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SummaryBackgroundWe aimed to report on long-term outcomes of patients with small, node-negative, HER2-positive breast cancer treated with adjuvant paclitaxel and trastuzumab and to establish potential biomarkers to predict prognosis. MethodsIn this open-label, single-arm, phase 2 study, patients aged 18 years or older, with small (≤3 cm), node-negative, HER2-positive breast cancer, and an Eastern Cooperative Oncology Group performance status of 0–1, were recruited from 16 institutions in 13 cities in the USA. Eligible patients were given intravenous paclitaxel (80 mg/m 2) with intravenous trastuzumab (loading dose of 4 mg/kg, subsequent doses 2 mg/kg) weekly for 12 weeks, followed by trastuzumab (weekly at 2 mg/kg or once every 3 weeks at 6 mg/kg) for 40 weeks to complete a full year of trastuzumab. The primary endpoint was 3-year invasive disease-free survival. Here, we report 10-year survival outcomes, assessed in all participants who received protocol-defined treatment, with exploratory analyses using the HER2DX genomic tool. This study is registered on ClinicalTrials.gov, NCT00542451, and is closed to accrual. FindingsBetween Oct 29, 2007, and Sept 3, 2010, 410 patients were enrolled and 406 were given adjuvant paclitaxel and trastuzumab and included in the analysis. Mean age at enrolment was 55 years (SD 10·5), 405 (99·8%) of 406 patients were female and one (0·2%) was male, 350 (86·2%) were White, 28 (6·9%) were Black or African American, and 272 (67·0%) had hormone receptor-positive disease. After a median follow-up of 10·8 years (IQR 7·1–11·4), among 406 patients included in the analysis population, we observed 31 invasive disease-free survival events, of which six (19·4%) were locoregional ipsilateral recurrences, nine (29·0%) were new contralateral breast cancers, six (19·4%) were distant recurrences, and ten (32·3%) were all-cause deaths. 10-year invasive disease-free survival was 91·3% (95% CI 88·3–94·4), 10-year recurrence-free interval was 96·3% (95% CI 94·3–98·3), 10-year overall survival was 94·3% (95% CI 91·8–96·8), and 10-year breast cancer-specific survival was 98·8% (95% CI 97·6–100). HER2DX risk score as a continuous variable was significantly associated with invasive disease-free survival (hazard ratio [HR] per 10-unit increment 1·24 [95% CI 1·00–1·52]; p=0·047) and recurrence-free interval (1·45 [1·09–1·93]; p=0·011). InterpretationAdjuvant paclitaxel and trastuzumab is a reasonable treatment standard for patients with small, node-negative, HER2-positive breast cancer. The HER2DX genomic tool might help to refine the prognosis for this population. FundingGenentech.

In the phase II NIMBUS trial, patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) and high tumor mutational burden (TMB ≥ 9 mut/Mb) received nivolumab (3 mg/kg biweekly) and low-dose ipilimumab (1 mg/kg every 6 weeks) for 2 years or until progression. The primary endpoint was objective response rate (ORR) per RECIST 1.1 criteria. Among 30 patients enrolled, the median TMB was 10.9 mut/Mb (range: 9–110) and the confirmed objective response rate was 20%. Secondary endpoints included progression-free survival, overall survival, clinical benefit rate, and safety and tolerability, including immune-related adverse events (irAEs). A prespecified correlative outcome was to evaluate the ORR in patients with a TMB ≥ 14 mut/Mb. Patients with TMB ≥ 14 mut/Mb (n = 6) experienced higher response rates (60% vs 12%; p = 0.041) and showed a trend towards improved progression-free survival and overall survival compared to patients with TMB < 14 mut/Mb. Exploratory genomic analyses suggested that ESR1 and PTEN mutations may be associated with poor response, while clinical benefit was associated with a decrease or no change in tumor fraction by serial circulating tumor DNA during treatment. Stool microbiome analysis revealed that baseline blood TMB, PD-L1 positivity, and immune-related diarrhea are associated with distinct taxonomic profiles. In summary, some patients with hypermutated HER2-negative MBC experience extended clinical benefit with a dual immunotherapy regimen; a higher TMB, and additional genomic and microbiome biomarkers may optimize patient selection for therapy with nivolumab plus low-dose ipilimumab. (Funded by Bristol Myers Squibb; ClinicalTrials.gov identifier, NCT03789110). The current use of immune checkpoint inhibitors without chemotherapy on patients with metastatic breast cancer (MBC) has not proven useful. Here this group reports a phase 2 NIMBUS trial evaluating the efficacy/safety of nivolumab + low dose ipilimumab in 30 patients with hypermutated HER2-negative MBC.

The efficacy of immune checkpoint inhibitors combined with chemotherapy varies among breast cancer subtypes and is particularly less effective in hormone receptor-positive (HR + ) breast cancers. Here, we analyze pre-, on-, and post-treatment biopsies from 20 female patients with stage II-III HR+ breast cancer who participated in a clinical trial of neoadjuvant chemo-immunotherapy with nab-paclitaxel and pembrolizumab. Through single-nucleus RNA and ATAC sequencing of these tumor biopsies, we identified gene expression metaprograms (MPs) associated with differential therapy responses. Here we show that favorable responders exhibit increased activity in pathways related to tumor state transition, T cell effector functions, and pro-inflammatory macrophage states. Unfavorable responders demonstrate increased tumor estrogen signaling and immunosuppressive tumor-immune interactions. In this work, we highlight the interplay between tumor and microenvironmental cells in treatment naïve and exposed HR+ breast cancers and reveal that pivotal shifts in tumor cell, macrophage, and T cell states may mediate response to chemo-immunotherapy. While anti-PD-1 therapy can be effective in patients with breast cancer, response varies and is often limited by the development of resistance. Here, the authors investigate molecular determinant of response and resistance using multiomic single nucleus sequencing of longitudinal biopsies taken from breast cancer patients on a phase II clinical trial investigating neoadjuvant chemotherapy and pembrolizumab (anti-PD-1).

Background Inflammatory breast cancer (IBC) is an aggressive and highly angiogenic disease. Eribulin is a microtubule inhibitor with anti-angiogenic properties. Methods In a phase II trial, we examined the efficacy of an eribulin-containing neoadjuvant regimen (eribulin- > doxorubicin plus cyclophosphamide (AC) or AC- > eribulin) for patients with newly diagnosed HER2-negative IBC. Pathologic complete response (pCR: ypT0/Tis ypN0) was the primary endpoint; residual cancer burden (RCB) categories were also recorded. Five patients from each cohort underwent dynamic contrast enhanced MRI (DCE-MRI) and diffusion weighted MRI. All patients had research breast biopsies for transcriptomic, differential gene expression, and cell subset analysis at baseline and one week after the first dose of therapy. Results 19/22 (86.4%) patients had hormone receptor-positive disease. All patients were able to undergo planned curative-intent surgery and radiation. One patient had a pCR, and long-term outcomes were encouraging: after median follow up of 76 months, 3 patients experienced disease recurrence. Five-year event-free survival (EFS) was 85.6%. The regimen was tolerated with expected side effects—the most common grade 1 or 2 AEs were fatigue (95.5%), nausea (68.2%), and alopecia (63.6%). Seven out of 22 (31.8%) patients experienced any grade 3 or 4 AE, with neutropenia (22.7%) being the most common. DCE-MRI showed decreased tumor vascularization after 1 week of treatment versus baseline. Transcriptomic analysis using quantification of synthesized dsDNA libraries and tumor microenvironment analysis of paired baseline and on-treatment samples showed residual cancer burden (RCB)-III tumors were more likely to have genes associated with adipogenesis/fatty acid metabolism and cells associated with immunosuppression. Conclusions Despite the low pCR rate, all patients were able to undergo curative surgery, and long-term outcomes were encouraging with 5-year EFS 85.6%. Decreases in tumor vascularization with treatment were detected by DCE-MRI parameters irrespective of initial chemotherapy received. Adipogenesis/fatty acid metabolism and cells associated with immunosuppression are potential mechanisms of resistance and targets for future investigation in this unique patient population. Trial registration ClinicalTrials.gov (NCT02623972)(Registration date: 12/02/15).

BackgroundPreclinical evidence suggests that cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors enhance antitumor immunity. We conducted a phase I trial of ribociclib (CDK4/6 inhibitor) plus spartalizumab (PD-1 inhibitor) in patients with hormone receptor (HR)-positive/HER2-negative metastatic breast cancer (MBC) or advanced ovarian cancer (AOC). The combination was also evaluated with fulvestrant in MBC.MethodsIn Cohort A, ribociclib was administered on Days 1–21 (28-day cycle) starting at 400 mg, and spartalizumab at 400 mg on Day 1. Dose escalation was followed by expansion in AOC. Fulvestrant was added (Cohort B) with a safety run-in followed by expansion in MBC. Primary objectives were to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D), and safety and tolerability of the combinations.Results33 patients enrolled (n=18, Cohort A; n=15, Cohort B). The RP2D of ribociclib in both cohorts was 600 mg. Treatment-related adverse events in >20% of patients in either cohort were neutropenia, fatigue, anemia, thrombocytopenia, hypertransaminasemia, maculopapular rash, fatigue, and nausea. Hypertransaminasemia occurred in 66.7% (AST) and 46.7% (ALT) of patients in Cohort B, including 46.7% and 40.0%, respectively, of grade 3 or 4 events. Two confirmed partial responses were observed (13.3%) in Cohort B, in patients with low baseline serum thymidine kinase activity, coupled with an increase on-treatment. Peripheral blood flow cytometry across patients demonstrated on-target drug binding with increases in PD-1 occupancy and activated CD8+ T cells during treatment, irrespective of response. PD-L1-positivity, tumor-infiltrating lymphocytes, or tumor mutational burden did not correlate with progression-free survival (PFS). Several copy-number variations detected with next-generation sequencing correlated with PFS.ConclusionsRibociclib with spartalizumab and fulvestrant showed limited efficacy and elevated hepatotoxicity, precluding further development. Correlative analyses revealed treatment-induced immunological effects, and genomic alterations associated with PFS.

The limited added benefit of immune checkpoint inhibitors in breast cancer indicates the pressing need to identify biomarkers of response to minimize risk and maximize benefit. We used single cell RNA sequencing and T cell receptor (TCR) sequencing of peripheral blood mononuclear cells (for 28 samples comprising 79,284 cells) to monitor the peripheral immune dynamic of an exploratory cohort of hormone receptor positive breast cancer patients treated with neoadjuvant nab-paclitaxel+pembrolizumab with the ultimate goal of identifying potential peripheral blood predictive biomarkers. In responsive patients, Granzyme B positive ( GZMB +) cytotoxic CD8 T cells expanded post-nab-paclitaxel+pembrolizumab, accompanied by rapid changes in TCR clones. In contrast, non-responders’ peripheral T cells may experience terminal exhaustion and are not significantly altered by treatment. In addition, B cell and monocyte-specific interferon response signatures were also associated with response. Our data suggests that peripheral immunological signatures may represent a facile way to monitor dynamic antitumor immune response.

HER2-low expression is associated with hormone receptor (HR) expression in HR-positive breast cancer. We aimed to evaluate its association with androgen receptor (AR) among 196 patients with metastatic triple-negative breast cancer (mTNBC). Central determination of AR showed significant enrichment in HER2-low compared with HER2-0 mTNBC (mean: 33.7% vs. 21.4%, p  = 0.038), whereas no significant immunological differences were observed. HER2-low/AR-positive patients trended towards longer overall survival, highlighting the potential relevance of these biomarkers.

BackgroundFew patients with hormone receptor positive (HR+) metastatic breast cancer (MBC) experience therapeutic benefit from inhibitors against programmed cell death 1 (PD-1) or its ligand (PD-L1). Using whole transcriptome and whole exome sequencing of tumor samples from a randomized phase 2 trial of eribulin +/- pembrolizumab for patients with HR+/HER2- MBC, we showed that patients who derived clinical benefit had increased antigen presentation machinery and IFNg-response genes compared to patients that did not achieve clinical benefit, which did not differ between treatment arms. This data suggests that baseline subsets of immune cells may predict primary response in patients with HR+ MBC.MethodsWe utilized single cell, multiplex cyclic immunofluorescence (CyCIF) on pretreatment, formalin-fixed paraffin-embedded tumor samples (n=29; table 1) from the trial of eribulin +/- pembrolizumab in HR+/HER- MBC to identify tumor cells, fibroblasts, and immune cells and to also characterize their states. We calculated cell type frequencies and associated them with measures of clinical outcome such as progression free survival (PFS) and overall survival (OS). Cox proportional hazard models were used to analyze PFS and OS in SAS 9.4.ResultsSpearman correlation between cell type frequencies across all samples revealed that T cells (Thelper, Treg, CD8T), B cells, and macrophages (CD163+, CD68+/CD163+ cells), were strongly correlated, indicating that these cell types are, on average, more abundant in samples with high immune infiltration. Survival analysis using cell type numbers as continuous variables showed higher baseline levels of immune cells in patients who experienced longer PFS and OS. Specifically, T cells were associated with better clinical outcome as those characterized as CD8+ or CD8+PD1- were associated with longer PFS, TregPD1- T cells were associated with longer OS and ThelperPD1- frequency was associated with longer PFS and OS independent of treatment arm (table 2). Macrophages were generally associated with worse clinical outcome. Macrophage subset CD68+CD163-PDL1+DPB1- was associated with shorter PFS and the CD68+CD163+PDL1-DPB1+ and CD68-CD163+PDL1-DPB1- subsets were associated with shorter OS, whereas the CD68-CD163+PDL1+DPB1+ population was marginally associated with longer PFS (table 3).ConclusionsOur work confirms that infiltration of T cells into metastatic HR+ breast cancers is a key feature associated with those who benefit from either chemotherapy or chemotherapy plus immunotherapy. The analysis also suggests that the presence of specific macrophage subsets is associated with worse clinical outcomes (figure 1). Deep phenotyping of the myeloid compartment on serial sections by CyCIF is ongoing.AcknowledgementsMerck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA provided financial support for this trial, and Eisai provided the drug eribulin for the trial. A Merck Oncology Translational Studies Program grant and a Dana-Farber Cancer Institute Medical Oncology grant funded part of the CyCIF analysis. Additional work was supported by the Susan G. Komen Foundation (CCR18547597), NCI Cancer Systems Biology Center of Excellence Grant (U54-CA225088), Terri Brodeur Breast Cancer Foundation, The Harvard Ludwig Center, NIH DF/HCC SPORE in Breast Cancer (P50 CA168504) and NIH NCI R01/R37 CA269499.Trial RegistrationClinicalTrials.gov Registration: NCT03051659.Ethics ApprovalThis study was approved by DFCI institution’s Ethics Board; approval number 16–577.Abstract 896 Table 1Pretreatment formalin-fixed paraffin-embedded tumor samples were collected retrospectively and prospectively as archival primary, metastatic or baseline prior to initiating protocol therapy.Abstract 896 Table 2T cell association with PFS and OS. n.s., not significant; Treg, Tregulatory cell.Abstract 896 Table 3Macrophage association with PFS and OS. n.s., not significant.Abstract 896 Figure 1Kaplan-Meier curves of PFS stratified by biomarkers, median values as cutoff points

Patients with hormone receptor-positive (HR + )/HER2- breast cancer may benefit from neoadjuvant immune checkpoint inhibitor (ICI) plus chemotherapy. The effect of chemotherapy or ICI run-in before combination therapy in this population is unexplored. In this randomized pilot trial, patients with HR + /HER2- breast cancer received two weeks of neoadjuvant nab-paclitaxel or pembrolizumab, with baseline and post-run-in tumor biopsy, followed by combined nab-paclitaxel/pembrolizumab. The primary endpoint was PD-L1 expression change between biopsies. Tumor whole exome/RNA sequencing were performed. Of 29 patients, 72% were node-positive. Residual cancer burden (RCB) 0-1 rate was 28% (inclusive of patients receiving additional neoadjuvant adriamycin/cyclophosphamide). No significant change in PD-L1 expression occurred following nab-paclitaxel or pembrolizumab run-in, thus the primary endpoint was not met. Other secondary outcome measures included overall response rate of 80% to the neoadjuvant regimen, and 3-year event-free survival of 86% (95% CI 69-100%); there were no unexpected safety signals. In exploratory biomarker analyses, higher baseline PD-L1 expression and inflammatory gene signatures were associated with favorable response (RCB 0-1); higher expression of estrogen response genes, with unfavorable response (RCB 2-3). Clinical Trial Number: NCT02999477 Despite often being poorly immunogenic, some subsets of patients with hormone receptor-positive, HER2-negative (HR + /HER2-) breast cancer benefit from immunotherapy. Here, the authors present a randomised pilot clinical trial comparing a neoadjuvant run-in of either nab-paclitaxel or pembrolizumab (anti-PD-1) monotherapy, followed by the combination, in patients with stage II-III HR + /HER2- breast cancer.

The intestinal microbiome shapes immune responses and is associated with patient outcomes in cancer following immunotherapy. We evaluated differences between the intestinal microbiome profiles of patients with early-stage invasive breast cancer (BC) and ductal carcinoma in situ (DCIS) by subtype using whole genome metagenomic sequencing. There were no significant differences in microbiome composition between DCIS and invasive BC as measured by alpha diversity (p = 0.20, ANOVA) or beta diversity (p = 0.52, PERMANOVA). Within invasive BC, patients with hormone receptor-positive (HR + )/HER2 + BC differed significantly in beta diversity relative to other subtypes (p < 0.05), with differences in six species (q < 0.25). Bacteroides ovatus was significantly more abundant in patients with stage III BC vs. stage I (p = 0.0003). Functional pathway analysis using HUMAnN3 revealed stage-specific enrichment of amino acid biosynthesis and nucleotide-related pathways. Altogether, these findings highlight potential microbial signatures associated with BC subtype and stage.