Tertiary lymphoid structures in pulmonary metastases of microsatellite stable colorectal cancer

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Tertiary lymphoid structures in pulmonary metastases of microsatellite stable colorectal cancer © 2023 the Authors Published version Karjula, Topias; Niskakangas, Anne; Mustonen, Olli; Puro, Iiris; Elomaa, Hanna; Ahtiainen, Maarit; Kuopio, Teijo; Mecklin, Jukka-Pekka; Seppälä, Toni T.; Wirta, Erkki-Ville; Sihvo, Eero; Yannopoulos, Fredrik; Helminen, Olli; Väyrynen, Juha P. Karjula, T., Niskakangas, A., Mustonen, O., Puro, I., Elomaa, H., Ahtiainen, M., Kuopio, T., Mecklin, J.-P., Seppälä, T. T., Wirta, E.-V., Sihvo, E., Yannopoulos, F., Helminen, O., & Väyrynen, J. P. (2023). Tertiary lymphoid structures in pulmonary metastases of microsatellite stable colorectal cancer. Virchows Archiv, 483, 21-32. https://doi.org/10.1007/s00428-023-03577-8 2023 Vol.:(0123456789) 1 3 Virchows Archiv https://doi.org/10.1007/s00428-023-03577-8 ORIGINAL ARTICLE Tertiary lymphoid structures inpulmonary metastases ofmicrosatellite stable colorectal cancer TopiasKarjula1 · AnneNiskakangas1· OlliMustonen1· IirisPuro1· HannaElomaa2,3· MaaritAhtiainen4· TeijoKuopio2,4· Jukka‑PekkaMecklin3,5· ToniT.Seppälä6,7,8· Erkki‑VilleWirta6,9· EeroSihvo10· FredrikYannopoulos1,11· OlliHelminen1· JuhaP.Väyrynen1 Received: 1 March 2023 / Revised: 8 May 2023 / Accepted: 5 June 2023 © The Author(s) 2023 Abstract Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates located at sites of chronic inflammation and recognized as prognosticators in several cancers. We aimed to analyse the prognostic effect of TLSs in colorectal cancer (CRC) pulmonary metastases and primary tumours, with a comparison to the CD3+ and CD8+ cell density-based immune cell score (ICS). For TLS density and TLS maximum diameter analysis, 67 pulmonary metastases and 63 primary tumours were stained with haematoxylin and eosin. For ICS scoring and analysis, CD3 and CD8 immunohistochemistry was performed. Excellent interobserver agreement was achieved in all TLS measurements. Of all patients, 36 patients had low TLS density (< 0.222 follicles/mm) and 31 patients had high TLS density (≥ 0.222 follicles/mm) in the first resected pulmonary metastases. TLS density (adjusted HR 0.91, 0.48–1.73) or maximum diameter (adjusted HR 0.78, 0.40–1.51) did not have prognostic value in pulmonary metastases. In primary tumours, higher TLS density (adjusted HR 0.39, 0.18–0.87) and maximum diameter (adjusted HR 0.28, 0.11–0.73) were associated with lower mortality. In the pulmonary metastases, ICS had superior prognostic value to TLSs; however, TLSs and ICS were significantly associated. In conclusion, TLSs in CRC pulmonary metastases had no prognostic value but correlated with the ICS. TLSs in primary tumours associated with favourable prognosis. Keywords Tertiary lymphoid structures· Pulmonary metastasis· Microsatellite stable colorectal cancer Fredrik Yannopoulos, Olli Helminen, and Juha P. Väyrynen have equal contribution as senior authors. * Topias Karjula [email protected] 1 Translational Medicine Research Unit, Medical Research Center Oulu, Oulu University Hospital andUniversity ofOulu, Aapistie 5a, 90220Oulu, Finland 2 Department ofBiological andEnvironmental Science, University ofJyväskylä, 40014Jyväskylä, Finland 3 Department ofEducation andResearch, Central Finland Health Care District, 40620Jyväskylä, Finland 4 Department ofPathology, Central Finland Health Care District, 40620Jyväskylä, Finland 5 Faculty ofSport andHealth Sciences, University ofJyväskylä, 40014Jyväskylä, Finland 6 Faculty ofMedicine andHealth Technology, Tampere University andTAYS Cancer Center, Tampere University Hospital, 33520Tampere, Finland 7 Department ofGastrointestinal Surgery, Helsinki University Central Hospital, University ofHelsinki, 00290Helsinki, Finland 8 Applied Tumor Genomics, Research Program Unit, University ofHelsinki, 00290Helsinki, Finland 9 Department ofGastroenterology andAlimentary Tract Surgery, Tampere University Hospital, 33520Tampere, Finland 10 Central Hospital ofCentral Finland, 40014Jyväskylä, Finland 11 Department ofCardiothoracic Surgery, Oulu University Hospital, Oulu, Finland Virchows Archiv 1 3 Introduction Lymphocyte populations in the tumour microenvironment have complex antiand pro-tumour interactions with the cancer cells, influencing cancer progression and survival [1]. The adaptive immune response is classically thought to be activated in secondary lymphoid organs such as the lymph nodes and the spleen. However, there is accumulating evidence that an adaptive immune response can be initiated ectopically outside the secondary lymphoid organs via lymphoid neogenesis [2]. In sites of chronic inflammation such as cancer, a spectrum of lymphoid cell aggregates can be found, varying from small lymphoid cell clusters to highly organized structures with germinal centres exhibiting lymph node-like characteristics [2, 3]. These tertiary lymphoid structures (TLSs) have been reported to have prognostic value in several cancer types [4–6]. In colorectal cancer (CRC)—one of the leading causes of cancer mortality globally [7]—peritumoural cancerassociated lymphoid aggregates were named Crohn’s-like lymphoid reaction (CLR) by Graham and Appelman due to their resemblance to the lymphocytic reaction of Crohn’s disease [8]. CLR can be assessed using haematoxylin and eosin (H&E) stained sections and the evaluation can be performed as part of standard routine diagnostics. Higher CLR has been found as a prognosticator for lower risk of regional lymph node metastasis and disease recurrence, as well as longer cancer-specific and overall survival [5, 8–10]. Of all CRC patients, around 5–10% have synchronous pulmonary metastases and around 5% have disease recurrence with pulmonary metastases within 5years after treatment of the primary tumour [11, 12]. Despite advances in cancer therapy, metastatic CRC remains a therapeutic challenge: patients with stage IV CRC at the time of diagnosis have a 5-year survival of only 14% [13]. There are few studies on TLSs in CRC metastases that demonstrate prognostic significance in pulmonary metastases [14] and liver metastases [15]; however, the studies are based on immunohistochemical analysis of individual cell types. Immunohistochemistrybased studies on tumour-infiltrating lymphocyte densities have revealed CD3+ and CD8+ T-cell density-based immune cell score (ICS) having significant prognostic value not only in the primary CRC tumour [16] but also in the liver and pulmonary metastases [17, 18]. TLSs evaluated from HEstained sections would have practical diagnostic advantages. A need for additional classification systems is recognized as the survival within TNM stages varies significantly [19]. This study aimed to analyse the prognostic value of TLSs in resected pulmonary metastases of CRC and corresponding primary tumours, with a comparison to the CD3+ and CD8+ T-cell density-based ICS. Material andmethods Study design All patients with histologically confirmed pulmonary metastases from CRC operated in Oulu University Hospital and Central Finland Central Hospital during 2000–2020 were included in the study. This was a population-based retrospective study. The study hospitals are the only hospitals offering thoracic surgery in their hospital districts. A total of 106 pulmonary metastasectomies for CRC were performed on 74 patients during the study period in the study hospitals. Patients were considered for pulmonary metastasectomy if surgical resection was evaluated to offer curative treatment. Data collection Patients were identified from the archives using surgical registries and pathology reports. All relevant clinical data were retrospectively collected from electronic patient record systems used in the study hospitals. Tumour classification was updated to the American Joint Committee on Cancer (AJCC) 8th edition of tumour-node-metastasis (TNM) classification [20]. Survival data until December 31, 2021, was received from Statistics Finland. The follow-up data were 100% complete. Prospectively collected diagnostic H&E-stained histopathological slides of the primary tumours and pulmonary metastases were retrieved from pathology archives and reviewed by a pathologist. In pulmonary metastases, the most representative slide was selected for further analysis. In primary tumours, the slide with the deepest invasion depth was selected. The slides were digitalized with a × 20 objective magnification and resolution of 50,000 pixels per inch using an Aperio digital scanner AT2 Console (Leica Biosystems Imaging Inc., Wetzlar, Germany) or NanoZoomer-XR (Hamamatsu Photonics, Hamamatsu City, Japan). Histopathological examination A TLS was defined as a dense lymphocyte aggregate separate from the tumour bulk but within a 3.0-mm distance of the invasive margin of the tumour. Germinal centres were not a requirement. Perivascular lymphoid aggregates were also interpreted as TLSs. The minimum diameter accepted as a TLS was 150µm. More diffuse peritumoural lymphocyte zones surrounding or in contact with the tumour bulk were not considered TLSs. TLS evaluation in a pulmonary metastasis and a primary tumour is illustrated in Fig.1. The TLSs were assessed using three criteria. First, TLS density was calculated as the number of lymphocyte follicles divided by the length of the invasive margin as suggested Virchows Archiv 1 3 by Väyrynen etal. [5]. Second, the diameter of the largest TLS was evaluated according to Ueno etal. [10]. Third, the number of TLSs in a hotspot was calculated using a field of view diameter of 5mm. Mismatch repair (MMR) and BRAF mutation status were determined by immunohistochemical analysis from the pulmonary metastases as described previously [17]. All patients were MMR proficient and 4.5% of all patients had a mutated BRAF. Tertiary lymphoid structure scoring Receiver operating characteristics (ROC) analysis was used for optimal cut-off determination. The cut-off points for TLS density, TLS maximum diameter, and TLS hotspot count were 0.222 follicles/mm, 315µm, and 3.50 follicles, respectively, in the pulmonary metastases (Supplementary Fig.1) and 0.161 follicles/mm, 453µm, and 3.50 follicles, respectively in the primary tumours (Supplementary Fig.2). For reproducibility assessment of the TLS evaluation in pulmonary metastases, two independent observers (T.Ka and J.P.V) independently conducted TLS evaluations on the first 20 pulmonary metastases. The interobserver agreement was measured as a continuous variable (Spearman rs) and a two-tiered variable (Cohen’s Kappa κ) using previously mentioned cut-offs. The interobserver agreement was excellent (TLS density rs = 0.87, κ = 0.88; TLS maximum diameter rs = 0.88, κ = 0.80; TLS hotspot count rs = 0.93; κ = 0.88). Immune cell score A CD3+ and CD8+ T-cell density-based ICS in the invasive margin and the tumour centre of the pulmonary metastases and primary CRC tumours was assessed as described earlier [17]. An example of an immune cell density analysis in a pulmonary metastasis is provided in Fig.2. A three-tiered ICS classification was performed with predefined cut-offs of 25% and 70%, following the main principles of the consensus Immunoscore validation article [16]. An additional CD8+ T-cell density-based ICS for pulmonary metastases was evaluated following similar scoring methods as a sensitivity analysis. Outcomes anddefinitions Royal College of Surgeons Charlson Score (RCS) was used for comorbidity classification [21]. The cancer under treatment was included as one comorbidity. Disease-free interval (DFI) was defined as the interval from surgery of CRC tumour to the date of detection of the first pulmonary Fig. 1 Haematoxylin and eosinophil–stained sections illustrating tertiary lymphoid structures (TLSs) in CRC primary tumours (A) and pulmonary metastases (B). Asterisks show examples of TLSs Virchows Archiv 1 3 metastasis. Pulmonary metastases that were detected less than 6months after primary cancer treatment were deemed as synchronous and metachronous if otherwise. The primary outcome of the study was 5-year overall survival from the date of pulmonary metastasectomy to death due to any cause before the end of follow-up. Only 1 patient died of another cause than cancer; therefore, cancer-specific survival was not analysed. Statistical analysis The chi-square test or Fisher’s exact test was used for group comparison in categorical variables. For continuous variable comparison, a Student’s t-test was used for normally distributed variables and a Mann–Whitney U-test was used for continuous variables with a skewed distribution. Spearman correlation coefficients were used for bivariate correlation analysis. ROC analysis was used to determine optimal Fig. 2 T-cell density analysis in a pulmonary metastasis of colorectal cancer. A Immune cell density analysis for representative sites of the tumour centre (TC) and the invasive margin (IM) using QuPath-bioimage software. The width of the invasive margin was 720µm spanning 360µm into the tumour and 360μm into the healthy tissue. B Examples of CD3+ and CD8+ staining patterns and the positive cell detection in the respective site of the tumour Virchows Archiv 1 3 cut-offs using 5-year mortality after pulmonary metastasectomy and 10-year mortality after primary cancer surgery as state variables for pulmonary metastases and primary tumours, respectively. The cut-off value was determined as the point with the shortest distance from the coordinate (0,1). In survival analysis, Kaplan–Meier survival curves were constructed from the first metastasectomy to death or end of the follow-up to visualize survival up to 5years after pulmonary metastasectomy and log-rank tests to compare survivals. The estimates for hazard ratios (HR) with 95% confidence intervals (CI) were calculated using Cox proportional hazards regression. For multivariate analysis of TLS in pulmonary metastases, Cox regression models were adjusted for sex (female/male), age (continuous variable), RCS (1/ ≥ 2), neoadjuvant chemotherapy (no/yes), the synchronicity of pulmonary metastases (synchronous/metachronous), number of pulmonary metastases at diagnosis (1/ ≥ 2), and former liver metastasectomy (no/yes). For multivariate analysis in primary tumours, Cox regression models were adjusted for sex (female/male), age (continuous variable), RCS (1/ ≥ 2), neoadjuvant chemotherapy (no/yes), CRC stage (I–II/III/IV), and CRC grade (1/2/3). Additional adjusted models were constructed including ICS and each TLS measure in pulmonary metastases and primary tumours. Statistical analysis was performed using IBM SPSS Version 28 (IBM Corp., Armonk, NY, USA). Ethical aspects The Oulu University Hospital Ethics Committee (EETMK 81/2008) approved the study. The Finnish National Authority of Medicolegal Affairs (VALVIRA) waived the need for informed consent due to the retrospective nature of the study. The study was performed in accordance with the Declaration of Helsinki. Results Patient characteristics A total of 106 pulmonary metastasectomies from CRC were performed on 70 patients during the study period. Of the metastasectomies, 36 cases were re-metastasectomies performed on 21 patients. Adequate samples for TLS analysis were available for 100 pulmonary metastases and 63 primary tumours. The final cohort of first pulmonary metastases and their corresponding primary CRCs consisted of 67 first resected pulmonary metastasis samples and 63 primary tumour samples. At the time of primary CRC treatment, five patients (7.1%) had stage I CRC, 18 patients (25.7%) had stage II CRC, 27 patients (38.6%) had stage III CRC, and 20 patients (28.6%) had stage IV CRC. The median DFI after primary CRC surgery was 337 (IQR 0–783) days. Twelve patients (17.1%) had bilateral pulmonary metastases and 35.7% of patients had more than one pulmonary metastasis. Four patients (5.7%) had an R1 resection of pulmonary metastases. Former liver metastases of CRC had been diagnosed and treated in 45.7% of all patients. Of all tumours, 50.0% were of rectal origin. The median follow-up time was 40.2months (IQR 20.9–56.3), ranging from 5 to 233months. The overall 5-year survival rate after pulmonary metastasectomy was 28.4%. BRAF mutation status was not associated with overall survival after pulmonary metastasectomy or primary tumour resection. Tertiary lymphoid structures The medians of TLS density, TLS maximum diameter, and TLS hotspot count in the first resected pulmonary metastases were 0.195 follicles/mm (IQR 0.054–0.362 follicles/mm), 325µm (IQR 200–520µm), and 3 follicles (IQR 1–5 follicles), respectively. Patient characteristics according to TLS density of metastases are presented in Table1. Thirty-six patients had low TLS density (< 0.222 follicles/mm) and 31 patients had high TLS density (≥ 0.222 follicles/mm) in the first pulmonary metastases of CRC. TLS density of resected pulmonary metastases was not associated with neoadjuvant chemotherapy or other clinical baseline parameters. In primary tumours, the medians of TLS density, TLS maximum diameter, and TLS hotspot count were 0.195 follicles/mm (IQR 0.073–0.418 follicles/mm), 383µm (IQR 260–577µm), and 3 follicles (IQR 1–5 follicles), respectively. Thirty patients had low TLS density (≤ 0.161 follicles/mm) and 33 patients high TLS density (> 0.161 follicles/mm). Patient characteristics according to TLSs of the primary tumour are presented in Supplementary table1. Higher TLS densities were more uncommon in rectal cancer (p = 0.009) and rarely associated with synchronous pulmonary metastases, although the difference was not statistically significant (p = 0.051). Patients who received neoadjuvant chemotherapy for primary tumours had significantly lower TLS density, maximum diameter, and hotspot density in primary tumours (Supplementary table1). There was no statistically significant difference in the median TLS measures between the first resected pulmonary metastases and primary tumours using the three criteria (TLS density 0.195 vs. 0.195 follicles/mm, p = 0.791; TLS maximum diameter 325 vs. 383µm, p = 0.158; TLS hotspot 3 vs. 3 follicles, p = 0.725). TLS assessments with the three different criteria were significantly correlated with each other both in pulmonary metastases and primary Virchows Archiv 1 3 Table 1 Patient characteristics (n = 67) according to the tertiary lymphoid structure (TLS) measures of the first pulmonary metastasis of colorectal cancer TLS density TLS maximum diameter TLS hotspot Low High p-value Low High p-value Low High p-value n (%) n (%) n (%) n (%) n (%) n (%) n36 31 32 35 38 29 Sex 0.067 0.389 0.067 Female 14 (38.9%) 19 (61.3%) 14 (43.8%) 19 (54.3%) 15 (39.5%) 18 (62.1%) Male 22 (61.1%) 12 (38.7%) 18 (56.3%) 16 (45.7%) 23 (60.5%) 11 (37.9%) Age (M; SD) 68.33 (9.70) 67 (11.15) 0.301 68.28 (9.98) 67.2 (10.77) 0.336 69.39 (9.37) 65.52 (11.27) 0.065 RCS 0.842 0.444 0.954 1 21 (58.3%) 20 (64.5%) 21 (65.6%) 20 (57.1%) 23 (60.5%) 18 (62.1%) 2 9 (25.0%) 6 (19.4%) 5 (15.6%) 10 (28.6%) 9 (23.7%) 6 (20.7%) ≥ 3 6 (16.7%) 5 (16.1%) 6 (18.8%) 5 (14.3%) 6 (15.8%) 5 (17.2%) Neoadjuvant chemotherapy 0.331 0.072 0.952 No 19 (52.8%) 20 (64.5%) 15 (46.9%) 24 (68.6%) 22 (57.9%) 17 (58.6%) Yes 17 (47.2%) 11 (35.5%) 17 (53.1%) 11 (31.4%) 16 (42.1%) 12 (41.4%) CRC stage 0.597 0.321 0.771 1–2 11 (30.6%) 12 (38.7%) 13 (40.6%) 10 (28.6%) 12 (31.6%) 11 (37.9%) 3 13 (36.1%) 12 (38.7%) 9 (28.1%) 16 (45.7%) 14 (36.8%) 11 (37.9%) 4 12 (33.3%) 7 (22.6%) 10 (31.3%) 9 (25.7%) 12 (31.6%) 7 (24.1%) Primary tumour location 0.895 0.273 0.724 Colon 18 (50.0%) 16 (51.6%) 14 (43.8%) 20 (57.1%) 20 (52.6%) 14 (48.3%) Rectum 18 (50.0%) 15 (48.4%) 18 (56.3%) 15 (42.9%) 18 (47.4%) 15 (51.7%) Former LM 0.558 0.726 0.941 No 20 (55.6%) 15 (48.4%) 16 (50.0%) 19 (54.3%) 20 (52.6%) 15 (51.7%) Yes 16 (44.4%) 16 (51.6%) 16 (50.0%) 16 (45.7%) 18 (47.4%) 14 (48.3%) DFI (MD, IQR) 288 (0–843.5) 427 (0–773) 0.569 325 (0–703.5) 482 (0–813) 0.417 288 (0–750) 482 (0–813) 0.351 No. of PM 0.627 0.239 0.977 1 21 (58.3%) 22 (71.0%) 18 (56.3%) 25 (71.4%) 24 (63.2%) 19 (65.5%) 2 12 (33.3%) 7 (22.6%) 10 (31.3%) 9 (25.7%) 11 (28.9%) 8 (27.6%) ≥ 3 3 (8.3%) 2 (6.5%) 4 (12.5%) 1 (2.9%) 3 (7.9%) 2 (6.9%) Laterality of metastases 0.321 0.864 0.443 Unilateral 28 (77.8%) 27 (87.1%) 26 (81.3%) 29 (82.9%) 30 (78.9%) 25 (86.2%) Bilateral 8 (22.2%) 4 (12.9%) 6 (18.8%) 6 (17.1%) 8 (21.1%) 4 (13.8%) Synchronicity 0.773 0.85 0.971 Synchronous 8 (22.2%) 6 (19.4%) 7 (21.9%) 7 (20.0%) 8 (21.1%) 6 (20.7%) Metachronous 28 (77.8%) 25 (80.6%) 25 (78.1%) 28 (80.0%) 30 (78.9%) 23 (79.3%) Size of largest PM (cm; MD; IQR) 1.5 (1–3.5) 2.2 (1.5–3.5) 0.303 2 (1.1–3.5) 2 (1.3–3.5) 0.746 2 (1–3.5) 2.2 (1.5–3.1) 0.376 ICS of metastases 0.031* 0.009* 0.24 Low 7 (19.4%) 2 (6.9%) 6 (18.8%) 3 (9.1%) 6 (15.8%) 3 (11.1%) Intermediate 24 (66.7%) 15 (51.7%) 23 (71.9%) 16 (48.5%) 25 (65.8%) 14 (51.9%) High 5 (13.9%) 12 (41.4%) 3 (9.4%) 14 (42.4%) 7 (18.4%) 10 (37.0%) ICS of primary tumour 0.004* 0.003* 0.221 Low 10 (29.4%) 2 (8.0%) 8 (29.6%) 4 (12.5%) 9 (25.0%) 3 (13.0%) Intermediate 21 (61.8%) 12 (48.0%) 18 (66.7%) 15 (46.9%) 21 (58.3%) 12 (52.2%) High 3 (8.8%) 11 (44.0%) 1 (3.7%) 13 (40.6%) 6 (16.7%) 8 (34.8%) prim TLS density 0.053 0.069 0.04* Low 18 (58.1%) 9 (30.0%) 15 (57.7%) 12 (34.3%) 19 (55.9%) 8 (29.6%) High 13 (41.9%) 21 (70.0%) 11 (42.3%) 23 (65.7%) 15 (44.1%) 19 (70.4%) Virchows Archiv 1 3 tumours (rs = 0.79–0.95, p < 0.001 in pulmonary metastases; rs = 0.717–0.887, p < 0.001 in primary tumours; Table2). TLS measures between the first pulmonary metastases and primary tumours were not significantly correlated in continuous variable analysis. However, after dichotomization using cut-off values selected using the ROC analysis, TLS measures in the pulmonary metastases were associated with those of the primary tumour (TLS maximum diameter: p = 0.007; Table1). Tertiary lymphoid structures andsurvival TLSs in the first resected pulmonary metastases had no statistically significant effect on 5-year survival in K-M analysis (TLS density low 22.2% vs. high 38.7%, p = 0.405; TLS maximum diameter low 17.1% vs. high 42.2%, p = 0.118; TLS hotspot low 20.0% vs. high 44.4%, p = 0.209; Fig.3A–C). In multivariable analysis, TLS did not affect 5-year overall survival (Table3). The variables were dichotomized using cut-offs selected based on the receiver operating characteristics analysis RCS Royal College of Surgeons Charlson Score, CRC colorectal carcinoma, DFI disease-free interval, ICS immune cell score, LM liver metastasectomy, PM pulmonary metastases, prim primary tumour * Statistically significant at the level of < 0.05 Table 1 (continued) TLS density TLS maximum diameter TLS hotspot Low High p-value Low High p-value Low High p-value n (%) n (%) n (%) n (%) n (%) n (%) prim TLS max diameter 0.054 0.007* 0.142 Low 23 (74.2%) 17 (56.7%) 22 (84.6%) 18 (51.4%) 25 (73.5%) 15 (55.6%) High 8 (25.8%) 13 (43.3%) 4 (15.4%) 17 (48.6%) 9 (26.5%) 12 (44.4%) prim TLS hotspot 0.249 0.087 0.21 Low 21 (67.7%) 16 (53.3%) 19 (73.1%) 18 (51.4%) 23 (67.6%) 14 (51.9%) High 10 (32.3%) 14 (46.7%) 7 (26.9%) 17 (48.6%) 11 (32.4%) 13 (48.1%) Table 2 Spearman correlation analysis of tertiary lymphoid structures (TLS) in the first resected pulmonary metastases of colorectal cancer and primary tumours rs, Spearman’s rank correlation coefficient ** Correlation is significant at the 0.01 level Pulmonary metastases Primary tumour TLS density TLS max diameter TLS hotspot TLS density TLS max diameter TLS hotspot Pulmonary metastases TLS density rs1.000 0.815** 0.949** 0.128 0.096 0.164 p < 0.001 < 0.001 0.327 0.461 0.207 N67 67 67 61 61 61 TLS max diameter rs0.815** 1.000 0.778** 0.130 0.144 0.131 p < 0.001 < 0.001 0.320 0.267 0.313 N67 67 67 61 61 61 TLS hotspot rs0.949** 0.778** 1.000 0.098 0.100 0.123 p < 0.001 < 0.001 0.451 0.444 0.343 N67 67 67 61 61 61 Primary tumour TLS density rs0.134 0.136 0.098 1.000 0.781** 0.887** p0.302 0.297 0.451 < 0.001 < 0.001 N61 61 61 63 63 63 TLS max diameter rs0.096 0.144 0.100 0.781** 1.000 0.717** p0.461 0.267 0.444 < 0.001 < 0.001 N61 61 61 63 63 63 TLS hotspot rs0.164 0.131 0.123 0.887** 0.717** 1.000 p0.207 0.313 0.343 < 0.001 < 0.001 N61 61 61 63 63 63 Virchows Archiv 1 3 TLSs in primary tumours were associated with longer survival in 10-year K-M survival analysis (TLS density low 6.0% vs. high 49.4%, p < 0.001; TLS maximum diameter low 10.0% vs. high 64.1%, p < 0.001; TLS hotspot low 14.1% vs. high 52.3%, p = 0.02; Fig.3D–F). In multivariate analysis, TLS density and maximum diameter had a statistically significant effect on 10-year overall survival (TLS density adjusted HR 0.39, 0.18–0.87, p = 0.022; TLS maximum diameter adjusted HR 0.28, 0.11–0.73, p = 0.01; Table3). Tertiary lymphoid structures andimmune cell score In the first resected pulmonary metastases, ICS was significantly associated with TLS density (p = 0.031) and the Time (years) 543210 Cumulative Survival Rate High-censored Low-censored High Low TLS density Time (years) 543210 Cumulative Survival Rate High-censored Low-censored High Low TLS max diameter Time (years) 543210 Cumulative Survival Rate High-censored Low-censored High Low TLS hotspot ABC Time (years) 1086420 Cumulative Survival Rate High-censored Low-censored High Low TLS density Time (years) 1086420 Cumulative Survival Rate High-censored Low-censored High Low TLS max diameter Time (years) 1086420 Cumulative Survival Rate High-censored Low-censored High Low TLS hotspot D E F 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 Fig. 3 K-M curves of 5-year overall survival after pulmonary metastasectomy according to A tertiary lymphoid structure (TLS) density (p = 0.405), B TLS maximum diameter (p = 0.118), C TLS hotspot count (p = 0.209) in the pulmonary metastases, and D TLS density (p = 0.002), E TLS maximum diameter (p < 0.001), and F the TLS hotspot count (p = 0.02) in the primary tumours. Log-rank tests were applied Table 3 Hazard ratios (HR) for 5-year all-cause mortality with 95% confidence intervals in the first pulmonary metastases and primary colorectal tumours according to tertiary lymphoid structure (TLS) measures TLS, tertiary lymphoid structure * Adjusted for sex (female/male), age (continuous), RCS (1/ ≥ 2), neoadjuvant chemotherapy (no/yes), synchronicity of pulmonary metastases (synchronous/metachronous), number of pulmonary metastases at diagnosis (1/ ≥ 2), former liver metastasectomy (no/yes) ** Adjusted for sex (female/male), age (continuous), RCS (1/ ≥ 2), neoadjuvant chemotherapy (no/yes), CRC stage (I–II/III/IV), CRC grade (1/2/3) TLS density TLS maximum diameter TLS hotspot nLow, HR (95% CI) High, HR (95% CI) Low, HR (95% CI) High, HR (95% CI) Low, HR (95% CI) High, HR (95% CI) Metastases Crude 67 1.00 (reference) 0.77 (0.41–1.44; p = 0.407) 1.00 (reference) 0.61 (0.33–1.14; p = 0.122) 1.00 (reference) 0.66 (0.35–1.27; p = 0.212) Adjusted* 67 1.00 (reference) 0.91 (0.48–1.73; p = 0.763) 1.00 (reference) 0.78 (0.40–1.51; p = 0.458) 1.00 (reference) 0.90 (0.50–1.80; p = 0.763) Primary tumours Crude 58 1.00 (reference) 0.38 (0.20–0.72, p = 0.003) 1.00 (reference) 0.25 (0.11–0.57; p < 0.001) 1.00 (reference) 0.44 (0.2–0.90; p = 0.024) Adjusted** 58 1.00 (reference) 0.39 (0.18–0.87; p = 0.022) 1.00 (reference) 0.28 (0.11–0.73; p = 0.01) 1.00 (reference) 0.51 (0.22–1.17; p = 0.112)