibb (BMS) and reports travel support from Novartis, Pierre Fabre Pharmaceuticals, BMS, Merck Sharp & Dohme (MSD) and 6DQR¿*HQ]\PHRXWVLGHWKHVXEPLWWHGZRUN7$UHSRUWVWUDYHO support from Novartis, personal fees and travel support from BMS, outside the submitted work. CG reports grants and personal fees from Novartis, personal fees from Pierre Fabre, grants and personal fees from Roche, during the conduct of the study; personal fees from Amgen, grants and personal fees from BMS, personal fees from MSD, grants and personal fees from Neracare, personal fees IURP3KLORJHQSHUVRQDOIHHVIURP6DQR¿RXWVLGHWKHVXEPLWWHG work. K-MT reports advisory roles for or has received honoraria from Roche, Novartis, Pierre Fabre, BMS, MSD and LEO; travel support from Roche, Novartis, Pierre Fabre, BMS and LEO; outVLGHWKHVXEPLWWHGZRUN30UHSRUWVSHUVRQDOIHHVQRQ¿QDQFLDO support and other from Pierre Fabre, GSK, MSD, Merk Germany, 5RFKH%061RYDUWLVDQG6DQR¿RXWVLGHWKHVXEPLWWHGZRUN AH has received clinical trial support from Amgen, BMS, Merck Serono, MSD, Novartis, Philogen, Pierre Fabre, Provectus, Regeneron and Roche and honoraria or consultancy fees from Amgen, BMS, Merck Serono, MSD, Novartis, OncoSec, Philogen, Pierre Fabre, Provectus, Regeneron and Roche. BS reports advisory roles for or has received honoraria from Pierre Fabre Pharmaceuticals, Incyte, Novartis, Roche, BMS and MSD, research funding from BMS, Pierre Fabre Pharmaceuticals and MSD, and travel support from Novartis, Roche, BMS, Pierre Fabre Pharmaceuticals and Amgen; outside the submitted work. 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"Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study." Heppt, M. V., T. Amaral, K. C. Kähler, L. Heinzerling, J. C. Hassel, M. Meissner, N. Kreuzberg, C. Loquai, L. Reinhardt, J. Utikal, E. Dabrowski, A. Gesierich, C. Pföhler, P. Terheyden, K.-M. Thoms, L. Zimmer, T. K. Eigentler, M. C. Kirchberger, H. M. Stege, F. Meier, M. Schlaak and C. Berking (2019). Journal for ImmunoTherapy of Cancer 7(1): 299. Manuscript 4 83 RESEARCH ARTICLE Open Access Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study Markus V. Heppt 1,2 , Teresa Amaral 3,4 , Katharina C. Kähler 5 , Lucie Heinzerling 2 , Jessica C. Hassel 6 , Markus Meissner 7 , Nicole Kreuzberg 8 , Carmen Loquai 9 , Lydia Reinhardt 10 , Jochen Utikal 11 , Evelyn Dabrowski 12 , Anja Gesierich 13 , Claudia Pföhler 14 , Patrick Terheyden 15 , Kai-Martin Thoms 16 , Lisa Zimmer 17 , Thomas K. Eigentler 3 , Michael C. Kirchberger 2 , Henner M. Stege 9 , Friedegund Meier 10 , Max Schlaak 1 and Carola Berking 1,2* Abstract Background: Uveal melanoma (UM) is highly refractory to treatment with dismal prognosis in advanced stages. The value of the combined checkpoint blockade with CTLA-4 and PD-1 inhibition in metastatic UM is currently unclear. Methods: Patients with metastatic or unresectable UM treated with ipilimumab in combination with a PD-1 inhibitor were collected from 16 German skin cancer centers. Patient records of 64 cases were analyzed for response, progression-free survival (PFS), overall survival (OS), and safety. Clinical parameters and serum biomarkers associated with OS and treatment response were determined with Cox regression modelling and logistic regression. Results: The best overall response rate to combined checkpoint blockade was 15.6% with 3.1 and 12.5% complete and partial response, respectively. The median duration of response was 25.5 months (range 9.0–65.0). Stable disease was achieved in 21.9%, resulting in a disease control rate of 37.5% with a median duration of the clinical benefit of 28.0 months (range 7.0–65.0). The median PFS was 3.0 months (95% CI 2.4–3.6). The median OS was estimated to 16.1 months (95% CI 12.9–19.3). Regarding safety, 39.1% of treated patients experienced a severe, treatment-related adverse event according to the CTCAE criteria (grade 3: 37.5%; grade 4: 1.6%). The most common toxicities were colitis (20.3%), hepatitis (20.3%), thyreoiditis (15.6%), and hypophysitis (7.8%). A poor ECOG performance status was an independent risk factor for decreased OS (p= 0.007). Conclusions: The tolerability of the combined checkpoint blockade in UM may possibly be better than in trials on cutaneous melanoma. This study implies that combined checkpoint blockade represents the hitherto most effective treatment option available for metastatic UM available outside of clinical trials. Keywords: Ipilimumab, Nivolumab, Combined immune checkpoint blockade, Uveal melanoma, Biomarker © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: [email protected] 1 Department of Dermatology and Allergy, Munich University Hospital (LMU), Frauenlobstr. 9-11, 80337 Munich, Germany 2 Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Ulmenweg 18, 91054 Erlangen, Germany Full list of author information is available at the end of the article Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 https://doi.org/10.1186/s40425-019-0800-0 Manuscript 4 84 Background Uveal melanoma (UM) is a malignant tumor of the eye that originates from the pigment cells of the choroid layer or the ciliary body which is clinically and biologically distinct from cutaneous melanoma. Although the incidence is much lower than that of cutaneous melanoma, UM belongs to the most common malignant intraocular tumors in adults [1]. In approximately 50% of all cases, patients develop distant metastasis during the course of the disease, which affects predominantly the liver. Clinical risk factors for metastases are posterior localization in the eye, tumor size of more than 10 mm, and presence of vascular loops. Molecular biomarkers associated with a higher risk of metastasis are monosomy 3 or genomic alterations of BAP-1 [2]. Once distant metastases have occurred, the prognosis is dismal with an average survival time of approximately 1 year across all therapeutic regimens [3]. Patients with metastatic UM have so far benefited little or not at all from the treatment innovations achieved in cutaneous melanoma in recent years. Neither targeted therapy with MEK inhibitors nor checkpoint blockade with ipilimumab or PD-1 inhibitors as monotherapy was able to significantly improve the prognosis of patients with UM [4,5]. The response rates were consistently in the single-digit percentage range in a panel of previous studies [6–9]. In cutaneous melanoma, combined checkpoint blockade with ipilimumab and nivolumab revealed response rates and survival outcomes superior to PD-1 inhibitor monotherapy, albeit at the cost of high immune-related toxicity [10]. However, the significance of combined checkpoint blockade in UM is unclear and has only been investigated in case reports and small case series [6,11,12]. In this study, we evaluate the clinical course of 64 patients with metastatic UM who received combined checkpoint blockade. We report clinical outcomes with respect to response, survival, and adverse events (AE). Furthermore, clinical and laboratory parameters were investigated which may have prognostic value in UM patients treated with checkpoint blockade. Patients and methods Patient population and study approval This study was designed as a retrospective multi-center explorative analysis. Patients were included if they had a diagnosis of stage IV UM and received combined checkpoint blockade of ipilimumab with a PD-1 inhibitor in any treatment line. A follow-up period of at least 3 months was required. The clinical data of 64 patients from 16 German skin cancer centers who met the inclusion criteria were investigated. The cases were collected from June 23, 2018 to October 4, 2019. Clinical data and the treatment outcomes of interest were extracted from the original patient records and merged into a central database prior to analysis. This study was approved by the institutional review board of the medical faculty of the Munich University Hospital (approval number 413– 16 UE) and was conducted in accordance with the principles of the Helsinki Declaration in its current version. Data collection and treatment outcomes The clinical data recorded at baseline prior to immunotherapy comprised demographics with Eastern Cooperative Oncology Group (ECOG) performance status, available information on the genotype, sites of metastasis, number of organ systems affected by metastases, and previous antineoplastic therapies. As potential serum biomarkers, lactate dehydrogenase (LDH), C-reactive protein (CRP), and the relative counts of lymphocytes (RLC), neutrophils (RNC), and eosinophils (REC) were specifically collected from patient charts and analyzed for their prognostic value [13,14]. Combined checkpoint blockade was carried out using different treatment schedules (Table 1). Ipilimumab was given at either 3 mg/kg or 1 mg/kg body weight for up to 4 treatment cycles. Nivolumab was applied at 1 mg/kg together with ipilimumab, followed by 3 mg/kg every 2 weeks (Q2W) as maintenance therapy. Treatment with pembrolizumab was applied every 3 weeks (Q3W) at 2 mg/kg. Patients were treated until disease progression or until the development of unacceptable toxicity. AE were retrospectively graded by the site investigators based on the patient records and clinical outcomes according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 published by the National Institutes of Health in 2017. Immune-related adverse events were managed according to pertinent guidelines and algorithms that were previously published [15,16]. Besides, fatal adverse events and events leading to permanent discontinuation of treatment were specifically recorded and evaluated. The best radiologic response to treatment was assessed by the site investigators and indicated as complete response, partial response, stable disease, or progressive disease based on the RECIST criteria version 1.1 [17]. Complete response and partial response were summarized as best overall response rate (ORR). Complete response, partial response, and stable disease were summarized as disease control rate (DCR). Statistical analyses Overall survival (OS) and progression-free survival (PFS) were calculated as the time from the initiation of the first cycle of combined checkpoint blockade until melanomaspecific or treatment-related death and disease progression, respectively. Time-to-event analyses were calculated where death or progression were considered as events. If neither occurred or if patients were lost to follow-up, the date of the last documented presentation was used as a Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 Page 2 of 9 Manuscript 4 85 Table 1 Baseline characteristics of the patient population Patient population n= 64 (100%) Gender Male 33 (51.6) Female 31 (48.4) Age < 60 years 28 (43.8) ≥60 years 36 (56.2) GNAQ Mutated 8 (12.5) Wildtype 8 (12.5) Unknown 48 (75.0) GNA11 Mutated 10 (15.6) Wildtype 5 (7.8) Unknown 49 (76.6) ECOG status 0 49 (76.6) 1 11 (17.2) 2 1 (1.6) 3 1 (1.6) Unknown 2 (3.1) Serum LDH Normal 28 (43.8) Elevated (>ULN) 33 (51.6) Unknown 3 (4.7) Previous systemic therapies 0 50 (78.1) 1 12 (18.8) ≥2 2 (3.1) Previous ipilimumab monotherapy 2 (3.1) Previous PD-1 inhibitor monotherapy 12 (18.8) Liver-directed therapies 0 33 (51.6) 1 30 (46.9) ≥2 1 (1.6) Metastatic sites a Liver 58 (90.6) Lung 23 (35.9) Bone 17 (26.6) Lymph nodes 12 (18.8) CNS 4 (6.3) Treatment regimen Ipilimumab 3 mg/kg + nivolumab 1 mg/kg Q3W, followed by nivolumab 3 mg/kg Q2W 59 (92.2%) Ipilimumab 1 mg/kg + pembrolizumab 2 mg/kg Q3W, followed by pembrolizumab 2 mg/kg Q3W 5 (7.8%) a Multiple metastatic sites per patient were possible (values do not sum up to 100%); abbreviations:CNS Central nervous system, Q2W Every two weeks, Q3W Every three weeks Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 Page 3 of 9 Manuscript 4 86 censored observation. The survival and progression probabilities were indicated with the Kaplan-Meier method for censored failure time data assuming proportional hazards. The survival curves were compared with the log-rank test [6]. The duration of the clinical response and clinical benefit was defined as time from treatment initiation to progressive disease if a response or stable disease was achieved, respectively. The time to response was defined as time from treatment start until a response was evident radiologically. Cox proportional hazards regression modelling was applied to investigate the relationship of clinical risk factors and serum biomarkers with OS. Cox regression was performed as a univariate and multivariate analysis in a stepwise approach [6]. Imputation of missing data was not allowed and patients with missing values of a given parameter were excluded from the analysis. Hazard ratios (HR) with 95% confidence intervals (CI) were calculated to quantify the impact on survival. P-values were calculated based on Wald statistics [6]. The association of treatment response as a categorical variable with clinical characteristics or serum biomarkers was investigated with the Chi-square test and logistic regression, as appropriate. In all cases, two-tailed p-values were calculated and considered significant with values p< 0.05. All analyses were carried out with SPSS statistics version 23.0 (IBM) or GraphPad Prism version 5.01 (GraphPad Software). Results A total of 64 (100%) patients with metastatic UM were included. Fifty patients (78.1%) were naïve to systemic treatment and received combined checkpoint blockade as first-line systemic therapy. Regarding genotype, the presence of monosomy 3 as risk factor was specifically investigated in 7 patients and identified in 2 of them. BRAF, NRAS and KIT were analyzed and reportedly wildtype as expected in 30, 22, and 20 patients, respectively. Mutations and inactivations of MBD4 which were previously linked to a hypermutator profile with high sensitivity to PD-1 inhibition were not investigated in any case [18,19]. Previous ipilimumab and PD-1 inhibitor monotherapy were applied in 2 (3.1%) and 12 (18.8%) cases, respectively. Both patients treated with ipilimumab before showed PD. Specifically, 4 patients (6.3%) had received nivolumab and 8 (12.5%) pembrolizumab before. In 4 cases, SD was achieved while 8 patients showed PD upon PD-1 inhibitor monotherapy. The median duration of the clinical benefit was 6.5 months in the 4 patients with SD. Liver-directed therapies were reported in 31 patients (48.4%). Most patients had an ECOG status of 0 (n= 49, 76.6%). Serum LDH was elevated in 33 cases (51.6%) at baseline. Other baseline characteristics are listed in detail in Table 1. Ipilimumab plus nivolumab was given in 59 patients (92.2%), while 5 patients (7.8%) received ipilimumab plus pembrolizumab. The median number of treatment cycles was 3 (range 1–4) for the combination of ipilimumab with a PD-1 inhibitor in the induction phase, and 0 (range 0–27) for PD-1 inhibitor maintenance therapy in the overall population. A total of 19 patients (29.7%) received a PD-1 inhibitor maintenance therapy. Among these, the median number of PD-1 inhibitor cycles was 3 (range 1–27). The best ORR to combined checkpoint blockade was 15.6% (n= 10) relating to the entire population (4 patients were not evaluable for a radiologic response). Two patients achieved a complete response (3.1%) and 8 (12.5%) a partial response. The median duration of response was 25.5 months (range 9.0–65.0). Stable disease was achieved in further 14 cases (21.9%), resulting in a disease control rate of 37.5% with a median duration of the clinical benefit of 28.0 months (range 7.0–65.0) (Table 2). The median PFS was 3.0 months (95% CI 2.4–3.6). The median OS was estimated to 16.1 months (95% CI 12.9–19.3) with a median follow-up period of 9.2 months (95% CI 7.8–10.6) (Fig. 1). The median time to response in patients with CR or PR after treatment initiation was 12 weeks (range 5–31). For the patients with SD, the median duration until the benefit was observed also amounted to 12 weeks (range 9–30). Interestingly, all 4 patients with SD after previous single PD-1 inhibitor blockade had PD to combined checkpoint blockade. Among the remaining 8 patients with PD after previous single PD-1 inhibitor blockade, one achieved a PR to combined checkpoint blockade. Thus, these data suggest that the effects of single and combined checkpoint blockade were observed independently from each other. A total of 78 AE were reported in 39 patients. Thus, the majority of patients developed any treatment-related AE (60.9%). Of all events, 37 AE were graded as severe (grade 3 + 4). They were observed in 25 patients (39.1%; grade 3: 37.5%; grade 4: 1.6%). The treatment was discontinued in 25 cases (39.1%) due to unacceptable toxicity. However, no treatment-related deaths occurred during treatment or the observation period. The most common events were colitis (20.3%), hepatitis (20.3%), Table 2 Best response rates to combined checkpoint blockade Cases (%) Cumulative percentage (%) Complete response 2 (3.1) 3.1 Partial response 8 (12.5) 15.6 (ORR) Stable disease 14 (21.9) 37.5 (DCR) Progressive disease 36 (56.3) 93.8 Unknown 4 (6.3) 100 Total 64 (100) 100 Abbreviations:ORR Objective response rate, DCR Disease control rate Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 Page 4 of 9 Manuscript 4 87 thyreoiditis (15.6%), hypophysitis (7.8%), fever (4.7%), and myalgia with myositis (4.7%). In all 5 cases with hypophysitis, the individual hormone axes including ACTH, cortisol, FSH, LH, TSH, and testosterone were investigated but not specifically graded. In 3 cases, the pituitary gland was enlarged in MRI examinations. All patients received systemic replacement of hydrocortisone. All AE are listed in Additional file 1. In univariate Cox regression, ECOG status (p= 0.000096), the presence of bone metastasis (p= 0.011), and the best response to checkpoint blockade (p= 0.002) were significantly associated with OS (Additional file 2). The risk factors ECOG status, serum LDH, serum levels of CRP, and presence of bone metastasis were further integrated into a multivariate Cox regression model. Of these factors, a significant association with OS was confirmed for ECOG status (p= 0.007) only (Table 3, Fig. 2a). We recently identified a prognostic score of the serum biomarkers LDH, CRP, and relative eosinophil count (REC) in a cohort of 94 UM patients receiving PD-1 inhibitors [6]. The score assigns one risk point for each unfavorable factor, i.e., elevated LDH, elevated CRP, and a REC < 1.5%, defining four distinct prognostic groups (low, intermediate, high, and very high risk). Each patient receiving combined checkpoint blockade was assigned to a risk group and the score was validated with Kaplan-Meier estimates. Due to a small sample size, patients with low and intermediate risk were pooled. The risk groups showed significantly different survival probabilities (p= 0.000005). The median survival times were superior for the low plus intermediate group (17.7 months, 95% CI 14.7–20.8) compared to the high (15.4 months, 95% CI 12.7–18.2) and very high risk group (7.1 months, 95% CI 0.0–16.2) (Fig. 2b). However, the score neither correlated with the response rate (p= 0.609) nor with the DCR (p= 0.446), suggesting that it was generally prognostic but not specifically predictive for the response to combined checkpoint blockade. Subgroup analysis were performed for patients with metastasis to the central nervous system (CNS) at treatment initiation and for the treatment responders. Four patients showed an involvement of the CNS. Two of them had neurological symptoms. Two patients achieved SD, 2 showed PD. The median PFS for the CNS subgroup was 3.0 months (95% CI 0.0–6.1) while the median OS was not reached. In contrast, none of the treatment responders (CR or PR) had CNS involvement when the treatment was initiated (Table 4). The median time from detection of the primary tumor to metastatic disease was 43 months among the responders. Data on the assessment of the risk of metastasis formation of the primary tumors were sparse, as e.g. the presence of monosomy 3 or the MBD4 status was not investigated in any of the responders. Fig. 1 Kaplan-Meier estimates of the patient population for aprogression-free survival (PFS) and boverall survival (OS). The median PFS and OS was estimated to 3.0 months (95% CI 2.4–3.6) and 16.1 months (95% CI 12.9–19.3), respectively. One patient was not included in the Kaplan-Meier analysis for PFS and OS due to missing data Table 3 Multivariate Cox regression analysis of clinical parameters and serum biomarkers Parameter Category HR (95% CI) P-value ECOG status n.a. (ordinal) 3.19 (1.36–7.47) 0.007* LDH normal 1 0.428 elevated (>ULN) 1.83 (0.41–8.08) CRP normal 1 0.534 elevated (>ULN) 1.73 (0.31–9.74) Bone metastasis no 1 0.331 yes 2.02 (0.49–8.27) Four parameters were included in the multivariate Cox regression analysis. Of these factors, ECOG status was significantly associated with overall survival in this model. Abbreviations:CI Confidence interval, n.a. not applicable, ULN Institutional upper limit of normal, LDH Lactate dehydrogenase, CRP C-reactive protein; *p<0.05. Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 Page 5 of 9 Manuscript 4 88 Discussion Here, we present a comparatively large cohort of patients with metastatic UM who were treated with combined checkpoint blockade. We detected a 15.6% ORR, with a 3.1% complete and 12.5% partial response rate. This response rate is in line with our previous report showing 16% ORR, although only 12 patients were evaluable for their radiologic response and the follow-up time was short [6]. Another case series was recently published from a single-center experience where 2 out of 8 patients treated with nivolumab and ipilimumab had a partial response [11]. Other preliminary data on the efficacy of the combined checkpoint blockade have been proposed as conference abstracts, but appear preliminary to date. Najjar et al. reported results from a multicenter, retrospective analysis in 66 patients from 11 U.S. centers, revealing an ORR of 13% and a DCR of 31% [20]. In addition to these estimates in a real-world setting, prospective trials are currently underway. A preliminary analysis of the Spanish phase II trial GEM1402 (NCT02626962) showed an ORR of 12% and disease stabilization in 52% of cases [21]. Another phase II trial is currently ongoing in the U.S. in 30 patients with UM (NCT01585194). A recently presented interim analysis revealed an ORR of 17% and disease control in 50% [22]. Thus, we conclude that the ORR of 15.6% identified in this population is a solid estimate for the efficacy of combined checkpoint blockade in UM and a good indicator of what we can expect from the final analyses of the prospective trials. This regimen appears to be significantly superior compared to the sobering efficacy values observed with ipilimumab and PD-1 inhibitor monotherapy [6–9,23–26]. Considering the data available so far, we conclude that the increase of ORR of the combined blockade versus PD-1 inhibition alone amounts to approximately 10%. Further evidence for a better efficacy of the combined regimen is supported by the observation of complete responders, albeit to a small extent. This is notable as UM is considered a “cold”tumor due to a low mutational burden and a unique immunosuppressive tumor microenvironment [27–29]. Further research is urgently needed to identify the radiologic, immunologic, and molecular determinants for treatment response in this small subset of patients. Regarding safety, the rate of severe AE was lower compared to the events reported in the pivotal trial in cutaneous melanoma (CheckMate-067) [30]. In particular, the occurrence of potentially life-threatening grade 4 AE was surprisingly low, suggesting that the regimen may be better tolerated in UM. However, it is also conceivable that the retrospective design and the small number of cases of this study causes an underreporting of AE. Among clinical parameters and serum biomarkers, only the ECOG performance status was a consistent prognostic factor in multivariate analysis. Other parameters such as serum LDH, CRP, and the REC showed a significant association neither with OS nor with the treatment response when they were considered as single factors. However, when integrated into a prognostic score, they were useful for risk stratification and discriminated groups with distinct survival probabilities. Thus, the risk score identified previously in a distinct Fig. 2 aKaplan-Meier estimates for overall survival (OS) according to ECOG performance status. The median OS was 17.7 months (95% CI 13.1– 22.3) for ECOG 0 versus 2.5 months (95% CI 0.0–9.6) for ECOG ≥1. Three patients were not included due to missing data. bKaplan-Meier estimates for OS according to the prognostic score based on the serum parameters LDH, CRP, and REC. The groups with low and intermediate risk were pooled due to a small number of cases. The median OS was 17.7 months (95% CI 14.7–20.8) in the low plus intermediate group versus 15.4 months (95% CI 12.7–18.2) in the high risk group versus 7.1 months (95% CI 0.0–16.2) in the very high risk group. The p-values indicated were calculated with the log-rank test. One patient was not included due to missing data Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 Page 6 of 9 Manuscript 4 89 Table 4 Characterization of the responders to combined checkpoint blockade (n= 10) Response Duration of response (months) Time to response (weeks) Treatment cycles (induction + maintenance) Gender Time to metastasis from primary tumor (months) Age at treatment onset (years) Available molecular/ genetic analysis ECOG LDH CRP Risk score Previous systemic treatment Previous liverdirected treatment Sites of metastasis AE ≥grade 3 (CTCAE v5.0) PR 14 5 4 + 0 female 9 46 mutated: GNAQ (Q209P) 0normalnormallow nivolumab (PD) TACE liver yes (colitis) PR 9 10 1 + 0 female 92 54 mutated: GNAQ (Q209P), ALK, MET; wildtype: BRAF, NRAS, GNA11, BAP1 0 normal normal low none none lung yes (colitis) PR 23 10 4 + 0 female 33 77 mutated: GNA11 0 elevated unknown low none chemosaturation liver, mesenteric fat tissue yes (GuillainBarré syndrome) PR 55 13 3 + 0 male 408 67 mutated: GNAQ 0 normal elevated high none surgery liver, nodal yes (colitis, hypophysitis) PR 65 19 4 + 0 female 168 60 mutated: GNAQ; wildtype: BRAF, NRAS, KIT, GNA11 0 elevated normal high none TACE liver, lung, ovarial, cervix, omentum no CR 53 12 4 + 5 male unknown 59 unknown 0 unknown unknown low none surgery lung no CR 50 12 4 + 16 male unknown 45 unknown 0 elevated elevated very high none none liver, bone, pelvic no PR 26 13 3 + 0 female 14 67 unknown 0 elevated normal intermediate none SIRT liver, lung yes (uveitis) PR 25 14 4 + 1 female 30 56 wildtype: BRAF, NRAS, KIT; expression PD-L1 20%; polysomia of chromosome 12 0 elevated elevated high none none liver, lung no PR 11 31 4 + 27 female 53 73 wildtype: BRAF, KIT, KRAS, NRAS, NF1, CDKN2A, CDK4 0 elevated unknown intermediate none chemosaturation liver, bone, nodal, renal no Abbreviations:CR Complete response, PR Partial response, ECOG Eastern Cooperative Oncology Group, LDH Lactate dehydrogenase, CRP C-reactive protein, TACE Transarterial chemoembolization, SIRT Selective internal radiation therapy, AE Adverse event(s), CTCAE Common Terminology Criteria for Adverse Events Heppt et al. Journal for ImmunoTherapy of Cancer (2019) 7:299 Page 7 of 9 Manuscript 5 96 Research Article Amaral, Tampouri, Eigentler et al. Table 1. Patients characteristics. All patients Overall survival p-value >12 m alive >12 m deceased ≤12 m N163 31 23 109 Age !55 y 44 19.4% 34.8% 27.5% 0.670 ≥55 y !70 y 58 45.2% 30.4% 33.9% ≥70 y 61 35.5% 34.8% 38.5% Gender Males 93 45% 57% 61% 0.311 Females 70 55% 43% 39% Extracerebral disease Yes 150 90% 87% 94% 0.526 No 13 10% 13% 6% Previous therapy Yes 81 32% 35% 58% 0.013 No 82 68% 65% 42% LDH Normal 66 55% 44% 36% 0.183† Elevated 69 39% 26% 47% Not available 28 6% 30% 17% Number of BM ≤3 92 77% 70% 48% 0.005 "3 71 23% 30% 52% BRAF status Mutated 84 45% 70% 49% 0.233† Wild-type 74 45% 30% 49% Not available 5 10% 0 2% ECOG PS 011584%74%66%0.147 ≥14816%26%34% GPA 0.0–1.0 33 7% 17% 25% 0.054 1.5–2.0 59 29% 26% 40% 2.5–3.0 25 19% 17% 14% 3.5–4.0 46 45% 39% 21% Clinical features and disease characteristics from all patients are presented, as well as their distribution in three subgroups used to perform the graphic representation in Figure 1 (patients with OS "12 months, still alive at the time of analysis; patients with OS !12 months and already dead at the time of analysis; patients with OS ≤12 months). To determine the relationship between these characteristics in the three different subgroups, we used crosstabs and results are presented in the last column. Bold p-values represent those that are significant. †Analysis performed only for patients with available data. BM: Brain metastasis; ECOG PS: Eastern Cooperative Oncology Group performance status; GPA: Melanoma-specific graded prognostic assessment; LDH: Lactate dehydrogenase; m: Month; Y: Year. Figure 2FshowsOSanalysisforthecombinationbetweenthedominanttherapyandlocaltherapies.Forpatients treated with S/RS +IT, the mOS was 25 months compared with 7 months for patients receiving the combination S/RS +TT. For each of the previous subgroups, the 1 y OS was 69.4 and 62.5% and the 2 y OS was 50.6 and 19.4%, respectively. The mOS for patients treated with WBRT ±systemic therapy was 5 months, and the 1 y OS and 2 y OS were 12.7 and 6.8%, respectively. Table 2 provides more information on OS analysis of systemic therapy and combination with local therapy. Figure 3 provides OS data stratified by the presence of symptoms and treatment with corticosteroids at the time of MBM diagnosis. The mOS for asymptomatic and symptomatic patients was 7 and 4 months (95% CI: 4.6–9.4 and 0.9–4.1; p = 0.359) and for patients not treated and treated with corticotherapy was 8 and 4 months (96% CI: 5.4–10.6 and 1.7–6.3; p = 0.053). 10.2217/imt-2018-0149 Immunotherapy (Epub ahead of print) future science group Manuscript 5 97 Immunotherapy +surgery/radiosurgery associated with favorable survival in MBM Research Article Time/years Patients 1-Y 754 737 634 625 598 590 515 507 497 462 454 446 440 436 430 422 417 404 403 385 384 368 356 2-Y 3-Y † † † † † † † † † † † † † † † † † † † † † † † » » » » » » » » » » » » » » » » » » † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † » 398 1-y 2-y 3-y Time / years Patients 398 414 415 463 468 469 522 585 594 602 626 654 671 693 725 765 765 795 937 967 967 997 1070 1075 1106 1138 1206 1298 1339 1371 » » » » » » » » » 130 1-Y6-M3-M Time/years Time/years Patients 131 132 138 138 143 143 143 144 148 153 155 166 169 173 177 180 186 192 195 196 197 199 215 217 221 227 227 233 240 244 245 256 257 284 296 299 301 306 310 311 316 320 328 332 338 347 349 351 355 356 BRAF-Inh WB-Rad. Ipilimumab PD-1 AB Chemoth. BRAF + MEK-Inh Treatment Symbols PD-1 + Ipi † » Complete response Continues Dead Female Male Progressive disease Stable disease/Partial response Stereotactic radiosurgery Surgery Immunotherapy Targeted therapy Chemotherapy No systemic therapy Dominant systemic treatment 8 7 1-Y6-M3-M Patients 13 15 16 18 19 20 20 20 22 25 28 33 34 36 37 39 39 46 50 52 52 52 52 53 53 55 61 62 67 68 70 71 72 76 77 78 83 86 89 91 93 94 97 97 101 101 105 108 110 111 111 114 114 114 † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † Figure 1. Multiple therapies swimmer´s plots. (A) Patients with OS >12 months, still alive. (B) Patients with OS >12 months deceased. (C) Patients with OS ≤12 months. The number displayed before each patient’s individual swimmers plot represents the OS in days. OS: Overall survival. Finally, the mOS for all patients was 7 months (95% CI: 5.4–8.6) and the 1 year and 2 years OS was 32.5 and 18.7%, respectively. Table 2 provides more information on the mOS for all therapy groups and on the 1 and 2 years OS. Classification & regression tree The classification and regression tree is shown in Figure 4. Node 1 represents the most significant prognostic factor for this population, which is S/RS. Two groups were then generated: Node 2 (patients treated with S/RS) and Node 5 (patients not treated with S/RS, that includes patients treated with WBRT and patients who did not receive radiotherapy). In both groups, the effect of the combination with dominant therapy is subsequently evaluated. The best mOS (17.4 months) was observed in the group of patients treated with S/RS and IT or TT (Node 3). For patients treated with S/RS +CT or no systemic therapy (Node 4) the mOS was 9.9 months. In the group of patients not treated with S/RS but receiving systemic therapy (Node 6), the mOS was 6.6 months for those treated with IT (Node 7) and 6.2 months for those treated with TT or CT (Node 8). The shortest mOS (2 months) was observed in the subgroup of patients that did not receive S/RS or systemic therapy (Node 9). future science group 10.2217/imt-2018-0149 Manuscript 5 98 Research Article Amaral, Tampouri, Eigentler et al. 1–3 mets. > 3 mets. 0.0 Months since brain metastasis diagnosis Overall survival 0 Number at risk 1–3 mets. > 3 mets. 1.0 0.2 0.4 0.6 0.8 6 12 18 24 30 36 92 64 43 20 12 7 5 71 33 15 11 6 2 1 36 3 3 LDH elevated LDH normal 0.0 Months since brain metastasis diagnosis Overall survival 0 Number at risk LDH elevated LDH normal 1.0 0.2 0.4 0.6 0.8 6 12 18 24 30 36 69 36 22 7 3 3 3 66 40 27 19 12 5 3 0.0 Months since brain metastasis diagnosis Overall survival 0 Number at risk No previous therapy Previous therapy 1.0 0.2 0.4 0.6 0.8 6 12 18 24 30 82 52 37 20 9 4 81 45 21 11 9 5 No previous therapy Previous therapy Months since brain metastasis diagnosis 0 Number at risk 0.0–1.0 1.5–2.0 6 12 18 24 30 36 33 14 7 3 2 0 0 59 31 16 11 5 3 1 2.5–3.0 25 19 12 6 4 1 1 3.5–4.0 46 33 23 11 7 5 4 0.0 Overall survival 1.0 0.2 0.4 0.6 0.8 0.0–1.0 1.5–2.0 2.5–3.0 3.5–4.0 Months since brain metastasis diagnosis 0 Number at risk S/S + IT S/S + TT 6 12 18 24 30 36 36 32 28 16 13 5 4 16 13 11 5 2 1 0 S/S + CT 7 7 3 0 0 0 0 S/S and no sys. Tx 14 7 3 2 1 1 1 WBR +/- sys. Tx 55 27 9 6 2 2 1 No RT 35 11 4 2 0 0 0 0.0 Overall survival 1.0 0.2 0.4 0.6 0.8 No RT S/S + CT S/S + IT S/S + TT S/S and no sys. Tx WBR +/- sys. Tx Months since brain metastasis diagnosis 0 Number at risk IT TT 6 12 18 24 30 36 64 46 37 22 15 7 5 44 27 16 7 2 1 0 CT 18 13 3 0 0 0 0 No sys. Tx 37 11 4 2 1 1 1 0.0 Overall survival 1.0 0.2 0.4 0.6 0.8 IT TT CT No sys. Tx Figure 2. Overall survival.Stratified by (A) number of brain metastases (1–3 vs >3), p smaller than 0.0001; (B) LDH (elevated vs normal), p=0.053; (C) prior systemic therapy (yes vs no), p =0.005; (D) melanoma-specificGPA,p=0.002; (E) dominant systemic therapy (IT vs TT vs CT vs No sys. Tx), p smaller than 0.0001; (F) combination dominant systemic therapy and local therapy (S/S+IT vs S/S+TT vs S/S+CT vs S/S and no sys. Tx vs WBR ±sys. Tx vs no RT), p smaller than 0.0001. CT: Chemotherapy; GPA: Graded prognostic assessment; LDH: Lactate dehydrogenase; IT: Immunotherapy; No sys. Tx: No systemic therapy; RT: Radiotherapy; S/S: Surgery/radiosurgery; TT: Targeted therapy; WBR: Whole brain irradiation. 10.2217/imt-2018-0149 Immunotherapy (Epub ahead of print) future science group Manuscript 5 99 Immunotherapy +surgery/radiosurgery associated with favorable survival in MBM Research Article Table 2. Median overall survival and 1 year and 2 years overall survival for dominant systemic therapies and combination with local therapies. Median OS (Months; 95% CI) OS (%; 95% CI) 1yearOS 2yearsOS Systemic therapy (p <0.0001) Immunotherapy 13 (8.1–17.8) 53.1 (40.9–62.2) 37.5 (25.3–49.6) Targeted therapy 7 (3.8–10.2) 29.5 (16–43) 8.4 (0–18) Chemotherapy 7 (5.6–8.4) 11.1 (0–25.6) – No systemic therapy 3 (1.5–4.8) 10.8 (0.8–20.8) – Radiotherapy +systemic therapy (p <0.0001) S/SRS +immunotherapy 25 (14.6–35.4) 69.4 (54.3–84.4) 50.6 (33.5–67.5) S/SRS +targeted therapy 14 (12.1–15.9) 62.5 (38.8–86.2) 19.4 (0–41.3) S/SRS +chemotherapy 11 (8.4–13.6) 28.6 (0–62.1) – S/SRS without systemic therapy 4 (0–2.1) 21.4 (0–43) – Whole brain irradiation ±systemic therapy 5 (3.9–6.1) 12.7 (3.9–21.5) 6.8 (0–13.7) No radiotherapy 3 (1.7–4.2) 11.4 (0.8–22) – CI: Confidence interval; OS: Overall survival; S/SRS: Surgery/radiosurgery. Multivariate analysis In the multivariate analysis (Figure 5), the type of therapy (combination of systemic therapy with surgery and/or radiotherapy) remained a significant prognostic factor (hazard ratio [HR] for S/RS +IT = 0.25) along with ECOG PS (HR: 1.67). The number of MBM was borderline significant (HR: 1.52). The other factors evaluated – gender, BRAF status, presence of extracerebral disease, LDH at the time of diagnosis and previous systemic therapy for stage IV disease were not significant in the multivariate analysis. Discussion The main finding of our study is the considerable impact of IT on OS of patients with MBM, particularly in combination with S/RS. The mOS of patients receiving predominantly IT was significantly longer (mOS = 13 months) than for those treated with other systemic therapies, namely TT and CT. Surprisingly, the mOS for patients predominantly treated with TT and CT was the same (mOS = 7 months) in our population. The combination of systemic immunotherapy and S/RS showed an impressive mOS of 25 months. The 1 year and 2 years OS rates were 69.4 and 50.6%, higher than previously reported [12–16]. However, it should be taken into consideration that these studies, contrary to ours, analyzed the outcomes of monotherapies with CTLA-4 and PD1 inhibitors only. The inclusion of S/RS and WBRT in the analysis already implies a selection bias. S/RS is mainly used in patients with ≤3 MBM while WBRT is used exclusively in patients with >3 MBM. By using this approach in the clinical practice, we are selecting patients with the best prognostic factors to receive predominantly S/RS upfront, which might partially explain the best outcomes with this approach. However, the subgroup of patients treated with S/RS and systemic therapies (Figure 2F, groups 1–4) also includes patients with >3MBM.Inthiscase,S/RS was not performed upfront but later on at the time of progression of individual brain metastases. In a retrospective analysis, this selection bias cannot be completely avoided, and prospective studies are required to answer the questions that remain unanswered here. As for the patients treated with S/RS +TT, the mOS was 14 months and the 1 year OS rate was 62.5% (95% CI: 38.8–86.2), which is better than compared with previously reported data [4,17]. 55 patients treated with WBRT were included (Figure 2F). In this subgroup, the mOS was only 5 months. In the two subgroups of patients that either did not receive systemic therapy or radiotherapy, the mOS was 3 months. In view of the fact that IT and TT are available in clinical practice, and also achieve good results in MBM, treatment with WBRT should be judged critically. For patients who did not receive S/RS but were treated with IT (Figure 4; Node 7), the mOS was 6.6 months, which does not exceed the mOS for patients receiving S/RS and CT or no systemic therapy (Node 4). However, future science group 10.2217/imt-2018-0149 Manuscript 5 100 Research Article Amaral, Tampouri, Eigentler et al. 0.0 Months since brain metastasis diagnosis Overall survival 036 1.0 0.2 0.4 0.6 0.8 6 12 18 24 30 49 2 24 16 7 5 3 114 4 73 42 24 13 6 Number at risk No symptoms Symptomatic No symptoms Symptomatic 0.0 Overall survival 1.0 0.2 0.4 0.6 0.8 Number at risk Corticotherapy No corticotherapy Months since brain metastasis diagnosis 036 6 12 18 24 30 51 1 24 15 6 4 2 109 5 71 43 25 14 7 Corticotherapy No corticotherapy Figure 3. Overall survival.Stratified by (A) presence of symptoms (no symptoms vs symptomatic), p =0.359; and (B) therapy with corticosteroids (no corticotherapy vs corticotherapy) at the time of MBM diagnosis, p =0.053. MBM: Melanoma brain metastasis. in the first group a higher percentage of the patients (>20%) were long-term survivors. We understand that this favors IT as first-line systemic therapy when S/RS is not possible. Our analysis shows that the previous therapies for stage IV disease have a prognostic significance in this population, as well as the number of MBM and performance status, already described by other authors [2,4,18,19]. In our population, LDH, presence of symptoms and corticotherapy at the time of MBM diagnosis were not significant. A considerable large subgroup (23% of the patients) did not receive any systemic treatment for MBM. The majority of these patients were either heavily pretreated for metastatic disease before developing MBM or were clinically unfit, resulting in an OS <12 months. Retrospective analysis including real-world patients’ data remain important since trials addressing specific questions associated with the treatment of patients with MBM are scarce. Results available from the ABC [3] and CheckMate 204 [20] trials showed that the combination of CTLA-4/PD-1 inhibitors is safe and active in patients with asymptomatic MBM, with similar intraand extra-cranial responses, and with better outcomes when given upfront. In the COMBI-MB [4] trial, the first interim analysis showed that, for BRAF mutated patients with 10.2217/imt-2018-0149 Immunotherapy (Epub ahead of print) future science group Manuscript 5 101 Immunotherapy +surgery/radiosurgery associated with favorable survival in MBM Research Article 0.0 0.2 10 040 1.0 0.4 0.6 0.8 20 30 Node 3 (n = 53) mOS = 17.4 months 0.0 0.2 10 040 1.0 0.4 0.6 0.8 20 30 Node 4 (n = 20) mOS = 9.9 months 0.0 0.2 10 040 1.0 0.4 0.6 0.8 20 30 Node 7 (n = 27 ) mOS = 6.6 months 0.0 0.2 10 040 1.0 0.4 0.6 0.8 20 30 Node 8 (n = 39) mOS = 6.2 months 0.0 0.2 10 040 1.0 0.4 0.6 0.8 20 30 Node 9 (n = 24) mOS = 2 months 1 25 6 S/RS S/RS No S/RS Systemic therapy Systemic therapy Systemic therapy No CT IT, TT CT; no ST IT TT, CT Figure 4. Classification and regression tree analysis.Y-axisrepresentstheOSprobabilityandX-axisrepresentstimeinmonths.InNode5 are included the patients who received WBRT and those who did not receive any radiotherapy. CT: Chemotherapy; IT: Immunotherapy; mOS: Median overall survival; n: Number of patient; no ST: No systemic therapy; OS: Overall survival; S/RS: Surgery/radiosurgery; TT: Targeted therapy; WBRT: Whole brain irradiation. asymptomatic MBM, ECOG PS 0/1andnopreviousintracraniallocaltherapy,themOSwas10.8months,with a 1 year OS of 46%, supporting the use of the combination dabrafenib/trametinib in this subgroup. The following limitations need to be considered when examining the results. This is a retrospective analysis from a single center. However, we present data from a rather large number of patients obtained from a very detailed dataset. We included patients diagnosed in 2014, for whom the currently first-line IT and TT were not yet available. The therapeutic approach changed considerably since then. A selection bias has to be considered. The patients with better prognostic factors (≤3 MBM, not progressing under previous therapy and with higher GPA score) mostly received S/RS and IT, which could explain the better outcomes observed. Hence, the results obtained by comparing groups 1–4 (which include patients treated with S/RS and therefore with better prognostic factors upfront) with groups 5 and 6 need to be interpreted with caution (Figure 2F). Our study also presents strengths. In this study, we present OS data from patients with MBM, for which very few data are available. Most of the information available is focused on intracranial response and intracranial disease control. The analysis based on multiple therapies swimmer plots, enabled us to identify the dominant therapy for the individual patient outcome. The results of our study show that this analytic approach can successfully identify effective combinations of systemic treatments with surgery and/or radiotherapy. Although a selection future science group 10.2217/imt-2018-0149 Manuscript 5 102 Research Article Amaral, Tampouri, Eigentler et al. 0.1 0.2 0.5 1.0 2.0 Hazard ratio Female (n = 70) Male (n = 93) S/S only (n = 14) No RT (n = 35) S/S + CT (n = 7) S/S + IT (n = 35) S/S + TT (n = 16) WBRT+-/ST (n = 55) Mutated (n = 84) Unknown (n = 5) Wild-type (n = 74) 1–3 (n = 92) 3+ (n = 71) No (n = 13) Yes (n = 150) 0 (n = 115) 1+ (n = 48) Normal (n = 66) Elevated (n = 69) Not available (n = 28) Naive (n = 82) RT + Sys. Tx Gender BRAF status Nr. of MBM Extracerebral disease ECOG LDH Previous sys. Tx Prev. therapy (n = 81) Reference 1.36 (0.930–1.98) reference 1.34 (0.647 – 2.79) 0.60 (0.219 – 1.63) 0.25 (0.112 – 0.56) 0.40 (0.163 – 1.00) 0.85 (0.399 – 1.81) reference 0.39 (0.091 – 1.69) 1.01 (0.675 – 1.50) reference 1.52 (0.987 – 2.34) reference 1.39 (0.640 – 3.02) reference 1.67 (1.095 – 2.54) reference 1.19 (0.787 – 1.81) 0.87 (0.515 – 1.49) reference 1.40 (0.945 – 2.07) 0.113 0.429 0.316 < 0.001*** 0.05 0.673 0.209 0.972 0.057 0.405 0.017 * 0.406 0.62 0.094 # Events: 132; Global p-value (log-rank): 7.196e-10 A/C: 1132.35; Concordance Index: 0.74 Figure 5. COX multivariate analysis. ECOG: Eastern Cooperative Oncology Group performance status; LDH: Lactate dehydrogenase; RT +Sys. Tx: Radiotherapy plus systemic therapy; S/S: Surgery/radiosurgery; S/S+CT: Surgery/radiosurgery plus chemotherapy; S/S+IT: Surgery/radiosurgery plus immunotherapy; S/S+TT: Surgery/radiosurgery plus targeted therapy; No RT: No radiotherapy; Nr. of MBM: Number of melanoma brain metastases; Previous Sys. Tx: Previous systemic therapy; WBRT ±ST: Whole brain irradiation with or without systemic therapy. bias was present regarding the prognostic factors previously mentioned, the multivariate analysis showed that the combination of S/RS +IT seems to be the best approach, with a clear survival advantage. The median FU after MBM diagnosed was 25 months, which is highly uncommon, supporting our long-term outcomes. Our results are based on data from the CMMR, which is a very detailed database, continuously updated, with well-documented therapy and FU information. Conclusion This retrospective study included 163 patients with MBM treated with a multidisciplinary approach in a tertiary center. This analysis confirmed the prognostic significance of the number of cerebral metastases, the ECOG performance status and GPA score that combines these two aspects. The LDH value was only marginally significant. The new systemic therapies, especially IT, improve the OS of patients with MBM, especially when combined with ablative therapies (S/RS). Finally, our results suggest that in MBM, local therapy should be considered as the first approach whenever possible. IT or TT should be the systemic therapies of choice. If upfront S/RS is not possible, first-line IT should be considered, which is also in line with the results from the ABC and CheckMate 204 clinical trials. 10.2217/imt-2018-0149 Immunotherapy (Epub ahead of print) future science group Manuscript 5 103 Immunotherapy +surgery/radiosurgery associated with favorable survival in MBM Research Article Future perspective The therapeutic approach for patients with MBM changed significantly in the last years. Previously, OS for these patients did not exceed 4–6 months, depending on the publications considered. However, IT and TT changed the spectrum of therapeutic options for these patients, which resulted in improved disease control and survival. Based on the results from clinical trials, combined IT (CTLA-4 +PD-1 inhibitors) seems to be the systemic therapy with the best outcomes in patients with MBM. The combination of dabrafenib and trametinib also showed an improvement in OS in patients with MBM and BRAF mutation. To be noted, intracranial and extracranial response do not seem to differ both for IT and TT. Nonetheless, some questions remain open. The best combination of systemic and local therapy is yet to be determined. Should they be given concurrently or sequential? Data show that concurrent therapy does not seem to increase intracranial toxicity, contrary to what might be expected. Ongoing clinical trials will show the role of the triple combination therapy (PD-1/PD-L1 inhibitors +BRAF/MEK inhibitors) in stage IV melanoma. The triple combination in MBM should also be investigated, particularly if no limiting toxicity is observed in the current ongoing trials. Currently, the cutoff of the MBM for receiving ablative therapy is also the subject of an intense discussion. However, the exact number is not consensual and depends on several factors including the experience of the treating multidisciplinary team. New concepts that are not limited by the number of MBM are being investigated. For patients with more than three MBM, should we treat locally the lesions that will most probably cause symptoms or are progressing, and initiate/continue systemic therapy or should we skip the local therapy and focus predominantly in systemic approaches? Finally, we need to mention the group of patients with symptomatic MBM that are normally excluded from clinical trials. Moreover, immunotherapy might be delayed in these patients since the therapy for symptomatic MBM includes corticosteroids. This aspect is particularly important for patients with BRAF wild-type melanoma who have no other valid systemic therapeutic options and other strategies that help mitigate this problem need to be addressed. Summary points •Treatment of patients with melanoma brain metastases (MBM) has changed significantly in the last years, but remains challenging. •Immunotherapy (particularly the combination of CTLA-4 and PD-1 inhibitors), and targeted therapy (BRAF +MEK inhibitors) have shown to improve disease control and survival in patients with asymptomatic MBM. •Atotalof163consecutivepatientsdiagnosedwithMBMbetween2014and2016wereincludedinthisanalysis. •The dominant therapy for each patient was defined based on the therapy duration and outcome of each systemic therapy. •Prognostic significance factors were number of cerebral metastases, Eastern Cooperative Oncology Group performance status, melanoma-specificgradedprognosticassessmentscoreandprevioussystemictherapies received for stage IV disease. In our population, lactate dehydrogenase, presence of symptoms and corticotherapy at the time of MBM diagnosis were not significant. •The median overall survival (mOS) of patients receiving IT was significantly longer (mOS =13 months) than for those receiving targeted therapy (TT) and chemotherapy (mOS =7monthsforbothTTandCT). •The combination of IT and surgery/radiosurgery (S/RS) showed an mOS of 25 months. The 1 year and 2 years OS rates were 69.4 and 50.6%. •In our analysis, IT and TT improve mOS in patients with MBM, particularly when combined with S/RS. •If upfront S/RS is not possible, first-line IT should be considered. Financial & competing interests disclosure CGarbereportsgrantsandpersonalfeesfromNovartis,grantsandpersonalfeesfromBMS,personalfeesfromMSD,grantsand personal fees from Roche, during the conduct of the study; personal fees from Amgen, personal fees from Philogen, personal fees from LEO, personal fees from Incyte, outside the submitted work. A Forschner serves as a consultant to Roche, Novartis, MSD; received travel grants from Roche, Novartis, BMS, and speaker fees from Roche, Novartis, BMS, MSD. G Tabatabai reports research grants from Roche Diagnostics and Medac, fees for advisory board participation from BMS, fees for lectures for Medac, travel grants from BMS and Medac. T Eigentler serves as consultant to Roche, Novartis, MSD and BMS and received speaker fees future science group 10.2217/imt-2018-0149 Manuscript 5 104 Research Article Amaral, Tampouri, Eigentler et al. from BMS. The remaining authors have declared no conflicts of interest. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Acknowledgments The authors would like to thank S Noor for her assistance with data retrieval. Ethical conduct of research All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study, formal consent is not required. References 1. Gershenwald JE, Scolyer RA, Hess KR et al. Melanoma staging: evidence-based changes in the American Joint Committee on Cancer Eighth Edition cancer staging manual. CA Cancer J. Clin. 67(6), 472–492 (2017). 2. Eigentler TK, Figl A, Krex D et al. Number of metastases, serum lactate dehydrogenase level, and type of treatment are prognostic factors in patients with brain metastases of malignant melanoma. Cancer 117(8), 1697–1703 (2011). 3. Long GV, Atkinson V, Lo S et al. Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: a multicentre randomised Phase II study. Lancet Oncol. 19(5), 672–681 (2018). 4. Davies MA, Saiag P, Robert C et al. Dabrafenib plus trametinib in patients with BRAFV600 mutant melanoma brain metastases (COMBI-MB): a multicentre, multicohort, open-label, Phase II trial. Lancet Oncol. 18(7), 863–873 (2017). 5. Long GV, Stroyakovskiy D, Gogas H et al. Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, Phase III randomised controlled trial. Lancet London 386(9992), 444–451 (2015). 6. Larkin J, Ascierto PA, Dreno B et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N. Eng. J. Med. 371(20), 1867–1876 (2014). 7. Larkin J, Chiarion-Sileni V, Gonzalez R et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N. Eng. J. Med. 373(1), 23–34 (2015). 8. Robert C, Schachter J, Long GV et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Eng. J. Med. 372(26), 2521–2532 (2015). 9. Sperduto PW, Kased N, Roberge D et al. Summary report on the graded prognostic assessment: an accurate and facile diagnosis-specific tool to estimate survival for patients with brain metastases. J. Clin. Oncol. 30(4), 419–425 (2012). 10. Zeileis A, Hothorn T. Partykit: a modular toolkit for recursive partytioning in R. J. Mach. Learn. Res. 16 (2015), pp. 3905–3909 (2015). http://jmlr.org/papers/v16/hothorn15a.html 11. Eisenhauer EA, Therasse P, Bogaerts J et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer 45(2), 228–247 (2009). 12. Ahmed KA, Abuodeh YA, Echevarria MI et al. Clinical outcomes of melanoma brain metastases treated with stereotactic radiosurgery and anti-PD-1 therapy, anti-CTLA-4 therapy, BRAF/MEK inhibitors, BRAF inhibitor, or conventional chemotherapy. Ann. Oncol. 27(12), 2288–2294 (2016). 13. Williams NL, Wuthrick EJ, Kim H et al. Phase I study of Ipilimumab combined with whole brain radiation therapy or radiosurgery for melanoma patients with brain metastases. Int. J. Radiat. Oncol. Biol. Phys. 99(1), 22–30 (2017). 14. Parakh S, Park JJ, Mendis S et al. Efficacy of anti-PD-1 therapy in patients with melanoma brain metastases. Br. J. Cancer 116(12), 1558–1563 (2017). 15. Patel KR, Shoukat S, Oliver DE et al. Ipilimumab and stereotactic radiosurgery versus stereotactic radiosurgery alone for newly diagnosed melanoma brain metastases. Am. J. Clin. Oncol. 40(5), 444–450 (2017). 16. Nardin C, Mateus C, Texier M et al. Tolerance and outcomes of stereotactic radiosurgery combined with anti-programmed cell death-1 (pembrolizumab) for melanoma brain metastases. Melanoma Res. 28(2), 111–119 (2018). 17. Pessina F, Navarria P, Tomatis S et al. Outcome evaluation of patients with limited brain metastasis from malignant melanoma, treated with surgery, radiation therapy, and targeted therapy. World Neurosurg. 105, 184–190 (2017). 18. Frinton E, Tong D, Tan J et al. Metastatic melanoma: prognostic factors and survival in patients with brain metastases. J. Neuro-Oncol. 135(3), 507–512 (2017). 19. Tio M, Wang X, Carlino MS et al. Survival and prognostic factors for patients with melanoma brain metastases in the era of modern systemic therapy. Pigm. Cell Melanoma Res. 31(4), 509–515 (2018). 10.2217/imt-2018-0149 Immunotherapy (Epub ahead of print) future science group Manuscript 5 105 Immunotherapy +surgery/radiosurgery associated with favorable survival in MBM Research Article 20. Tawbi HA, Forsyth PA, Algazi A et al. Combined Nivolumab and Ipilimumab in melanoma metastatic to the brain. N. Engl. J. Med. 379(8), 722–730 (2018). future science group 10.2217/imt-2018-0149 Manuscript 6 112 6AmaralT, etal. J Immunother Cancer 2020;8:e000333. doi:10.1136/jitc-2019-000333 Open access Table 3 Median OS and 1year, 2year and 3year OS rates mOS (months) (95% CI) 1year OS (%; 95% CI) 2year OS (%; 95% CI) 3year OS (%; 95% CI) All patients 19 (15.9 to 22.0) 69 (63.5 to 74.5) 41.1 (34.9 to 47.9) 30.1 (22.2 to 37.9) Number of MBM 1–3 29 (16.9 to 41.4) 71.2 (63.6 to 78.8) 57.0 (46.8 to 67.2) 42.3 (28.6 to 56.0) >3 14 (10.2 to 17.9) 52.1 (44.3 to 59.9) 32.2 (23.9 to 40.4) 22.7 (13.5 to 31.9) BRAF status BRAF wild type 19 (14.9 to 23.0) 61.3 (51.9 to 70.7) 40.1 (28.3 to 51.9) N/A BRAF mutant 18 (14.1 to 21.9) 60.7 (53.8 to 67.6) 42.0 (34.2 to 49.8) 27.3 (18.1 to 36.5) LDH level Normal 21 (15.1 to 26.9) 69.3 (61.6 to 76.7) 45.9 (36.3 to 55.5) 32.6 (20.4 to 44.6) Elevated 19 (12.8 to 25.1) 58.4 (48.8 to 68.0) 40.1 (29.1 to 51.1) 32.9 (19.9 to 45.8) 2×>ULN 7 (6.1 to 7.9) 32.1 (17.6 to 46.6) 22.9 (8.0 to 37.8) 8.6 (5.5 to 22.7) S100B level Normal 22 (18.2 to 25.8) 78.4 (68.6 to 88.2) 44.7 (30.8 to 58.6) 36.1 (20.6 to 51.6) Elevated 17 (9.6 to 24.4) 57.0 (48.2 to 65.8) 42.8 (32.8 to 52.8) 32.3 (20.5 to 44.6) 10×>ULN 17 (8.2 to 25.8) 53.5 (40.4 to 66.6) 30.9 (15.8 to 45.9) 20.6 (1.2 to 40.0) Best intracerebral response CR Not reached 92.7 (82.9 to 100) 85.6 (69.3 to 100) N/A PR 42 (22.6 to 61.4) 86.9 (76.9 to 96.9) 62.9 (46.0 to 79.8) 55.1 (34.5 to 75.7) SD Not reached 93.6 (86.5 to 100) 83.6 (71.1 to 96.1) 50.2 (19.0 to 81.4) PD 10 (16.7 to 23.3) 39.0 (31.4 to 46.6) 20.0 (13.1 to 26.9) 12.8 (6.0 to 19.3) CombiIT First line 17 (10.7 to 23.9) 56.4 (48.9 to 63.8) 44.7 (35.9 to 53.5) 27.9 (11.2 to 44.6) Not first line 21 (17.8 to 24.2) 67.9 (59.9 to 75.9) 41.9 (32.7 to 51.1) 31.6 (21.8 to 41.4) BRAF mutant patients Firstline targeted therapy 22 (17.2 to 26.77) 65.6 (55.2 to 76) 44.3 (34.5 to 57.7) 32.0 (20 to 44) Firstline CombiIT 16 (7 to 25) 53.6 (43.2 to 64) 42.9 (30.7 to 55.1) N/A BRAF wildtype patients Firstline CombiIT 21 (10.2 to 31.8) 59.6 (52.6 to 73.4) 47 (33.8 to 60.1) 47 (33.8 to 60.1) Firstline not CombiIT 19 (16.3 to 21.7) 68.3 (50.1 to 74.2) 31.9 (11.5 to 52.3) 31.9 (11.5 to 52.3) STR/surgery (at any time point) Yes 24 (19.6 to 28.4) 70.6 (63.7 to 77.5) 49.5 (40.9 to 58.1) 36.5 (26.3 to 46.7) No 16 (7.6 to 24.4) 53.2 (41.0 to 65.4) 40.9 (26.6 to 55.2) N/A WBRT 8 (4.9 to 11.0) 40.7 (28.4 to 53.0) 20.8 (9.4 to 32.2) 10.4 (1.4 to 22.2) STR/surgery Upfront 26 (21.1 to 30.9) 72.5 (65.1 to 79.9) 50.9 (41.3 to 60.5) 39.5 (28.3 to 50.7) Later 16 (10.8 to 21.2) 63.7 (47.6 to 79.8) 44.3 (24.9 to 63.7) 22.2 (1.5 to 45.9) ECOGPS 0 22 (16.4 to 27.6) 65.7 (59.0 to 72.4) 47.1 (39.1 to 55.1) 36.4 (26.4 to 46.4) 1 18 (7.3 to 28.7) 52.3 (40.1 to 64.5) 38.0 (34.1 to 519) 22.2 (6.1 to 38.3) >1 8 (7.3 to 17.1) 49.3 (31.8 to 66.7) 23.5 (5.5 to 41.5) 5.9 (5.1 to 16.9) Presence of symptomatic MBM No 19 (10.7 to 27.2) 62.5 (53.4 to 71.8) 45.4 (34.6 to 56.2) 35.1 (21.8 to 48.4) Continued on March 31, 2020 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2019-000333 on 26 March 2020. Downloaded from Manuscript 6 113 7 AmaralT, etal. J Immunother Cancer 2020;8:e000333. doi:10.1136/jitc-2019-000333 Open access mOS (months) (95% CI) 1year OS (%; 95% CI) 2year OS (%; 95% CI) 3year OS (%; 95% CI) Yes 12 (7.0 to 17.0) 46 (32.1 to 59.9) 28.1 (13.8 to 42.4) 15.0 (0 to 30.7) CombiIT, nivolumab plus ipilimumab; CR, complete response; ECOGPS, Eastern Cooperative Oncology Group performance status; MBM, melanoma brain metastases; mOS, median overall survival; PD, progressive disease; PR, partial response; SD, stable disease; STR, stereotactic radiosurgery; ULN, upper level normal; WBRT, whole brain radiotherapy. Table 3 Continued Figure 1 KaplanMeier curves for overall survival (A) and considering the different factors: (B) BRAF status; (C) LDH level; (D) number of melanoma brain metastases (MBM) at the time of therapy with nivolumab+ipilimumab; (E) protein S100B level; (F) best intracranial response. CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease. Figure 2 KaplanMeier curves for overall survival (OS) according to the following factors: (A) local therapy (STR/ surgery, stereotactic radiosurgery or surgery); (B) time of local therapy (before or after combined immunotherapy with nivolumab+ipilimumab); (C) for patients receiving whole brain radiotherapy (WBRT); (D) combined immunotherapy for melanoma brain metastasis (MBM) in first line or later; (E) firstline therapy in patients harboring a BRAF mutation and (F) combined immunotherapy first line or later in BRAF wildtype patients. Patients treated with WBRT were excluded from the analysis in figure2A. Ten patients (4.5%) from the STR/surgery group (n=220) received only surgery. In the KaplanMeier analysis in figure2B, four patients receiving STR/surgery before combined immunotherapy and two patients receiving STR/surgery after combined immunotherapy were treated with the two techniques in an interval of 2 weeks. due to irAEs, we again found no significant difference between the two groups (p=0.913). DISCUSSION The present study shows that combined immunotherapy with NIVO+IPI can result in improved survival of patients with MBM, comparable to results in other stage IV patients. This is particularly true if intracranial CR, PR or SD has been achieved. The type of intracranial response is a strong predictor for OS. In our cohort, the 2year OS rates of patients with SD, PR and CR ranged from 63% to 86%, whereas patients with PD had a 2year OS rate of only 20% (table 3). Similar favorable results have been reported in the ABC trial, a randomized phase II study of nivolumab or NIVO+IPI in patients with MBM.16 The 3year intracranial PFS was above 90% for patients with asymptomatic, treatmentnaïve MBM achieving an intracranial CR, and above 50% for patients with PR. We have no explanation why in our cohort patients with SD did better than patients with PR. In our study, the 1year and 2year OS rate were 69% and 41%, respectively, in line with previous reports.9 10 In the already mentioned ABC trial, patients who received on March 31, 2020 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2019-000333 on 26 March 2020. Downloaded from Manuscript 6 114 8AmaralT, etal. J Immunother Cancer 2020;8:e000333. doi:10.1136/jitc-2019-000333 Open access combined immunotherapy had a 1year and 2year OS rate of 63%16 and in the Checkmate-204 trial the reported 1year OS rate was even higher (81.5%).10 The survival rates in these trials are higher than those reported in our cohort. Compared with the ABC trial and the Checkmate-204 trial, which included patients with asymptomatic MBM and treatmentnaïve BRAF wildtype patients, 31% (60/193) of the patients in our trial had symptomatic MBM and 20% of the BRAF wildtype patients were pretreated. In the Checkmate-204 trial, 17% of the patients had received previous systemic therapy for MBM and 52% had only one MBM compared with 34% pretreated patients and 53% patients with more than three MBM in our cohort. Two studies evaluating pembrolizumab in patients with MBM also reported similar outcomes.17 18 The first study evaluated treatment with pembrolizumab monotherapy in 23 patients with one or more asymptomatic and untreated MBM. With a longer followup of 38 months, the mOS time was 17 months (95% CI: 10 months to not reached) and the 2year OS was 48%. These are in line with our results for patients who did not receive STR/ surgery for whom the mOS was 16 months (95% CI: 7.6 to 24.4) and the 2year OS rate was 41%. However, in this trial, only asymptomatic patients were included and 87% had <3 MBM, a population with potentially better outcome that the one included in our report. In the second study, Anderson et al reported the results of the combination from pembrolizumab and radiation therapy in 21 patients with MBM. Despite the low number of patients included, the percentage of lesions that had a CR (>30%), was higher than previously reported with systemic therapy or STR alone. The combination of immunotherapy and local therapy with stereotactic irradiation or surgery improved patients’ survival compared with patients who only received NIVO+IPI. This benefit might be related to a synergic effect between radiotherapy and immunotherapy that has been demonstrated both in preclinical and clinical studies.19–23 The combination of radiation and immune checkpoint inhibitors seems to be effective both in the irradiated and nonirradiated lesions, and this effect might be associated with the activation of cytotoxic Tcells and reduction of myeloidderived suppressor cells.18 24 25 The benefit of combining local and systemic therapy in MBM has been previously shown by our group and others, with mOS that range from 14 to 25 months and 1year OS rates between 58% and 78% in the groups that received local and systemic therapy, clearly superior to the outcomes of patients receiving only systemic therapy (mOS between 6 and 13 months and 1year OS rates ranging from 34% to 53%).14 15 26–33 In our study, the time point at which the patients received local therapy did not seem to play a significant role in OS: local therapy performed upfront or after initiation of NIVO+IPI resulted in similar OS rates, with a trend benefiting local therapy upfront (mOS 26 months vs 16 months). Different retrospective studies have also addressed this question, and, similar to our cohort, upfront local therapy seems to have better outcomes (mOS of 11–23 months in the group receiving local therapy upfront and 3–9 months in patients receiving local therapy after systemic therapy).34 35 There is still an ongoing debate whether some patients might be better served with systemic therapy alone, as we see very positive outcomes.9–11 36 Not applying local therapy reduces local complications, potential cognitive impairment and might be particularly adequate for patients with a low number of asymptomatic MBM. This question along with the best sequence regarding local therapy is being addressed in ongoing clinical trials, and in the future, we might be better equipped to decide which patients to treat with the different modalities.37 38 In this study, there was a high proportion of patients with BRAFV600mutated melanoma (63%), but similar to other publications where this subgroup represents between 52% and 65% of the patients.14 15 26 28 Previously, it has been postulated that even in patients with BRAFV600mutated MBM, firstline systemic treatment should consist of combined immunotherapy. Our analysis showed that there was no difference in OS of patients receiving firstline NIVO+IPI or firstline targeted therapy followed by combined immunotherapy (p=0.085). The two subgroups did not differ significantly (online supplementary table S4), except for the number of MBM, where a higher proportion of patients with >3 MBM received firstline targeted therapy (p=0.002). Our results in this subgroup need to be interpreted with caution since we have not included patients with BRAFV600 mutation who only received targeted therapy. In the multivariate Cox regression analysis, we identified LDH, S100B, ECOGPS and number of MBM as independent prognostic factors. These prognostic factors have already been described in previous analyses,8 14 39–41 but to the best of our knowledge, S100B has only been described as independent prognostic factor for checkpoint inhibitor immunotherapy in one monocentric study.42 It is interesting, however, that both tumor markers, LDH and S100B, remained independent prognostic factors in the multivariate analysis, suggesting that these noninvasive and easy to determine blood parameters can and should be used early in the course of the disease to inform about patients’ prognosis. Regarding the presence of symptomatic MBM, there was no OS differences between patients with and without symptoms (p=0.065), but a trend can be seem showing that patients with symptomatic MBM have worse prognosis that those who are asymptomatic (1year OS rate 46% and 63%, respectively). In other prospective studies investigating similar cohorts, the OS rate ranged from 66% at 6 months43 to 31% at 12 months.16 Unfortunately, information regarding the presence of symptomatic MBM is missing in approximately 50% of the patients in our study, and therefore, definitive conclusions cannot be drawn from our data. Strengths of this investigation are that data from 23 Germancertified skin cancer centers with high standards on March 31, 2020 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2019-000333 on 26 March 2020. Downloaded from Manuscript 6 115 9 AmaralT, etal. J Immunother Cancer 2020;8:e000333. doi:10.1136/jitc-2019-000333 Open access for data quality were included. Threehundred and eighty patients were analyzed which is thus far the largest published cohort of patients with MBM managed in a routine clinical setting. This high number of patients allowed us to perform subgroup analyses, with results of reasonable sensitivity. Furthermore, this study provides longterm followup data of patients with MBM covering a period of up to 18 months. The study limitations are related to its retrospective design. Patients were included regardless of previous systemic and local therapies prior to the combined immunotherapy and thus some heterogeneity of the study population might have contributed to differences in survival outcomes observed in our cohort. The decision to offer local therapy or not was probably influenced by the number and size of MBM. Additionally, the maximum number of MBM considered to be treated individually by STR/surgery might vary between different centers. We have not evaluated intracranial toxicities. However, this aspect might have been considered when planning local therapy and targeted therapy in patients with BRAFV600mutated melanoma, influencing the systemic therapy offered as well as the therapy sequence in this subgroup. In conclusion, our study shows that treatment with NIVO+IPI, particularly in combination with STR/surgery improves survival of patients with MBM. Results presented herein also suggest that local therapy with STR/surgery either before or after starting combined immunotherapy might be advantageous to prolonging OS. Author affiliations 1Center for Dermatooncology, Department of Dermatology, Eberhard Karls University of Tuebingen, Eberhard Karls University of Tuebingen, Tubingen, Germany 2Skin Cancer Center, Department of Dermatology, Charité Universitätsmedizin Berlin, Berlin, Germany 3Skin Cancer Center, Department of Dermatology and National Center for Tumor Diseases (NCT), University Hospital Heidelberg, Heidelberg, Germany 4Department of Dermatology, University Medical Center Mainz, Mainz, Germany 5Skin Cancer Center, Department of Dermatology, University Hospital Kiel, Kiel, Germany 6Skin Cancer Center, Department of Dermatology, University of Lübeck, Lübeck, Germany 7Department of Dermatology, University Hospital Wuerzburg, Wuerzburg, Germany 8Skin Cancer Center Hannover, Department of Dermatology, Hannover Medical School, Hannover, Germany 9Department of Dermatology, University of Regensburg, Regensburg, Germany 10Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany 11Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, RuprechtKarl University of Heidelberg, Mannheim, Germany 12Department of Dermatology, Universitätsklinikum Erlangen, FriedrichAlexanderUniversität (FAU), ErlangenNürnberg, Germany 13Department of Dermatology and Allergy, University Hospital Munich, Ludwig Maximilian University, Munich, Germany 14Skin Cancer Center, Department of Dermatology and Venerology, Medical Centre University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany 15Skin Cancer Center at the University Cancer Center and National Center for Tumor Diseases Dresden, Department of Dermatology, University Hospital Carl Gustav Carus at the TU Dresden, Dresden, Germany 16Department of Dermatology, University Hospital Magdeburg, Magdeburg, Germany 17Department of Dermatology and Allergy, Technical University of Munich, School of Medicine, Munich, Germany 18Sigmund Freud Universität Wien, Faculty of Medicine, Wien, Austria 19Skin Cancer Center, Department of Dermatology, RuhrUniversity Bochum, Bochum, Germany 20Department of Dermatology, Saarland University Medical School, Homburg/Saar, Germany 21Department of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany 22Department of Dermatology and Allergology, Augsburg Medical Center, Augsburg, Germany 23Skin Cancer Center, Department of Dermatology, Paracelsus Medical University, General Hospital Nuremberg, Nuremberg, Germany 24HELIOS Klinikum Erfurt, Erfurt, Germany 25Clinic for Dermatology and Venereology, University Medical Center, Rostock, Germany 26Clinic of Radiation Oncology, Eberhard Karls University of Tuebingen, Tuebingen, Germany 27Department of Dermatology, University Hospital Essen, Essen, Germany Twitter Teresa Amaral @TeresaSAmaral Contributors Study concept: TA, TE, CG, LZ. Data collection: all authors. Data analysis: TA, TE, CG, LZ. Data interpretation: TA, RG, CB, ChP, TG, JH, FM, TE, CG, LZ. Writing: all authors. Final approval: all authors. Agreement to be accountable for all aspects of the work: all authors. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or notforprofit sectors. Competing interests TA: reports personal fees and travel grants from BMS, grants, personal fees and travel grants from Novartis, personal fees from Pierre Fabre, grants from Neracare, grants from Sanofi, outside the submitted work. FK: reports personal fees from Amgen, personal fees from BMS, personal fees from MSD, grants and personal fees from Novartis, personal fees from Pierre Fabre, personal fees from Roche, personal fees from Sanofi, outside the submitted work. CL: reports personal fees from BMS, personal fees from MSD, personal fees from Merck, personal fees from Novartis, personal fees from Roche, personal fees from Pierre Fabre, personal fees from Sanofi, personal fees from Amgen, personal fees from Biontech, personal fees from Sun Pharma, other from Kiowa Kirin, outside the submitted work. KK: reports grants and personal fees from BMS, personal fees from MSD, during the conduct of the study; personal fees from Amgen, grants and personal fees from NeraCare, grants and personal fees from Novartis, personal fees from Philogen, grants and personal fees from Roche, grants and personal fees from Sanofi, outside the submitted work. PT: reports personal fees from BMS, Novartis, MSD, Pierre Fabre, CureVac and Roche, personal fees from BMS, Novartis, Pierre Fabre, Merck Serono, Sanofi and Roche, nonfinancial support from BMS, Pierre Fabre and Roche, outside the submitted work. AG: reports personal fees from BMS, personal fees from MSD, personal fees from Novartis, personal fees from Pierre Fabre, personal fees from Pfizer, personal fees from Roche, personal fees from Sanofi, outside the submitted work. RG: reports honoraria: Almirall Hermal, Amgen, BristolMyers Squibb (BMS), Incyte, Merck Serono, MSD, Novartis, Pierre Fabre, Pfizer, Roche, SUN; research funding: Amgen, Johnson & Johnson, MerckSerono, Novartis, Pfizer; travel and accommodations: BMS, Merck Serono, Pierre Fabre, Roche, SUN, outside the submitted work. SH: reports grants and personal fees from BMS, personal fees from MSD, during the conduct of the study; personal fees from Amgen, personal fees from Novartis, personal fees from Roche, personal fees from Sanofi, personal fees from Pierre Fabre, outside the submitted work. JU: is on the advisory board or has received honoraria and travel support from Amgen, BMS, GSK, LeoPharma, Merck Sharp & Dohme (MSD), Novartis, Pierre Fabre, Roche, Sanofi outside the submitted work. CB: has been investigator of clinical trials sponsored by Amgen, Array Pharma, BMS, ImmunoCore, MSD, Novartis, Regeneron and Roche; has received speaker’s and/or consultant’s fees by Amgen, BMS, ImmunoCore, Merck, MSD, Novartis, Pierre Fabre, Roche, SanofiAventis and SunPharma, outside the submitted work. DRS: reports personal fees from Novartis, personal fees from Roche, outside the submitted work. AK: reports advisory board honoraria from Novartis Pharma, Roche, travel grants from Amgen and BMS, personal fees from AbbVie and Medac Pharma, outside the submitted work. ChP: reports personal fees from BMS, MSD, Roche, Pierre Fabre, Novatris and SUNPharma for advisory roles during the conduct of the study. TG: reports receiving speakers and/or advisory board honoraria from BMS, SanofiGenzyme, MSD, Novartis Pharma, Roche, AbbVie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, outside the submitted work. ClP: reports personal fees from BMS, personal fees from MSD, during the conduct of the study; personal fees from Amgen, personal fees from Merck Serono, personal fees from Novartis, personal fees from Roche, personal fees from Sanofi, personal fees from Pierre Fabre, outside the submitted on March 31, 2020 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2019-000333 on 26 March 2020. Downloaded from Manuscript 6 116 10 AmaralT, etal. J Immunother Cancer 2020;8:e000333. doi:10.1136/jitc-2019-000333 Open access work. DD: reports consulting and speaking fees and/or payment of travel expenses/ participation fees: Amgen, BMS, MSD, Mylan, Novartis, Pierre Fabre, Roche, Sanofi, outside the submitted work. RH: served as consultant and/or has received speakers’ honoraria from Roche, BMS, MSD, Novartis and Pierre Fabre outside the submitted work. SE: reports advice and speakers’ honoraria from MSD, BMS, Pierre Fabre, Sanofi, Amgen, Novartis, LEO Pharm, ROCHE and Genzyme Corporation, outside the submitted work. JCH: reports personal fees and travel grants from BMS, MSD, Novartis, Roche, Pfizer, Pierre Fabre and Sanofi, grants for scientific projects from BMS, personal fees for participation in advisory boards from MSD and Pierre Fabre, outside the submitted work. FM: reports personal fees and nonfinancial support from Novartis, personal fees and nonfinancial support from Roche, personal fees from MSD, personal fees and nonfinancial support from BMS, personal fees and nonfinancial support from Pierre Fabre, outside the submitted work. TT: reports grants and personal fees from Novartis, grants and personal fees from Roche, outside the submitted work. TE: reports personal fees from Amgen, grants and personal fees from BMS, personal fees from MSD, grants and personal fees from Novartis, personal fees from Pierre Fabre, grants and personal fees from Roche, grants and personal fees from Sanofi, outside the submitted work. CG: reports grants and personal fees from BMS, personal fees from MSD, during the conduct of the study; personal fees from Amgen, grants and personal fees from NeraCare, grants and personal fees from Novartis, personal fees from Philogen, grants and personal fees from Roche, grants and personal fees from Sanofi, outside the submitted work. LZ: served as consultant and/or has received honoraria from Roche, BMS, MSD, Novartis, Pierre Fabre, Sanofi, and travel support from MSD, BMS, Amgen, Pierre Fabre, Sanofi and Novartis, outside the submitted work. Patient consent for publication Not required. Ethics approval and consent to participate The current study was submitted and approved by the Ethics commission of the Eberhard′s Karls University Tuebingen (approval number: 766/2018BO2). Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available on reasonable request. The data that support the findings of this study are available but restrictions apply to the availability of these data, which were used according to the Ethics Commission vote and recommendations for the current study, and so are not publicly available. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BYNC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is noncommercial. See http:// creativecommons. org/ licenses/ bync/ 4. 0/. ORCID iDs TeresaAmaral http:// orcid. org/ 0000000225165181 DavidRafeiShamsabadi http:// orcid. org/ 0000000278503199 REFERENCES 1 Eigentler TK, Figl A, Krex D, etal. Number of metastases, serum lactate dehydrogenase level, and type of treatment are prognostic factors in patients with brain metastases of malignant melanoma. Cancer 2011;117:1697–703. 2 Staudt M, Lasithiotakis K, Leiter U, etal. Determinants of survival in patients with brain metastases from cutaneous melanoma. Br J Cancer 2010;102:1213–8. 3 Avril MF, Aamdal S, Grob JJ, etal. Fotemustine compared with dacarbazine in patients with disseminated malignant melanoma: a phase III study. J Clin Oncol 2004;22:1118–25. 4 Schadendorf D, Hauschild A, Ugurel S, etal. DoseIntensified biweekly temozolomide in patients with asymptomatic brain metastases from malignant melanoma: a phase II DeCOG/ADO study. Ann Oncol 2006;17:1592–7. 5 Gershenwald JE, Scolyer RA, Hess KR, etal. Melanoma staging: evidencebased changes in the American joint Committee on cancer eighth edition cancer staging manual. CA Cancer J Clin 2017;67:472–92. 6 Larkin J, ChiarionSileni V, Gonzalez R, etal. FiveYear survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl J Med 2019;381:1535–46. 7 Robert C, Grob JJ, Stroyakovskiy D, etal. FiveYear outcomes with dabrafenib plus trametinib in metastatic melanoma. N Engl J Med 2019;381:626–36. 8 Franken MG, Leeneman B, Gheorghe M, etal. A systematic literature review and network metaanalysis of effectiveness and safety outcomes in advanced melanoma. Eur J Cancer 2019;123:58–71. 9 Long GV, Atkinson V, Lo S, etal. Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: a multicentre randomised phase 2 study. Lancet Oncol 2018;19:672–81. 10 Tawbi HA, Forsyth PA, Algazi A, etal. Combined nivolumab and ipilimumab in melanoma metastatic to the brain. N Engl J Med 2018;379:722–30. 11 Davies MA, Saiag P, Robert C, etal. Dabrafenib plus trametinib in patients with BRAFV600mutant melanoma brain metastases (COMBIMB): a multicentre, multicohort, openlabel, phase 2 trial. Lancet Oncol 2017;18:863–73. 12 Long GV, Trefzer U, Davies MA, etal. Dabrafenib in patients with Val600Glu or Val600Lys BRAFmutant melanoma metastatic to the brain (BREAKMB): a multicentre, openlabel, phase 2 trial. Lancet Oncol 2012;13:1087–95. 13 Rulli E, Legramandi L, Salvati L, etal. The impact of targeted therapies and immunotherapy in melanoma brain metastases: a systematic review and metaanalysis. Cancer 2019;125:3776–89. 14 Amaral T, Tampouri I, Eigentler T, etal. Immunotherapy plus surgery/ radiosurgery is associated with favorable survival in patients with melanoma brain metastasis. Immunotherapy 2019;11:297–309. 15 Rauschenberg R, Bruns J, Brütting J, etal. Impact of radiation, systemic therapy and treatment sequencing on survival of patients with melanoma brain metastases. Eur J Cancer 2019;110:11–20. 16 Long VGA GV, Lo S, Sandhu SK, etal. Longterm Outcomes from the Randomized Ph 2 Study of Nivolumab or Nivo+Ipilimumab in Patients with Melanoma Brain Metastases - The ABC trial. Ann Oncol 2019;30:v533–63. 17 Kluger HM, Chiang V, Mahajan A, etal. LongTerm survival of patients with melanoma with active brain metastases treated with pembrolizumab on a phase II trial. J Clin Oncol 2019;37:52–60. 18 Anderson ES, Postow MA, Wolchok JD, etal. Melanoma brain metastases treated with stereotactic radiosurgery and concurrent pembrolizumab display marked regression; efficacy and safety of combined treatment. J Immunother Cancer 2017;5:76. 19 Dovedi SJ, Cheadle EJ, Popple AL, etal. Fractionated radiation therapy stimulates antitumor immunity mediated by both resident and infiltrating polyclonal Tcell populations when combined with PD-1 blockade. Clin Cancer Res 2017;23:5514–26. 20 Sharabi AB, Nirschl CJ, Kochel CM, etal. Stereotactic radiation therapy augments antigenspecific PD-1Mediated antitumor immune responses via crosspresentation of tumor antigen. Cancer Immunol Res 2015;3:345–55. 21 Roger A, Finet A, Boru B, etal. Efficacy of combined hypofractionated radiotherapy and antiPD-1 monotherapy in difficulttotreat advanced melanoma patients. Oncoimmunology 2018;7:e1442166. 22 Chandra RA, Wilhite TJ, Balboni TA, etal. A systematic evaluation of abscopal responses following radiotherapy in patients with metastatic melanoma treated with ipilimumab. Oncoimmunology 2015;4:e1046028. 23 Saiag P, Baghad B, Fort M, etal. Efficacy of hypofractionated radiotherapy (RX) in melanoma patients who failed antiPD-1 monotherapy: assessing the abscopal effect. J Clin Oncol 2019;37:9537. 24 TwymanSaint Victor C, Rech AJ, Maity A, etal. Radiation and dual checkpoint blockade activate nonredundant immune mechanisms in cancer. Nature 2015;520:373–7. 25 Ngiow SF, McArthur GA, Smyth MJ. Radiotherapy complements immune checkpoint blockade. Cancer Cell 2015;27:437–8. 26 Tétu P, Allayous C, Oriano B, etal. Impact of radiotherapy administered simultaneously with systemic treatment in patients with melanoma brain metastases within MelBase, a French multicentric prospective cohort. Eur J Cancer 2019;112:38–46. 27 Stera S, Balermpas P, Blanck O, etal. Stereotactic radiosurgery combined with immune checkpoint inhibitors or kinase inhibitors for patients with multiple brain metastases of malignant melanoma. Melanoma Res 2019;29:187–95. 28 Minniti G, Anzellini D, Reverberi C, etal. Stereotactic radiosurgery combined with nivolumab or ipilimumab for patients with melanoma brain metastases: evaluation of brain control and toxicity. J Immunother Cancer 2019;7:102. 29 Tio M, Wang X, Carlino MS, etal. Survival and prognostic factors for patients with melanoma brain metastases in the era of modern systemic therapy. Pigment Cell Melanoma Res 2018;31:509–15. 30 Ahmed KA, Stallworth DG, Kim Y, etal. Clinical outcomes of melanoma brain metastases treated with stereotactic radiation and antiPD-1 therapy. Annals of Oncology 2016;27:434–41. on March 31, 2020 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2019-000333 on 26 March 2020. Downloaded from Manuscript 6 117 11 AmaralT, etal. J Immunother Cancer 2020;8:e000333. doi:10.1136/jitc-2019-000333 Open access 31 Nardin C, Mateus C, Texier M, etal. Tolerance and outcomes of stereotactic radiosurgery combined with antiprogrammed cell death-1 (pembrolizumab) for melanoma brain metastases. Melanoma Res 2018;28:111–9. 32 Chen L, Douglass J, Kleinberg L, etal. Concurrent immune checkpoint inhibitors and stereotactic radiosurgery for brain metastases in nonsmall cell lung cancer, melanoma, and renal cell carcinoma. International Journal of radiation oncology, biology. Physics 2018;100:916–25. 33 Qian JM, Yu JB, Kluger HM, etal. Timing and type of immune checkpoint therapy affect the early radiographic response of melanoma brain metastases to stereotactic radiosurgery. Cancer 2016;122:3051–8. 34 Schmidberger H, Rapp M, Ebersberger A, etal. LongTerm survival of patients after ipilimumab and hypofractionated brain radiotherapy for brain metastases of malignant melanoma: sequence matters. Strahlenther Onkol 2018;194:1144–51. 35 AlvarezBreckenridge C, GiobbieHurder A, Gill CM, etal. Upfront surgical resection of melanoma brain metastases provides a bridge toward ImmunotherapyMediated systemic control. Oncologist 2019;24:671–9. 36 Tawbi HA, Boutros C, Kok D, etal. New era in the management of melanoma brain metastases. Am Soc Clin Oncol Educ Book 2018;38:741–50. 37 Gonzalez M, Hong AM, Carlino MS, etal. A phase II, open label, randomized controlled trial of nivolumab plus ipilimumab with stereotactic radiotherapy versus ipilimumab plus nivolumab alone in patients with melanoma brain metastases (ABCX trial). J Clin Oncol 2019;37:TPS9600–TPS. 38 Stereotactic radiosurgery added to binimetinib and Encorafenib in patients with BRAFV600 melanoma with brain metastasis (BECOMEMB), 2019. Available: https://clinicaltrialsgov/ct2/show/ NCT04074096 39 Choong ES, Lo S, Drummond M, etal. Survival of patients with melanoma brain metastasis treated with stereotactic radiosurgery and active systemic drug therapies. Eur J Cancer 2017;75:169–78. 40 da Silva IP, Lo S, Carlino MS, etal. Clinical factors and overall survival (OS) associated with patterns of metastases (Mets) in melanoma patients (PTS). Ann Oncol 2019;30:v540. 41 ܇uteu P, Todor N, Ignat RM, etal. Clinical prognostic factors associated with survival and a survival score for patients with brain metastases. Future Oncol 2019;15:2619–34. 42 Gambichler T, Brown V, Steuke AK, etal. Baseline laboratory parameters predicting clinical outcome in melanoma patients treated with ipilimumab: a singlecentre analysis. J Eur Acad Dermatol Venereol 2018;32:972–7. 43 Tawbi HAH, Forsyth PAJ, Hodi FS, etal. Efficacy and safety of the combination of nivolumab (NIVO) plus ipilimumab (IPI) in patients with symptomatic melanoma brain metastases (CheckMate 204). J Clin Oncol 2019;37:9501. on March 31, 2020 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2019-000333 on 26 March 2020. Downloaded from Discussion 118 7. Discussion 7.1. Systemic treatment of stage IV cutaneous melanoma Treatment of advanced melanoma has changed amazingly in the last decade, and the results presented in this thesis confirm that. Ten years ago, only chemotherapy with dacarbazine, was approved for treating stage IV melanoma. 49 Non-approved chemotherapies, namely temozolomide, and different combinations of cytotoxic therapies were also used, with marginal benefit. Objective responses to dacarbazine were reported in approximately 25% of the patients in phase II trials. However, later investigations showed that the real percentage was about 5%-12%, and the percentage of patients that derived a long-term benefit was extremely disappointing between 1%-2%. 133-135 Intense investigation was already on the way for other therapeutic options as mentioned by Garbe et al 49, namely CTLA-4 inhibitors and BRAF/MEK inhibitors, but the data available was not enough to grasp the future impact of these therapies. The first manuscript included in this thesis shows that, in our population of patients treated between 2011 and 2014, there was still an important percentage of patients treated with chemotherapy (51.5%; n=132), particularly dacarbazine and carboplatin plus paclitaxel. 136 Immunotherapy with checkpoint inhibitors was mostly represented by ipilimumab monotherapy. In our analysis, 42 patients treated with ipilimumab were included, representing 80.8% of all the patients treated with immunotherapy and 20% of all patients treated with first-line systemic therapy. Finally, 49 patients were treated with BRAF inhibitors, namely vemurafenib or dabrafenib, which represented 72% of the patients treated with targeted therapy, and 23% of all patients treated with first-line systemic therapy. We showed that the OS for stage IV melanoma patients has steadily improved over the years: the 1-year (1-y) OS rate for M1a patients increased from 62% to 85%, for M1b patients improved from 53 to 74%, and for M1c patients from 33 to 52%, when compared Discussion 119 with data from Balch et al from 2009. 137 As the indication, for starting systemic therapy, and performing surgery in stage IV melanoma didn’t change in the period analyzed, the improvement of OS in each M sub-stage was due to the availability of more effective systemic therapies. When looking into the type of therapy received, we also saw a significant improvement compared with the historical data referred by Garbe et al. 49 The median OS (mOS) was 33 months, 16 months, and 11 months for immunotherapy, targeted therapy, and chemotherapy (95% CI: 21.7-44.3; 10.6-21.4 and 7.6-14.4, respectively) and the difference between the three groups was statistically significant (p= 0.003). Our OS results for patients treated with immunotherapy and targeted therapy were in line with the previously reported in clinical trials for treatment naïve patients receiving ipilimumab, and patients treated with dabrafenib monotherapy, i.e., the mOS was 30 months and 19 months, respectively (95% CI: 16.6-not reached and 15.2–23.7). 138,139 The 3-y OS rate for the patients treated with immunotherapy, in our cohort mostly ipilimumab, was 37.4% (95%CI: 16.6-58.2). These OS rates were slightly better than the ones from a pooled analysis reporting the 10-y OS rates of patients treated with ipilimumab monotherapy in different clinical trials.64 However, in this pooled analysis the patients included were both treatment naïve patients and patients that have already received other systemic therapies, which might explain the better outcomes n our cohort, which included only treatment naïve patients. Schadendorf et al. showed that an OS plateau forms around the three years, with a 3-y OS rate of 21% (95% CI: 17%-24%). These results confirmed that approximately one third to one fourth of the patients receiving ipilimumab alone can derive long survival benefit, and led for the first time to a discussion whether stage IV melanoma patients could be cured. This was an historical in the melanoma field. Discussion 120 7.2. Primary resistance to PD-1 based immunotherapy The immune-checkpoint inhibitors pembrolizumab, nivolumab, ipilimumab, and the combination of nivolumab plus ipilimumab are approved in Europe for the treatment of stage IV melanoma. 65,86,87 The EMA’s approval was based on the results from phase II and phase III trials, showing sustained benefits in both PFS and OS. 78 60 79-82,84,138 The 5-y OS rate for nivolumab plus ipilimumab, nivolumab monotherapy and pembrolizumab is 52%, 44% and 34-41%, and the 5-y PFS rate is 36%, 26% and 21-29%, respectively. Despite these very positive outcomes, primary resistance to immunotherapy, defined as the absence of benefit from immunotherapy, still is observed in a rather high percentage of patients. It is estimated to be between 40% and 65%, depending on whether patients receive first-line immunotherapy or immunotherapy after progression under other systemic therapies. 76 90,140 Higher percentages were observed when patients were treated with ipilimumab monotherapy. 141 142 For patients with primary resistance to immune checkpoint therapy, there are not many therapeutic options available, particularly if they have BRAF wild-type tumors. In manuscript number 2, we analyzed the survival outcomes of stage IV melanoma patients treated with first-line PD-1 based immunotherapy between 2015 and 2018.143 By then, pembrolizumab, nivolumab, the combination of nivolumab plus ipilimumab, and two combinations of BRAF/MEK inhibitors were already available in the clinical practice. We addressed the following questions: (1) Which factors are associated with the development of primary resistance? (2) How does survival of patients with primary resistance compare to those with disease control (CR, PR, and SD)? (3) Did the patients with primary resistance to PD-1-based immunotherapy receive further therapies, and if so, which therapies were offered and what was the outcome? We defined primary resistance as the presence of PD using the RECIST 1.1 criteria 144 at the time of the first radiological evaluation, after starting first-line PD-1 based immunotherapy. Discussion 121 In our cohort, we found that 40% of the patients (127/319) were primary resistant according to our definition. Our results confirm the percentages previously reported by Robert et al., and Larkin et al. 76,90,140 We further showed that patients with primary resistance have a highly significantly unfavorable PFS and OS as compared to those who achieve CR, PR or SD (p < 0.0001). The mOS in patients with primary resistance was only 11 months (95% CI: 9.0–13.0), which is similar to what was observed in patients receiving first-line chemotherapy in our series from 2011-2014. 136 These unfavorable outcomes stress the need for further investigation on the mechanisms associated to primary resistance to immunotherapy. The percentage of patients achieving a CR or PR in our study, approximately 40%, was very similar to the one reported in the Checkmate 067 trial, 78 and achieving an objective response (CR or PR) was decisive for favorable OS. This seems to be true not only for the metastatic setting, as in our cohort, but also in the neo-adjuvant setting, as we saw in the OpACIN-neo trial. 145 Here, patients with an objective response, in this case a pathologic objective response, had a numerically higher long-term benefit compared to those with no pathologic objective response. The estimated 24-months relapse free survival was 97% and 36% for patients with and without a pathological objective response, respectively (95% CI: 93-100% and 17-74%). We saw no differences in terms of OS for patients receiving PD-1 monotherapy or combination of nivolumab plus ipilimumab, but the two groups were not homogenous, and therefore no definitive conclusions can be drawn. Furthermore, the follow-up of 22 months is shorter than the one from the Checkmate 067 trial, and in this study the clinically significant OS differences were seen only after 48 months of follow-up. 78 Finally, and similar to other series, only half of the patients with primary resistance received a second-line therapy. 146 The median PFS and the mOS were 3 months and 10 months, respectively (95%CI: 2.4-3.6 and 6.9-13.1). The response rate was extremely Discussion 128 Moreover, the reported 1-y and 2y OS rates (69% and 41%) are comparable to those of the ABC trial (1-y and 2-y OS rate of 63%) and Checkmate 204 trial (1-y OS rate 81.5%). The minor differences can be explained by the different collectives evaluated in each study. Compared with the ABC and the Checkmate 204 trials, which included patients with asymptomatic MBM and treatment-naïve BRAF wild-type patients, 31% (60/193) of the patients in our study had symptomatic MBM, and 20% of the BRAF wildtype patients were pretreated. In the Checkmate 204 trial, 17% of the patients had received previous systemic therapy for MBM and 52% had only one MBM, compared with 34% pretreated patients and 53% patients with more than three MBM in our cohort. The survival outcomes from combining local therapy and systemic therapy were in line with what we have shown in manuscript number 5, and favored the combination of local plus systemic therapy (p=0.009). Receiving local therapy upfront or later had no statistically significant impact in survival, but a trend was seen favoring local therapy upfront, confirming data previously reported by us and other groups. 121,122 Currently, several centers use the “treatment on demand” approach, i. e., patients receive local therapy only for the progressing brain metastasis, while the same systemic therapy is continued, if the extracerebral disease is stable. In our cohort of patients treated with checkpoint inhibition first-line or later, in the subgroup of patients with BRAFV60 mutated melanoma we found no differences in terms of OS when receiving first-line either BRAF and MEK inhibitors or nivolumab plus ipilimumab (p=0.085). Based on our results we cannot produce a definitive conclusion whether BRAFV600 mutated patients should be treated upfront with targeted or immunotherapy, as we did not include the patients that only received targeted therapy. However, results from trials evaluating systemic therapy in stage IV melanoma, showed a higher survival benefit of nivolumab plus ipilimumab in the sub-group of BRAFV600 Discussion 129 mutated melanoma.78 In BRAF wild-type patients, we saw no difference in OS when receiving nivolumab plus ipilimumab in first-line or later (p=0.996). 7.6. Safety profile of immune checkpoint inhibitors and BRAF/MEK inhibitors Targeted and immunotherapies have different safety profiles. This aspect has not been extensively addressed in any of the publications generated by this thesis. Yet, we have presented data on the toxicity induced by PD-1 based immunotherapy in patients treated in our center and others, and a publication is in preparation addressing toxicity associated with BRAF/MEK inhibitors, particularly cardiovascular toxicity. 203 Table 2 and Table 3 refer to the different toxicity in the most important clinical trials. Figure 4 and Figure 5 illustrate the potential mechanism associated with immune-related adverse events (irAE) and propose an algorithm to managing irAE. 204 In clinical trials, we see no significant differences between the toxicity from nivolumab and pembrolizumab, and choosing one or the other has more to do with the patients’ and treating physician preferences, and the frequency of the cycles – every 2 or 4 weeks for nivolumab, and every 3 or 6 weeks for pembrolizumab. For the combination of nivolumab plus ipilimumab versus nivolumab and ipilimumab alone, the differences are related not only to the frequency of the AE but also to their severity. The combination induces a higher percentage and more severe AE than both monotherapies isolated. 78 For ipilimumab, the onset of AE is normally between week 2 and 8, but later onset of AE has been documented. The most frequent AE reported for PD-1 monotherapy was fatigue, and grade 3 and 4 AE are less common with PD-1 monotherapy than with ipilimumab. With the combination of nivolumab plus ipilimumab, almost 95% of patients reported treatment related AE, and more than half of these AEs Discussion 130 were CTCAE grade 3 or higher. 90 These toxicities may develop earlier, and be apparent over a longer period of time. 205 Regarding targeted therapy, the frequency of AE between the three combinations available is similar. 157 However, the type of AE is different, and this aspect is the one that frequently leads to choosing one or the other combination. Dabrafenib induces almost no photosensitivity compared to vemurafenib (41%). It also induces fewer keratoacanthomas and squamous cell carcinomas (7% versus 20-30%). Arthralgia (56%), fatigue (46%) and rash (41%) were commonly reported with vemurafenib treatment. 95 On the other hand, pyrexia is the most common problem associated with dabrafenib treatment, with almost 50% of the patients reporting pyrexia that leads to treatment interruption. 157 As for encorafenib plus binimetinib, the most frequent AE were gastrointestinal (28% to 40%). The cutaneous AE were manageable, and in a percentage between 3% to 13%, similar to dabrafenib plus trametinib and lower than for vemurafenib plus cobimetinib. 103 Finally, MEK inhibitors are associated with ophthalmological toxicity, which is a class effect and normally requires treatment delay. Patients with previous history of ophthalmological issues should be evaluated before treatment start. 206 As always, patients’ collaboration is the backbone for a successful management of toxicity. A structured first appointment, experienced nurses and medical doctors, as well as a dedicated ambulatory support, is one of the reasons why CTACE grade 3 and 4 AE are not so frequently seen in our population of patients. Discussion 131 Figure 4: Mechanism of immune-related adverse events associated with immune checkpoint inhibition The mechanisms of irAE associated with ICI depend on the type of ICI, i.e., anti-PD-1/PD-L1 or antiCTLA4. CTLA-4 inhibitors are able to induce as T cell activation and proliferation, impaired CD4+CD25+ regulatory T cell (Treg cell) survival, and increased counts of type 17 T helper cells. CTLAS-4 inhibitors are also able to induce cross-reactivity between anti-tumor T cells and antigens on normal cells, as well as autoantibody production. PD-1 and PD-L1 inhibitors are able to reduce Treg cell survival and Treg cell inhibitory function. They also increase cytokine production. TCR, T cell receptor; TNF tumor necrosis factor. From RamosCasals et al. 204 who received combination immunotherapy (CTLA-4 and PD-1 inhibitors) with fatal myocarditis had robust Tcell infiltration and clonal expansion with shared Tcell receptors in both the myocardium and the tumour60. In one of the patients, a tenfold increase in expression of PDL1 was demonstrated in affected cardiac tissue compared with nondiseased muscle tissue60. B cellmediated autoantibody production Owing to increased Tcell activation with ICIs, augmented Tcell–B cell interactions can result in autoantibody production. Indeed, interactions between follicular Tcells and B cells in germinal centres are vital for humoral immunity and abnormal interactions have been associated with autoimmunity61. The production of autoantibodies in mouse models of antiCTLA4induced irAEs is common62. Indeed, wildtype mice administered with repeated injections of antiCTLA-4 antibodies develop antipituitary antibodies, and hypophysitis (inflammation of the pituitary gland) is an irAE commonly observed with ipilimumab but not with PD-1 and PDL1 inhibitors62. Further evidence supporting a potential role for B cells in immunotoxicity comes from recent data showing that patients treated with ICIs had B cell changes after a single dose, including reduced numbers of circulating B cells and increased numbers of CD21low B cells and plasmablasts. These early changes were strong predictors of subsequent irAEs63. The detection of autoantibodies during an adverse event would support an immunemediated aetiology and could assist in guiding specific therapeutic intervention. To this end, several autoantibodies have been identified in some patients with specific irAEs, although their presence is not universal across patients. For example, T cell ↑ T cell activation T cell KPȮNVTCVKQP B cell CTLA-4 PD-1 TCR Anti-PD-1 antibody Anti-CTLA-4 antibody ↓ Treg cell function and survival ↑ 2TQKPȯCOOCVQT[ cytokines 1TICPURGEKȮEEGNNU Tumour cell Autoantibody production Autoantibody-mediated damage Direct ICI binding to cell surface (CTLA-4-mediated) IL-17, CXCL10, TNF ↑ T cell proliferation Autoantigen Cross-reactivity Fig. 2 | Mechanism of immune-related adverse events. The mechanisms of immunerelated adverse events owing to immune checkpoint inhibitors (ICIs) depend on the type of ICI therapy used (antiPD-1 or antiPDL1 inhibitors versus anti- %6.#KPJKDKVQTU%6.#KPJKDKVQTUECPKPFWEGUGXGTCNEGNNWNCTCNVGTCVKQPUUWEJCU6|EGNNCEVKXCVKQPCPFRTQNKHGTCVKQP impaired CD4+CD25+TGIWNCVQT[6|EGNN6reg|EGNNUWTXKXCNCPFKPETGCUGFEQWPVUQHV[RG6JGNRGTEGNNUKPCFFKVKQPVQVJG KPFWEVKQPQHETQUUTGCEVKXKV[DGVYGGPCPVKVWOQWT6|EGNNUCPFCPVKIGPUQPJGCNVJ[EGNNUCPFCWVQCPVKDQF[RTQFWEVKQP PD-1 and PDL1 inhibitors lead to a reduction in Treg|EGNNUWTXKXCNCPF6reg|EGNNKPJKDKVQT[HWPEVKQPCPFCPKPETGCUGKPE[VQMKPG RTQFWEVKQP6%46|EGNNTGEGRVQT60(VWOQWTPGETQUKUHCEVQT 6 | Article citation ID: (2020) 6:38 www.nature.com/nrdp PRIMER 0123456789(); Discussion 132 Figure 5: Proposed therapeutic algorithm for the management of immune related adverse events The first-line therapy for patients who develop irAE while receiving treatment with ICI is glucocorticoids, except for adverse events affecting the endocrine system. Other therapies can be considered for severe or refractory cases, including other immunosuppressive therapies, intravenous immunoglobulin, plasma exchange and monoclonal antibodies. These therapeutic suggestions are based on official guidelines, retrospective analysis, published case reports, and the authors personal experience. a) Avoid etanercept owing to the risk of autoimmune inflammatory colitis. b) Consider abatacept or alemtuzumab. c) Consider infliximab or tocilizumab. AIHA/ITP, autoimmune hemolytic anemia/immune thrombocytopenic purpura; ILD, interstitial lung disease; SAD, systemic autoimmune diseases; TNF, tumor necrosis factor. From Ramos-Casals et al. 204 inhibitors and increased survival, providing clinically feasible strategies to dissociate efficacy and toxicity in human trials177. Before administering TNF inhibitors, tests to identify infectious disease, such as a tuberculosis spot test, should be performed as TNF inhibitors can increase the risk of reactivation of certain infections89,174. Vedolizumab (a monoclonal antibody to the integrin α4β7 that inhibits the migration of Tcells into inflamed gastrointestinal mucosa) can be used instead of infliximab for immunerelated colitis178. The theoretical advantage of using vedolizumab is that the immunosuppression would be limited to the gastrointestinal tract and, therefore, spares the systemic immune suppression. In a retrospective study of patients refractory to steroids (n = 19) and infliximab (n = 9) who received vedolizumab, 86% achieved a sustained clinical remission and 54% achieved an endoscopic remission179. Tocil izumab (a n ant iIL-6 antibody) has been suggested for the management of some steroidrefractory irAEs180. One study in patients with nivolumabassociated grades 3–4 irAEs (n = 34; predominantly pneumonitis, serum sickness and systemic inflammatory response syndrome or cerebritis) reported a clinical improvement in 80% of those who received tocilizumab, which, in most cases, required only 1–2 doses to cause clinical improvement181. Another study has reported the effective use of tocilizumab in three cases of severe polyarthritis56. Other monoclonal antibodies have also shown some promise for the treatment of some steroidrefractory irAEs. Rituximab has shown efficacy for the treatment of glucocorticoidrefractory cases of severe encephalitis182, autoimmune cytopenias183 or severe bullous skin disease184. In addition, two cases of successful response to abatacept185 or alemtuzumab186 have been reported in patients with steroidrefractory autoimmune myocarditis. Despite the benefits of monoclonal antibodies for the treatment of steroidrefractory irAEs, they are associated with specific adverse effects that may preclude their use for some irAEs (FIG.5). For example, antiTNF antibodies should be used with caution for treating pneumonitis because there is a risk of exacerbating interstitial lung disease187, whereas using etanercept and tocilizumab to irAE Glucocorticoids Synthetic immunodepressants Intravenous immunoglobulins Plasma exchanges Monoclonal antibody target Hormonal supplementation irAE Other options First line Avoid and/or caution B cell Various c IL-6 TNF a TNF B cell TNF TNF Integrin Integrin SAD Colitis Arthritis AIHA/ITP Nephritis Hepatitis Pancreatitis Endocrine ILD Uveitis Neuropathy Myelitis Encephalitis Myasthenia Myocarditis B cell IL-6 Various b Integrin TNF Fig. 5 | Suggested therapeutic algorithm for the organ-by-organ management of irAEs.9JGPCU[UVGOKEVJGTCR[KU considered in patients presenting with immunerelated adverse events (irAEs) owing to immune checkpoint inhibitors, the firstline treatment is glucocorticoids with an exception for adverse events that affect the endocrine system. Other VJGTCRKGUVQDGEQPUKFGTGFKPUGXGTGTGHTCEVQT[ECUGUFGRGPFQPVJGCHHGEVGFQTICPU[UVGODWVECPKPENWFGU[PVJGVKE immunosuppressants, intravenous immunoglobulin, plasma exchange and monoclonal antibodies. These therapeutic suggestions are based on recommendations included in official guidelines, data from some retrospective studies, isolated published cases and personal experience of the authors. aAvoid etanercept owing to the risk of autoimmune inflammatory colitis. bConsider abatacept or alemtuzumab. c%QPUKFGTKPHNKZKOCDQTVQEKNK\WOCD#+*#+62CWVQKOOWPGJCGOQN[VKE CPCGOKCKOOWPGVJTQODQE[VQRGPKERWTRWTC+.&KPVGTUVKVKCNNWPIFKUGCUG5#&U[UVGOKECWVQKOOWPGFKUGCUGU 60(VWOQWTPGETQUKUHCEVQT 14 | Article citation ID: (2020) 6:38 www.nature.com/nrdp PRIMER 0123456789(); Limitations and conclusions 133 8. Limitations and conclusions The limitations of this study were extensively described and discussed in the respective publications. They include the observational nature of the data, the absence of a population-based sample, the potential referral bias to the centers from which patients were included, the monocentric analysis in three publications, and the potentially limited follow-up time. Nonetheless, we provided insights on patterns of care in distinctive centers in Germany, that might be different from those in other European countries, namely Portugal. This can be explained by the earlier availability of the new systemic therapies, compared to other countries, and the elevated number of German patients included in clinical trials. By using observational data, we were able to inform on the reproducibility of clinical trials’ survival data in a real-world setting. Observational data can further advise on the best strategy to be used in the design of future clinical trials. Finally, we highlighted the need of having a well-designed and continuously updated population-based and therapy-based registry. Data derived from such registries enable clinicians and investigators to examine regional and international differences with an educated opinion on the potential causes of these disparities, and facilitate discussions on how to reduce the obstacles to optimal care. References 134 9. References 1. Apalla Z, Nashan D, Weller RB, et al: Skin Cancer: Epidemiology, Disease Burden, Pathophysiology, Diagnosis, and Therapeutic Approaches. Dermatol Ther (Heidelb) 7:5-19, 2017 2. Bray F, Ferlay J, Soerjomataram I, et al: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68:394-424, 2018 3. Garbe C, Keim U, Eigentler TK, et al: Time trends in incidence and mortality of cutaneous melanoma in Germany. J Eur Acad Dermatol Venereol 33:12721280, 2019 4. Coory M, Baade P, Aitken J, et al: Trends for in situ and Invasive Melanoma in Queensland, Australia, 1982–2002. Cancer Causes & Control 17:21-27, 2006 5. Australia AG-C: Melanoma of the skin statistics. https://melanoma.canceraustralia.gov.au/statistics, 2020 6. Cormier JN, Xing Y, Ding M, et al: Ethnic differences among patients with cutaneous melanoma. Arch Intern Med 166:1907-14, 2006 7. Arnold M, Holterhues C, Hollestein LM, et al: Trends in incidence and predictions of cutaneous melanoma across Europe up to 2015. J Eur Acad Dermatol Venereol 28:1170-8, 2014 8. Cancer IAfRo: International Agency for Research on Cancer report 2020. https://gco.iarc.fr/today/home, 2020 9. Blum A, Garbe C: Epidemiologie, Prävention und Nachsorge maligner Melanome. Der Onkologe 7:18-35, 2001 10. Minini R, Rohrmann S, Braun R, et al: Incidence trends and clinicalpathological characteristics of invasive cutaneous melanoma from 1980 to 2010 in the Canton of Zurich, Switzerland. Melanoma Res 27:145-151, 2017 11. Sacchetto L, Zanetti R, Comber H, et al: Trends in incidence of thick, thin and in situ melanoma in Europe. Eur J Cancer 92:108-118, 2018 12. Teramoto Y, Keim U, Gesierich A, et al: Acral lentiginous melanoma: a skin cancer with unfavourable prognostic features. A study of the German central malignant melanoma registry (CMMR) in 2050 patients. Br J Dermatol 178:443-451, 2018 13. Dennis LK, Vanbeek MJ, Beane Freeman LE, et al: Sunburns and risk of cutaneous melanoma: does age matter? A comprehensive meta-analysis. Annals of epidemiology 18:614-627, 2008 14. Gandini S, Sera F, Cattaruzza MS, et al: Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure. Eur J Cancer 41:45-60, 2005 15. Oliveria SA, Saraiya M, Geller AC, et al: Sun exposure and risk of melanoma. Archives of disease in childhood 91:131-138, 2006 16. Leiter U, Garbe C: Epidemiology of melanoma and nonmelanoma skin cancer--the role of sunlight. Adv Exp Med Biol 624:89-103, 2008 17. Arnold M, de Vries E, Whiteman DC, et al: Global burden of cutaneous melanoma attributable to ultraviolet radiation in 2012. Int J Cancer 143:1305-1314, 2018 18. Blum A, Garbe C, Bauer J: Epidemiologie und Risikofaktoren des malignen Melanoms. Der Onkologe 10:688-700, 2004 19. Pleasance ED, Cheetham RK, Stephens PJ, et al: A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 463:191-6, 2010 References 135 20. Alexandrov LB, Nik-Zainal S, Wedge DC, et al: Signatures of mutational processes in human cancer. Nature 500:415-21, 2013 21. Forschner A, Battke F, Hadaschik D, et al: Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma – results of a prospective biomarker study. Journal for ImmunoTherapy of Cancer 7:180, 2019 22. Chan TA, Yarchoan M, Jaffee E, et al: Development of tumor mutation burden as an immunotherapy biomarker: utility for the oncology clinic. Annals of Oncology 30:44-56, 2019 23. Mihajlovic M, Vlajkovic S, Jovanovic P, et al: Primary mucosal melanomas: a comprehensive review. Int J Clin Exp Pathol 5:739-53, 2012 24. Postow MA, Hamid O, Carvajal RD: Mucosal melanoma: pathogenesis, clinical behavior, and management. Curr Oncol Rep 14:441-8, 2012 25. Moreno MA, Hanna EY: Management of mucosal melanomas of the head and neck: did we make any progress? Curr Opin Otolaryngol Head Neck Surg 18:101-6, 2010 26. Yde SS, Sjoegren P, Heje M, et al: Mucosal Melanoma: a Literature Review. Curr Oncol Rep 20:28, 2018 27. Boer FL, Ten Eikelder MLG, Kapiteijn EH, et al: Vulvar malignant melanoma: Pathogenesis, clinical behaviour and management: Review of the literature. Cancer Treat Rev 73:91-103, 2019 28. D'Angelo SP, Larkin J, Sosman JA, et al: Efficacy and Safety of Nivolumab Alone or in Combination With Ipilimumab in Patients With Mucosal Melanoma: A Pooled Analysis. J Clin Oncol 35:226-235, 2017 29. Shoushtari AN, Munhoz RR, Kuk D, et al: The efficacy of anti-PD-1 agents in acral and mucosal melanoma. Cancer 122:3354-3362, 2016 30. Moya-Plana A, Herrera Gomez RG, Rossoni C, et al: Evaluation of the efficacy of immunotherapy for non-resectable mucosal melanoma. Cancer Immunol Immunother 68:1171-1178, 2019 31. Shoushtari AN, Wagstaff J, Ascierto PA, et al: CheckMate 067: Longterm outcomes in patients with mucosal melanoma. Journal of Clinical Oncology 38:10019-10019, 2020 32. Eschelman DJ, Gonsalves CF, Sato T: Transhepatic therapies for metastatic uveal melanoma. Semin Intervent Radiol 30:39-48, 2013 33. Rowcroft A, Loveday BPT, Thomson BNJ, et al: Systematic review of liver directed therapy for uveal melanoma hepatic metastases. HPB (Oxford), 2019 34. Ho C, McCormack S: CADTH Rapid Response Reports, Radioembolization with yttrium-90 Microspheres for the Management of Uveal Melanoma Liver Metastases: A Review of Clinical Effectiveness and CostEffectiveness. Ottawa (ON), Canadian Agency for Drugs and Technologies in Health Copyright (c) 2018 Canadian Agency for Drugs and Technologies in Health., 2018 35. Huppert PE, Fierlbeck G, Pereira P, et al: Transarterial chemoembolization of liver metastases in patients with uveal melanoma. Eur J Radiol 74:e38-44, 2010 36. Kennedy AS, Nutting C, Jakobs T, et al: A first report of radioembolization for hepatic metastases from ocular melanoma. Cancer Invest 27:682-90, 2009 37. Schelhorn J, Richly H, Ruhlmann M, et al: A single-center experience in radioembolization as salvage therapy of hepatic metastases of uveal melanoma. Acta Radiol Open 4:2047981615570417, 2015 References 136 38. Klingenstein A, Haug AR, Zech CJ, et al: Radioembolization as locoregional therapy of hepatic metastases in uveal melanoma patients. Cardiovasc Intervent Radiol 36:158-65, 2013 39. Heppt MV, Amaral T, Kahler KC, et al: Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study. J Immunother Cancer 7:299, 2019 40. Heppt MV, Heinzerling L, Kahler KC, et al: Prognostic factors and outcomes in metastatic uveal melanoma treated with programmed cell death-1 or combined PD-1/cytotoxic T-lymphocyte antigen-4 inhibition. Eur J Cancer 82:56-65, 2017 41. Heppt MV, Steeb T, Schlager JG, et al: Immune checkpoint blockade for unresectable or metastatic uveal melanoma: A systematic review. Cancer Treat Rev 60:44-52, 2017 42. Wessely A, Steeb T, Erdmann M, et al: The Role of Immune Checkpoint Blockade in Uveal Melanoma. Int J Mol Sci 21, 2020 43. Heppt MV, Amaral T, Kähler KC, et al: Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study. Journal for ImmunoTherapy of Cancer 7:299, 2019 44. Gershenwald JE, Scolyer RA, Hess KR, et al: Melanoma staging: Evidence-based changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA: a cancer journal for clinicians 67:472-492, 2017 45. Grob JJ, Schadendorf D, Lorigan P, et al: Eighth American Joint Committee on Cancer (AJCC) melanoma classification: Let us reconsider stage III. Eur J Cancer 91:168-170, 2018 46. Kanaki T, Stang A, Gutzmer R, et al: Impact of American Joint Committee on Cancer 8th edition classification on staging and survival of patients with melanoma. Eur J Cancer 119:18-29, 2019 47. Isaksson K, Katsarelias D, Mikiver R, et al: A Population-Based Comparison of the AJCC 7th and AJCC 8th Editions for Patients Diagnosed with Stage III Cutaneous Malignant Melanoma in Sweden. Annals of Surgical Oncology 26:28392845, 2019 48. Garbe C, Keim U, Suciu S, et al: Prognosis of Patients With Stage III Melanoma According to American Joint Committee on Cancer Version 8: A Reassessment on the Basis of 3 Independent Stage III Melanoma Cohorts. Journal of Clinical Oncology 0:JCO.19.03034, 2020 49. Garbe C, Eigentler TK, Keilholz U, et al: Systematic review of medical treatment in melanoma: current status and future prospects. Oncologist 16:5-24, 2011 50. Walker LS, Sansom DM: The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses. Nat Rev Immunol 11:852-63, 2011 51. Camacho LH, Antonia S, Sosman J, et al: Phase I/II Trial of Tremelimumab in Patients With Metastatic Melanoma. Journal of Clinical Oncology 27:1075-1081, 2009 52. Hodi FS, O'Day SJ, McDermott DF, et al: Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:711-23, 2010 53. Snyder A, Makarov V, Merghoub T, et al: Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 371:2189-2199, 2014 54. Van Allen EM, Miao D, Schilling B, et al: Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science 350:207-211, 2015 55. Warner AB, Postow MA: Combination Controversies: Checkpoint Inhibition Alone or in Combination for the Treatment of Melanoma? Oncology (Williston Park) 32:228-34, 2018 References 137 56. Guo Q, Huang F, Goncalves C, et al: Translation of cancer immunotherapy from the bench to the bedside. Adv Cancer Res 143:1-62, 2019 57. Amaral T, Meraz-Torres F, Garbe C: Immunotherapy in managing metastatic melanoma: which treatment when? Expert Opinion on Biological Therapy 17:1523-1538, 2017 58. Robert C, Thomas L, Bondarenko I, et al: Ipilimumab plus Dacarbazine for Previously Untreated Metastatic Melanoma. New England Journal of Medicine 364:2517-2526, 2011 59. O'Day SJ, Maio M, Chiarion-Sileni V, et al: Efficacy and safety of ipilimumab monotherapy in patients with pretreated advanced melanoma: a multicenter single-arm phase II study. Ann Oncol 21:1712-1717, 2010 60. Wolchok JD, Neyns B, Linette G, et al: Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study. Lancet Oncol 11:155-64, 2010 61. Weber J, Thompson JA, Hamid O, et al: A randomized, double-blind, placebo-controlled, phase II study comparing the tolerability and efficacy of ipilimumab administered with or without prophylactic budesonide in patients with unresectable stage III or IV melanoma. Clin Cancer Res 15:5591-8, 2009 62. Prieto PA, Yang JC, Sherry RM, et al: CTLA-4 blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. Clin Cancer Res 18:2039-47, 2012 63. Wolchok JD, Weber JS, Maio M, et al: Four-year survival rates for patients with metastatic melanoma who received ipilimumab in phase II clinical trials. Annals of Oncology 24:2174-2180, 2013 64. Schadendorf D, Hodi FS, Robert C, et al: Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma. Journal of Clinical Oncology 33:1889-1894, 2015 65. YERVOY - Summary of Product Characteristics. https://www.ema.europa.eu/en/medicines/human/EPAR/yervoy Last access July 2020, 2020 66. Dong H, Zhu G, Tamada K, et al: B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5:13659, 1999 67. Freeman GJ, Long AJ, Iwai Y, et al: Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192:1027-34, 2000 68. Dong H, Strome SE, Salomao DR, et al: Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8:793800, 2002 69. Topalian SL, Drake CG, Pardoll DM: Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 24:207-12, 2012 70. Iwai Y, Ishida M, Tanaka Y, et al: Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 99:12293-7, 2002 71. Tumeh PC, Harview CL, Yearley JH, et al: PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515:568-71, 2014 72. Topalian SL, Hodi FS, Brahmer JR, et al: Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366:2443-54, 2012 73. Hamid O, Robert C, Daud A, et al: Safety and Tumor Responses with Lambrolizumab (Anti–PD-1) in Melanoma. New England Journal of Medicine 369:134144, 2013 References 144 168. Nathan FE, Berd D, Sato T, et al: BOLD+interferon in the treatment of metastatic uveal melanoma: first report of active systemic therapy. J Exp Clin Cancer Res 16:201-8, 1997 169. Kivelä T, Suciu S, Hansson J, et al: Bleomycin, vincristine, lomustine and dacarbazine (BOLD) in combination with recombinant interferon alpha-2b for metastatic uveal melanoma. Eur J Cancer 39:1115-20, 2003 170. Guenterberg KD, Grignol VP, Relekar KV, et al: A pilot study of bevacizumab and interferon-α2b in ocular melanoma. Am J Clin Oncol 34:87-91, 2011 171. Croce M, Ferrini S, Pfeffer U, et al: Targeted Therapy of Uveal Melanoma: Recent Failures and New Perspectives. Cancers 11, 2019 172. Carvajal RD, Sosman JA, Quevedo JF, et al: Effect of Selumetinib vs Chemotherapy on Progression-Free Survival in Uveal Melanoma: A Randomized Clinical Trial. JAMA 311:2397-2405, 2014 173. Steeb T, Wessely A, Ruzicka T, et al: How to MEK the best of uveal melanoma: A systematic review on the efficacy and safety of MEK inhibitors in metastatic or unresectable uveal melanoma. Eur J Cancer 103:41-51, 2018 174. Carvajal RD, Piperno-Neumann S, Kapiteijn E, et al: Selumetinib in Combination With Dacarbazine in Patients With Metastatic Uveal Melanoma: A Phase III, Multicenter, Randomized Trial (SUMIT). J Clin Oncol 36:1232-1239, 2018 175. Olson D, Bao R, Allred JB, et al: Correlates of overall survival (OS) in metastatic uveal melanoma (mUM) and a randomized trial of cabozantinib (cabo) versus chemotherapy (chemo). Journal of Clinical Oncology 37:9506-9506, 2019 176. Buchbinder EI, Cohen JV, Haq R, et al: A phase II study of ERK inhibition by ulixertinib (BVD-523) in metastatic uveal melanoma. Journal of Clinical Oncology 38:10036-10036, 2020 177. Algazi AP, Tsai KK, Shoushtari AN, et al: Clinical outcomes in metastatic uveal melanoma treated with PD-1 and PD-L1 antibodies. Cancer 122:3344-3353, 2016 178. Rossi E, Zizzari I, Schinzari G, et al: Immune profile of metastatic uveal melanoma during treatment with pembrolizumab. Journal of Clinical Oncology 37:95369536, 2019 179. Park JJ, Diefenbach RJ, Byrne N, et al: Circulating tumor DNA (ctDNA) in patients (pts) with metastatic uveal melanoma (UM) treated with protein kinase C inhibitor (PKCi). Journal of Clinical Oncology 38:e22054-e22054, 2020 180. Najjar YG, Navrazhina K, Ding F, et al: Ipilimumab plus nivolumab for patients with metastatic uveal melanoma: a multicenter, retrospective study. Journal for immunotherapy of cancer 8, 2020 181. Piulats Rodriguez JM DLCM, Espinosa E, Alonso Carrión L, Martin Algarra S, López-Castro R, Curiel García MT, Rodriguez Abreu D, Rullan Iriarte AJ, Berrocal JA.: 1247PD Phase II multicenter, single arm, open label study of nivolumab in combination with ipilimumab in untreated patients with metastatic uveal melanoma (GEM1402.NCT02626962). Ann Oncol. 2018;29(suppl_8):mdy289.003., 2018 182. Pelster M, Gruschkus SK, Bassett R, et al: Phase II study of ipilimumab and nivolumab (ipi/nivo) in metastatic uveal melanoma (UM). Journal of Clinical Oncology 37:9522-9522, 2019 183. The UM CURE 2020 Project. https://www.umcure2020.org/en/; accessed 27.07.2020, 2020 184. Yang J, Orloff MM, Sacco JJ, et al: Resensitization of uveal melanoma (UM) to immune checkpoint inhibition (ICI) by IMCgp100 (IMC). Journal of Clinical Oncology 37:9592-9592, 2019 References 145 185. Phillips S, Lizee G, Brown C, et al: A phase Ib study of endogenous SLC45A2-specific cytotoxic T cells for the treatment of patients with metastatic uveal melanoma. Journal of Clinical Oncology 38:TPS10086-TPS10086, 2020 186. Minor DR, Sato T, Orloff MM, et al: Initial report of treatment of uveal melanoma with hepatic metastases with yttrium90 internal radiation followed by ipilimumab and nivolumab. Journal of Clinical Oncology 38:10025-10025, 2020 187. Sato T, Orloff MM, Valsecchi ME, et al: A randomized phase II study of adjuvant sunitinib or valproic acid in high-risk patients with uveal melanoma. Journal of Clinical Oncology 38:e22059-e22059, 2020 188. Khan S, Lutzky J, Shoushtari AN, et al: Adjuvant crizotinib in high-risk uveal melanoma following definitive therapy. Journal of Clinical Oncology 38:1007510075, 2020 189. Gutzmer R, Vordermark D, Hassel JC, et al: Melanoma brain metastases – Interdisciplinary management recommendations 2020. Cancer Treatment Reviews 89:102083, 2020 190. Leitlinienprogramm Onkologie (Deutsche Krebsgesellschaft, Deutsche Krebshilfe, AWMF): Diagnostik, Therapie und Nachsorge des Melanoms, Langversion 3.2, 2019,. AWMF Registernummer: 032/024OL, http://www.leitlinienprogrammonkologie.de/leitlinien/melanom/ Last access July 2020, 2019 191. Michielin O, van Akkooi ACJ, Ascierto PA, et al: Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up††Approved by the ESMO Guidelines Committee: February 2002, last update September 2019. Annals of Oncology 30:1884-1901, 2019 192. Fife KM, Colman MH, Stevens GN, et al: Determinants of Outcome in Melanoma Patients With Cerebral Metastases. Journal of Clinical Oncology 22:12931300, 2004 193. Davies MA, Liu P, McIntyre S, et al: Prognostic factors for survival in melanoma patients with brain metastases. Cancer 117:1687-1696, 2011 194. Sperduto PW, Jiang W, Brown PD, et al: Estimating Survival in Melanoma Patients With Brain Metastases: An Update of the Graded Prognostic Assessment for Melanoma Using Molecular Markers (Melanoma-molGPA). International Journal of Radiation Oncology*Biology*Physics 99:812-816, 2017 195. Ahmed KA, Abuodeh YA, Echevarria MI, et al: Clinical outcomes of melanoma brain metastases treated with stereotactic radiosurgery and anti-PD-1 therapy, anti-CTLA-4 therapy, BRAF/MEK inhibitors, BRAF inhibitor, or conventional chemotherapy. Ann Oncol 27:2288-2294, 2016 196. Williams NL, Wuthrick EJ, Kim H, et al: Phase 1 Study of Ipilimumab Combined With Whole Brain Radiation Therapy or Radiosurgery for Melanoma Patients With Brain Metastases. Int J Radiat Oncol Biol Phys 99:22-30, 2017 197. Parakh S, Park JJ, Mendis S, et al: Efficacy of anti-PD-1 therapy in patients with melanoma brain metastases. Br J Cancer 116:1558-1563, 2017 198. Patel KR, Shoukat S, Oliver DE, et al: Ipilimumab and Stereotactic Radiosurgery Versus Stereotactic Radiosurgery Alone for Newly Diagnosed Melanoma Brain Metastases. Am J Clin Oncol 40:444-450, 2017 199. Eigentler TK, Figl A, Krex D, et al: Number of metastases, serum lactate dehydrogenase level, and type of treatment are prognostic factors in patients with brain metastases of malignant melanoma. Cancer 117:1697-703, 2011 200. Brown PD, Ballman KV, Cerhan JH, et al: Postoperative stereotactic radiosurgery compared with whole brain radiotherapy for resected metastatic brain disease (NCCTG N107C/CEC·3): a multicentre, randomised, controlled, phase 3 trial. The Lancet Oncology 18:1049-1060, 2017 References 146 201. Garbe C, Amaral T, Peris K, et al: European consensus-based interdisciplinary guideline for melanoma. Part 2: Treatment – Update 2019. European Journal of Cancer, 2019 202. Mahajan A, Ahmed S, McAleer MF, et al: Post-operative stereotactic radiosurgery versus observation for completely resected brain metastases: a singlecentre, randomised, controlled, phase 3 trial. Lancet Oncol 18:1040-1048, 2017 203. Immunotherapy Bridge 2018 and Melanoma Bridge 2018: meeting abstracts. Journal of Translational Medicine 17:1-18, 2019 204. Ramos-Casals M, Brahmer JR, Callahan MK, et al: Immune-related adverse events of checkpoint inhibitors. Nature Reviews Disease Primers 6:38, 2020 205. Haanen JBAG, Carbonnel F, Robert C, et al: Management of toxicities from immunotherapy: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Annals of Oncology 28:iv119-iv142, 2017 206. Stjepanovic N, Velazquez-Martin JP, Bedard PL: Ocular toxicities of MEK inhibitors and other targeted therapies. Annals of Oncology 27:998-1005, 2016 Curriculum vitae 147 10. Curriculum vitae Name Teresa Amaral Address Universitäts-Hautklink Tübingen, Liebermeisterstr. 25, 72076 Tübingen, Germany Telephone + 49 – (0)7071-298-4553 Fax + 49 – (0)7071-29-4599 Email [email protected]-tuebingen.de; [email protected] Birth date 24.11.1980 Nationality Portuguese Family status Single, no children Medical Education 2000 - 2006 Medical degree – Medical Faculty Lisbon University 2013 European Society of Medical Oncology (ESMO) Medical Oncology Examination Amsterdam 06.2014 Specialist in Medical Oncology Medical Experience 2007-2008 General Internship - General Training in Internal Medicine, General Surgery, Gynecology, Pediatrics and Primary Care Medicine – Centro Hospitalar Lisboa Norte (CHLN) – Hospital Pulido Valente - Lisbon 2008 - 2014 Medical Oncology Internship – CHLN –Hospital de Santa Maria – Lisbon 2008 - 2014 Collaboration as co-investigator in trials in skin and ovarian cancers 2011 - 2016 Medical Oncology Unit - Lusíadas Hospital – Lisbon 2012 3 months fellowship (October – December) in the Skin Cancer Center in Tuebingen - Prof. Dr. med Claus Garbe working group (WG) Since 01.2016 Skin Cancer Center - Tuebingen University, Prof. Claus Garbe WG Curriculum vitae 148 Other Activities 2002 Invited teacher/monitor for normal anatomy in the Medical Faculty of Lisbon University Faculdade de Medicina Universidade de Lisboa 2003 Invited teacher/monitor for neuroanatomy 2008 - 2009 Collaboration with Centro de Estudos de Medicina Baseados na Evidência (CEMBE) - Evidence Based Medicine Study Center - Lisbon 2012 - 2013 Invited speaker at the Coimbra University Medicine PhD program 2013 Invited speaker at the Lisbon University Medicine master’s program June 2018 German Approbation Medical Association of Baden-Württemberg (Ärztekammer Baden-Württemberg) Since 2018 Elected - Member of the Portuguese Young Oncologists Group (Núcleo de Internos e Jovens Oncologistas da Sociedade Portuguesa de Oncologia - NIJE) Since 2019 By competitive selection - Allumna of the European Society of Medical Oncology (ESMO) Leaders Generation Program (LGP) Since 2019 By invitation - Member of the ESMO magnitude of clinical benefit scale (ESMO-MCBS) WG, ESMO clinical research observatory (ECRO) task force Since 2019 By invitation - Member of theESMO clinical research observatory (ECRO) task force Since 2019 By invitation - ESMO faculty member for the Melanoma faculty 2019-2023 Since 2020 By invitation - Member of the ESMO Social Media WG Since 01.2020 Elected and nominated - Chair of the ESMO Young Oncologists Committee (YOC) Memberships Since 2016 ESMO member Since 2019 Member of the Sociedade Portuguesa de Oncologia (SPO) Since 2020 Member of American Society Clinical Oncology (ASCO) Military Experience Curriculum vitae 149 2000 - 2006 Portuguese Air Force Academy – Aeronautic Medical Sciences 2006 Aeronautic Medicine Level 1 2006 - 2008 Portuguese Air Force Health Care Direction 2008 - 2009 Azores Air Base 4 / Aeromedical Evacuation Team 2009 - 2014 Military Hospital Lisbon 2014 - 2016 Monte Real Air Base 5 10-12.2014 NATO - Baltic Air Policing 14 Lithuania 06-07.2015 NATO – Falcon Defense 15 Romania 2016 - 2017 Portuguese Air Force Health Care Direction Full list of publications 150 11. Full list of publications Publication IF 1. Sarac E, Wilhelmi J, Thomas I, Leiter U, Keim U, Eigentler TK, Garbe C, Amaral T. Late recurrence of melanoma after 10 years - Is the course of the disease different from early recurrences? Journal of the European Academy of Dermatology and Venereology : JEADV. 2020; 34: 977-83. 5,248 2. Sarac E, Amaral T, Keim U, Leiter U, Forschner A, Eigentler TK, Garbe C. Prognostic factors in 161 patients with mucosal melanoma: a study of German Central Malignant Melanoma Registry. Journal of the European Academy of Dermatology and Venereology : JEADV. 2020 5,248 3. Leiter U, Heppt MV, Steeb T, Amaral T, Bauer A, Becker JC, Breitbart E, Breuninger H, Diepgen T, Dirschka T, Eigentler T, Flaig M, Follmann M, Fritz K, Greinert R, Gutzmer R, Hillen U, Ihrler S, John SM, Kölbl O, Kraywinkel K, Löser C, Nashan D, Noor S, Nothacker M, Pfannenberg C, Salavastru C, Schmitz L, Stockfleth E, Szeimies RM, Ulrich C, Welzel J, Wermker K, Garbe C, Berking C. S3 guideline for actinic keratosis and cutaneous squamous cell carcinoma (cSCC) - short version, part 2: epidemiology, surgical and systemic treatment of cSCC, follow-up, prevention and occupational disease. Journal der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG. 2020; 18: 400-13. 3,664 4. Heppt MV, Leiter U, Steeb T, Amaral T, Bauer A, Becker JC, Breitbart E, Breuninger H, Diepgen T, Dirschka T, Eigentler T, Flaig M, Follmann M, Fritz K, Greinert R, Gutzmer R, Hillen U, Ihrler S, John SM, Kölbl O, Kraywinkel K, Löser C, Nashan D, Noor S, Nothacker M, Pfannenberg C, Salavastru C, Schmitz L, Stockfleth E, Szeimies RM, Ulrich C, Welzel J, Wermker K, Berking C, Garbe C. S3 guideline for actinic keratosis and cutaneous squamous cell carcinoma - short version, part 1: diagnosis, interventions for actinic keratoses, care structures and quality-of-care indicators. Journal der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG. 2020; 18: 27594. 3,664 5. Hilke FJ, Sinnberg T, Gschwind A, Niessner H, Demidov G, Amaral T, Ossowski S, Bonzheim I, Röcken M, Riess O, Garbe C, Schroeder C, Forschner A. Distinct Mutation Patterns Reveal Melanoma Subtypes and Influence Immunotherapy Response in Advanced Melanoma Patients. Cancers. 2020; 12. 6,126 6. Glutsch V, Amaral T, Garbe C, Thoms KM, Mohr P, Hauschild A, Schilling B. Indirect Comparison of Combined BRAF and MEK Inhibition in Melanoma Patients with Elevated Baseline Lactate Dehydrogenase. Acta dermato-venereologica. 2020; 100: adv00174. 4,016 7. Garbe C, Keim U, Suciu S, Amaral T, Eigentler TK, Gesierich A, Hauschild A, Heinzerling L, Kiecker F, Schadendorf D, Stadler R, Sunderkötter C, Tüting T, Utikal J, Wollina U, Zouboulis CC, Keilholz U, Testori A, Martus P, Leiter U, Eggermont AMM. Prognosis of Patients With Stage III Melanoma According to American Joint Committee on 32,956 Full list of publications 151 Cancer Version 8: A Reassessment on the Basis of 3 Independent Stage III Melanoma Cohorts. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2020; 38: 2543-51. 8. Garbe C, Amaral T, Peris K, Hauschild A, Arenberger P, Bastholt L, Bataille V, Del Marmol V, Dréno B, Fargnoli MC, Grob JJ, Höller C, Kaufmann R, Lallas A, Lebbé C, Malvehy J, Middleton M, MorenoRamirez D, Pellacani G, Saiag P, Stratigos AJ, Vieira R, Zalaudek I, Eggermont AMM. European consensus-based interdisciplinary guideline for melanoma. Part 1: Diagnostics - Update 2019. European journal of cancer (Oxford, England : 1990). 2020; 126: 141-58. 7,275 9. Garbe C, Amaral T, Peris K, Hauschild A, Arenberger P, Bastholt L, Bataille V, Del Marmol V, Dréno B, Fargnoli MC, Grob JJ, Höller C, Kaufmann R, Lallas A, Lebbé C, Malvehy J, Middleton M, MorenoRamirez D, Pellacani G, Saiag P, Stratigos AJ, Vieira R, Zalaudek I, Eggermont AMM. European consensus-based interdisciplinary guideline for melanoma. Part 2: Treatment - Update 2019. European journal of cancer (Oxford, England : 1990). 2020; 126: 159-77 7,275 10. Forschner A, Hilke FJ, Bonzheim I, Gschwind A, Demidov G, Amaral T, Ossowski S, Riess O, Schroeder C, Martus P, Klumpp B, GonzalezMenendez I, Garbe C, Niessner H, Sinnberg T. MDM2, MDM4 and EGFR Amplifications and Hyperprogression in Metastatic Acral and Mucosal Melanoma. Cancers. 2020; 12. 6,126 11. Amaral TMS, Hoffmann MC, Sinnberg T, Niessner H, Sülberg H, Eigentler TK, Garbe C. Clinical validation of a prognostic 11-gene expression profiling score in prospectively collected FFPE tissue of patients with AJCC v8 stage II cutaneous melanoma. European journal of cancer (Oxford, England : 1990). 2020; 125: 38-45. 7,275 12. Amaral T, Seeber O, Mersi E, Sanchez S, Thomas I, Meiwes A, Forschner A, Leiter U, Eigentler T, Keim U, Garbe C. Primary Resistance to PD-1-Based Immunotherapy-A Study in 319 Patients with Stage IV Melanoma. Cancers. 2020; 12. 6,126 13. Amaral T, Schulze M, Sinnberg T, Nieser M, Martus P, Battke F, Garbe C, Biskup S, Forschner A. Are Pathogenic Germline Variants in Metastatic Melanoma Associated with Resistance to Combined Immunotherapy? Cancers. 2020; 12. 6,126 14. Amaral T, Kiecker F, Schaefer S, Stege H, Kaehler K, Terheyden P, Gesierich A, Gutzmer R, Haferkamp S, Uttikal J, Berking C, RafeiShamsabadi D, Reinhardt L, Meier F, Karoglan A, Posch C, Gambichler T, Pfoehler C, Thoms K, Tietze J, Debus D, Herbst R, Emmert S, Loquai C, Hassel JC, Meiss F, Tueting T, Heinrich V, Eigentler T, Garbe C, Zimmer L. Combined immunotherapy with nivolumab and ipilimumab with and without local therapy in patients with melanoma brain metastasis: a DeCOG* study in 380 patients. Journal for immunotherapy of cancer. 2020; 8. 9,913 15. Perez-Gracia JL, Awada A, Calvo E, Amaral T, Arkenau H-T, Gruenwald V, Bodoky G, Lolkema MP, Di Nicola M, Penel N, Vera R, Sanmamed 5,329 Full list of publications 152 MF, Douillard J-Y. ESMO Clinical Research Observatory (ECRO): improving the efficiency of clinical research through rationalisation of bureaucracy. ESMO Open. 2020; 5: e000662. 16. Kaesler S, Wölbing F, Kempf WE, Skabytska Y, Köberle M, Volz T, Sinnberg T, Amaral T, Möckel S, Yazdi A, Metzler G, Schaller M, Hartmann K, Weide B, Garbe C, Rammensee HG, Röcken M, Biedermann T. Targeting tumor-resident mast cells for effective antimelanoma immune responses. JCI insight. 2019; 4. 6,014 17. Heppt MV, Amaral T, Kähler KC, Heinzerling L, Hassel JC, Meissner M, Kreuzberg N, Loquai C, Reinhardt L, Utikal J, Dabrowski E, Gesierich A, Pföhler C, Terheyden P, Thoms K-M, Zimmer L, Eigentler TK, Kirchberger MC, Stege HM, Meier F, Schlaak M, Berking C. Combined immune checkpoint blockade for metastatic uveal melanoma: a retrospective, multi-center study. Journal for immunotherapy of cancer. 2019; 7: 299. 9,913 18. Garbe C, Keim U, Eigentler TK, Amaral T, Katalinic A, Holleczek B, Martus P, Leiter U. Time trends in incidence and mortality of cutaneous melanoma in Germany. Journal of the European Academy of Dermatology and Venereology : JEADV. 2019; 33: 1272-80. 5,248 19. Forschner A, Battke F, Hadaschik D, Schulze M, Weißgraeber S, Han CT, Kopp M, Frick M, Klumpp B, Tietze N, Amaral T, Martus P, Sinnberg T, Eigentler T, Keim U, Garbe C, Döcker D, Biskup S. Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma - results of a prospective biomarker study. Journal for immunotherapy of cancer. 2019; 7: 180 9,913 20. Bochem J, Zelba H, Amaral T, Spreuer J, Soffel D, Eigentler T, Wagner NB, Uslu U, Terheyden P, Meier F, Garbe C, Pawelec G, Weide B, Wistuba-Hamprecht K. Peripheral PD-1+CD56+ T-cell frequencies correlate with outcome in stage IV melanoma under PD-1 blockade. PloS one. 2019; 14: e0221301. 2,870 21. Amaral T, Tampouri I, Eigentler T, Keim U, Klumpp B, Heinrich V, Zips D, Paulsen F, Gepfner-Tuma I, Skardelly M, Tatagiba M, Tabatabai G, Garbe C, Forschner A. Immunotherapy plus surgery/radiosurgery is associated with favorable survival in patients with melanoma brain metastasis. Immunotherapy. 2019; 11: 297-309. 2,964 22. Amaral T, Osewold M, Presser D, Meiwes A, Garbe C, Leiter U. Advanced cutaneous squamous cell carcinoma: real world data of patient profiles and treatment patterns. Journal of the European Academy of Dermatology and Venereology : JEADV. 2019; 33 Suppl 8: 44-51. 5,248 23. Amaral T, Tampouri I, Garbe C. How to use neoadjuvant medical treatment to maximize surgery in melanoma. Expert review of anticancer therapy. 2018; 18: 121-30. 3,573 24. Spänkuch I, Gassenmaier M, Tampouri I, Noor S, Forschner A, Garbe C, Amaral T. Severe hepatitis under combined immunotherapy: 7,275 Full list of publications 153 Resolution under corticosteroids plus anti-thymocyte immunoglobulins. European journal of cancer (Oxford, England : 1990). 2017; 81: 203-05. 25. Forschner A, Eichner F, Amaral T, Keim U, Garbe C, Eigentler TK. Improvement of overall survival in stage IV melanoma patients during 2011-2014: analysis of real-world data in 441 patients of the German Central Malignant Melanoma Registry (CMMR). Journal of cancer research and clinical oncology. 2017; 143: 533-40. 3,295 26. Amaral T, Sinnberg T, Meier F, Krepler C, Levesque M, Niessner H, Garbe C. The mitogen-activated protein kinase pathway in melanoma part I - Activation and primary resistance mechanisms to BRAF inhibition. European journal of cancer (Oxford, England : 1990). 2017; 73: 85-92. 7,275 27. Amaral T, Sinnberg T, Meier F, Krepler C, Levesque M, Niessner H, Garbe C. MAPK pathway in melanoma part II-secondary and adaptive resistance mechanisms to BRAF inhibition. European journal of cancer (Oxford, England : 1990). 2017; 73: 93-101. 7,275 28. Amaral T, Meraz-Torres F, Garbe C. Immunotherapy in managing metastatic melanoma: which treatment when? Expert opinion on biological therapy. 2017; 17: 1523-38. 3,540 29. Amaral T, Leiter U, Garbe C. Merkel cell carcinoma: Epidemiology, pathogenesis, diagnosis and therapy. Reviews in endocrine & metabolic disorders. 2017; 18: 517-32. 5,910 30. Amaral T, Garbe C. Reply to 'Recent advances in systemic targeted therapy for cutaneous T-cell lymphoma'. Expert opinion on pharmacotherapy. 2017; 18: 1537. 2,878 31. Amaral T, Garbe C. Non-melanoma skin cancer: new and future synthetic drug treatments. Expert opinion on pharmacotherapy. 2017; 18: 689-99. 2,878 32. Amaral T, Nouri N, Garbe C. The safety and efficacy of cobimetinib for the treatment of BRAF V600E or V600K melanoma. Expert review of anticancer therapy. 2016; 16: 705-15. 3,573 33. Amaral T, Garbe C. Acquired resistance mechanisms to immunotherapy. Annals of translational medicine. 2016; 4: 547. 3,270 34. Macedo D, Amaral T, Fernandes I, Sousa AR, Costa AL, Távora I, Quintela A, Cortes P, Costa L. The Treatment of Liver Metastases in Patients with Neuroendocrine Tumors in 2012. ISRN hepatology. 2013; 2013: 702167. 0 35. Amaral TM, Macedo D, Fernandes I, Costa L. Castration-resistant prostate cancer: mechanisms, targets, and treatment. Prostate cancer. 2012; 2012: 327253. 0 Total 209,279