Qualification of a multiplexed tissue imaging assay and detection of novel patterns of HER2 heterogeneity in breast cancer.

Emerging data suggests that HER2 intratumoral heterogeneity (ITH) is associated with therapy resistance, highlighting the need for new strategies to assess HER2 ITH. A promising approach is leveraging multiplexed tissue analysis techniques such as cyclic immunofluorescence (CyCIF), which enable visualization and quantification of 10-60 antigens at single-cell resolution from individual tissue sections. In this study, we qualified a breast cancer-specific antibody panel, including HER2, ER, and PR, for multiplexed tissue imaging. We then compared the performance of these antibodies against established clinical standards using pixel-, cell- and tissue-level analyses, utilizing 866 tissue cores (representing 294 patients). To ensure reliability, the CyCIF antibodies were qualified against HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) data from the same samples. Our findings demonstrate the successful qualification of a breast cancer antibody panel for CyCIF, showing high concordance with established clinical antibodies. Subsequently, we employed the qualified antibodies, along with antibodies for CD45, CD68, PD-L1, p53, Ki67, pRB, and AR, to characterize 567 HER2+ invasive breast cancer samples from 189 patients. Through single-cell analysis, we identified four distinct cell clusters within HER2+ breast cancer exhibiting heterogeneous HER2 expression. Furthermore, these clusters displayed variations in ER, PR, p53, AR, and PD-L1 expression. To quantify the extent of heterogeneity, we calculated heterogeneity scores based on the diversity among these clusters. Our analysis revealed expression patterns that are relevant to breast cancer biology, with correlations to HER2 ITH and potential relevance to clinical outcomes.

Survival Outcomes in Premenopausal Patients With Invasive Lobular Carcinoma.

Importance: The disparate prognostic implications between invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) have been demonstrated. However, information on premenopausal patients remains insufficient. Objective: To examine long-term survival outcomes of ILC and IDC in premenopausal patients using national databases. Design, Setting, and Participants: This cohort study used the Surveillance, Epidemiology, and End Results (SEER), Korean Breast Cancer Registry (KBCR), and Asan Medical Center Research (AMCR) databases to identify premenopausal patients with stage I to III ILC or IDC between January 1, 1990, and December 31, 2015. The median follow-up time was 90 (IQR, 40-151) months in the SEER database, 94 (IQR, 65-131) months in the KBCR database, and 120 (IQR, 86-164) months in the AMCR database. Data were analyzed from January 1 to May 31, 2023. Main Outcomes and Measures: The primary outcome was breast cancer-specific survival (BCSS), which was analyzed according to histological type, and the annual hazard rate was evaluated. Survival rates were analyzed using a log-rank test and a Cox proportional hazards regression model with time-varying coefficients. Multivariable analysis was performed by adjusting for tumor characteristics and treatment factors. Results: A total of 225 938 women diagnosed with IDC or ILC and younger than 50 years were identified. Mean (SD) age at diagnosis was 42.7 (5.3) years in the SEER database, 41.8 (5.5) years in the KBCR database, and 41.8 (5.5) years in the AMCR database. In terms of race (available for the SEER database only), 12.4% of patients were Black, 76.1% were White, 11.0% were of other race (including American Indian or Alaska Native, Asian, and Native Hawaiian or Other Pacific Islander), and 0.5% were of unknown race). Patients with ILC had better BCSS in the first 10 years after diagnosis than those with IDC (hazard ratios [HRs], 0.73 [95% CI, 0.68-0.78] in the SEER database, 1.20 [95% CI, 0.91-1.58] in the KBCR database, and 0.50 [95% CI, 0.29-0.86] in the AMCR database), although BCSS was worse after year 10 (HRs, 1.80 [95% CI, 1.59-2.02] in the SEER database, 2.79 [95% CI, 1.32-5.88] in the KBCR database, and 2.23 [95% CI, 1.04-4.79] in the AMCR database). Similar trends were observed for hormone receptor-positive tumors (HRs, 1.55 [95% CI, 1.37-1.75] in the SEER database, 2.27 [95% CI, 1.01-5.10] in the KBCR database, and 2.12 [95% CI, 0.98-4.60] in the AMCR database). Considering the annual hazard model of BCSS, IDC events tended to decline steadily after peaking 5 years before diagnosis. However, the annual peak event of BCSS was observed 5 years after diagnosis for ILC, which subsequently remained constant. Conclusions and Relevance: These findings suggest that premenopausal women with ILC have worse BCSS estimates than those with IDC, which can be attributed to a higher late recurrence rate of ILC than that of IDC. Histological subtypes should be considered when determining the type and duration of endocrine therapy in premenopausal women.

Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor-Positive Breast Cancer.

Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated ß2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of ß2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer. SIGNIFICANCE: Adding SMAC mimetics to endocrine therapy enhances tumor regression in a cell autonomous manner while increasing tumor immunogenicity, indicating that this combination could be an effective treatment for HR+ patients with breast cancer.

Assessment of the HER2DX Assay in Patients With ERBB2-Positive Breast Cancer Treated With Neoadjuvant Paclitaxel, Trastuzumab, and Pertuzumab.

Importance: Patients with early-stage ERBB2 (formerly HER2)-positive breast cancer (ERBB2+ BC) who experience a pathologic complete response (pCR) after receiving neoadjuvant therapy have favorable survival outcomes. Predicting the likelihood of pCR may help optimize neoadjuvant therapy. Objective: To test the ability of the HER2DX assay to predict the likelihood of pCR in patients with early-stage ERBB2+ BC who are receiving deescalated neoadjuvant therapy. Design, Setting, and Participants: In this diagnostic/prognostic study, the HER2DX assay was administered on pretreatment tumor biopsy samples from patients enrolled in the single-arm, multicenter, prospective phase 2 DAPHNe clinical trial who had newly diagnosed stage II to III ERBB2+ BC that was treated with neoadjuvant paclitaxel weekly for 12 weeks plus trastuzumab and pertuzumab every 3 weeks for 4 cycles. Interventions and Exposures: The HER2DX assay is a classifier derived from gene expression and limited clinical features that provides 2 independent scores to predict prognosis and likelihood of pCR in patients with early-stage ERBB2+ BC. The assay was administered on baseline tumor samples from 80 of 97 patients (82.5%) in the DAPHNe trial. Main Outcomes and Measures: The primary aim was to test the ability of the HER2DX pCR likelihood score (as a continuous variable from 0-100) to predict pCR (ypT0/isN0). Results: Of 80 participants, 79 (98.8%) were women and there were 4 African American (5.0%), 6 Asian (7.5%), 4 Hispanic (5.0%), and 66 White individuals (82.5%); the mean (range) age was 50.3 (26.0-78.0) years. The HER2DX pCR score was significantly associated with pCR (odds ratio, 1.05; 95% CI, 1.03-1.08; P < .001). The pCR rates in the HER2DX high, medium, and low pCR score groups were 92.6%, 63.6%, and 29.0%, respectively (high vs low odds ratio, 30.6; P < .001). The HER2DX pCR score was significantly associated with pCR independently of hormone receptor status, ERBB2 immunohistochemistry score, HER2DX ERBB2 expression score, and prediction analysis of microarray 50 ERBB2-enriched subtype. The correlation between the HER2DX pCR score and prognostic risk score was weak (Pearson coefficient, -0.12). Performance of the risk score could not be assessed due to lack of recurrence events. Conclusions and Relevance: The results of this diagnostic/prognostic study suggest that the HER2DX pCR score assay could predict pCR following treatment with deescalated neoadjuvant paclitaxel with trastuzumab and pertuzumab in patients with early-stage ERBB2+ BC. The HER2DX pCR score might guide therapeutic decisions by identifying patients who are candidates for deescalated or escalated approaches.

The prevalence and predictors of adjuvant chemotherapy use among patients treated with neoadjuvant endocrine therapy.

PURPOSE: Neoadjuvant endocrine therapy (NET) facilitates clinical response and breast conservation in hormone receptor-positive (HR-positive) breast cancer. Patient selection for adjuvant chemotherapy (CT) post-NET is unclear and potentially evolving with use of genomic assays. We evaluated post-NET CT use in a national dataset. METHODS: Using the National Cancer DataBase, we identified patients with cT2-3N0-3M0 HR-positive/human epidermal growth factor receptor 2-negative breast cancer treated between 2010 and 2017 with 3-12 months of NET prior to breast surgery. CT use was evaluated in the overall population, in patients with a pathologic complete response (pCR) and in patients with ypT1-2N0 disease (approximating PEPI 0). Exploratory analysis included patients > 50 years with ypN0-1, and 21-gene recurrence score (RS) ≤ 25 (approximating TAILORx/RxPONDER populations not benefiting from CT). Multivariable logistic regression was used to identify factors associated with CT. RESULTS: Among 3624 eligible patients, 20.4% (740/3624) received CT. On multivariable analysis, age ≤ 50, lobular histology, grade 2, progesterone receptor negativity, ypT3, ypN + and RS ≥ 18 were associated with CT receipt. Co-morbidity, longer NET duration, ypT4, ypNx, and RS < 18 were associated with CT omission. CT was administered to 3.3% (1/30) of patients experiencing pCR and 5.5% (82/1483) with ypT1-2N0 disease. Among patients > 50 years with ypT0-3N0-1 residual disease, 13.8% (355/2569) received CT; RS was available for 24.8% (88/355) and 60% (53/88) had a score 0-25. CONCLUSION: A minority of patients receive CT post-NET. This decision appears to be driven by younger age, RS and pathological nodal status. Increased consideration of these factors prior to neoadjuvant treatment choice may be warranted.

A prospective trial of treatment de-escalation following neoadjuvant paclitaxel/trastuzumab/pertuzumab in HER2-positive breast cancer.

De-escalating adjuvant therapy following pathologic complete response (pCR) to an abbreviated neoadjuvant regimen in human epidermal growth factor receptor 2-positive (HER2+) breast cancer is the focus of international research efforts. However, the feasibility of this approach and its appeal to patients and providers had not been formally investigated. We aimed to assess adherence to de-escalated adjuvant antibody doublet therapy (trastuzumab and pertuzumab [HP], without chemotherapy) among patients with pCR following neoadjuvant paclitaxel/HP (THP). In this single-arm prospective trial, patients with treatment-naïve stage II-III HER2+ breast cancer received neoadjuvant weekly paclitaxel ×12 and HP every 3 weeks ×4. The primary endpoint was receipt of adjuvant non-HER2-directed cytotoxic chemotherapy. Ninety-eight patients received ≥1 dose of THP on study. Patients had median age of 50 years, 86% had stage II tumors, and 34% were hormone receptor-negative. Five patients had incomplete clinical response following THP and received doxorubicin and cyclophosphamide before surgery; they were classified as non-pCR and censored from further analyses. The overall pCR rate was 56.7%. Among patients with pCR, the adherence rate to de-escalated antibody-only therapy (HP) was 98.2% (95% CI 90.3-100.0%), and the primary feasibility endpoint was reached. The majority of patients felt positive or neutral about their adjuvant treatment plans. With brief follow-up (median 19.1 months), there were no breast cancer recurrences. De-escalation of adjuvant chemotherapy among patients who experience pCR in early-stage HER2+ breast cancer is a practicable approach for both patients and physicians. Planned and ongoing prospective trials will determine the long-term efficacy of this approach.Trial registration clinicaltrials.gov, NCT03716180, https://clinicaltrials.gov/ct2/show/NCT03716180 .

Temporal and spatial topography of cell proliferation in cancer.

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

Prognostic Utility of Breast Cancer Index to Stratify Distant Recurrence Risk in Invasive Lobular Carcinoma.

PURPOSE: The prognostic utility of Breast Cancer Index (BCI) for risk assessment of overall (0-10 years), early (0-5 years), and late (5-10 years) distant recurrence (DR) in hormone receptor-positive (HR+) invasive lobular carcinoma (ILC) was evaluated. EXPERIMENTAL DESIGN: BCI gene expression analysis was performed blinded to clinical outcome utilizing tumor specimens from patients with HR+ ILC from a multi-institutional cohort. The primary endpoint was time to DR. Kaplan-Meier analyses of overall, early, and late DR risk were performed, and statistical significance was evaluated by log-rank test and Cox proportional hazards regression. The prognostic contribution of BCI in addition to clinicopathologic factors was evaluated by likelihood ratio analysis. RESULTS: Analysis of 307 patients (99% ER+, 53% T1, 42% N+, 70% grade II) showed significant differences in DR over 10 years based on BCI risk categories. BCI low- and intermediate-risk patients demonstrated similar DR rates of 7.6% and 8.0%, respectively, compared with 27.0% for BCI high-risk patients. BCI was a significant independent prognostic factor for overall 10-year DR [HR = 4.09; 95% confidence interval (CI), 2.00-8.34; P = 0.0001] as well as for both early (HR = 8.19; 95% CI, 1.85-36.30; P = 0.0042) and late (HR = 3.04; 95% CI, 1.32-7.00; P = 0.0224) DR. In multivariate analysis, BCI remained the only statistically significant prognostic factor for DR (HR = 3.49; 95% CI, 1.28-9.54; P = 0.0150). CONCLUSIONS: BCI is an independent prognostic factor for ILC and significantly stratified patients for cumulative risk of 10-year, early, and late DR. BCI added prognostic value beyond clinicopathologic characteristics in this distinct subtype of breast cancer.

How Researchers, Clinicians and Patient Advocates Can Accelerate Lobular Breast Cancer Research.

Breast cancer research and therapies have significantly advanced in recent years. However, Invasive Lobular Carcinoma (ILC), the second most common histological type of breast cancer and the sixth most frequently diagnosed cancer of women, has not always benefited from critical analysis, missing opportunities to better understand this important subtype. Recent progress understanding the biological and behavioral differences of ILC demonstrates that it is a unique subtype of breast cancer which can respond differently to common therapies. These new insights have increased interest in researching lobular breast disease. Concurrently, the formation of motivated patient-led advocacy organizations working in partnership with basic, translational and clinical researchers creates new opportunities, including connecting a dispersed patient population to research, encouraging new research funding and connecting patient advocates to researchers to advance common goals. This commentary will explore the unprecedented opportunity to drive multidisciplinary, multicenter and international collaborative research into lobular breast cancer that builds on recent research progress. Collaborative research partnerships that include advocates can result in a better understanding of ILC, identify targeted therapies and refine standard of care therapies that are currently equally applied to all breast cancers, resulting in improvements in the diagnosis, treatment and follow-up care for patients with ILC.

Prognostic significance of residual nodal disease after neoadjuvant endocrine therapy for hormone receptor-positive breast cancer.

Axillary management after NET has not been well studied and the significance of residual axillary node disease after NET remains uncertain. We used the National Cancer Data Base to examine the prognostic significance of residual nodal disease after NET. From 2010-2016, 4,496 patients received NET for cT1-3N0-1M0 hormone receptor-positive, HER2-negative breast cancer. Among cN0 patients treated with NET, final node status was ypN0 in 65%, isolated tumor cells (ITCs) in 3%, ypN1mi in 6%, and ypN1 in 26%. In cN1 patients, nodal pathologic complete response was uncommon (10%), and residual nodal disease included ITCs in 1%, ypN1mi in 3%, and ypN1 in 86%. There were no differences in 5-year overall survival (OS) between patients with pathologic node-negative disease, ITCs, or micrometastases after NET. When compared to a matched cohort of upfront surgery patients, there were also no differences in 5-year OS between NET and upfront surgery patients for any residual nodal disease category. These findings suggest NET patient outcomes mirror those of upfront surgery patients and present an opportunity to consider de-escalation of axillary management strategies in NET patients.

Hormone Receptor-Positive/Human Epidermal Growth Receptor 2-Negative Metastatic Breast Cancer in Young Women: Emerging Data in the Era of Molecularly Targeted Agents.

Breast cancer is the most common malignancy in young women worldwide, accounting for an estimated 30% of new cancer diagnoses and 25% of cancer deaths. Approximately two thirds of young women with breast cancer have hormone receptor-positive (HR+)/human epidermal growth receptor 2-negative (HER2-) tumors. Numerous studies, primarily in early-stage breast cancer, have demonstrated that young age is an independent risk factor for more aggressive disease and worse outcomes. Although more limited data are available regarding outcomes in young patients with advanced disease, these age-related disparities suggest that breast cancer in premenopausal women has distinct clinicopathologic and molecular features that can impact treatment outcomes. Until recently, limited data were available on the intrinsic molecular subtypes and genetics of young patients with HR+/HER2- metastatic breast cancer (mBC). In this review, we explore insights into the clinical and pathologic features of HR+/HER2- mBC in younger women derived from recent clinical trials of the cyclin-dependent kinase 4/6 inhibitors palbociclib (PALOMA-3), ribociclib (MONALEESA-7), and abemaciclib (MONARCH 2) and the implications of these findings for clinical practice, guideline development, and future research. IMPLICATIONS FOR PRACTICE: This review provides clinicians with an overview of emerging data on the unique clinicopathologic and molecular features of hormone receptor-positive/human epidermal growth receptor 2-negative metastatic breast cancer (mBC) in premenopausal women, summarizes findings from the most recent clinical trials of endocrine-based treatment in this patient population, and explores the implications of these findings for clinical practice, guideline development, and future research. Improved understanding of the key factors influencing disease course and treatment response in premenopausal patients with mBC may lead to more timely incorporation of evidence-based treatment approaches, thereby improving patient care and outcomes.

Patterns of Axillary Management in Stages 2 and 3 Hormone Receptor-Positive Breast Cancer by Initial Treatment Approach.

BACKGROUND: Data regarding axillary management after neoadjuvant endocrine therapy (NET) are lacking. This study examined axillary management of hormone receptor-positive (HR+) patients based on initial treatment with NET, neoadjuvant chemotherapy (NAC), or upfront surgery. METHODS: Patients with stage 2 or 3 HR+/HER2- breast cancer treated between 2012 and 2015 were identified in the National Cancer Database. The study examined axillary surgery [sentinel lymph node biopsy (SLNB), SLNB followed by axillary lymph node dissection (ALND), or upfront ALND] by initial treatment stratified by cN0/N1 using pairwise comparisons and multivariable logistic regression. RESULTS: Of 92,204 eligible patients, 2138 (2.3%) received NET, 11,014 (12%) received NAC, and 79,052 (85.7%) received surgery. Among 60,998 cN0 patients, attempted SLNB was more likely for surgery patients (86.2%, 47,159/54,684) and NET patients (85.8%, 1342/1564) than for NAC patients (79.9%, 3793/4750) (both p < 0.001). Among 31,206 cN1 patients, attempted SLNB was more likely for the surgery patients (46.0%, 11,201/24,368) than for the NET patients (41.8%, 240/574; p = 0.05) or the NAC patients (39.8%, 2491/6264; p < 0.0001). The differences between surgery and NET did not persist in the adjusted analyses. Among both the cN0 patients (n = 13,856) and the cN1 patients (n = 8688) with pN1 disease shown by SLNB, the NET patients were treated with ALND less frequently than those receiving NAC or surgery (p < 0.0001 for all comparisons). In the multivariate analysis, for the patients with pN1 disease shown by SLNB, NET use was associated with increased odds of undergoing SLNB alone [cN0 patients: odds ratio (OR), 1.31, 95% confidence interval (CI), 1.04-1.64; cN1 patients: OR 1.45; 95% CI 1.00-2.10]. CONCLUSIONS: For stages 2 and 3 HR+/HER2- patients, SLNB use after NET was similar to that for upfront surgery. Among those with pN1 disease, the NET patients were less likely to undergo ALND. Additional outcomes data are needed to guide axillary management after NET.

Genomic Characterization of Primary Invasive Lobular Breast Cancer.

PURPOSE: Invasive lobular breast cancer (ILBC) is the second most common histologic subtype after invasive ductal breast cancer (IDBC). Despite clinical and pathologic differences, ILBC is still treated as IDBC. We aimed to identify genomic alterations in ILBC with potential clinical implications. METHODS: From an initial 630 ILBC primary tumors, we interrogated oncogenic substitutions and insertions and deletions of 360 cancer genes and genome-wide copy number aberrations in 413 and 170 ILBC samples, respectively, and correlated those findings with clinicopathologic and outcome features. RESULTS: Besides the high mutation frequency of CDH1 in 65% of tumors, alterations in one of the three key genes of the phosphatidylinositol 3-kinase pathway, PIK3CA, PTEN, and AKT1, were present in more than one-half of the cases. HER2 and HER3 were mutated in 5.1% and 3.6% of the tumors, with most of these mutations having a proven role in activating the human epidermal growth factor receptor/ERBB pathway. Mutations in FOXA1 and ESR1 copy number gains were detected in 9% and 25% of the samples. All these alterations were more frequent in ILBC than in IDBC. The histologic diversity of ILBC was associated with specific alterations, such as enrichment for HER2 mutations in the mixed, nonclassic, and ESR1 gains in the solid subtype. Survival analyses revealed that chromosome 1q and 11p gains showed independent prognostic value in ILBC and that HER2 and AKT1 mutations were associated with increased risk of early relapse. CONCLUSION: This study demonstrates that we can now begin to individualize the treatment of ILBC, with HER2, HER3, and AKT1 mutations representing high-prevalence therapeutic targets and FOXA1 mutations and ESR1 gains deserving urgent dedicated clinical investigation, especially in the context of endocrine treatment.

DNA methylation profiling reveals a predominant immune component in breast cancers.

Breast cancer is a molecularly, biologically and clinically heterogeneous group of disorders. Understanding this diversity is essential to improving diagnosis and optimizing treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease are still poorly understood. By means of DNA methylation profiling of 248 breast tissues, we have highlighted the existence of previously unrecognized breast cancer groups that go beyond the currently known 'expression subtypes'. Interestingly, we showed that DNA methylation profiling can reflect the cell type composition of the tumour microenvironment, and in particular a T lymphocyte infiltration of the tumours. Further, we highlighted a set of immune genes having high prognostic value in specific tumour categories. The immune component uncovered here by DNA methylation profiles provides a new perspective for the importance of the microenvironment in breast cancer, holding implications for better management of breast cancer patients.