Clinicopathological and prognostic correlations of HER3 expression and its degradation regulators, NEDD4-1 and NRDP1, in primary breast cancer 11 Medical and Health Sciences 1112 Oncology and Carcinogenesis

RESEARCH ARTICLE Open Access Clinicopathological and prognostic correlations of HER3 expression and its degradation regulators, NEDD4–1 and NRDP1, in primary breast cancer Satu Luhtala 1* , Synnöve Staff 1,2 , Anne Kallioniemi 1 , Minna Tanner 3 and Jorma Isola 1 Abstract Background: Human epidermal growth factor receptor HER3 (ErbB3), especially in association with its relative HER2 (ErbB2), is known as a key oncogene in breast tumour biology. Nonetheless, the prognostic relevance of HER3 remains controversial. NEDD4–1 and NRDP1 are signalling molecules closely related to the degradation of HER3 via ubiquitination. NEDD4–1 and NRDP1 have been reported to contribute to HER3-mediated signalling by regulating its localization and cell membrane retention. We studied correlations between HER3, NEDD4–1, and NRDP1 protein expression and their association with tumour histopathological characteristics and clinical outcomes. Methods: The prevalence of immunohistochemically detectable expression profiles of HER3 (n= 177), NEDD4–1 (n= 145), and NRDP1 (n= 145) proteins was studied in primary breast carcinomas on archival formalin-fixed paraffin-embedded (FFPE) samples. Clinicopathological correlations were determined statistically using Pearson’s Chi-Square test. The Kaplan-Meier method, log-rank test (Mantel-Cox), and Cox regression analysis were utilized for survival analysis. Results: HER3 protein was expressed in breast carcinomas without association with HER2 gene amplification status. Absence or low HER3 expression correlated with clinically aggressive features, such as triple-negative breast cancer (TNBC) phenotype, basal cell origin (cytokeratin 5/14 expression combined with ER negativity), large tumour size, and positive lymph node status. Low total HER3 expression was prognostic for shorter recurrence-free survival time in HER2-amplified breast cancer (p= 0.004, p= 0.020 in univariate and multivariate analyses, respectively). The majority (82.8%) of breast cancers demonstrated NEDD4–1 protein expression - while only a minor proportion (8. 3%) of carcinomas expressed NRDP1. NEDD4–1 and NRDP1 expression were not associated with clinical outcomes in HER2-amplified breast cancer, irrespective of adjuvant trastuzumab therapy. Conclusions: Low HER3 expression is suggested to be a valuable prognostic biomarker to predict recurrence in HER2-amplified breast cancer. Neither NEDD4–1 nor NRDP1 demonstrated relevance in prognostics or in the subclassification of HER2-amplified breast carcinomas. Keywords: HER3, ErbB3, NEDD4–1, NRDP1, FLRF, RNF41, Prognostic biomarker, Survival, Breast cancer * Correspondence: [email protected] 1 BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Arvo Ylpön katu 34, 33520 Tampere, Finland Full list of author information is available at the end of the article © The Author(s). 2018 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. Luhtala et al. BMC Cancer (2018) 18:1045 https://doi.org/10.1186/s12885-018-4917-1 Background Human epidermal growth factor receptor HER3 (ErbB3), a cell membrane-associated protein encoded by the ERBB3 gene, is a promising target for cancer therapy, especially in HER2-positive (carrying ERBB2/HER2 gene amplification) breast carcinoma [1]. Both HER3 and HER2 belong to a family of epidermal growth factor receptor (EGFR, HER) tyrosine kinases that activate after receptor dimerization. This culminates in the initiation of signal transduction pathways that markedly regulate cellular viability [1]. When catalytically defective, HER3 is unable to homodimerize and orchestrate its own activation [2,3]. HER3 is known to interact most preferably with its structurally homologous relative HER2 once bound with its ligand heregulin (HRG), also called neuregulin-1 [4–6]. Heterodimerization between HER2 and HER3 induces subsequent PI3K/AKT and Ras/Raf/MAPK signalling cascades [7]. The presence of HER3, as an allosteric activator, is required to maintain active HER2-mediated signalling [8,9], and aberrantly intensified HER2-HER3 signalling is hence critically associated with breast carcinogenesis and tumour cell proliferation [4,10–12]. HER3 protein overexpression has been shown to commonly co-occur with HER2 gene amplification and HER2 overexpression, therefore, HER3 is thought to contribute markedly to the pathogenesis of HER2-amplified breast cancer subtype [4,13,14]. The co-expression of HER2 and HER3 proteins [15,16] and abundance of HER2-HER3 heterodimers in situ have also been associated with adverse clinical outcomes in breast cancer [17– 19]. The formation of HER2-HER3 heterodimers also inhibits HER3 downregulation [20]. Due to the close interaction between HER2 and HER3, dual inhibitory therapy is preferred and clinically relevant treatment for carcinomas with altered HER2 signalling [8,11,21]. In addition to HER2-positive breast carcinomas, therapeutic targeting of HER3 receptors has been suggested also in the treatment of HER3-dependent, HER2-negative breast cancers to prevent cell growth-promoting signalling triggered by intensified HER3-HER1 heterodimerization [22]. Several HER3-targeting molecules have been developed as therapeutics, and many of them are currently being tested in clinical trials [23,24]. After a careful survey of the literature, it appears that the prognostic value of HER3 expression (at the protein or mRNA level) in breast cancer is controversial (Table 1). Overexpressed HER3 is mostly associated with a worse survival [16,25–35], but conflicting results have also been published [36–40]. Many studies did not find any demonstrable relationships between HER3 and patient survival [15,41–53]. Studies focusing on HER3 specifically in HER2-amplified breast cancer [16,25,26, 29,31,32,37,38,41,44,45,48,49,52,54] have not drawn conclusive results either. Interestingly, HER3 activation has been implicated as a molecular mechanism inducing inherent or acquired de novo resistance to anti-HER2 therapy [19,31,55,56]. Continuous inhibition of HER2 signalling may lead to compensatory HER3 activation, which results from heterodimerization between HER3 and its alternative dimerization partner HER1 [57,58]. The exact mechanisms behind aberrant HER3 protein expression have not been fully elucidated [13]. Unlike HER2, HER3 does not undergo gene amplification during breast carcinogenesis [16,59,60]. Cancer-related ERBB3 mutations are relatively uncommon, except for colon and gastric carcinomas [59,61]. One hypothesis is that excessive cellular HER3 expression may be due to defects in downstream signalling mechanisms that regulate HER3 membrane trafficking [13]. Aberrant expression of HER3 degradation regulators may lead to an abnormal accumulation or deficit of membrane-bound HER3 receptors, consequently influencing HER3 signalling efficiency. Here, we studied the expression of two proteins, NEDD4–1( neural precursor cell expressed developmentally downregulated 4–1) and NRDP1 (neuregulin receptor degradation protein 1, also known as FLRF and RNF41), which are known to be necessary for HER receptor quantity control [62]. NEDD4–1[63] and NRDP1 [64–67] are both E3 ubiquitin protein ligases suggested to crucially downregulate HER3 and its subcellular localization by mediating HER3 receptors to degradation via the ubiquitin-proteasome-pathway. Defects in ubiquitination are critical and lead to aberrant receptor activity and signalling [68]. Hypothetically, HER3 overexpression may be associated with the concurrent absence of its ubiquitination regulators, NEDD4–1 and NRDP1. Low NEDD4–1 expression due to NEDD4–1knockdown has been demonstrated to activate HER3 and increase cancer cell proliferation in vivo and in vitro [63]. Conversely, NEDD4–1 overexpression has resulted in decreased HER3 expression and increased HER3 ubiquitination [63]. Aberrant expression of NEDD4–1 has been implicated in the pathogenesis and adverse prognosis of several human malignancies [69–72]. Despite the frequent overexpression in breast cancer [73,74], the prognostic value of NEDD4–1 remains unclear in the clinical context. NRDP1, in turn, is less frequently overexpressed than NEDD4–1 in breast carcinoma [75,76]. NRDP1 overexpression has been shown to cause a decrease in HER3 expression and an inhibition of breast cancer cell growth in vitro [75]. Conversely, a loss of NRDP1 followed by NRDP1 knockdown suppressed HRG-induced HER3 ubiquitination and degradation in MCF7 breast cancer cells [64]. An inverse correlation between NRDP1 and HER3 expression in situ has been demonstrated in breast tumours derived from ERBB2 transgenic mice Luhtala et al. BMC Cancer (2018) 18:1045 Page 2 of 19 Table 1 Literature review of studies relating to HER3 prognostics in human breast cancer Publication by Laboratory Methodology Cohort Characteristics Prognostic Implications Takada et al. [91] IHC (RTJ2) met-HER2+ BCA (n= 29), TPD ↓Low HER3 expression was associated with shortened PFS Adamczyk et al. [25] IHC (SP71) HER2+ BCA (n= 97), Adj.T ↑High HER3 expression (only with concurrent PTEN negativity) was associated with shorten MFS Duchnowska et al. [44] VeraTag assay HER2+ BCA (n= 189), Adj.T - No correlation between HER3 expression and OS in advanced stage HER2 + BCA Nishimura et al. [54] VeraTag assay met-HER2+ BCA (n= 47), T - HER3 expression did not has any influence on PFS in trastuzumab-refractory advanced HER2 + BCA Koutras et al. [39] qRT-PCR BCA (n= 663, HER2 + BCA n= 143) ↓Low HER3 mRNA (only with concurrently high EGFR, high HER2, low HER4 mRNA) was associated with worse DFS Baselga et al. [38] qRT-PCR * , IHC ** (DAK-H3-IC) HER2+ BCA (n= 740 * /497 ** ), Adj.T ↓High HER3 mRNA was associated with better prognosis in metastatic HER2 + BCA Berghoff et al. [16] IHC (DAK-H3-IC) met-BCA (n= 110, met-HER2 + BCA n= 34) ↑High HER3 expression was associated with shorter OS in initially metastatic HER2 + BCA subgroup Park et al. [31] IHC (DAK-H3-IC) met-HER2+ BCA (n= 125), T ↑High HER3 expression was associated with worse PFS in initially metastatic HER2 + BCA Bae et al. [26] IHC (DAK-H3-IC) HR-BCA (n= 886, HER2 + BCA n= 221) ↑High HER3 expression was associated with poorer DFS in HER2 + BCA subgroup and poorer DFS and OS in TNBC Czopek et al. [48] IHC (DAK-H3-IC) HER2+ BCA (n= 35) - No correlation between HER3 expression and DFS or OS Lipton et al. [29] VeraTag assay met-HER2+ BCA (n= 89), T ↑High HER3 expression was associated with shorter PFS in initially metastatic HER2 + BCA Gori et al. [41] IHC (RTJ1) met-HER2+ BCA (n= 61), T - HER3 was not significantly associated with clinical outcome in initially metastatic HER2 + BCA Han et al. [37] VeraTag assay met-HER2+ BCA (n= 50), T ↓High HER3 expression was related to longer TTP in advanced HER2 + BCA Larsen et al. [43] IHC (DAK-H3-IC) ER+ BCA (n= 1062) - HER3 expression did not shown any association to DFS Chiu et al. [27] IHC (Ab-10 pAb) BCA (n= 3123) ↑High HER3 expression was associated with decreased BCSS Yonemori et al. [45] IHC (DAK-H3-IC) HER2+ BCA (n= 44), neoAdj.T - HER3 expression did not significantly correlate with pCR Giltnane et al. [28] AQUA BCA (n= 550) ↑High HER3 expression was associated with decreased survival Haas et al. [42] IHC (SGP1) HER2BCA (n= 171) - No prognostic value for HER3 Sassen et al. [50] IHC (5A12), FISH BCA (n= 173) - No prognostic value for HER3 expression, HER3 gene amplification was related to decreased DFS Luhtala et al. BMC Cancer (2018) 18:1045 Page 3 of 19 [75] and in human breast carcinomas [76]. The prognostic and clinical significance of NRDP1 remains unknown. In the current study, we studied the association between HER3, NEDD4–1, and NRDP1 protein expression, clinicopathological characteristics and clinical outcomes in primary breast cancer, especially in the HER2-amplified subtype. Methods Clinical sample material Two separate archival sample collections of formalin-fixed paraffin-embedded (FFPE) primary breast carcinomas were used for biomarker analyses conducted in compliance with the REMARK guidelines [77]. The first sample collection, “the BCA cohort”, consisted of Table 1 Literature review of studies relating to HER3 prognostics in human breast cancer (Continued) Publication by Laboratory Methodology Cohort Characteristics Prognostic Implications Giuliani et al. [52] IHC (RTJ1) met-HER2+ BCA (n= 103), T - No prognostic value for HER3 Lee et al. [36] IHC (pAb) BCA (n= 378) ↓High HER3 expression correlated with longer DFS Bianchi et al. [53] IHC (RTJ1) BCA (n= 145) - No prognostic value for HER3 expression singly, but high co-expression of HER2/3/4 predicted worse prognosis Fuchs et al. [34] IHC (C-17 pAb) BCA (n= 48) ↑High HER3 expression singly and in co-expression with high HER1 and HER2 was associated with poor prognosis Robinson et al. [32] IHC (polyclonal) met-HER2+ BCA (n= 104), T ↑High HER3 expression was associated with worse OS Wiseman et al. [33] IHC (2-18C9) BCA (n= 242) ↑High HER3 expression independently and with high HER1 and/or HER2 was associated with decreased DSS Abd El-Rehim et al. [15] IHC (RTJ1) BCA (n= 1499) - No prognostic value for HER3 singly, but in co-expression with high HER2 predicted unfavorable DFS and OS Smith et al. [49] IHC met-HER2+ BCA (n= 77), T - No prognostic value for HER3 Bièche et al. [35] qRT-PCR BCA (n= 130) ↑High HER3 mRNA was associated with shorten RFS Witton et al. [30] IHC (H3.105.5) BCA (n= 220) ↑High HER3 expression was associated with reduced BCSS survival Suo et al. [47] IHC (sc-415), RT-PCR BCA (n= 100) - High HER3 expression was predictive for reduced DFS or BCSS only in co-overexpression with HER2 or HER1 + HER2 Pawlowski et al. [40] qRT-PCR BCA (n= 365) ↓Elevated HER3 mRNA expression was associated with a better prognosis in terms of OS, but did not relate to RFS Travis et al. [46] IHC (RTJ1) BCA (n= 346), met-BCA (n= 145) - No prognostic value for HER3 expression neither in primary nor metastatic breast cancer Lemoine et al. [51] IHC (49.3 pAb) BCA (n= 195) - No demonstrable relationship between HER3 expression and survival Abbreviations: Adj.T = adjuvant trastuzumab therapy; BCA = primary breast cancer; BCSS = breast cancer-specific survival; DFS = disease-free survival; DSS = diseasespecific survival; ER+ BCA = oestrogen receptor-positive breast cancer; HER2BCA = HER2-negative breast cancer; HER2+ BCA = HER2-positive primary breast cancer; HRBCA = hormone receptor-negative breast cancer; IHC = immunohistochemistry (antibody clone); met- = breast cancer diagnosed at advanced stage; MFS = metastasis-free survival; neoAdj.T = neoadjuvant trastuzumab therapy; n = number of patients being determined for HER3 status and followed for survival; OS = overall survival; pAb = polyclonal antibody; PFS = progression-free survival; pCR = pathologically complete response; qRT-PCR = quantitative reverse transcription polymerase chain reaction; RFS = recurrence-free survival; T = trastuzumab therapy after metastasis; TNBC = triple-negative breast cancer; TPD = trastuzumab, pertuzumab, docetaxel regimen; TTP = time to progression; ↑= high HER3 mRNA or protein expression associated with worse clinical outcome; ↓= low HER3 mRNA or protein expression associated with worse clinical outcome Luhtala et al. BMC Cancer (2018) 18:1045 Page 4 of 19 308 primary, invasive breast carcinomas that were diagnosed in the area served by Tampere University Hospital between 1990 and 1999. Of these carcinomas, 47 (15.3%) were characterized as HER2-positive based on HER2 protein overexpression. Lobular carcinomas were overrepresented in this cohort compared to the overall prevalence of this type of carcinoma (Table 2). This sample set was prepared as tissue microarray (TMA) sections and was originally established for another study, which has been described in more detail in publications by Korhonen et al. [78,79]. Primary treatment for patients was conducted according to the existing clinical practice: surgery, post-operative radiotherapy, adjuvant cytotoxic chemotherapy (mostly CMF) and endocrine therapy (Table 3). The other sample collection, specified as the “HER2+ BCA cohort”, consisted exclusively of 177 HER2-amplified invasive breast carcinomas diagnosed during the years 2003–2007 in the Pirkanmaa Hospital District. The status of hormone receptors, oestrogen receptor (ER) and Table 2 Clinicopathological characteristics of primary breast cancer patients in BCA cohort and HER2+ BCA cohort Characteristic nBCA cohort, n (%) n HER2-amplified BCA cohort, n (%) Follow-up period for RFS (range) Mean 10.4 yr. (1 mo.-22 yr.) Mean 5.3 yr. (1 mo.-9 yr.) Age (range) 308 Median 61 yr. (32–93 yr.) 177 Median 60 yr. (29–91 yr.) < 50 years 64 (20.8) 36 (20.3) ≥50 years 244 (79.2) 141 (79.7) HER2 status 308 177 Positive 47 (15.3) 177 (100.0) Negative 261 (84.7) 0 (0.0) ER status 307 177 Positive (≥10%) 248 (80.8) 113 (63.8) Negative (< 10%) 59 (19.2) 64 (36.2) PR status 307 177 Positive (≥10%) 201 (65.5) 74 (41.8) Negative (< 10%) 106 (34.5) 103 (58.2) Triple negativity 307 177 TNBC (HER2−/ER-/PR-) 30 (9.8) 0 (0.0) No TNBC 277 (90.2) 177 (100.0) Histological grade 232 174 I-II 179 (77.2) 41 (23.6) III 53 (22.8) 133 (76.4) Ki67 proliferation index 230 177 Low (< 20%) 165 (71.7) 33 (18.6) High (≥20%) 65 (28.3) 144 (81.4) Histological type 304 168 Ductal 173 (56.9) 156 (92.9) Lobular 131 (43.1) 12 (7.1) Tumour size 177 142 < 2 cm 57 (32.2) 68 (47.9) ≥2 cm 120 (67.8) 74 (52.1) Tumour size 308 172 pT1-pT2 282 (91.6) 161 (93.6) pT3-pT4 26 (8.4) 11 (6.4) Lymph nodal spread 286 169 Positive pN+ 114 (39.9) 73 (43.2) Negative pN0 172 (60.1) 96 (56.8) Number of patient cases with available data (n) for each character is marked within the columns Luhtala et al. BMC Cancer (2018) 18:1045 Page 5 of 19 progesterone receptor (PR), HER2 gene amplification, and Ki67 proliferation index were determined during the diagnostic procedure, and related data were retrieved from the clinical records. HER2 gene amplification status was previously determined by the chromogenic in situ hybridization (CISH) technique. This sample set was prepared as whole tissue sections. Approximately half (n= 82) of the carcinomas, primarily patients diagnosed after June 2005, were treated with conventional chemotherapy combined with adjuvant trastuzumab during 9-wk schema as a first-line therapy [80] for primary disease. The remaining patients (n= 95) did not receive any adjuvant HER2-targeted therapy for primary disease. In addition to surgery and adjuvant cytotoxic chemotherapy (mostly consisting of taxanes, CEF), post-operative radiotherapy and adjuvant endocrine therapy were given when necessary (Table 3). Samples were selected for the current study according to the following inclusion criteria: availability of representative tumour tissue (FFPE), adequate pathological characterization, and clinical follow-up data. Clinicopathological data and follow-up information were collected, retrospectively. The mean follow-up period for recurrence-free survival (RFS) in the HER2+ BCA cohort was 5.3 years (range: 1 month to 9 years) and 10.4 years (range: 1 month to 22 years) for the BCA cohort. NEDD4–1 and NRDP1 expression was studied in a smaller fraction of the HER2+ BCA cohort representing available HER2-amplified cases (n= 145). Table 2describes the clinicopathological characteristics of the study cohorts. Immunohistochemical stainings For immunohistochemistry (IHC), serial four-μm-thick sections were cut from FFPE sample blocks and mounted on Super Frost Plus® slides followed by deparaffinization and dehydration. Heat-induced epitope retrieval (HIER) was performed in TE buffer (50 mM Tris 1 mM EDTA, pH 9) at 98 °C for 15 min. To determine HER3 protein expression, we used the optimized IHC staining protocol described in our earlier study [81]. We used a mouse monoclonal (clone DAK-H3-IC) antibody against the human HER3 protein at a dilution of 1:100. Table 3 Primary treatments of patients in BCA and HER2+ BCA study cohorts Primary treatment BCA cohort (n= 308) HER2-amplified BCA cohort (n= 177) n% n % Breast surgery Mastectomy (ablation) 161 52.4 101 57.1 Conservative surgery (resection) 146 47.6 72 40.7 No operation 3 0.6 Unknown 1 Post-operative radiotherapy 198 65.3 110 62.1 No 105 34.7 67 37.9 Unknown 5 Adjuvant endocrine therapy 97 32.1 104 58.8 No 205 67.9 73 41.2 Unknown 6 Adjuvant chemotherapy 40 13.4 133 75.1 No 259 86.6 44 24.9 Unknown 9 Adjuvant trastuzumab 82 46.3 No 308 100.0 95 53.7 Table 4 Details of antibodies used in the IHC-protocols of the current study Antibody Host species Catalog No. Clonality Dilution Manufacturer/distributor Anti-Human HER3 Mouse M7297 DAK-H3-IC 1:100 DAKO A/S, Glostrup, Denmark FLRF/RNF41 Antibody Rabbit A300-049A polyclonal 1:3000 Bethyl Laboratories, Inc., Montgomery, Texas, USA Anti-Nedd4, WW2 domain Rabbit #07–049 polyclonal 1:750 Merck KGaA, Darmstadt, Germany Cytokeratin 5 Antibody Mouse NCL-L-CK5 XM26 1:150 Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK Cytokeratin 14 Antibody Mouse NCL-L-LL0022 LL0022 1:150 Leica Biosystems, Newcastle Ltd., Newcastle Upon Tyne, UK Anti-human Ki67 Mouse BSH-7302 BS4 1:100 Nordic BioSite AB, Täby, Sweden Luhtala et al. BMC Cancer (2018) 18:1045 Page 6 of 19 The expression of basal epithelium cytokeratins 5 and 14 was determined using the same IHC protocol with an antibody cocktail composed of anti-human mouse monoclonal antibodies CK14 (clone LL002) and CK5 (clone XM26), both diluted at 1:150. Ki-67 expression was determined similarly in BCA cohort samples with mouse monoclonal Ki-67 antibody (clone BS4) at a dilution of 1:100. For NEDD4–1 IHC, we used rabbit polyclonal anti-NEDD4 WW2 domain antibody (dilution 1:750) to detect NEDD4–1 proteins. Bright Vision+ Poly-HRPAnti-mouse/rabbit IgG kit (ImmunoLogic, AD Duiven, the Netherlands) and 3,3′-diaminobenzidine tetrahydrochloride DAB-2V kit (Nichirei Biosciences Inc., Tsukiji, Chuo-ku, Tokyo, Japan) were used for the detection of immunoreactivity according to manufacturers’instructions. To detect the NRDP1 protein, we used rabbit polyclonal FLRF/RNF41 antibody (dilution 1:3000), EnVision™FLEX High pH HRP and EnVision™FLEX DAB + reagents (Dako, Glostrup, Denmark), according to manufacturers’protocols. After staining, slides were counterstained with Mayer’s Hematoxylin (Oy FF-Chemicals Ab, Haukipudas, Finland) with 1:4 addition of 2% copper sulfate to intensify the DAB reaction. Slides were then dehydrated, cleared with xylene and sealed with DePeX mountant. All staining reactions were conducted using the LabVision™Autostainer 480S platform. As positive control samples, we used human FFPE tissues known to express the specified proteins: normal prostate ductal cells for HER3 [82], kidney proximal tubule cells for NEDD4–1[83], testicular cells in seminiferous ducts and mononuclear blood cells for NRDP1 [84]. A negative staining control was prepared by omitting and replacing the primary antibody with diluent reagent and was included in each staining batch. An additional file 1and Table 4present detailed information on antibodies and IHC-staining protocols used in the current study. Microscopic analysis and interpretation of immunoreactivity Samples stained for HER3, NEDD4–1 and NRDP1 were scanned with SlideStrider (Jilab Inc., Tampere, Finland) into digital images that were examined virtually with JVSview JPEG2000 [85] and SlideVantage 1.2 (Jilab Inc., Tampere, Finland) viewer applications. The ImmunoRatio 2.5 application was used for automated cell counting of distinct cancer cells with nuclear immunoreactivity [86]. Staining patterns were analysed within the invasive cancerous tissue area displaying the most intense brown DAB reaction (region of interest, ROI). For HER3 appearance, both membranous and cytoplasmic staining reactions were inspected on a computer screen. Samples were classified according to the staining intensity and proportion of specifically stained cancer cells as previously described [81]. Briefly, HER3 staining localized to the cancer cell outer membrane was considered ‘membranous’and was scored according to the following criteria: (0) absent/low staining (< 10% of cells), (1+) intermediate circumferential staining (10–30% of cells) and (2+) strong circumferential staining (> 30% of cells). The staining reaction observed in the cancer cell cytoplasm was considered ‘cytoplasmic’and was categorized as (0) no/faint staining, (1+) overall low-intensity staining, and (2+) prevalent high-intensity staining covering most of the cancer cells. Score 1+ was set as a threshold to define HER3 positivity both for membranous and cytoplasmic staining. Total HER3 staining was designated as negative for cases with low (0/1+) membranous staining concurrently with low (0/1+) cytoplasmic staining and as positive for cases with high (2+) membranous and/or (2+) cytoplasmic staining. The NEDD4–1 protein expression pattern was analysed by scoring the staining intensity as follows: 0 (no staining), 1+ (weak), 2+ (moderate), and 3+ (strong). Samples with scores < 3+ were seen as NEDD4–1negative‘low expressing’and samples with score 3+ as NEDD4–1positive‘high expressing’. Overall, the NEDD4–1 staining pattern in cancerous areas was homogenous, and therefore, the percentage of stained cells was not evaluated. NRDP1 staining was analysed by applying a scoring system presented in a study by Jiao et al. [76]. We analysed nuclear and cytoplasmic staining separately. Staining intensity was scored accordingly: 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). Based on the percentage of stained cancer cell nuclei, samples were classified as 0 (< 1%), 1 (1–24%), 2 (25–49%), 3 (50–74%), and 4 (75–100%).Thegradeswerethen multiplied to determine a score for low and high nuclear expression. Cases with scores ≤3 were defined as ‘low expressing’and those with scores ≥4as‘high expressing’. Cytoplasmic NRDP1 expression was categorized as high if the staining intensity in the tumour cells was moderate or strong. Expression patterns of basal epithelium cytokeratins 5 and 14 and Ki67 protein were analysed with Olympus System Microscope BX43. Carcinomas were interpreted as positive for CK5 and CK14 expression if more than 20% of the malignant cells displayed clear cytoplasmic staining [87]. For Ki67 protein expression, we used a 20% cut-off value to determine low (< 20%) and high (≥20%) cell proliferation activity [86]. Statistical analysis All statistical data analyses were performed using IBM® SPSS® Statistics version 23 (IBM Corp.). Generally, p-values < 0.05 were considered statistically Luhtala et al. BMC Cancer (2018) 18:1045 Page 7 of 19 Fig. 1 HER3 immunohistochemistry. aPositive control (prostate), bConcurrently high (score 3+) membranous and cytoplasmic HER3 expression (breast carcinoma), cHigh (score 3+) membranous HER3 expression with negative/low (score 0) cytoplasmic HER3 status, dNegative/low total cellular HER3 staining. Mayer's Hematoxylin used as a counterstain Fig. 2 NEDD4–1 immunohistochemistry. aPositive control (kidney), bNegative/low NEDD4–1 expression (score 1+, breast carcinoma), c Moderate NEDD4–1 expression (score 2+, breast carcinoma), dHigh NEDD4–1 expression (score 3+, breast carcinoma). Mayer's Hematoxylin used as a counterstain Luhtala et al. BMC Cancer (2018) 18:1045 Page 8 of 19 significant for any relationship being considered. Proportions among categorical variables were compared using Pearson’s Chi-Square test to determine clinicopathological correlations. Kaplan-Meier survival analysis and log-rank test (Mantel-Cox) were used to compare survival differences for each categorical variable. RFS time was chosen as the endpoint for the current study. To determine RFS, patients were followed from the date of surgery for initial diagnosis to the date of disease progression as local recurrence or distant metastasis. Patients who did not experience recurrence during the follow-up were censored at the time of death or last date of medical record inspection. Results HER3 protein expression in breast carcinomas In the BCA sample set consisting of HER2-positive and -negative breast carcinomas (BCA cohort), high membranous HER3 expression was observed in half of the cases (51.9%, 160 of 308). Nearly all (95.8%, 295 of 308) carcinomas showed HER3 protein expression localized in the cancer cell cytoplasm. When the total cellular HER3 expression pattern was evaluated, the majority (75.3%, 232 of 308) of carcinomas were classified as HER3-positive, ‘high total HER3 expressing’. One-fourth of the carcinomas (24.7%, 76 of 308) were determined to be HER3-negative, ‘low total HER3 expressing’. Figure 1 shows examples of membranous and cytoplasmic HER3 IHC staining patterns observed in the present study. HER3, NEDD4–1, and NRDP1 protein expression in HER2amplified breast carcinomas To determine whether HER3 protein expression is common in HER2-amplified breast cancer subtype, we also studiedHER3expressionintheHER2+BCAcohortestablished for this purpose. We noticed that 80.2% (142 of 177) of HER2-amplified breast carcinomas showed complete circumferential membrane staining for HER3. Cytoplasmic HER3 staining was more common, since only a small fraction (8.5%, 15 of 177) of these carcinomas were completely unstained. High total HER3 expression was demonstrated in 75.7% of cases (134 of 177), and one-fourth of carcinomas were designated as HER3-negative. Overall, HER3 protein was heterogeneously expressed within the cancerous areas represented in whole tissue sections. The HER3 staining pattern was, therefore, equally evaluated from the ROI showing the most intense DAB reaction (Fig. 1). Next, we studied NEDD4–1 and NRDP1 protein expression in a cohort of HER2-amplified breast carcinomas. Most of the cases (82.8%, 120 of 145) demonstrated strong-to-moderate NEDD4–1 staining localized predominantly in the cytoplasmic region (Fig. 2). Approximately one-fifth (17.2%, 25 of 145) of the cases were categorized as NEDD4–1 low expression based on faint IHC staining reaction. The staining intensity and subcellular localization of NEDD4–1 protein were homogenous within the cancerous areas. Cells in histologically normal breast ducts were also positive for NEDD4–1. NRDP1 protein expression was uncommon in HER2-amplified breast carcinomas. NRDP1 localization in carcinoma cells was clearly nuclear or cytoplasmic (Fig. 3). The high presence of nuclear or cytoplasmic NRDP1 protein was observed in a minor proportion (8.3%, 12 of 145) of samples, while the majority of carcinomas (91.7%, 133 of 145) were classified as low for NRDP1 expression. Association of HER3, NEDD4–1 and NRDP1 with clinicopathological characteristics In the BCA cohort, we noticed that HER3 protein expression was not dependent on HER2 status Fig. 3 NRDP1 immunohistochemistry. aPositive control (testis, cells in seminiferous ducts), bPositive control (mononuclear blood cells), cAbsent NRDP1 expression (breast carcinoma), dCytoplasmic NRDP1 expression (breast carcinoma), eand fNuclear NRDP1 expression (breast carcinoma). Mayer's Hematoxylin used as a counterstain Luhtala et al. BMC Cancer (2018) 18:1045 Page 9 of 19 anti-HER2 therapy, such as trastuzumab, would elucidate the concept of intensified HER3 signalling due to HER2 downregulation. HER3 upregulation has been related to trastuzumab resistance in studies [19,106] showing that breast cancers driven primarily by HER2 homodimerization are more susceptible to trastuzumab therapy than tumours with a predominance of HER2-HER3 heterodimers. From this context, it would be interesting to determine HER3 expression in breast carcinomas that are confirmed intrinsically resistant to trastuzumab. To elucidate the therapeutic predictive potential of HER3, one intriguing thought is to clarify HER3 expression retrospectively in breast cancer patients who were subsequently treated with adjuvant pertuzumab or novel HER3-targeting antibodies. Conclusions The results of the current study suggest HER3 as a novel versatile biomarker to predict recurrence of HER2-amplified breast cancer. Irrespective of its subcellular localization, absent or low HER3 expression was associated with shorter RFS time when compared to HER3-overexpressing breast carcinomas. Low HER3 expression was associated with clinicopathological characteristics related to more aggressive and therapeutically unfavourable breast cancer types, such as axillary lymph nodal infiltration, larger tumour size, young patient age, negative ER status, triple-negative subtype, and basal phenotype. HER3 did not show any predictive value for the benefit of short-term (9-wk) adjuvant trastuzumab therapy as a first-line therapy. The HER3 degradation regulators NEDD4–1 and NRDP1 did not show any clinically meaningful correlations or predictive or prognostic applicability in HER2-amplified breast cancer subtype. Additional file Additional file 1: IHC-staining protocols provide detailed information on reagents used in the current study to demonstrate HER3, NEDD4–1, NRDP1, and Cytokeratin 5/14 protein expression on FFPE breast cancer tissues. (PDF 283 kb) Abbreviations ER: Oestrogen receptor; FFPE: Formalin-fixed paraffin-embedded tissue; FLRF: Fetal liver ring finger; HER1/2/3: Human epidermal growth factor receptor 1/2/3; HIER: Heat-induced epitope retrieval; HRG: Heregulin; IHC: Immunohistochemistry; NEDD4–1: Neural precursor cell expressed developmentally downregulated 4–1; NRDP1: Neuregulin receptor degradation protein-1; PR: Progesterone receptor; RFS: Recurrence free survival; RNF41: RING finger protein 41; ROI: Region of interest; TMA: Tissue microarray; TNBC: Triple-negative breast cancer Acknowledgements The authors thank Mrs. Kristiina Salonoja and Mrs. Sari Toivola for their excellent technical assistance. Funding This research was financially supported by the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital, and Doctoral Program in Medicine and Life Sciences at University of Tampere (to S.L.). The funding bodies had no role in the study design, data collection, analysis and interpretation, or in writing the manuscript. Availability of data and materials The datasets used and analyzed in the current study are available from the corresponding author on reasonable request. The datasets supporting the conclusions of this article are included within the article and its additional files. Authors’contributions SL, MT and JI designed the study. SL and SS harvested retrospectively clinical follow-up data and diagnostic information from the pathological review of cancer samples. AK provided the BCA sample set and derived clinical data. SL conceived the study in practice, performed data analysis and wrote the manuscript. All authors contributed to data interpretation, read and approved the final manuscript. Ethics approval and consent to participate The use of the tumour samples and patient records in this study was approved by the Ethics Committee of Pirkanmaa Hospital District (references no. R07082, R00143), the Ministry of Social Affairs and Health, and the National Authority for Medicolegal Affairs in Finland. The need for informed consent was waived by the aforementioned authorities due to the large number of samples and the fact that a sizeable portion of the patients were already deceased. Individual patient data has not been made available and the dataset has been handled anonymized. Consent for publication Not applicable. Competing interests None of the authors declares any conflicts of interest. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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