Aranzazu Fernandez-Martinez

Purpose: To better characterize the therapeutic benefits of the addition of carboplatin to anthracycline- and taxane-based neoadjuvant chemotherapy (NACT) in early-stage triple-negative breast cancer (TNBC), we performed a pooled analysis of three randomized clinical trials: BrighTNess (ClinicalTrials.gov identifier: NCT02032277 ), Cancer and Leukemia Group B 40603 (ClinicalTrials.gov identifier: NCT00861705 ), and GeparSixto (ClinicalTrials.gov identifier: NCT01426880 ). Methods: We evaluated the impact of the addition of carboplatin to NACT on pathologic complete response (pCR) rates, event-free survival (EFS), and overall survival (OS) across a combined total of 1,084 patients. We additionally looked for associations of outcome by germline BRCA1 and BRCA2 (g BRCA ) mutation status, eight gene expression signatures, and molecular subtypes (PAM50, MD Anderson Cancer Center/Baylor College of Medicine [MDACC/BCM]). Results: In the pooled data set, the addition of carboplatin to NACT was associated with a higher pCR rate (odds ratio, 1.89 [95% CI, 1.41 to 2.55]; P < .001) and improved EFS (hazard ratio, 0.71 [95% CI, 0.54 to 0.93]; P = .01), but not OS. In patients with g BRCA mutations (n = 137), we found that the addition of carboplatin conveys a significant EFS benefit but had no significant association with pCR rate or OS. We found six immune gene expression signatures that were significantly associated with a higher pCR rate and four that were significantly associated with improved EFS and OS. In addition, we found that the PAM50 luminal and MDACC/BCM luminal androgen receptor molecular subtypes were significantly associated with lower pCR rates in the combined data set. Conclusion: Our findings support the recent addition of carboplatin to NACT within the standard-of-care treatment of stage II to III TNBC and emphasize the importance of the TNBC immune microenvironment to patient outcomes.

Patients with hormone receptor–positive/HER2-positive (HR+/HER2+) breast cancer represent a historically underrecognized subgroup demonstrating poor response to combined endocrine and HER2-targeted therapies. In this study, using single-cell transcriptomic and epigenomic sequencing of estrogen receptor–positive (ER+)/HER2+ models, we identified BRD8, an acetyl-lysine reader in the EP400 histone acetyltransferase complex, as a critical mediator of ER/HER2 signaling cross-talk. BRD8 expression rapidly increased following anti-HER2 treatment, whereas its depletion disrupted ER–HER2 interaction and enhanced drug sensitivity. Single-nucleus assay for transposase-accessible chromatin using sequencing revealed that chromatin regions opening after anti-HER2 treatment were enriched for ER, FOX, and ETS transcription factor motifs, coinciding with BRD8-dependent gene activation through EP400-mediated acetylated H2AZ deposition. BRD8 regulated ER-dependent and ER-independent growth pathways, and depletion of BRD8 abolished neratinib-induced ER activation and restored drug sensitivity in resistant cells. A three-gene BRD8 signature successfully predicted anti-HER2 therapy response in two human clinical trials. Together, these findings establish BRD8 as both a predictive biomarker for anti-HER2 response and a therapeutic target to overcome resistance in HR+/HER2+ breast cancer. Significance: Multiomic single-cell profiling of breast cancer identified BRD8 as a critical mediator of resistance to HER2-targeted therapies that enables ER-HER2 cross-talk and serves as a predictive biomarker for treatment response.

PURPOSE: For hormone receptor–positive/human epidermal growth factor receptor 2–negative (HR+/HER2–) metastatic breast cancer (MBC), first-line cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) + endocrine therapy (ET) is the standard of care. They are also used after progression on first-line aromatase inhibitors (AIs), but some patients may respond better to chemotherapy-based options. We examined tumor features associated with survival from GEICAM/2013-02 PEARL, a phase III trial of palbociclib + ET versus capecitabine in AI-resistant HR+/HER2– MBC. METHODS: For 158 and 155 patients from each arm, 878 previously published gene expression signatures were derived using RNA sequencing on pretreatment tumor specimens, both primary and metastatic. Multivariable Cox models for progression-free survival (PFS) and overall survival (OS) were constructed with 16 preselected signatures related to proliferation, loss of retinoblastoma, and immune infiltration, and via Elastic Net using all signatures. RESULTS: Significant PFS difference by PAM50 intrinsic subtype was observed with palbociclib + ET. Comparing treatment arms, luminal A subtype trended toward longer PFS with palbociclib + ET, and luminal B and nonluminal subtypes had significantly longer PFS with capecitabine. Three B-cell (B-lymphocyte)–associated signatures correlated with shorter OS with palbociclib + ET. The immune-activated Immune1 TCGA breast cancer signature had significant treatment arm interaction for OS. Elastic Net iteratively selected B-cell–associated signatures independently associated with shorter OS with palbociclib + ET. CONCLUSION: PAM50 intrinsic subtype predicted PFS differences between palbociclib + ET and capecitabine. Lower B-cell–associated gene expression predicted longer OS with palbociclib + ET versus capecitabine. These features may help identify HR+/HER2– tumors resistant to further ET-based treatment with CDK4/6i.

Importance: Biologic features may affect pathologic complete response (pCR) and event-free survival (EFS) after neoadjuvant chemotherapy plus ERBB2/HER2 blockade in ERBB2/HER2-positive early breast cancer (EBC).Objective: To define the quantitative association between pCR and EFS by intrinsic subtype and by other gene expression signatures in a pooled analysis of 3 phase 3 trials: CALGB 40601, NeoALTTO, and NSABP B-41.Design, Setting, and Participants: In this retrospective pooled analysis, 1289 patients with EBC received chemotherapy plus either trastuzumab, lapatinib, or the combination, with a combined median follow-up of 5.5 years. Gene expression profiling by RNA sequencing was obtained from 758 samples, and intrinsic subtypes and 618 gene expression signatures were calculated. Data analyses were performed from June 1, 2020, to January 1, 2023.Main Outcomes and Measures: The association of clinical variables and gene expression biomarkers with pCR and EFS were studied by logistic regression and Cox analyses.Results: In the pooled analysis, of 758 women, median age was 49 years, 12% were Asian, 6% Black, and 75% were White. Overall, pCR results were associated with EFS in the ERBB2-enriched (hazard ratio [HR], 0.45; 95% CI, 0.29-0.70; P < .001) and basal-like (HR, 0.19; 95% CI, 0.04-0.86; P = .03) subtypes but not in luminal A or B tumors. Dual trastuzumab plus lapatinib blockade over trastuzumab alone had a trend toward EFS benefit in the intention-to-treat population; however, in the ERBB2-enriched subtype there was a significant and independent EFS benefit of trastuzumab plus lapatinib vs trastuzumab alone (HR, 0.47; 95% CI, 0.27-0.83; P = .009). Overall, 275 of 618 gene expression signatures (44.5%) were significantly associated with pCR and 9 of 618 (1.5%) with EFS. The ERBB2/HER2 amplicon and multiple immune signatures were significantly associated with pCR. Luminal-related signatures were associated with lower pCR rates but better EFS, especially among patients with residual disease and independent of hormone receptor status. There was significant adjusted HR for pCR ranging from 0.45 to 0.81 (higher pCR) and 1.21-1.94 (lower pCR rate); significant adjusted HR for EFS ranged from 0.71 to 0.94.Conclusions and relevance: In patients with ERBB2/HER2-positive EBC, the association between pCR and EFS differed by tumor intrinsic subtype, and the benefit of dual ERBB2/HER2 blockade was limited to ERBB2-enriched tumors. Immune-activated signatures were concordantly associated with higher pCR rates and better EFS, whereas luminal signatures were associated with lower pCR rates.

In this study, we performed genomic analyses of cell cycle and tumor microenvironment changes during and after ribociclib and letrozole or chemotherapy in the CORALLEEN trial. 106 women with untreated PAM50-defined Luminal B early breast cancers were randomly assigned to receive neoadjuvant ribociclib and letrozole or standard-of-care chemotherapy. Ki67 immunohistochemistry, tumor-infiltrating lymphocytes quantification, and RNA sequencing were obtained from tissue biopsies pre-treatment, on day 14 of treatment, and tumor specimens from surgical resection. Results showed that at surgery, Ki67 and the PAM50 proliferation scores were lower after ribociclib compared to chemotherapy. However, consistent reactivation of tumor cell proliferation from day 14 to surgery was only observed in the ribociclib arm. In tumors with complete cell cycle arrest (CCCA) at surgery, PAM50 proliferation scores were lower in the ribociclib arm compared to chemotherapy (p < 0.001), whereas the opposite was observed with tumor cellularity (p = 0.002). Gene expression signatures (GES) associated with antigen-presenting cells (APCs) and innate immune system activity showed increased expression post-chemotherapy but decreased expression post-ribociclib. Interferon-associated GES had decreased expression with CCCA and increased expression with non-CCCA. Our findings suggest that while both treatment strategies decreased proliferation, the depth and the patterns over time differed by treatment arm. Immunologically, ribociclib was associated with downregulated GES associated with APCs and the innate immune system in Luminal B tumors, contrary to existing preclinical data. Further studies are needed to understand the effect of CDK4/6 inhibition on the tumor cells and microenvironment, an effect which may vary according to tumor subtypes.

PURPOSE: CALGB 40603 ( NCT00861705 ), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS: The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS: Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION: Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

PURPOSE: CALGB 40601 assessed whether dual versus single human epidermal growth factor receptor 2 (HER2) –targeting drugs added to neoadjuvant chemotherapy increased pathologic complete response (pCR). Here, we report relapse-free survival (RFS), overall survival (OS), and gene expression signatures that predict pCR and survival. PATIENTS AND METHODS: Three hundred five women with untreated stage II and III HER2-positive breast cancer were randomly assigned to receive weekly paclitaxel combined with trastuzumab plus lapatinib (THL), trastuzumab (TH), or lapatinib (TL). The primary end point was pCR, and secondary end points included RFS, OS, and gene expression analyses. mRNA sequencing was performed on 264 pretreatment samples. RESULTS: One hundred eighteen patients were randomly allocated to THL, 120 to TH, and 67 to TL. At more than 7 years of follow-up, THL had significantly better RFS and OS than did TH (RFS hazard ratio, 0.32; 95% CI, 0.14 to 0.71; P = .005; OS hazard ratio, 0.34; 95% CI, 0.12 to 0.94; P = .037), with no difference between TH and TL. Of 688 previously described gene expression signatures, significant associations were found in 215 with pCR, 45 with RFS, and only 22 with both pCR and RFS (3.2%). Specifically, eight immune signatures were significantly correlated with a higher pCR rate and better RFS. Among patients with residual disease, the immunoglobulin G signature was an independent, good prognostic factor, whereas the HER2-enriched signature, which was associated with a higher pCR rate, showed a significantly shorter RFS. CONCLUSION: In CALGB 40601, dual HER2-targeting resulted in significant RFS and OS benefits. Integration of intrinsic subtype and immune signatures allowed for the prediction of pCR and RFS, both overall and within the residual disease group. These approaches may provide means for rational escalation and de-escalation treatment strategies in HER2-positive breast cancer.

HER2-positive (HER2+) breast cancer (BC) comprises all the intrinsic molecular subtypes, with the HER2-enriched (HER2-E) usually being the most represented. Data coming from neoadjuvant trials of HER2+ BC treated with anti-HER2 containing regimens, with or without chemotherapy (CT), have shown that HER2-E tumors are more likely to achieve pathologic complete response (pCR) than non-HER2-E tumors. We decided to perform a meta-analysis combining all the available data in attempt to validate the ability of the HER2-E signature to predict pCR. A systematic literature search was performed to identify clinical studies exploring the correlation between BC subtypes and pCR after neoadjuvant therapy (NAT) with anti-HER2 containing regimens in patients affected by HER2+ early BC. Primary analysis compared the association of gene signatures with pCR. Secondary analyses compared the association of gene signatures with pCR within hormone receptor (HR) positive (+) or negative (-) BC. Odds Ratio (OR) and 95% confidence intervals (CI) for pCR were extracted from each trial. A random-effect model was applied. The Higgins’ I² was used to quantify heterogeneity. Sixteen studies (2,857 patients) were included; 4 investigated CT-free regimens. Various methods for assessing BC intrinsic subtypes were used across all trials. HER2-E subtype was significantly associated with pCR in all patients (OR: 3.32, 95% CI: 2.70-4.07, p < 0.001, I²=25%) and in HR + (OR: 3.40, 2.51-4.61, p < 0.001, I²=0%) and HR- (OR: 1.97, 1.10-3.54, p = 0.02, I²=46%) disease. In CT-free studies, HER2-E subtype was significantly associated with pCR in all patients (OR: 4.43, 2.34-8.38, p < 0.001, I²=0%) and in HR+ disease (OR: 4.79, 2.23-10.29, p < 0.001, I²=0%), but not within HR- tumors (OR: 2.18, 0.66-7.26, p = 0.20). HER2-E subtype identifies patients with a higher likelihood of achieving a pCR following anti-HER2-based NAT, with or without CT. In the latter case, albeit limited by small casuistry, the association seems stronger in HR+ tumors. This suggests that strategies to escalate or de-escalate systemic therapy in HER2+ tumors would benefit from incorporating intrinsic subtypes. The authors. Has not received any funding. F. Schettini: Travel / Accommodation / Expenses: Pfizer and Celgene. T. Pascual: Advisory / Consultancy: Roche. P.F. Conte: Honoraria (self): BMS, Roche, EliLilly, Novartis, AstraZeneca; Advisory / Consultancy: Novartis, EliLilly, AstraZeneca, Tesaro; Research grant / Funding (self): Novartis, Roche, BMS, Merck-KGa; Research grant / Funding (self): Italian Ministry of Health, Veneto Secretary of Health, University of Padua. S. De Placido: Honoraria (self): Roche, Pfizer, AstraZeneca, Novartis, Celgene, Lilly and Eisai. L. Carey: Research grant / Funding (institution): Genentech / Roche, Novartis, Seattle Genetics, G1 Therapeutics, Immunomedics, Innocrin. C.M. Perou: Shareholder / Stockholder / Stock options: BioClassifier LLC; Advisory / Consultancy: BioClassifier LLC; Licensing / Royalties: Breast PAM50. A. Prat: Full / Part-time employment, An immediate family member employed: Novartis; Honoraria (self): Pfizer, Novartis, Roche, MSD Oncology, Lilly and Daiichi Sankyo; Advisory / Consultancy: NanoString Technologies, Amgen, Roche, Novartis, Pfizer and Bristol-Myers Squibb; Research grant / Funding (self): Roche, Novartis; Travel / Accommodation / Expenses: Daiichi Sankyo. All other authors have declared no conflicts of interest.