FGFR4 regulates tumor subtype differentiation in luminal breast cancer and metastatic disease

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4- induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification

A multivariable prognostic score to guide systemic therapy in early-stage HER2-positive breast cancer: a retrospective study with an external evaluation

Background: In early-stage HER2-positive breast cancer, escalation or de-escalation of systemic therapy is a controversial topic. As an aid to treatment decisions, we aimed to develop a prognostic assay that integrates multiple data types for predicting survival outcome in patients with newly diagnosed HER2-positive breast cancer. Methods: We derived a combined prognostic model using retrospective clinical–pathological data on stromal tumour-infiltrating lymphocytes, PAM50 subtypes, and expression of 55 genes obtained from patients who participated in the Short-HER phase 3 trial. The trial enrolled patients with newly diagnosed, node-positive, HER2-positive breast cancer or, if node negative, with at least one risk factor (ie, tumour size >2 cm, histological grade 3, lymphovascular invasion, Ki67 >20%, age ≤35 years, or hormone receptor negativity), and randomly assigned them to adjuvant anthracycline plus taxane-based combinations with either 9 weeks or 1 year of trastuzumab. Trastuzumab was administered intravenously every 3 weeks (8 mg/kg loading dose at first cycle, and 6 mg/kg thereafter) for 18 doses or weekly (4 mg/kg loading dose in the first week, and 2 mg/kg thereafter) for 9 weeks, starting concomitantly with the first taxane dose. Median follow-up was 91·4 months (IQR 75·1–105·6). The primary objective of our study was to derive and evaluate a combined prognostic score associated with distant metastasis-free survival (the time between randomisation and distant recurrence or death before recurrence), an exploratory endpoint in Short-HER. Patient samples in the training dataset were split into a training set (n=290) and a testing set (n=145), balancing for event and treatment group. The training set was further stratified into 100 iterations of Monte-Carlo cross validation (MCCV). Cox proportional hazard models were fit to MCCV training samples using Elastic-Net. A maximum of 92 features were assessed. The final prognostic model was evaluated in an independent combined dataset of 267 patients with early-stage HER2-positive breast cancer treated with different neoadjuvant and adjuvant anti-HER2-based combinations and from four other studies (PAMELA, CHER-LOB, Hospital Clinic, and Padova) with disease-free survival outcome data. Findings: From Short-HER, data from 435 (35%) of 1254 patients for tumour size (T1 vs rest), nodal status (N0 vs rest), number of tumour-infiltrating lymphocytes (continuous variable), subtype (HER2-enriched and basal-like vs rest), and 13 genes composed the final model (named HER2DX). HER2DX was significantly associated with distant metastasis-free survival as a continuous variable (p<0·0001). HER2DX median score for quartiles 1–2 was identified as the cutoff to identify low-risk patients; and the score that distinguished quartile 3 from quartile 4 was the cutoff to distinguish medium-risk and high-risk populations. The 5-year distant metastasis-free survival of the low-risk, medium-risk, and high-risk populations were 98·1% (95% CI 96·3–99·9), 88·9% (83·2–95·0), and 73·9% (66·0–82·7), respectively (low-risk vs high-risk hazard ratio [HR] 0·04, 95% CI 0·0–0·1, p<0·0001). In the evaluation cohort, HER2DX was significantly associated with disease-free survival as a continuous variable (HR 2·77, 95% CI 1·4–5·6, p=0·0040) and as group categories (low-risk vs high-risk HR 0·27, 0·1–0·7, p=0·005). 5-year disease-free survival in the HER2DX low-risk group was 93·5% (89·0–98·3%) and in the high-risk group was 81·1% (71·5–92·1). Interpretation: The HER2DX combined prognostic score identifies patients with early-stage, HER2-positive breast cancer who might be candidates for escalated or de-escalated systemic treatment. Future clinical validation of HER2DX seems warranted to establish its use in different scenarios, especially in the neoadjuvant setting. Funding: Instituto Salud Carlos III, Save the Mama, Pas a Pas, Fundación Científica, Asociación Española Contra el Cáncer, Fundación SEOM, National Institutes of Health, Agenzia Italiana del Farmaco, International Agency for Research on Cancer, and the Veneto Institute of Oncology, and Italian Association for Cancer Research

Oestrogen receptor activity in hormone-dependent breast cancer during chemotherapy: ER activity in pre-menopausal HR+ breast cancer during CT

Background: Chemotherapy efficacy in early-stage hormone receptor-positive (HR+) breast cancer (BC) according to menopausal status needs a biological explanation. Methods: We compared early-stage HR+ BC biological features before and after (neo)adjuvant chemotherapy or endocrine therapy (ET), and assessed oestrogen receptor (ER) pathway activity in both pre- and post-menopausal patients. The nCounter platform was used to detect gene expression levels. Findings: In 106 post-menopausal patients with HR+/HER2-negative BC randomized to neoadjuvant chemotherapy or ET (letrozole+ribociclib), a total of 19 oestrogen-regulated genes, including progesterone receptor (PGR), were found downregulated in the ET-based arm-only. We confirmed this finding in an independent dataset of 20 letrozole-treated post-menopausal patients and found, conversely, an up-regulation of the same signature in HR+/HER2-negative MCF7 cell line treated with estradiol. PGR was found down-regulated by 2 weeks of ET+anti-HER2 therapy in pre-/post-menopausal patients with HR+/HER2-positive (HER2+) BC, while anti-HER2 therapy alone increased PGR expression in HR-negative/HER2+ BC. In 88 pre- and post-menopausal patients with newly diagnosed HR+/HER2-negative BC treated with chemotherapy, the 19 oestrogen-regulated genes were found significantly downregulated only in pre-menopausal patients. In progesterone receptor (PR)+/HER2-negative BC treated with neoadjuvant chemotherapy (n=40), tumours became PR-negative in 69.2% of pre-menopausal patients and 14.8% of post-menopausal patients (p=0.001). Finally, a mean decrease in PGR levels was only observed in pre-menopausal patients undergoing anti-HER2-based multi-agent chemotherapy. Interpretation: Chemotherapy reduces the expression of ER-regulated genes in pre-menopausal women suffering from hormone-dependent BC by supressing ovarian function. Further studies should test the value of chemotherapy in this patient population when ovarian function is suppressed by other methods. Funding: Instituto de Salud Carlos III, Breast Cancer Now, the Breast Cancer Research Foundation, the American Association for Cancer Research, Fundació La Marató TV3, the European Union's Horizon 2020 Research and Innovation Programme, Pas a Pas, Save the Mama, Fundación Científica Asociación Española Contra el Cáncer, PhD4MDgrant of “Departament de Salut”, exp SLT008/18/00122, Fundación SEOM and ESMO. Any views, opinions, findings, conclusions, or recommendations expressed in this material are those solely of the author(s

HER2-Enriched Subtype and ERBB2 Expression in HER2-Positive Breast Cancer Treated with Dual HER2 Blockade

Background: Identification of HER2-positive breast cancers with high anti-HER2 sensitivity could help de-escalate chemotherapy. Here, we tested a clinically applicable RNA-based assay that combines ERBB2 and the HER2-enriched (HER2-E) intrinsic subtype in HER2-positive disease treated with dual HER2-blockade without chemotherapy. Methods: A research-based PAM50 assay was applied in 422 HER2-positive tumors from five II-III clinical trials (SOLTI-PAMELA, TBCRC023, TBCRC006, PER-ELISA, EGF104090). In SOLTI-PAMELA, TBCRC023, TBCRC006, and PER-ELISA, all patients had early disease and were treated with neoadjuvant lapatinib or pertuzumab plus trastuzumab for 12-24 weeks. Primary outcome was pathological complete response (pCR). In EGF104900, 296 women with advanced disease were randomized to receive either lapatinib alone or lapatinib plus trastuzumab. Progression-free survival (PFS), overall response rate (ORR), and overall survival (OS) were evaluated. Results: A total of 305 patients with early and 117 patients with advanced HER2-positive disease were analyzed. In early disease, HER2-E represented 83.8% and 44.7% of ERBB2-high and ERBB2-low tumors, respectively. Following lapatinib and trastuzumab, the HER2-E and ERBB2 (HER2-E/ERBB2)-high group showed a higher pCR rate compared to the rest (44.5%, 95% confidence interval [CI] = 35.4% to 53.9% vs 11.6%, 95% CI = 6.9% to 18.0%; adjusted odds ratio [OR] = 6.05, 95% CI = 3.10 to 11.80, P <. 001). Similar findings were observed with neoadjuvant trastuzumab and pertuzumab (pCR rate of 66.7% in HER2-E/ERBB2-high, 95% CI = 22.3% to 95.7% vs 14.7% in others, 95% CI = 4.9% to 31.1%; adjusted OR = 11.60, 95% CI = 1.66 to 81.10, P =. 01). In the advanced setting, the HER2-E/ERBB2-high group was independently associated with longer PFS (hazard ratio [HR] = 0.52, 95% CI = 0.35 to 0.79, P <. 001); higher ORR (16.3%, 95% CI = 8.9% to 26.2% vs 3.7%, 95% CI = 0.8% to 10.3%, P =. 02); and longer OS (HR = 0.66, 95% CI = 0.44 to 0.97, P =. 01). Conclusions: Combining HER2-E subtype and ERBB2 mRNA into a single assay identifies tumors with high responsiveness to HER2-targeted therapy. This biomarker could help de-escalate chemotherapy in approximately 40% of patients with HER2-positive 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 &lt;.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 &lt;.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

HER2-enriched subtype and pathological complete response in HER2-positive breast cancer: A systematic review and meta-analysis

Background: HER2-positive (HER2+) breast cancer (BC) comprises all the four PAM50 molecular subtypes. Among these, the HER2-Enriched (HER2-E) appear to be associated with higher pathological complete response (pCR) rates following anti-HER2-based regimens. Here, we present a meta-analysis to validate the association of the HER2-E subtype with pCR following anti-HER2-based neoadjuvant treatments with or without chemotherapy (CT). Methods: A systematic literature search was performed in February 2019. The primary objective was to compare the association between HER2-E subtype (versus others) and pCR. Selected secondary objectives were to compare the association between 1) HER2-E subtype and pCR in CT-free studies, 2) HER2-E subtype within hormone receptor (HR)-negative and HR+ disease and 3) HR-negative disease (versus HR+) and pCR in all patients and within HER2-E subtype. A random-effect model was applied. The Higgins’ I2 was used to quantify heterogeneity. Results: Sixteen studies were included, 5 of which tested CT-free regimens. HER2-E subtype was significantly associated with pCR in all patients (odds ratio [OR] = 3.50, p < 0.001, I2 = 33%), in HR+ (OR = 3.61, p < 0.001, I2 = 1%) and HR-negative tumors (OR = 2.28, p = 0.01, I2 = 47%). In CT-free studies, HER2-E subtype was associated with pCR in all patients (OR = 5.52, p < 0.001, I2 = 0%) and in HR + disease (OR = 4.08, p = 0.001, I2 = 0%). HR-negative status was significantly associated with pCR compared to HR + status in all patients (OR = 2.41, p < 0.001, I2 = 30%) and within the HER2-E subtype (OR = 1.76, p < 0.001, I2 = 0%). Conclusions: The HER2-E biomarker identifies patients with a higher likelihood of achieving a pCR following neoadjuvant anti-HER2-based therapy beyond HR status and CT use. Future trial designs to escalate or de-escalate systemic therapy in HER2+ disease should consider this genomic biomarker

Assessment of a Genomic Assay in Patients with ERBB2 -Positive Breast Cancer Following Neoadjuvant Trastuzumab-Based Chemotherapy with or Without Pertuzumab

Importance: Biomarkers to guide the use of pertuzumab in the treatment of early-stage ERBB2 (formerly HER2)-positive breast cancer beyond simple ERBB2 status are needed. Objective: To determine if use of the HER2DX genomic assay (Reveal Genomics) in pretreatment baseline tissue samples of patients with ERBB2-positive breast cancer is associated with response to neoadjuvant trastuzumab-based chemotherapy with or without pertuzumab. Design, Setting, and Participants: This is a retrospective diagnostic/prognostic analysis of a multicenter academic observational study in Spain performed during 2018 to 2022 (GOM-HGUGM-2018-05). In addition, a combined analysis with 2 previously reported trials of neoadjuvant cohorts with results from the assay (DAPHNe and I-SPY2) was performed. All patients had stage I to III ERBB2-positive breast cancer, signed informed consent, and had available formalin-fixed paraffin-embedded tumor specimens obtained prior to starting therapy. Exposures: Patients received intravenous trastuzumab, 8 mg/kg, loading dose, followed by 6 mg/kg every 3 weeks in combination with intravenous docetaxel, 75 mg/m2, every 3 weeks and intravenous carboplatin area under the curve of 6 every 3 weeks for 6 cycles, or this regimen plus intravenous pertuzumab, 840 mg, loading dose, followed by an intravenous 420-mg dose every 3 weeks for 6 cycles. Main Outcome and Measures: Association of baseline assay-reported pathologic complete response (pCR) score with pCR in the breast and axilla, as well as association of baseline assay-reported pCR score with response to pertuzumab. Results: The assay was evaluated in 155 patients with ERBB2-positive breast cancer (mean [range] age, 50.3 [26-78] years). Clinical T1 to T2 and node-positive disease was present in 113 (72.9%) and 99 (63.9%) patients, respectively, and 105 (67.7%) tumors were hormone receptor positive. The overall pCR rate was 57.4% (95% CI, 49.2%-65.2%). The proportion of patients in the assay-reported pCR-low, pCR-medium, and pCR-high groups was 53 (34.2%), 54 (34.8%), and 48 (31.0%), respectively. In the multivariable analysis, the assay-reported pCR score (as a continuous variable from 0-100) showed a statistically significant association with pCR (odds ratio [OR] per 10-unit increase, 1.43; 95% CI, 1.22-1.70; P &lt;.001). The pCR rates in the assay-reported pCR-high and pCR-low groups were 75.0% and 28.3%, respectively (OR, 7.85; 95% CI, 2.67-24.91; P &lt;.001). In the combined analysis (n = 282), an increase in pCR rate due to pertuzumab was found in the assay-reported pCR-high tumors (OR, 5.36; 95% CI, 1.89-15.20; P &lt;.001) but not in the assay-reported pCR-low tumors (OR, 0.86; 95% CI, 0.30-2.46; P =.77). A statistically significant interaction between the assay-reported pCR score and the effect of pertuzumab in pCR was observed. Conclusions and Relevance: This diagnostic/prognostic study demonstrated that the genomic assay predicted pCR following neoadjuvant trastuzumab-based chemotherapy with or without pertuzumab. This assay could guide therapeutic decisions regarding the use of neoadjuvant pertuzumab

Identification of cell surface targets for CAR-T cell therapies and antibody-drug conjugates in breast cancer

Two promising therapeutic strategies in oncology are chimeric antigen receptor-T cell (CAR-T) therapies and antibody-drug conjugates (ADCs). To be effective and safe, these immunotherapies require surface antigens to be sufficiently expressed in tumors and less or not expressed in normal tissues. To identify new targets for ADCs and CAR-T specifically targeting breast cancer (BC) molecular and pathology-based subtypes, we propose a novel in silico strategy based on multiple publicly available datasets and provide a comprehensive explanation of the workflow for a further implementation

Gene Expression Analysis of the Bone Marrow Microenvironment Reveals Distinct Immunotypes in Smoldering Multiple Myeloma Associated to Progression to Symptomatic Disease

Background: We previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression. Methods: Gene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology. Results: BM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival. Conclusions: Our results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression