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Aims: Digital image analysis (DIA) is used increasingly as an assisting tool to evaluate biomarkers, including human epidermal growth factor receptor 2 (HER2) in invasive breast cancer (BC). DIA can assist pathologists in HER2 evaluation by presenting quantitative information about the HER2 staining in APP assisted reading (AR). Concurrently, the HER2-low category (HER2-1+/2+ without HER2 gene amplification) has gained prominence due to newly developed antibody-drug conjugates. However, major inter- and intraobserver variability have been observed for the entity. The present quality assurance study investigated the concordance between DIA and AR in clinical use, especially concerning the HER2-low category. Methods and results: HER2 immunohistochemistry (IHC) in 761 tumours from 727 patients was evaluated in tissue microarray (TMA) cores by DIA (Visiopharm HER2-CONNECT) and AR. Overall concordance between HER2-scores were 73% (n = 552, weighted-κ: 0.66), and 88% (n = 669, weighted-κ: 0.70), when combining HER2-0/1+. A total of 205 scores were discordant by one category, while four were discordant by two categories. A heterogeneous HER2 pattern was relatively common in the discordant cases and a pitfall in the categorisation of HER2-low BC. AR more commonly reassigned a lower HER2 score (from HER2-1+ to HER2-0) within the HER2-low subgroup (n = 624) compared with DIA. Conclusion: DIA and AR display moderate agreement with heterogeneous and aberrant staining, representing a source of discordance and a pitfall in the evaluation of HER2.

Background: Several Breast Cancer Multigene Signatures (BCMS) are available to profile early breast cancer (eBC) that according to current evidence, can provide reliable information on the risk of recurrence. However, knowledge regarding their perceived value and use in clinical practice is scarce. The first results regarding the panelists’ opinions on the clinical utility of BCMS were presented at SABCS Symposium 2021 (Poster P4-06-08). Here we discuss present and future recommendations for improving the use of BCMS resulting from the PROCURE Project. Methods: The Delphi survey developed by the 8 members of the Scientific Committee was administered twice to oncologists, pathologists, and surgeons across Europe. The questionnaire included 5 sections: 1) Participants’ profile and experience with BCMS, 2) Current clinical practice in eBC and use of BCMS, 3) Participants’ opinion on the utility of the BCMS in eBC according to patient profiles, 4) Agreement with a set of recommendations on the use of BCMS in clinical practice and 5) Identification of unmet needs and future applications of BCMS. 70% agreement was used to determine consensus on a topic. Results: 133 panelists from 11 European countries completed both rounds of the survey. Experts were mostly medical oncologists (72.2%) with extensive experience (more than 5 years) in the management of BC patients (97.0%) and in the use of BCMS (73.4%). Most of them worked in university hospitals (86.5%). Regarding recommendations for improving BCMS utility, according to panelists’ opinion, these genomic tests should provide prognostic as well as predictive information (82.0%), especially to help physicians when deciding on the most appropriate adjuvant chemotherapy (81.2%). Also, their evidence should be based mostly on prospective randomized clinical trials (85.0%) and they should evaluate the clinical and pathological features of the disease (91.0%). Another aspect agreed by panelists is that patients should have the right to access their BCMS results to take part in treatment decisions (85.7%). Finally, when thinking about future applications of BCMS, consensus was only reached after the second Delphi round, and according to panelists, there is a need for validating BCMS in order to know if they can predict treatment benefits in patients with ER-positive advanced/metastatic BC (78.2%) and in triple-negative eBC patients (72.2%). Likewise, panelists also agreed on the need of developing newly validated tests to evaluate the risk of distant recurrence and predict treatment benefits in the neoadjuvant setting (71.4% and 81.2%, respectively). However, no consensus was reached on the need for BCMS to give an accurate prognosis for patients with ER+ advanced and/or metastatic BC (50.4%), HER2+ advanced BC (21.8%), or triple-negative advanced BC (36.8%). Conclusions: The panelists who participated in the PROCURE Project had an extensive experience in the management of eBC patients as well as using BCMS; however, the high degree of importance that they attributed to both prognostic and predictive information reflects a misconception on the correct interpretation of BCMS results because, even if those test can be used to predict the benefit of adjuvant chemotherapy, they are primarily prognostic tools but not predictive about the effect of a specific cytotoxic intervention. These results in conjunction with the agreements regarding treatment benefit prediction reached in the neoadjuvant, the triple-negative eBC, and the metastatic settings reinforce the idea that more training is needed to highlight that BCMS should be used in the context of risk assessment in intermediate-risk eBC as well as the possibility of running those tests in core biopsies. Citation Format: Michael Gnant, Anne-Vibeke Lænkholm, Aleix Prat, Judy King, Giuseppe Curigliano, Nadia Harbeck, Frédérique Penault-Llorca, Fatima Cardoso. Unmet needs and future applications of breast cancer multigene signatures in routine clinical practice: results from the European PROCURE Project [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-07-03.

The purpose of this study was to examine the effect of chemotherapy on invasive disease-free survival (iDFS) and overall survival (OS) in a nationwide cohort of patients with estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative, T1abN0 breast cancer.Patients with ER-negative/HER2-negative, T1abN0 breast cancer registered in the Danish Breast Cancer Group database between 2007 and 2016 were identified. The effect of adjuvant chemotherapy on iDFS and OS was analyzed with Cox proportional hazards analysis.In total, 296 patients were included in the statistical analyses. Of these, 235 (79.4%) received chemotherapy and 61 patients (20.6%) did not. Patients treated with chemotherapy were significantly younger, had a significantly higher proportion of grade 3 tumors, T1b tumors, and tumors of ductal subtype. With 7.7 years of median follow-up, treatment with chemotherapy was associated with a significant improvement in OS in the adjusted analysis, Hazard Ratio 0.35 (95% Confidence Interval (0.15-0.81), p = 0.02), chemotherapy vs. no chemotherapy. In the unadjusted analyses, patients with both T1a and T1b tumors had significantly improved OS with chemotherapy. At 5 years, OS was 100% vs. 94.4% and 93.8% vs. 81.3% for patients with T1a and T1b tumors, respectively, chemotherapy vs. no chemotherapy. With 4.9 years of median follow-up, iDFS was not significantly improved with chemotherapy.Patients with ER-negative/HER2-negative, T1abN0 breast cancer had significantly improved OS when treated with chemotherapy. This improvement was significant in patients with both T1a and T1b tumors, respectively. The effect was, however, limited in patients with T1a tumors.

The purpose of this study was to evaluate the effect of chemotherapy and trastuzumab on invasive disease-free survival (iDFS) and overall survival (OS) in patients with human epidermal growth factor receptor 2 (HER2) positive, T1abN0 breast cancer.In the Danish Breast Cancer Group database, patients with HER2-positive, T1abN0 tumors diagnosed between 2007 and 2016 were identified. Cox proportional hazards analysis was performed to analyze the association between adjuvant chemotherapy and trastuzumab and iDFS and OS.Of 605 patients included in the analyses, 465 patients received chemotherapy and trastuzumab and 140 patients did not. Chemotherapy and trastuzumab did not improve iDFS or OS significantly in adjusted analyses. 5-year iDFS was 92.3% vs. 89.9%, Hazard ratio (HR) 1.01 (p = 0.98), and 5-year OS was 97.4% vs. 94.3%, HR 0.60 (p = 0.15), chemotherapy and trastuzumab vs. no chemotherapy/trastuzumab. In unadjusted analyses, significant treatment benefit on OS was found in patients with T1b tumors. The largest absolute treatment benefits were found in patients with T1b tumors and estrogen receptor (ER) negative tumors, respectively, whereas treatment effects in patients with T1a tumors and ER-positive tumors, respectively, were limited.Adjuvant chemotherapy and trastuzumab did not improve OS or iDFS significantly in patients with HER2-positive, T1abN0 breast cancers in adjusted analyses. In unadjusted analyses, significant OS benefit was found in patients with T1b tumors. The largest absolute benefit was observed in patients with T1b tumors and ER-negative tumors, respectively, whereas the effect was limited in patients with T1a tumors and ER-positive tumors, respectively.

Purpose:PIK3CA mutations are frequent in breast cancer and activate the PI3K/Akt pathway. Unexpectedly, PIK3CA mutation appears in general to be associated with better outcome. In a cohort of patients where both primary and metastatic lesions were available, the objective was to assess changes in PIK3CA mutations. We wished to discern whether selective pressures occur and the influence of PIK3CA mutation on time to recurrence.Experimental Design: Formalin-fixed paraffin-embedded tumor blocks were obtained from 104 patients with paired samples from primary tumors and corresponding asynchronous metastatic breast tumors. Samples were analyzed for PIK3CA mutations (exons 9 and 20) as well as immunohistochemical evaluation for PTEN, pAKT, Ki67, ER, and HER2.Results:PIK3CA mutation was detected in 45% of the primary tumors. Overall, there was a net gain in mutation in metastatic disease, to 53%; nonetheless, there were instances where metastases were wild type in patients with PIK3CA mutant primary tumors. Laser capture microdissection on a subset of cases revealed microheterogeneity for PIK3CA mutational status in the primary tumor. PIK3CA mutants overall showed a significantly longer time to first recurrence than wild type cases (P = 0.03).Conclusion:PIK3CA mutations occur at high frequency in primary and metastatic breast cancer; these may not necessarily confer increased aggressiveness as mutants had a longer time to recurrence. Because PIK3CA status quite frequently changes between primary and metastatic disease, it emphasizes the necessity of assessing the PIK3CA status in the metastatic lesion for selection of PIK3CA inhibitor therapy. Clin Cancer Res; 17(4); 667–77. ©2010 AACR.

Supplementary Tables S1-S4.

Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large naïve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically recognize cancer-associated cell-surface proteins. One of the isolated human antibody fragments, Ab39, recognizes a cell-surface antigen expressed on a subpopulation of cancer cell lines of different origins. Immunohistochemical analysis of a large panel of cancerous and normal tissues showed that Ab39 bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast two-hybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized by Ab39. The interaction was confirmed by colocalization studies and antibody competition experiments that also mapped the epitope recognized by Ab39 to the COOH terminus of GRP78. The expression of GRP78 on the surface of cancer cells, but not normal cells, makes it an attractive target for cancer therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers. [Cancer Res 2007;67(20):9507–17]

Background: A striking heterogeneity combined with an absence of highly efficient targeted treatments necessitates a continued effort to optimize the choice of chemotherapy for patients with triple negative breast cancer (TNBC). Neoadjuvant therapy has become the standard of care for high risk TNBC in order to potentially reduce breast and axillary surgery, start medical treatment early, allow genomic screening and guide post neoadjuvant therapy. Much attention is drawn to the use of immunotherapy in early TNBC, but there is also interesting data suggesting a potential to optimize the use of conventional chemotherapy. According to one meta-analysis including data from nine randomized controlled trials, the addition of platinum salts in the preoperative setting increases the pathologic complete response (pCR) rate in TNBC (Poggio 2018). A second meta-analysis, evaluating the effect of capecitabine in early breast cancer indicates an additional benefit from adding capecitabine to conventional chemotherapy in terms of an increased recurrence-free survival in the subset of TNBC (Mackelenbergh SABCS 2019). An adjuvant trial in TNBC suggests that the beneficial effect of capecitabine may be confined to patients with a non-basal like phenotype of TNBC (Lluch 2020), a subset that correlates with the homologous repair non-deficient subset which constitutes 41 % of TNBC in a mainly south Swedish population based cohort (Staaf 2019). The effect of capecitabine in platinum treated early TNBC is unknown. Objectives: We are conducting a multi-center randomized controlled trial evaluating the effect on the pCR rate by the addition of capecitabine to optimal platinum based and dose dense preoperative chemotherapy in early TNBC. Method: 820 patients (pts) with early TNBC stage 1 (≥20 mm) - 3 will be randomized 1:1 between two treatment arms A and B. A: Epirubicine Cyclophosphamide (EC) q2w x 4 followed by Carboplatin AUC 5 q3w with weekly paclitaxel x 12. B: Epirubicine Cyclophosphamide with capecitabine daily for two weeks (CEX) q3w x 4 followed by Carboplatin AUC 5 q3w with weekly paclitaxel x 12 The primary endpoint is pCR rate in the different treatment strata, and the primary translational endpoint will be the potential difference of treatment effect stratified for HRD-status in the primary tumor. Secondary endpoints include IDFS in subsets of TNBC, eg according to gene-expression-based subtypes of TNBC (Lehmann 2011, Burstein 2015), and germline mutation status of hereditary breast cancer genes. Tumor material for genotypig is collected at baseline, at surgery and optionally after two treatment cycles. Sequential blood samples are collected for tsDNA analysis during and after treatment termination. Results: The trial, which is a collaborative effort between the national breast cancer groups in the participating countries, has been postponed by the pandemic but is now recruiting according to plan. So far 56 patients out of 820 have been recruited at 15 sites in Sweden and Denmark. Additional Sites in Sweden, Denmark, Iceland and Finland are preparing to join the study. For update please see: www.NordicTrip.se; E-mail address: Nordictrip.onkologi@skane.se Citation Format: Niklas Loman, Barbro Linderholm, Johan Ahlgren, Siker Kimbung, Lina Zander, Heidi Grill Magnusson, Eva-Christin Kjellman, Ann Julia Raaberg, Maj-Britt Raaby Jensen, Åke Borg, Johan Staaf, Anders Ståhlberg, Pär-Ola Bendahl, Anne-Vibeke Laenkholm, Minna Tanner, Olöf Bjarnadóttir, Oskar Johannsson, Bent Ejlertsen, Henrik Lindman. Nordictrip, a translational randomized phase-3study exploring the effect of the addition of capecitabine to carboplatinum-based chemotherapy in early “triple negative” breast cancer, ClinicalTrials.gov Identifier: NCT04335669 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-30-01.

Background: The Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA), now a part of CanRisk, is a comprehensive risk prediction tool for breast and/or ovarian cancer (BOC) with a high accuracy to predict the likelihood of carrying pathogenic variants (PVs) in BRCA1 and BRCA2. BOADICEA version 6 also includes PVs in PALB2, CHEK2, ATM, BARD1, RAD51C and RAD51D, but the accuracy of its predictions remains to be investigated. Methods: The study included 2,033 individuals counselled at clinical genetics departments in Denmark on suspicion of hereditary susceptibility to BOC. All counselees underwent comprehensive genetic testing by next generation sequencing of BRCA1, BRCA2, PALB2, CHEK2, ATM, BARD1, RAD51C and RAD51D. Predicted likelihoods of PVs were obtained from BOADICEA v6.1.0. The accuracy of predictions was examined by calibration using the observed-to-expected ratio (O/E) and by discrimination using the area under the receiver-operating characteristics curve (AUC). Results: BOADICEA remained well-calibrated after addition of the additional genes. Thus, the O/E was 1.07 (95% CI 0.94-1.22) for all genes in the model combined. At sub-categories of predicted likelihood, the model performed well with only limited misestimation at the extremes of predicted likelihood. The ability to discriminate between carriers and non-carriers of PVs was acceptable with an AUC of 0.70 (95% CI 0.66-0.74), although discrimination was better for BRCA1 and BRCA2 (AUC 0.79) than for the other genes (AUC 0.59). Conclusion: BOADICEA remains a valid decision-making aid for determining which individuals to offer comprehensive genetic testing on the suspicion of hereditary susceptibility to BOC.

International audience; Abstract Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error ( p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections ( p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.; v2.5, 25 March 2021.

Background: Several genomic assays are available to profile early breast cancer (eBC) that according to current evidence, can provide reliable information on the risk of recurrence. However, little is known regarding their current clinical use and the perception of their utility among European breast cancer (BC) specialists. The PROCURE project aims to develop a consensus on the utility of BC multigene signatures (BCMS) in treatment decision making for different BC patient profiles. Methods: A Scientific Committee of 8 experts in BC from 8 European countries developed a Delphi questionnaire that was administered in two waves to experienced clinicians across Europe with expertise in the use of BCMS. The questionnaire included 5 sections: 1) Participants’ profile and experience with BCMS. 2) Current clinical practice in eBC and use of BCMS. 3) Participants’ opinion on the utility of the BCMS in eBC according to patient profiles. 4) Agreement with a set of recommendations on the use of BCMS in clinical practice. 5) Identification of unmet needs and future applications of BCMS. 70% agreement was used to determine consensus on a topic. Results: 163 BC experts were invited to answer anonymously the online Delphi questionnaire. At wave 2 closure (1st of June 2021) 133 panelists from 11 European countries (grouped in 5 regions) completed both rounds of the survey. Panelists were 48.8 years old (± 9.5). 86.5% of them worked in a teaching hospital, 72.2% were medical oncologists and 12.0% pathologists, the distribution of the specialities of the participants differed by region. The mean number of patients seen, treated or diagnosed on a weekly basis was 36.4 with statistical differences among regions and specialities. The ratio of eBC on BC patients was 63.5%, similar between regions but showed statistical differences among specialities. 93.2% of the panelists used BCMS routinely or in selected patients, with statistical differences among regions, and 73.4% had more than 5 years of experience using BCMS, however the experience of participants varies by regions and specialities. After the 2nd wave, experts panelists in BC management reached a consensus on 50% (35/70) of the items asked on clinical utility, recommendations and future applications of BCMS. Conclusions: European panelists that participated in the PROCURE study showed an extensive experience in the management of BC patients and on the use of BCMS. Detailed results from the 2nd wave will be presented during the 2021 SABCS meeting to engage larger discussion with symposium participants about the agreement or disagreement reached on the main topics. Citation Format: Giuseppe Curigliano, Fatima Cardoso, Michael Gnant, Nadia Harbeck, Judy King, Anne-Vibeke Laenkholm, Frédérique Penault-Llorca, Aleix Prat. Consensus on the utility of breast cancer multigene signatures in routine clinical practice among European breast cancer specialists - 1st results of the PROCURE project [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-06-08.

Background The natural history of breast cancer among BRCA2 carriers has not been clearly established. In a previous study from Iceland, positive ER status was a negative prognostic factor. We sought to identify factors that predicted survival after invasive breast cancer in an expanded cohort of BRCA2 carriers. Methods We studied 608 women with invasive breast cancer and a pathogenic BRCA2 mutation (variant) from four Nordic countries. Information on prognostic factors and treatment was retrieved from health records and by analysis of archived tissue specimens. Hazard ratios (HR) were estimated for breast cancer-specific survival using Cox regression. Results About 77% of cancers were ER-positive, with the highest proportion (83%) in patients under 40 years. ER-positive breast cancers were more likely to be node-positive (59%) than ER-negative cancers (34%) (P P = 0.03); thereafter, the effect was adverse (HR = 1.91; 95% CI 1.07–3.39, P = 0.03). The adverse effect of positive ER status was limited to women who did not undergo endocrine treatment (HR = 2.36; 95% CI 1.26–4.44, P = 0.01) and patients with intact ovaries (HR = 1.99; 95% CI 1.11–3.59, P = 0.02). Conclusions The adverse effect of a positive ER status in BRCA2 carriers with breast cancer may be contingent on exposure to ovarian hormones.

Estrogen receptor positive (ER+) breast cancer constitutes almost 85% of all breast cancer patients and are a genetically highly heterogenic group. Data on the association of somatic alterations to outcome and prognosis are however sparse. In this neoadjuvant endocrine phase II trial including postmenopausal breast cancer patients with ER+, HER2 normal breast cancer, we investigated the rate of pathogenic mutations before and after treatment as well as the association with treatment response and survival. Pretreatment and posttreatment tumour samples from 109 patients treated with neoadjuvant letrozole were collected and analysed with Next Generation Sequencing utilizing a panel of 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, and RAF1). Residual disease was assessed by a modified Miller Payne scale and the Residual Cancer Burden index. Survival data were collected prospectively. Among the 109 patients, 52 had at least one pathogenic mutation in the pretreatment sample and 60 in the posttreatment sample. The most frequently mutated gene was PIK3CA, followed by EGFR and KRAS. Twelve different pathogenic PIK3CA mutations were identified, primarily in exon 20 and exon 9. An altered PIK3CA mutation profile from the pre- to the posttreatment specimen was significantly associated to improved pathological outcome. Overall and Disease-Free Survival benefits in PIK3CA mutated patients was observed. Considerable heterogeneity was identified both among patients and between pre- and posttreatment samples. PIK3CA has the potential to be a predictive biomarker. To further assess the implications of a treatment related altered PIK3CA mutation profile, more data are needed.

Purpose The Prosigna-PAM50 risk of recurrence (ROR) score has documented clinical utility for the prediction of 10-year distant recurrence (DR). The present study investigated the value of Prosigna-PAM50 for predicting 10-year DR and overall survival after 5 years of endocrine treatment for postmenopausal patients with invasive lobular carcinoma. Patients and Methods Using the Danish Breast Cancer Group database, we identified patients with a diagnosis from 2000 to 2003 of estrogen receptor-positive, human epidermal growth factor receptor 2-negative invasive ductal (n = 1570) or lobular (n = 341) cancer > 20 mm or 1 to 3 positive lymph nodes and applied multivariate Cox models. Results The median follow-up for DR was 9.3 years and for overall survival 15.2 years. Of the 341 lobular and 1570 ductal cases, 140 (41%) and 349 (22%) were classified as low ROR, with a 10-year DR rate of 7.7% (95% confidence interval [CI], 3.7%-13.6%) and 3.5% (95% CI, 1.8%-6.2%), respectively. The 10-year DR rate for the intermediate ROR group for those with lobular cancer was 18% (95% CI, 10.1%-27.9%) compared with 9.7% (95% CI, 6.7%-13.4%) for those with ductal cancer. Luminal B tumors had a significantly worse outcome than luminal A tumors in both lobular (hazard ratio, 1.89; 95% CI, 1.03%-3.45%; P = .04) and ductal (hazard ratio, 3.18; 95% CI, 2.29%-4.43%; P Conclusion Prosigna PAM-50 provides significant prognostic information beyond the clinicopathologic factors in patients with invasive lobular breast cancer. Those with lobular cancer had worse 10-year DR rates compared with those with ductal cancer in the same ROR category. Our results could have an effect on the treatment decisions regarding the addition of chemotherapy for those in the intermediate ROR group.

Background: Stromal Tumour Infiltrating Lymphocytes (sTILs) has been established as a predictive biomarker for response to neoadjuvant chemotherapy irrespective of subtype. While increased sTIL levels is associated with prolonged survival among patients with triple-negative and HER2 positive breast cancer increased sTILs has in Estrogen Receptor positive (ER+) breast cancer been suggested to be an adverse prognostic factor. It has been hypothesised that differences in the cellular composition of immune cells may explain the dissimilarity in prognostic impact according to breast cancer subtype. Here, we report data from a neoadjuvant phase II study, treating postmenopausal patients with primary ER+, HER2 negative, operable breast cancer with letrozole for four months by the Danish Breast Cancer Group (DBCG). We analysed the association of sTILs, and changes in sTILs during neoadjuvant endocrine treatment (NET), with pathological response and correlation with other clinicopathological variables such as Ki67, as potential biomarkers for risk-stratification of patients following NET. Method: 113 postmenopausal patients with ER+, HER2 negative breast cancer were treated with NET for four months. Endpoint was pathological response assessed by a modified Miller Payne scale and the Residual Cancer Burden (RCB) score. Pretherapeutic core biopsies and post therapeutic surgical specimens were assessed centrally for the percentage of sTILs on HE sections according to the International TILs working group guidelines. sTILs association to pathological response were assessed with univariate logistic regression. Odds Ratio (OR) was estimated with a 95 % confidence interval. Correlation of sTILs and Ki67 were tested with Pearson´s correlation coefficient. Changes in sTILs with a paired t-test. Level of significance was set to 5 %. Results: sTILs concentration increased with mean 6.8 % (p The correlation between pre-therapeutic sTILs and Ki67 was moderate (Pearson 0.4; p = 0.0004), however the association grew stronger post treatment (Pearson 0.5; p Conclusion: Increased sTILs activity during NET was associated with poor treatment response. An increase in sTILs during NET could be considered indictive for potential immunogenic tumours, suggesting that patients with increasing sTIL in residual disease after NET might derive benefit from the addition of immunotherapy. We found a significant correlation between sTILs and Ki67 levels. Ki67 has been established as a window of opportunity biomarker for antiproliferative response. Combining biomarkers of antiproliferative response such as Ki67 with biomarkers of immunogenetic significance might be important to determine optimal combination of adjuvant therapy. A window of opportunity study with an aromatase inhibitor could show if it is feasible to detect a rise in sTILs early enough to guide adjuvant treatment in the perioperative setting. Citation Format: Signe Korsgaard Skriver, Maj-Britt Jensen, Ann Soegaard Knoop, Bent Ejlertsen, Anne-Vibeke Laenkholm. Changes in tumour infiltrating lymphocytes during neoadjuvant endocrine therapy and possible clinical implications for guiding therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-18.

The DBCG89D trial randomized high-risk early breast cancer patients to adjuvant CMF (cyclophosphamide, methotrexate and fluorouracil) or CEF (cyclophosphamide, epirubicin and fluorouracil). Prosigna assays were performed by researchers with no access to clinical data. Time to distant recurrence (DR) was the primary endpoint, time to recurrence (TR) and overall survival (OS) secondary. Among the 980 Danish patients enrolled, Prosigna results were obtained in 686. Continuous ROR score was associated with DR for CMF (adjusted hazard ratio (HR) 1.20, 95% CI 1.09–1.33), and for CEF (HR 1.04, 95% CI 0.92–1.18), Pinteraction = 0.06. DR was significantly longer in CEF compared to CMF treated patients with Her2-enriched tumors (HR 0.58, 95% CI 0.38–0.86), but not in patients with luminal tumors. Heterogeneity of treatment effect was significant for TR and OS. In this prospective-retrospective analysis, patients with Her2-enriched breast cancer derived substantial benefit from anthracycline chemotherapy whereas anthracyclines are not an essential component of chemotherapy for patients with luminal subtypes. The benefit of CEF vs. CMF correlated with increasing ROR Score.

Genome-wide analysis identifies 32 loci associated with breast cancer susceptibility, accounting for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 status and tumor grade. Breast cancer susceptibility variants frequently show heterogeneity in associations by tumor subtype(1-3). To identify novel loci, we performed a genome-wide association study including 133,384 breast cancer cases and 113,789 controls, plus 18,908 BRCA1 mutation carriers (9,414 with breast cancer) of European ancestry, using both standard and novel methodologies that account for underlying tumor heterogeneity by estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 status and tumor grade. We identified 32 novel susceptibility loci (P < 5.0 x 10(-8)), 15 of which showed evidence for associations with at least one tumor feature (false discovery rate < 0.05). Five loci showed associations (P < 0.05) in opposite directions between luminal and non-luminal subtypes. In silico analyses showed that these five loci contained cell-specific enhancers that differed between normal luminal and basal mammary cells. The genetic correlations between five intrinsic-like subtypes ranged from 0.35 to 0.80. The proportion of genome-wide chip heritability explained by all known susceptibility loci was 54.2% for luminal A-like disease and 37.6% for triple-negative disease. The odds ratios of polygenic risk scores, which included 330 variants, for the highest 1% of quantiles compared with middle quantiles were 5.63 and 3.02 for luminal A-like and triple-negative disease, respectively. These findings provide an improved understanding of genetic predisposition to breast cancer subtypes and will inform the development of subtype-specific polygenic risk scores.

Tumor infiltrating lymphocytes (TILs) have predictive and prognostic potential in HER2-positive breast cancer (HER2 + BC). Due to tumor heterogeneity, guidelines recommend evaluation on full tumor-sections over biopsies, but aren't clear regarding tissue microarrays (TMAs). Herein, we investigate the inter-observer agreement of TILs assessment in HER2 + BC on full-sections and TMAs using a standardized method. Two pathologists assessed TILs using HE full-sections and TMAs from 244 patients with HER2 + BC. TILs were assessed in 10% intervals; values

Recent studies have shown that immune infiltrates in the tumor microenvironment play a role in response to therapy, with some suggesting that patients with immunogenic tumors may receive increased benefit from chemotherapies. We evaluated this hypothesis in early breast cancer by testing the interaction between immune biomarkers and chemotherapy using materials from DBCG77B, a phase III clinical trial where high-risk premenopausal women were randomized to receive chemotherapy or no chemotherapy. Tissue microarrays were evaluated for tumor-infiltrating lymphocytes (TILs) assessed morphologically on hematoxylin and eosin-stained slides, and by immunohistochemistry for CD8, FOXP3, LAG-3, PD-1 and PD-L1. Following REMARK reporting guidelines, data analyses were performed according to a prespecified statistical plan, using 10-year invasive disease-free survival as the endpoint. Differences in survival probabilities between biomarker groups were evaluated by Kaplan–Meier and Cox proportional hazard ratio analyses and prediction for treatment benefit by an interaction test. Our results showed that stromal TILs were associated with an improved prognosis (HR = 0.93; p-value = 0.03), consistent with previous studies. However, none of the immune biomarkers predicted benefit from chemotherapy in the full study set nor within major breast cancer subtypes. Our study indicates that primary tumors with higher immune infiltration do not derive extra benefit from cyclophosphamide-based cytotoxic chemotherapy.

Chronic encapsulated seroma following breast cancer surgery is a rare entity, and management is challenging. We present clinical and pathologic findings in a patient with previous node-negative breast cancer and an extensive history of chronic bilateral seromas, successfully treated with capsulectomy. This is the first report of fibrous encapsulated breast seroma with bilateral presentation and late onset, following mastectomy with no prior axillary dissection. When managing breast seroma refractory to conventional treatment, the diagnosis of encapsulated seroma should be considered, followed by prompt capsulectomy. Chronic encapsulated seroma following breast cancer surgery is a rare entity, and management is challenging. We present clinical and pathologic findings in a patient with previous node-negative breast cancer and an extensive history of chronic bilateral seromas, successfully treated with capsulectomy. This is the first report of fibrous encapsulated breast seroma with bilateral presentation and late onset, following mastectomy with no prior axillary dissection. When managing breast seroma refractory to conventional treatment, the diagnosis of encapsulated seroma should be considered, followed by prompt capsulectomy.

Background: The prognostic value of tumour-infiltrating lymphocytes (TILs) in breast cancer is well-established. However, the investigation of specific T-cell subsets exclusively in BRCA-associated breast cancer is sparse. Methods: Tumour tissues from 414 BRCA-mutated breast cancer patients were analysed by immunohistochemistry and digital image analysis for expression of CD4, CD8 and FOXP3 immune markers. Distribution of CD4-, CD8- and FOXP3-positive cells and clinicopathological characteristics were assessed according to groups of low or high expression. The prognostic value was evaluated as continuous variables in univariate and multivariate analyses of overall survival and disease-free survival. Results: Both CD4 and CD8 expression are associated with histological diagnosis, tumour grade and oestrogen and progesterone receptor expression status. CD4 expression is associated with BRCA gene status. A high percentage of tumour-infiltrating CD4-, CD8- or FOXP3-positive cells is significantly associated with lower mortality in BRCA1- and BRCA2-associated breast cancer and CD8-positive cells are associated with disease-free survival. No heterogeneity according to BRCA gene status was found for the prognostic value of the immune markers. Conclusions: The results support a prognostic role of specific T-cell subsets in BRCA-associated breast cancer and the promising potential of targeting the immune system in the treatment of these patients.

Stromal tumour infiltrating lymphocytes (sTILs) are a strong prognostic marker in triple negative breast cancer (TNBC). Consistency scoring sTILs is good and was excellent when an internet-based scoring aid developed by the TIL-WG was used to score cases in a reproducibility study. This study aimed to evaluate the reproducibility of sTILs assessment using this scoring aid in cases from routine practice and to explore the potential of the tool to overcome variability in scoring. Twenty-three breast pathologists scored sTILs in digitized slides of 49 TNBC biopsies using the scoring aid. Subsequently, fields of view (FOV) from each case were selected by one pathologist and scored by the group using the tool. Inter-observer agreement was good for absolute sTILs (ICC 0.634, 95% CI 0.539–0.735, p &lt, 0.001) but was poor to fair using binary cutpoints. sTILs heterogeneity was the main contributor to disagreement. When pathologists scored the same FOV from each case, inter-observer agreement was excellent for absolute sTILs (ICC 0.798, 95% CI 0.727–0.864, p &lt, 0.001) and good for the 20% (ICC 0.657, 95% CI 0.561–0.756, p &lt, 0.001) and 40% (ICC 0.644, 95% CI 0.546–0.745, p &lt, 0.001) cutpoints. However, there was a wide range of scores for many cases. Reproducibility scoring sTILs is good when the scoring aid is used. Heterogeneity is the main contributor to variance and will need to be overcome for analytic validity to be achieved.

Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. Using genome sequencing, we first noted for non-BRCA1/BRCA2 tumours, only a small proportion (3/23) demonstrated features of BRCAness, with high HRDetect scores and concomitant somatic BRCA1 mutation or promoter hypermethylation to explain their BRCAness. Second, a small proportion (4/23) showed no features of BRCAness but had mutationally active tumours. Third, the remaining tumours lacked features of BRCAness and were mutationally quiescent. Only few families could be explained by pathogenic germline variants in other genes or polygenic risk score.

Aims: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. Methods and results: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799–0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74–0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively. Conclusions: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

Background: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown. Material and methods: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (

e13639 Background: Several Breast Cancer Multigene Signatures (BCMS) are available to profile early breast cancer (eBC) that according to current evidence, can provide reliable information on the risk of recurrence but the knowledge regarding their real use in clinical practice is scarce. The PROCURE Project aims to establish recommendations on the use of BCMS in clinical practice. Methods: A Scientific Committee consisting of 8 European experts in BC, including oncologists, pathologists, and surgeons, developed a Delphi questionnaire that was administered twice to clinicians across different European centers. The questionnaire included 5 sections: 1) Panelists’ profile and experience with BCMS, 2) Current clinical practice in eBC and use of BCMS, 3) Panelists’ opinion on the utility of the BCMS in eBC according to patient profiles, 4) Agreement with a set of recommendations on the use of BCMS in clinical practice and 5) Identification of unmet needs and future applications of BCMS. 70% agreement was used to determine consensus on a topic. Results: 133 panelists from 11 European countries completed both rounds of the survey. Most of the panelists worked in a teaching hospital (86.5%) and 72.2% of them were medical oncologists. 93.6% of the panelists used BCMS routinely or in selected patients and 73.4% had more than 5 years of experience using BCMS. According to panelists, for being useful in clinical practice BCMS should consider clinical and pathological features of the disease (91%). Additionally, BCMS must provide both, prognostic and predictive information (82%), and information on the risk of late distant recurrence from 5 to 10 years (74%), all based on prospective randomized clinical trials (85%). BCMS must also provide accurate results to guide adjuvant chemotherapy decision (81%). Regarding recommendations for improving BCMS utility, 76% of the panelists agreed that hospitals policies on the use of BCMS must be based on clinical practice guidelines. Panelists also agreed that patients should have the right to access their BCMS results to take part in the treatment decision (86%) and more training is needed for patients (79%) as well as oncologists (84%) and pathologists (82%). Finally, panelists mostly disagree on the fact that BCMS must be performed in all patients when BC is suspected (76%). Conclusions: The high degree of importance that panelists attributed to the fact that a BCMS should provide both prognostic and predictive information, reflects a misconception on the utility of BCMS as they are primarily prognostic tools but not predictive about the effect of a therapeutic intervention. Also, consensus reached on the need of providing risk of late recurrence from 5 to 10 years shows that panelists evaluate the long-term treatment even in the early setting.

In a formal prospective-retrospective analysis of the phase III SBG0102 clinical trial randomizing metastatic breast cancer patients to gemcitabine-docetaxel or to single agent docetaxel, patients with basal-like tumors by PAM50 gene expression had significantly better overall survival in the gemcitabine arm. By immunohistochemistry (IHC), triple negative status was not predictive, but more specific biomarkers have since become available defining basal-like by nestin positivity or loss of inositol-polyphosphate-4-phosphate (INPP4B). Here, we evaluate their capacity to identify which patients benefit from gemcitabine in the metastatic setting. Nestin and INPP4B staining and interpretation followed published methods. A prespecified statistical plan evaluated the primary hypothesis that patients with basal-like breast cancer, defined as "nestin+ or INPP4B-", would have superior overall survival on gemcitabine-docetaxel when compared to docetaxel. Interaction tests, Kaplan-Meier curves and forest plots were used to assess prognostic and predictive capacities of biomarkers relative to treatment. Among 239 cases evaluable for our study, 36 (15%) had been classified as basal-like by PAM50. "Nestin+ or INPP4B-" was observed in 41 (17%) of the total cases and was significantly associated with PAM50 basal-like subtype. Within an estimated median follow-up of 13 years, patients assigned as IHC basal "nestin+ or INPP4B-" had significantly better overall survival on gemcitabine-docetaxel versus docetaxel monotherapy (HR = 0.31, 95%CI: 0.16-0.60), whereas no differences were observed for other patients (HR = 0.99), p-interaction < 0.01. In the metastatic setting, women with IHC basal breast cancers defined as "nestin+ or INPP4B-" have superior overall survival when randomized to gemcitabine-containing chemotherapy compared to docetaxel alone. These findings need to be validated using larger prospective-retrospective phase III clinical trials series.

Background Cancer development is among other factors driven by tumor immune escape and tumor-mediated changes in the immune response. Investigating systemic immune changes may provide important knowledge for the improvement of patient prognosis and treatment opportunities. Methods The systemic immune profile of patients with ER-positive breast cancer (n = 22) and healthy controls (n = 30) was investigated based on complete blood counts, flow cytometric analysis of T cell subsets including regulatory T cells (Tregs), and immune assays investigating soluble (s)HLA-G and the cytokine profile in plasma. We further examined the correlation between the immune markers and clinical parameters including tumor size, tumor grade and lymph node involvement. Results Results indicated that breast cancer patients possessed a higher amount of neutrophils and monocytes and fewer lymphocytes and eosinophils compared with healthy controls. Breast cancer patients had significantly more CD25+CD127low Tregs than controls, and both lymphocyte and Treg numbers were negatively correlated with tumor size. Furthermore, Treg numbers were elevated in grade I tumors compared with grade II tumors and with healthy controls. No difference in sHLA-G levels was observed between patients and controls. Higher levels of IL-6 and TNF-α were observed in breast cancer patients. Cytokine and sHLA-G levels were not associated with clinical parameters. Conclusion The results of this exploratory study contribute to the elucidation of the systemic immune response in breast cancer indicating a potential use of peripheral immune cell counts and Tregs to distinguish patients from healthy controls and as potential diagnostic and prognostic biomarkers to be investigated in future studies.

Background: Chronic encapsulated seroma following breast cancer surgery is a rare entity, and management is challenging. We present clinical and pathologic findings in a patient with extensive history of bilateral refractory breast seroma successfully treated with capsulectomy. Case presentation: A 71-year-old woman with previous early BRCA2 associated, node-negative breast cancer, developed late onset seroma following endocrine therapy, unilateral breast-conserving surgery, and completion of adjuvant radiotherapy. Non-simultaneous bilateral mastectomy was later performed to treat infection and reduce risk of contralateral breast cancer. Each surgery was followed by chronic seroma. After six years of ineffective treatment attempts with repeated aspirations, the patient successfully underwent bilateral capsulectomy. She remains seroma-free nine months from time of capsulectomy. Conclusion: This is the first report of late onset bilateral encapsulated breast seroma following mastectomy without axillary dissection. When managing breast seroma refractory to conventional treatment encapsulated seroma and prompt capsulectomy should be considered.