Breast Cancer Histopathology in the Age of Molecular Oncology.

For more than a century, microscopic histology has been the cornerstone for cancer diagnosis, and breast carcinoma is no exception. In recent years, clinical biomarkers, gene expression profiles, and other molecular tests have shown increasing utility for identifying the key biological features that guide prognosis and treatment of breast cancer. Indeed, the most common histologic pattern-invasive ductal carcinoma of no special type-provides relatively little guidance to management beyond triggering grading, biomarker testing, and clinical staging. However, many less common histologic patterns can be recognized by trained pathologists, which in many cases can be linked to characteristic biomarker and gene expression patterns, underlying mutations, prognosis, and therapy. Herein we describe more than a dozen such histomorphologic subtypes (including lobular, metaplastic, salivary analog, and several good prognosis special types of breast cancer) in the context of their molecular and clinical features.

High inter-laboratory variability in the assessment of HER2-low breast cancer: a national registry study on 50,714 Danish patients.

BACKGROUND: Considering the recent advancements in the treatment of breast cancer with low expression of human epidermal growth factor receptor 2 (HER2), we aimed to examine inter-laboratory variability in the assessment of HER2-low breast cancer across all Danish pathology departments. METHODS: From the Danish Breast Cancer Group, we obtained data on all women diagnosed with primary invasive breast cancer in 2007-2019 who were subsequently assigned for curatively intended treatment. RESULTS: Of 50,714 patients, HER2 score and status were recorded for 48,382, among whom 59.2% belonged to the HER2-low group (score 1+ or 2+ without gene amplification), 26.8% had a HER2 score of 0, and 14.0% were HER2 positive. The proportion of HER2-low cases ranged from 46.3 to 71.8% among pathology departments (P < 0.0001) and from 49.3 to 65.6% over the years (P < 0.0001). In comparison, HER2 positivity rates ranged from 11.8 to 17.2% among departments (P < 0.0001) and from 12.6 to 15.7% over the years (P = 0.005). In the eight departments with the highest number of patients, variability in HER2-low cases increased from 2011 to 2019, although the same immunohistochemical assay was used. By multivariable logistic regression, the examining department was significantly related to both HER2 score 0 and HER2 positivity (P < 0.0001) but showed greater dispersion in odds ratios in the former case (range 0.25-1.41 vs. 0.84-1.27). CONCLUSIONS: Our data showed high inter-laboratory variability in the assessment of HER2-low breast cancer. The findings cast doubt on whether the current test method for HER2 is robust and reliable enough to select HER2-low patients for HER2-targeted treatment in daily clinical practice.

Evaluation of tumor-infiltrating lymphocytes, PD-L1, and PIK3CA mutations and association with prognosis in HER2-positive early stage breast cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) have predictive and prognostic potential in HER2-positive breast cancer (HER2+ BC). Programmed death-ligand 1 (PD-L1) is an immune checkpoint protein, with important roles in the tumor microenvironment, possibly in both tumor and immune cells (ICs), providing rationale for targeting with immune-checkpoint therapy. PIK3CA mutations are oncogenic, activating mutations, which are also of relevance in breast cancer. Herein, we investigate the frequency of TILs, PD-L1 and PIK3CA mutations, and whether these factors influence outcome, in early HER2+ BC. MATERIALS AND METHODS: Stromal TILs (sTILs) and PD-L1 expressions were assessed using full tumor-sections and TMA, respectively, from 236 patients with HER2+ BC. TILs were assessed, according to a standardized method, as continuous measurement and according to three predefined categories: low (0-10%), intermediate (11-59%), and high (60-100%). PD-L1 immunohistochemistry (Ventana SP263) was evaluated and positivity defined as ≥1% expression in tumor and ICs. PIK3CA mutations (exons 9 and 20) were determined by pyrosequencing. RESULTS: Fourteen percent of patients had high sTILs and 25% had a PIK3CA mutation. PD-L1 expression was more frequent in ICs (68%) than tumor cells (24%). Patients with low sTILs had a significantly worse overall survival (multivariate: HR 2.80; 95% CI 1.36-5.78; p = .02). DISCUSSION: Patients with low sTILs had a significantly poorer survival, despite adequate treatment with adjuvant therapy.

A Multi-Stain Breast Cancer Histological Whole-Slide-Image Data Set from Routine Diagnostics.

The analysis of FFPE tissue sections stained with haematoxylin and eosin (H&E) or immunohistochemistry (IHC) is essential for the pathologic assessment of surgically resected breast cancer specimens. IHC staining has been broadly adopted into diagnostic guidelines and routine workflows to assess the status of several established biomarkers, including ER, PGR, HER2 and KI67. Biomarker assessment can also be facilitated by computational pathology image analysis methods, which have made numerous substantial advances recently, often based on publicly available whole slide image (WSI) data sets. However, the field is still considerably limited by the sparsity of public data sets. In particular, there are no large, high quality publicly available data sets with WSIs of matching IHC and H&E-stained tissue sections from the same tumour. Here, we publish the currently largest publicly available data set of WSIs of tissue sections from surgical resection specimens from female primary breast cancer patients with matched WSIs of corresponding H&E and IHC-stained tissue, consisting of 4,212 WSIs from 1,153 patients.

Neo-train: study protocol and feasibility results for a two-arm randomized controlled trial investigating the effect of supervised exercise during neoadjuvant chemotherapy on tumour response in patients with breast cancer.

BACKGROUND: Prehabilitation with exercise interventions during neoadjuvant chemotherapy (NACT) is effective in reducing physical and psychosocial chemotherapy-related adverse events in patients with cancer. In preclinical studies, data also support a growth inhibitory effect of aerobic exercise on the tumour microenvironment with possible improved chemotherapy delivery but evidence in human patients is limited. The aim of the study here described is to investigate if supervised exercise with high-intensity aerobic and resistance training during NACT can improve tumour reduction in patients with breast cancer. METHODS: This parallel two-armed randomized controlled trial is planned to include 120 women aged ≥ 18 years with newly diagnosed breast cancer starting standard NACT at a university hospital in Denmark (a total of 90 participants needed according to the power calculation and allowing 25% (n = 30) dropout). The participants will be randomized to usual care or supervised exercise consisting of high-intensity interval training on a stationary exercise bike and machine-based progressive resistance training offered three times a week for 24 weeks during NACT, and screening-based advice to seek counselling in case of moderate-severe psychological distress (Neo-Train program). The primary outcome is tumour size change (maximum diameter of the largest lesion in millimetre) measured by magnetic resonance imaging prior to surgery. Secondary outcomes include clinical/pathological, physical and patient-reported measures such as relative dose intensity of NACT, hospital admissions, body composition, physical fitness, muscle strength, health-related quality of life, general anxiety, depression, and biological measures such as intratumoural vascularity, tumour infiltrating lymphocytes, circulating tumour DNA and blood chemistry. Outcomes will be measured at baseline (one week before to 1-2 weeks after starting NACT), during NACT (approximately week 7, 13 and 19), pre-surgery (approximately week 21-29), at surgery (approximately week 21-30) and 3 months post-surgery (approximately 33-42 weeks from baseline). DISCUSSION: This study will provide novel and important data on the potential benefits of supervised aerobic and resistance exercise concomitant to NACT on tumour response and the tumour microenvironment in patients with breast cancer, with potential importance for survival and risk of recurrence. If effective, our study may help increase focus of exercise as an active part of the neoadjuvant treatment strategy. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov (NCT04623554) on November 10, 2020.

Non-BRCA1/BRCA2 high-risk familial breast cancers are not associated with a high prevalence of BRCAness.

BACKGROUND: Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. The somatic mutational landscape and in particular the extent of BRCA-like tumour features (BRCAness) in these familial breast cancers where germline BRCA1 or BRCA2 mutations have not been identified is to a large extent unknown. METHODS: We performed whole-genome sequencing on matched tumour and normal samples from high-risk non-BRCA1/BRCA2 breast cancer families to understand the germline and somatic mutational landscape and mutational signatures. We measured BRCAness using HRDetect. As a comparator, we also analysed samples from BRCA1 and BRCA2 germline mutation carriers. RESULTS: We noted for non-BRCA1/BRCA2 tumours, only a small proportion displayed high HRDetect scores and were characterized by concomitant promoter hypermethylation or in one case a RAD51D splice variant previously reported as having unknown significance to potentially explain their BRCAness. Another small proportion showed no features of BRCAness but had mutationally active tumours. The remaining tumours lacked features of BRCAness and were mutationally quiescent. CONCLUSIONS: A limited fraction of high-risk familial non-BRCA1/BRCA2 breast cancer patients is expected to benefit from treatment strategies against homologue repair deficient cancer cells.

Validation of the BOADICEA model for predicting the likelihood of carrying pathogenic variants in eight breast and ovarian cancer susceptibility genes.

BOADICEA is a comprehensive risk prediction model for breast and/or ovarian cancer (BC/OC) and for carrying pathogenic variants (PVs) in cancer susceptibility genes. In addition to BRCA1 and BRCA2, BOADICEA version 6 includes PALB2, CHEK2, ATM, BARD1, RAD51C and RAD51D. To validate its predictions for these genes, we conducted a retrospective study including 2033 individuals counselled at clinical genetics departments in Denmark. All counselees underwent comprehensive genetic testing by next generation sequencing on suspicion of hereditary susceptibility to BC/OC. Likelihoods of PVs were predicted from information about diagnosis, family history and tumour pathology. Calibration was examined using the observed-to-expected ratio (O/E) and discrimination using the area under the receiver operating characteristics curve (AUC). The O/E was 1.11 (95% CI 0.97-1.26) for all genes combined. At sub-categories of predicted likelihood, the model performed well with limited misestimation at the extremes of predicted likelihood. Discrimination was acceptable with an AUC of 0.70 (95% CI 0.66-0.74), although discrimination was better for BRCA1 and BRCA2 than for the other genes in the model. This suggests that BOADICEA remains a valid decision-making aid for determining which individuals to offer comprehensive genetic testing for hereditary susceptibility to BC/OC despite suboptimal calibration for individual genes in this population.

ONEST (Observers Needed to Evaluate Subjective Tests) Analysis of Stromal Tumour-Infiltrating Lymphocytes (sTILs) in Breast Cancer and Its Limitations.

Tumour-infiltrating lymphocytes (TILs) reflect antitumour immunity. Their evaluation of histopathology specimens is influenced by several factors and is subject to issues of reproducibility. ONEST (Observers Needed to Evaluate Subjective Tests) helps in determining the number of observers that would be sufficient for the reliable estimation of inter-observer agreement of TIL categorisation. This has not been explored previously in relation to TILs. ONEST analyses, using an open-source software developed by the first author, were performed on TIL quantification in breast cancers taken from two previous studies. These were one reproducibility study involving 49 breast cancers, 23 in the first circulation and 14 pathologists in the second circulation, and one study involving 100 cases and 9 pathologists. In addition to the estimates of the number of observers required, other factors influencing the results of ONEST were examined. The analyses reveal that between six and nine observers (range 2-11) are most commonly needed to give a robust estimate of reproducibility. In addition, the number and experience of observers, the distribution of values around or away from the extremes, and outliers in the classification also influence the results. Due to the simplicity and the potentially relevant information it may give, we propose ONEST to be a part of new reproducibility analyses.

Adjuvant treatment with trastuzumab of patients with HER2-positive, T1a-bN0M0 breast tumors: A systematic review and meta-analysis.

The benefit of adjuvant trastuzumab treatment in patients with HER2-positive breast tumors ≤ 10 mm without lymph node involvement (T1abN0) is insufficiently investigated. The aim of this systematic review and meta-analysis was to examine if adjuvant trastuzumab improves the prognosis in these patients. Databases were searched to identify interventional and observational studies evaluating the effect of trastuzumab on breast cancer specific survival (BCSS), disease free survival (DFS), distant recurrence free survival (DRFS), overall survival (OS) or recurrence free survival (RFS). Twelve studies examining the effect of trastuzumab and nine control studies without trastuzumab were identified (n = 6927). Median follow-up was 36-123 months. Significantly improved DFS (Hazard Ratio (HR) 0.14, p < 0.0001) and OS (HR 0.17, p = 0.011) were found for patients receiving trastuzumab and chemotherapy compared to no trastuzumab/chemotherapy based on four and two studies. The prognosis was good even for patients without trastuzumab treatment: 5-year DFS 88.3% and 5-year OS 95.9%.

Digital image analysis and assisted reading of the HER2 score display reduced concordance: pitfalls in the categorisation of HER2-low breast cancer.

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.

PROCURE European consensus on breast cancer multigene signatures in early breast cancer management.

Breast cancer multigene signatures (BCMS) have changed how patients with early-stage breast cancer (eBC) are managed, as they provide prognostic information and can be used to select patients who may avoid adjuvant chemotherapy. Clinical guidelines make recommendations on the use of BCMS; however, little is known on the current use of BCMS in clinical practice. We conduct a two-round Delphi survey to enquire about current use and perceived utility for specific patient profiles, and unmet needs of BCMS. Overall, 133 panellists experienced in breast cancer across 11 European countries have participated, most using BCMS either routinely (66.2%) or in selected cases (27.1%). Our results show that BCMS are mainly used to assess the risk of recurrence and to select patients for adjuvant chemotherapy; notably, no consensus has been reached on the lack of utility of BCMS for selecting the type of chemotherapy to administer. Also, there are discrepancies between the recommended and current use of BCMS in clinical practice, with use in certain patient profiles for which there is no supporting evidence. Our study suggests that physician education initiatives are needed to ensure the correct use and interpretation of BCMS to, ultimately, improve management of patients with eBC.

Adjuvant chemotherapy in patients with ER-negative/HER2-negative, T1abN0 breast cancer: a nationwide study.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Benefit of adjuvant chemotherapy and trastuzumab in patients with HER2-positive, node-negative breast tumors ≤ 10 mm: a nationwide study.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Neither Tumor-Infiltrating Lymphocytes nor Cytotoxic T Cells Predict Enhanced Benefit from Chemotherapy in the DBCG77B Phase III Clinical Trial.

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.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

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.

Inter-observer agreement of tumor infiltrating lymphocytes in primary HER2-positive breast cancer and correlation between tissue microarray and full tumor-sections.

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 <10% evaluated as 0%, 1% or 5%. Levels of agreement were evaluated using intraclass-coefficient (ICC), Kappa statistics and concordance analysis. For inter-observer agreement for full-sections, ICC was 0.93 (95% CI 0.89-0.95) and Kappa was 0.75, corresponding to acceptable and moderate agreement respectively. For TMAs, ICC was 0.73 (95% CI: 0.62-0.81) and Kappa 0.33, corresponding to unacceptable agreement. For association in matched TMA and full-sections, ICC was 0.64 (95% CI 0.55-0.71) and Lin's concordance correlation coefficient was 0.63 (95% CI 0.55-0.71), corresponding to unacceptable agreement. There is acceptable inter-observer agreement of TILs assessment on full-sections but not TMAs and discrepancy between full-sections and TMAs. TMA preparation must include consideration for representation of both entire tumor area and tumor-microenvironment to correctly define prognostic and predictive values of potential immuno-related biomarkers.

Capsulectomy Can Successfully Treat Chronic Encapsulated Breast Seroma: A Case Report.

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.

Incorporation of TILs in daily breast cancer care: how much evidence can we bear?

One of the most important developments in the breast cancer field has been an improved understanding of prognostic and predictive biomarkers, of which TILs are increasingly gaining importance. The evaluation of TILs by light microscopy on a H&E-stained section is workable in a daily practice setting. Reproducibility of reporting TILs is good, but heterogeneity is a cause of variation. TILs provide clinicians with important prognostic information for patients with TNBC, as early-stage TNBC with high TILs have > 98% 5-year survival and TILs predict benefit to immunotherapy. Importantly, while TILs do not have level of evidence IA, TILs should be used as a prognostic factor with caution and with other accepted prognostic variables, such as tumour size and lymph node status, to inform clinicians and patients on their treatment options. A framework on how to use the TILs in daily practice is proposed, including a co-assessment with PD-L1 for its predictive role to immunotherapy.