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Background: Identification of biomarkers to optimize treatment strategies for early-stage triple-negative breast cancer (TNBC) is crucial. This study presents the development and validation of TNBC-DX, a novel test aimed at predicting both short- and long-term outcomes in early-stage TNBC., Methods: Information from 1,259 patients with early-stage TNBC (SCAN-B, CALGB-40603, and BrighTNess) were used to establish the TNBC-DX scores. Independent validation of TNBC-DX was carried out in 3 studies: i) WSG-ADAPT-TN, ii) MMJ-CAR-2014-01, and iii) NeoPACT, including 527 patients with stage I-III TNBC undergoing neoadjuvant chemotherapy. In WSG-ADAPT-TN, patients were randomized to receive nab-paclitaxel plus gemcitabine or carboplatin. In MMJ-CAR-2014-01, patients received carboplatin plus docetaxel. In NeoPACT, patients received carboplatin plus docetaxel and pembrolizumab. The objective of this study was to evaluate the association between TNBC-DX and efficacy outcomes (pCR, distant disease-free survival [DDFS] or event-free survival [EFS], and overall survival [OS]) in the validation cohorts., Results: TNBC-DX test was created incorporating 10-gene core immune gene module, 4-gene tumor cell proliferation signature, tumor size, and nodal staging. In the 2 independent validation cohorts without pembrolizumab, the TNBC-DX pCR score was significantly associated with pCR after adjustment for clinicopathological variables and treatment regimen (odds ratio per 10-units increment=1.34, 95% CI 1.20-1.52, p<0.001). pCR rates for the TNBC-DX pCR-high, -medium, and -low categories were 56.3%, 53.6%, and 22.5% respectively (odds ratio for pCR-high vs pCR-low=3.48 [95% CI 1.72-7.15], p<0.001). Additionally, the TNBC-DX risk score was significantly associated with DDFS (hazard ratio [HR] high-risk vs low-risk=0.24, 95% CI 0.15-0.41, p<0.001) and OS (HR=0.19, 95% CI 0.11-0.35, p<0.001). In the validation cohort with pembrolizumab, the TNBC-DX scores were significantly associated with pCR, EFS, and OS., Conclusions: TNBC-DX predicts pCR to neoadjuvant taxane-carboplatin in stage I-III TNBC and helps to forecast the patient´s long-term survival in the absence of neoadjuvant anthracycline/cyclophosphamide, and independent of pembrolizumab use., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: The purpose of this study was to assess the predictive capability of HER2DX assay following (neo)adjuvant trastuzumab-pertuzumab (HP)-based therapy in HER2-positive (HER2+) early breast cancer., Experimental Design: HER2DX was analyzed in baseline pretreatment tumors from the PHERGain trial. Patients with stage I-IIIA HER2+ early breast cancer were randomized to group A [docetaxel, carboplatin, and HP (TCHP)] and group B (HP ± endocrine therapy). PET response was evaluated after two cycles. Group A received TCHP for six cycles regardless of PET response. Group B continued with HP ± endocrine therapy for six cycles (PET responders) or with TCHP for six cycles (PET nonresponders). The primary objective of this retrospective study was to associate the HER2DX pathologic complete response (pCR) score with pCR. The secondary objective was the association of the HER2DX risk score with 3-year invasive disease-free survival (iDFS)., Results: HER2DX was performed on 292 (82.0%) tumors. The overall pCR rate was 38.0%, with pCR rates of 56.4% in group A and 33.8% in group B. In multivariable analysis including treatment and clinicopathologic factors, the HER2DX pCR score (continuous variable) significantly correlated with pCR [OR, 1.29; 95% confidence interval (CI), 1.10-1.54; P < 0.001]. HER2DX-defined pCR-high, -med, and -low groups exhibited pCR rates of 50.4%, 35.8%, and 23.2%, respectively (pCR-high vs. pCR-low OR, 3.27; 95% CI, 1.54-7.09; P < 0.001). In patients with residual disease, the HER2DX high-risk group demonstrated numerically worse 3-year iDFS than the low-risk group (89.8% vs. 100%; HR, 2.70; 95% CI, 0.60-12.18; P = 0.197)., Conclusions: HER2DX predicts pCR in the context of neoadjuvant HP-based therapy, regardless of chemotherapy addition, and might identify patients at higher risk of recurrence among patients with residual disease., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Development of a computational biomarker to predict, prior to treatment, the response to CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy in patients with breast cancer., Experimental Design: A mechanistic mathematical model that accounts for protein signaling and drug mechanisms of action was developed and trained on extensive, publicly available data from breast cancer cell lines. The model was built to provide a patient-specific response score based on the expression of six genes (CCND1, CCNE1, ESR1, RB1, MYC, and CDKN1A). The model was validated in five independent cohorts of 148 patients in total with early-stage or advanced breast cancer treated with endocrine therapy and CDK4/6i. Response was measured either by evaluating Ki67 levels and PAM50 risk of relapse (ROR) after neoadjuvant treatment or by evaluating progression-free survival (PFS)., Results: The model showed significant association with patient's outcomes in all five cohorts. The model predicted high Ki67 [area under the curve; AUC (95% confidence interval, CI) of 0.80 (0.64-0.92), 0.81 (0.60-1.00) and 0.80 (0.65-0.93)] and high PAM50 ROR [AUC of 0.78 (0.64-0.89)]. This observation was not obtained in patients treated with chemotherapy. In the other cohorts, patient stratification based on the model prediction was significantly associated with PFS [hazard ratio (HR) = 2.92 (95% CI, 1.08-7.86), P = 0.034 and HR = 2.16 (1.02 4.55), P = 0.043]., Conclusions: A mathematical modeling approach accurately predicts patient outcome following CDK4/6i plus endocrine therapy that marks a step toward more personalized treatments in patients with Luminal B breast cancer., (©2024 American Association for Cancer Research.)

The incidence of breast cancer in ≤ 40 yr-old women (YWBC) has been steadily increasing in recent decades. Although this group of patients represents less than 10 % of all newly diagnosed BC cases it encompasses a significant burden of disease. Usually underrepresented in clinical trials, YWBCs are also characterized by late diagnoses and poorly differentiated, aggressive-subtype disease, partly explaining its poor prognosis along with a high recurrence risk, and high mortality rates. On the other hand, YWBC treatment poses unique challenges such as preservation of fertility, and long-term toxicity and adverse events. Herein, we summarize the current evidence in hormone receptor-positive YWBC including specific risk factors, clinicopathologic and genomic features, and available evidence on response to chemotherapy and endocrine therapy. Overall, we advocate for a more comprehensive multidisciplinary healthcare model to improve the outcomes and the quality of life of this subset of younger patients., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [BW reports travel expenses from Astra Zeneca and BMS. IGF has declared honoraria for presentations from Novartis, Daiichi Sankyo, Esteve, GSK; and travel expenses from Novartis, Gilead, Daiichi Sankyo, Lilly, and BMS. OMS has declared travel expenses and consulting fees from Roche and Reveal, and speaker fees from Eisai, Daiichi-Sankyo, and Novartis. FS reports honoraria and travel expenses from Novartis, Gilead and Daiichy Sankyo, and advisory role for Pfizer. CS has received honorary for lectures from Roche, Novartis, Astra Zeneca, Pfizer, consulting/advisory role for Roche, Novartis, Pfizer, MSD and Astra Zeneca, and travel expenses from Roche and Pfizer. MM reports grants from Novartis, Breast Cancer Research Foundation- AACR, Breast Cancer Now Career Catalyst, during the study and advisors from Novartis, Roche, Pfizer and Lilly, outside of the submitted work. FBM reports patent application (PCT/EP2022/086493, PCT/EP2023/060810, EP23382703 and EP23383369). AP has declared personal honoraria from Pfizer, Novartis, Roche, MSD Oncology, Lilly and Daiichi Sankyo, travel, accommodations, and expenses paid by Daiichi Sankyo, research funding from Nanostring Technologies, Roche and Novartis, consulting/advisory role for Nanostring Technologies, Roche, Novartis, Pfizer, Oncolytics Biotech, Amgen, Lilly, MSD, PUMA and Daiichi Sankyo, Inc. outside the submitted work. MVL has declared honoraria for presentations from Novartis, Roche, Pfizer, and Daichii, and travel expenses from Roche and Pfizer. Additionally, she has participated on a Data Safety Monitoring Board or Advisory Board for Novartis and Roche.], (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Inflammation and neutrophils play a central role in both COVID-19 disease and cancer. We aimed to assess the impact of pre-existing tumor-related inflammation on COVID-19 outcomes in patients with cancer and to elucidate the role of circulating neutrophil subpopulations., Methods: We conducted a multicenter retrospective analysis of 524 patients with cancer and SARS-CoV-2 infection, assessing the relationship between clinical outcomes and circulating inflammatory biomarkers collected before and during COVID-19 infection. Additionally, a single-center prospective cohort study provided data for an exploratory analysis, assessing the immunophenotype of circulating neutrophils and inflammatory cytokines. The primary endpoints were 30-day mortality and the severity of COVID-19 disease., Results: Prior to COVID-19, 25% of patients with cancer exhibited elevated dNLR, which increased to 55% at the time of COVID-19 diagnosis. We developed the FLARE score, incorporating both tumor- and infection-induced inflammation, which categorized patients into four prognostic groups. The poor prognostic group had a 30-day mortality rate of 68%, significantly higher than the 23% in the favorable group ( p < 0.0001). This score proved to be an independent predictor of early mortality. This prospective analysis revealed a shift towards immature forms of neutrophils and higher IL-6 levels in patients with cancer and severe COVID-19 infection., Conclusions: A pre-existing tumor-induced pro-inflammatory state significantly impacts COVID-19 outcomes in patients with cancer. The FLARE score, derived from circulating inflammatory markers, emerges as an easy-to-use, globally accessible, effective tool for clinicians to identify patients with cancer at heightened risk of severe COVID-19 complications and early mortality who might benefit most from immediate and intensive treatment strategies. Furthermore, our findings underscore the significance of immature neutrophils in the progression of COVID-19 in patients with cancer, advocating for further investigation into how these cells contribute to both cancer and COVID-19 disease.

This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated β-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated β-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation., (© 2024. The Author(s).)

Patritumab deruxtecan (HER3-DXd) exhibits promising efficacy in breast cancer, with its activity not directly correlated to baseline ERBB3/HER3 levels. This research investigates the genetic factors affecting HER3-DXd's response in women with early-stage hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer. In the SOLTI-1805 TOT-HER3 trial, a single HER3-DXd dose was administered to 98 patients across two parts: 78 patients received 6.4 mg/kg (Part A), and 44 received a lower 5.6 mg/kg dose (Part B). The CelTIL score, measuring tumor cellularity and infiltrating lymphocytes from baseline to day 21, was used to assess drug activity. Part A demonstrated increased CelTIL score after one dose of HER3-DXd. Here we report CelTIL score and safety for Part B. In addition, the exploratory analyses of part A involve a comprehensive study of gene expression, somatic mutations, copy-number segments, and DNA-based subtypes, while Part B focuses on validating gene expression. RNA analyses show significant correlations between CelTIL responses, high proliferation genes (e.g., CCNE1, MKI67), and low expression of luminal genes (e.g., NAT1, SLC39A6). DNA findings indicate that CelTIL response is significantly associated with TP53 mutations, proliferation, non-luminal signatures, and a distinct DNA-based subtype (DNADX cluster-3). Critically, low HER2DX ERBB2 mRNA, correlates with increased HER3-DXd activity, which is validated through in vivo patient-derived xenograft  models. This study proposes chemosensitivity determinants, DNA-based subtype classification, and low ERBB2 expression as potential markers for HER3-DXd activity in HER2-negative breast cancer., (© 2024. The Author(s).)

Background: The PHERGain study (NCT03161353) is assessing early metabolic responses to neoadjuvant treatment with trastuzumab-pertuzumab and chemotherapy de-escalation using a [ 18 Fluorine]fluorodeoxyglucose-positron emission tomography ([ 18 F]FDG-PET) and a pathological complete response-adapted strategy in HER2-positive (HER2+) early breast cancer (EBC). Herein, we present RESPONSE, a PHERGain substudy, where clinicopathological and molecular predictors of [ 18 F]FDG-PET disease detection were evaluated., Methods: A total of 500 patients with HER2 + EBC screened in the PHERGain trial with a tumor size > 1.5 cm by magnetic resonance imaging (MRI) were included in the RESPONSE substudy. PET[-] criteria entailed the absence of  ≥ 1 breast lesion with maximum standardized uptake value (SUVmax) ≥ 1.5 × SUVmean liver + 2 standard deviation. Among 75 PET[-] patients screened, 21 with SUVmax levels < 2.5 were randomly selected and matched with 21 PET[+] patients with SUVmax levels ≥ 2.5 based on patient characteristics associated with [ 18 F]FDG-PET status. The association between baseline SUVmax and [ 18 F]FDG-PET status ([-] or [+]) with clinicopathological characteristics was assessed. In addition, evaluation of stromal tumor-infiltrating lymphocytes (sTILs) and gene expression analysis using PAM50 and Vantage 3D™ Cancer Metabolism Panel were specifically compared in a matched cohort of excluded and enrolled patients based on the [ 18 F]FDG-PET eligibility criteria., Results: Median SUVmax at baseline was 7.2 (range, 1-39.3). Among all analyzed patients, a higher SUVmax was associated with a higher tumor stage, larger tumor size, lymph node involvement, hormone receptor-negative status, higher HER2 protein expression, increased Ki67 proliferation index, and higher histological grade (p < 0.05). [ 18 F]FDG-PET [-] criteria patients had smaller tumor size (p = 0.014) along with the absence of lymph node involvement and lower histological grade than [ 18 F]FDG-PET [+] patients (p < 0.01). Although no difference in the levels of sTILs was found among 42 matched [ 18 F]FDG-PET [-]/[+] criteria patients (p = 0.73), [ 18 F]FDG-PET [-] criteria patients showed a decreased risk of recurrence (ROR) and a lower proportion of PAM50 HER2-enriched subtype than [ 18 F]FDG-PET[+] patients (p < 0.05). Differences in the expression of genes involved in cancer metabolism were observed between [ 18 F]FDG-PET [-] and [ 18 F]FDG-PET[+] criteria patients., Conclusions: These results highlight the clinical, biological, and metabolic heterogeneity of HER2+ breast cancer, which may facilitate the selection of HER2+ EBC patients likely to benefit from [ 18 F]FDG-PET imaging as a tool to guide therapy., Trial Registration: Clinicaltrials.gov; NCT03161353; registration date: May 15, 2017., (© 2024. The Author(s).)

Background: The characterization and comparison of gene expression and intrinsic subtype (IS) changes induced by neoadjuvant chemotherapy (NACT) and endocrine therapy in hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-low versus HR+/HER2-0 breast cancer (BC) has not been conducted so far. Most evidence on the association of HER2 status with pathologic responses and prognosis in HR+/HER2-negative BC is controversial and restricted to NACT-treated disease. Similarly, a temporal heterogeneity in HER2 status has been described only with NACT., Methods: We retrospectively recruited a consecutive cohort of 186 patients with stage I-IIIB HR+/HER2-negative BC treated with neoadjuvant therapy (NAT). Available diagnostic biopsies and surgical samples were characterized for main pathological features, PAM50 IS and ROR-P score, and gene expression. Associations with pathologic complete response, residual cancer burden-0/I, event-free survival (EFS) and overall survival (OS) based on HER2 status were assessed. Pre/post pathologic/molecular changes were analyzed in matched samples., Results: The HER2-low (62.9%) and HER2-0 (37.1%) cohorts did not differ significantly in main baseline features, treatments administered, breast-conserving surgery, pathologic complete response and residual cancer burden-0/I rates, EFS, and OS. NAT induced, regardless of HER2 status, a significant reduction of estrogen receptor/progesterone receptor and Ki67 levels, a down-regulation of PAM50 proliferation- and luminal-related genes/signatures, an up-regulation of selected immune genes, and a shift towards less aggressive IS and lower ROR-P. Moreover, 25% of HER2-0 changed to HER2-low and 34% HER2-low became HER2-0. HER2 shifts were significant after NACT (P < 0.001), not neoadjuvant endocrine therapy (P = 0.063), with consistent ERBB2 mRNA level dynamics. HER2 changes were not associated with EFS/OS., Conclusions: HER2-low and HER2-0 status change after NAT in ∼30% of cases, mostly after NACT. Targeted adjuvant strategies should be investigated accordingly. Molecular downstaging with current chemo/endocrine agents and immunotherapy should not rely on HER2 immunohistochemical levels in HR+/HER2-negative BC. Instead, HER2-low-targeted approaches should be explored to pursue more effective and/or less toxic dimensional downstaging., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The optimal treatment approach for hormone receptor-positive/HER2-negative metastatic breast cancer (HR+/HER2-negative MBC) with aggressive characteristics remains controversial, with lack of randomized trials comparing cyclin-dependent kinase (CDK)4/6-inhibitors (CDK4/6i) + endocrine therapy (ET) with chemotherapy + ET., Materials and Methods: We conducted an open-label randomized phase II trial (NCT03227328) to investigate whether chemotherapy + ET is superior to CDK4/6i + ET for HR+/HER2-negative MBC with aggressive features. PAM50 intrinsic subtypes (IS), immunological features, and gene expression were assessed on baseline samples., Results: Among 49 randomized patients (median follow-up: 35.2 months), median progression-free survival (mPFS) with chemotherapy + ET (11.2 months, 95% confidence interval [CI]: 7.7-15.4) was numerically shorter than mPFS (19.9 months, 95% CI: 9.0-30.6) with CDK4/6i + ET (hazard ratio: 1.41, 95% CI: 0.75-2.64). Basal-like tumors under CDK4/6i + ET exhibited worse PFS (mPFS: 11.4 months, 95% CI: 3.00-not reached [NR]) and overall survival (OS; mOS: 18.8 months, 95% CI: 18.8-NR) compared to other subtypes (mPFS: 20.7 months, 95% CI: 9.00-33.4; mOS: NR, 95% CI: 24.4-NR). In the chemotherapy arm, luminal A tumors showed poorer PFS (mPFS: 5.1 months, 95% CI: 2.7-NR) than other IS (mPFS: 13.2 months, 95% CI: 10.6-28.1). Genes/pathways involved in BC cell survival and proliferation were associated with worse outcomes, as opposite to most immune-related genes/signatures, especially in the CDK4/6i arm. CD24 was the only gene significantly associated with worse PFS in both arms. Tertiary lymphoid structures and higher tumor-infiltrating lymphocytes also showed favorable survival trends in the CDK4/6i arm., Conclusions: The KENDO trial, although closed prematurely, adds further evidence supporting CDK4/6i + ET use in aggressive HR+/HER2-negative MBC instead of chemotherapy. PAM50 IS, genomic, and immunological features are promising biomarkers to personalize therapeutic choices., (© The Author(s) 2024. Published by Oxford University Press.)

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors., (© 2024. The Author(s).)

Background: Early-stage triple-negative breast cancer (TNBC) displays clinical and biological diversity. From a biological standpoint, immune infiltration plays a crucial role in TNBC prognosis. Currently, there is a lack of genomic tools aiding in treatment decisions for TNBC. This study aims to assess the effectiveness of a B-cell/immunoglobulin signature (IGG) alone, or in combination with tumor burden, in predicting prognosis and treatment response in patients with TNBC., Methods: Genomic and clinical data were retrieved from 7 cohorts: SCAN-B (N = 874), BrighTNess (n = 482), CALGB-40603 (n = 389), METABRIC (n = 267), TCGA (n = 118), GSE58812 (n = 107), GSE21653 (n = 67). IGG and a risk score integrating IGG with tumor/nodal staging (IGG-Clin) were assessed for event-free survival (EFS) and overall survival (OS) in each cohort. Random effects model was used to derive pooled effect sizes. Association of IGG with pathological complete response (pCR) was assessed in CALGB-40603 and BrighTNess. Immune significance of IGG was estimated through CIBERSORTx and EcoTyper., Findings: IGG was associated with improved EFS (pooled HR = 0.77, [95% CI = 0.70-0.85], I 2  = 18%) and OS (pooled HR = 0.79, [0.73-0.85], I 2  = 0%) across cohorts, and was predictive of pCR in CALGB-40603 (OR 1.25, [1.10-1.50]) and BrighTNess (OR 1.57 [1.25-1.98]). IGG-Clin was predictive of recurrence (pooled HR = 2.11, [1.75-2.55], I 2  = 0%) and death (pooled HR = 1.99, 95% [0.84-4.73], I 2  = 79%) across cohorts. IGG was associated with adaptive immune response at CIBERSORTx and EcoTyper analysis., Interpretation: IGG is linked to improved prognosis and pCR in early-stage TNBC. The integration of IGG alongside tumor and nodal staging holds promise as an approach to identify patients benefitting from intensified or de-intensified treatments., Funding: This study received funding from: Associació Beca Marta Santamaria, European Union's Horizon 2020 research and innovation and Marie Skłodowska-Curie Actions programs, Fundación FERO, Fundación CRIS contra el cáncer, Agència de Gestó d'Ajuts Universitaris i de Recerca, Instituto de Salud Carlos III, Fundación Contigo, Asociación Cáncer de Mama Metastásico IV, Breast Cancer Research Foundation, RESCUER, Fundación científica AECC and FSEOM., Competing Interests: Declaration of interests B. Conte reports speaker fees from Veracyte and payment for educational events from Medsite and Novartis. A. Prat reports consulting fees from Roche, Pfizer, Novartis, Amgen, BMS, Puma, Oncolytics Biotech, MSD, Guardant Health, Peptomyc and Lilly, lecture fees from Roche, Pfizer, Novartis, Amgen, BMS, Nanostring Technologies and Daiichi Sankyo; patents filed PCT/EP2016/080056, PCT/EP2022/086493, PCT/EP2023/060810, EP23382703 and EP23383369; stockholder and consultant of Reveal Genomics, SL; and institutional financial interests from Boehringer, Novartis, Roche, Nanostring, Sysmex Europa GmbH, Medica Scientia Innovation Research, SL, Celgene, Astellas and Pfizer. F. Schettini has declared consulting fees from Pfizer, honoraria for lectures from Novartis, Gilead and Daiichy Sankyo, and travel expenses from Novartis and Daiichy Sankyo. O. Martínez-Sáez has declared institutional grants from Roche; personal consulting fees from Roche and Reveal Genomics; honoraria for presentations by Daiichi Sankyo, Pierre Fabre, and Reveal Genomics; and travel expenses by Gilead, Pierre Fabre, Novartis, and MSD. A. Vivancos has declared institutional grants from Bristol Meyers Squibb, Incyte, and Roche; personal consulting fees from Bayer, Bristol Meyers Squibb (BMS); Guardant, Incyte, and Roche; and personal stock options from Reveal Genomics. F. Brasó-Maristany has patents filed: PCT/EP2022/086493, PCT/EP2023/060810, EP23382703 and EP23383369. J. Parker as declared individual and institutional royalties from Veracyte–Prosigna, consulting fees from Bristol Meyers Squibb, Reveal Genomics, and GeneCentric, and holds a patent for breast cancer subtyping. Additionally, he has an equity interest in Reveal Genomics. M. Vidal has declared honoraria for presentations from Novartis, Roche, Pfizer, and Daichii, and travel expenses from Roche and Pfizer. Additionally, she has participated on a Data Safety Monitoring Board or Advisory Board for Novartis and Roche. C. Perou has declared consulting fees and personal stock options from Reveal Genomics. T. Pascual has declared consulting fees from Novartis; honoraria from Novartis, Astra-zeneca, Veracyte, and Argenetics. I. Garcia-Fructuoso has declared honoraria for presentations from Novartis, Daiichi Sankyo, Esteve, GSK; and travel expenses from Novartis, Gilead, Daiichi Sankyo, Lilly, and BMS. L. Paré has declared contract from Reveal Genomics, a HER2DX patent filed (PCT/EP2022/086493), and the TNBCDX patent filed (EP23382703.9). M. Marín-Aguilera has declared contract from Reveal Genomics. P. Villagrasa has declared contract and personal stock options from Reveal Genomics, the HER2DX patent filed (PCT/EP2022/086493), and the DNADX patent filed (EP22382387.3). G. Villacampa has received a speaker's fee from Merck Sharp & Dohme, Pfizer, GlaxoSmithKline and Pierre Fabrer, and received consultant fees from Reveal Genomics. C. Falato is currently employed in AstraZeneca. The remaining authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

Background: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance., Materials and Methods: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations., Results: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80)., Conclusions: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer., Competing Interests: Disclosure MMA is an employee at Reveal Genomics; GV has received a speaker’s fee from MSD, Pfizer, GSK, and Pierre Fabrer, has held an advisory role with AstraZeneca, and received consultant fees from Reveal Genomics; FBM has a HER2DX patent application EP21383165; PG is an employee at Reveal Genomics; OMS has declared travel expenses and consulting fees from Roche and Reveal, and speaker fees from Eisai, Daiichi, and Novartis; KP serves on the Scientific Advisory Board of Novartis, Ideaya Biosciences, and Scorpion Therapeutics, holds equity options in Scorpion Therapeutics and Ideaya Biosciences, and receives sponsored research funding from Novartis through DFCI; and has patents on S100A7 antibody and BET inhibitor resistance held by DFCI; JM is an employee at Reveal Genomics; WB is an employee at Reveal Genomics; CMP is an equity stockholder and consultant of BioClassifier LLC, and for Reveal Genomics, is also listed as an inventor on patent applications for the Breast PAM50 assay; PVG is one of the stockholders of Reveal Genomics; AP reports advisory and consulting fees from Roche, Pfizer, Novartis, Amgen, BMS, Puma, Oncolytics Biotech, MSD, Guardant Health, Peptomyc, and Lilly, lecture fees from Roche, Pfizer, Novartis, Amgen, BMS, and Daiichi Sankyo, institutional financial interests from Novartis, Roche, and Pfizer, stockholder and consultant of Reveal Genomics, SL; AP is also listed as an inventor on patent applications for the HER2DX assay; JSP is an equity stockholder and consultant for Reveal Genomics and is also listed as an inventor on patent applications for the Breast PAM50 assay; LP is listed as an inventor on HER2DX patent PCT/EP2021/070788 and is an employee at Reveal Genomics. All other authors have declared no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The HER2DX risk-score has undergone rigorous validation in prior investigations involving patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer. In this study, we present the outcomes of the HER2DX risk-score within the most recent release of the Sweden Cancerome Analysis Network-Breast (SCAN-B) HER2+ cohort. This updated examination benefits from a larger patient sample, an extended follow-up duration, and detailed treatment information., Materials and Methods: Clinical and RNAseq data from the SCAN-B dataset were retrieved from Gene Expression Omnibus (GSE81538). Among the 6600 patients, 819 had HER2+ breast cancer, with 757 individuals with research-based HER2DX risk-scores and corresponding survival outcomes. The HER2DX risk-score was evaluated (i) as a continuous variable and (ii) using predefined cut-offs. The primary endpoint for this study was overall survival (OS). The Kaplan-Meier method and Cox models were used to estimate OS and a multistate model with four states was fitted to better characterize patients' follow-up., Results: The median follow-up time was 7.5 years (n = 757). The most common systemic therapy was chemotherapy with trastuzumab (82.0%) and most tumors were classified as T1-T2 (97.1%). The HER2DX risk-score as a continuous variable was significantly associated with OS after adjustment for clinical variables and treatment regimen [hazard ratios (HR) per 10-unit increment = 1.31, 95% confidence interval (CI) 1.13-1.51, P < 0.001] as well as within predefined risk groups (high versus low; HR = 2.57, 95% CI 1.36-4.85, P < 0.001). Patients classified as HER2DX high-risk also had higher risk of (i) breast cancer recurrence and (ii) death without previous recurrence. Within the subgroup of HER2+ T1N0 tumors (n = 297), those classified as high-risk demonstrated inferior OS compared to low-risk tumors (7-year OS 77.8% versus 96.8%, P < 0.001). The HER2DX mRNA ERBB2 score was associated with clinical HER2 status (area under the receiver operating characteristic curve = 0.91)., Conclusions: In patients with early-stage HER2+ breast cancer, HER2DX risk-score provides prognostic information beyond clinicopathological variables, including treatment regimen with or without trastuzumab., Competing Interests: Role of the funder In addition to financial support, the funders actively contributed to various aspects of the research, including study design, data collection, analysis, interpretation, and report writing. Disclosure GV has received a speaker’s fee from MSD, Pfizer, GSK, and Pierre Fabre; has held an advisory role with AstraZeneca; and received consultant fees from Reveal Genomics. TP has received honoraria for speaker activities from AstraZeneca, Pfizer, Novartis, Veracyte, and Argenetics, and has held an advisory role with Novartis. FBM has a patent application (EP21383165). LP is listed as an inventor on patent PCT/EP2021/070788. OMS has declared travel expenses and consulting fees from Roche and Reveal, and speaker fees from Eisai, Daiichi-Sankyo, and Novartis. JC has ownership interests in MedSIR, Nektar, and Leuko; has held consulting or advisory roles in Celgene, Cellestia Biotech, AstraZeneca, Roche, Seattle Genetics, Daiichi-Sankyo, ERYTECH Pharma, Polyphor, Athenex, Lilly, Servier, Merck Sharp & Dohme, GlaxoSmithKline, Leuko, Clovis Oncology, Bioasis, Boehringer Ingelheim, Ellipses Pharma, HiberCell, Bioinvent, GEMoaB, Gilead Sciences, Menarini, Zymeworks, and Reveal Genomics; has received research funding from ARIAD, AstraZeneca, Baxalta, Bayer, Eisai, Guardant Health, Merck Sharp & Dohme, Pfizer, Puma Biotechnology, Queen Mary University of London, Roche, and Piqur; has patents, royalties, or other intellectual property from pharmaceutical combinations of a Pi3k Inhibitor and a microtubule destabilizing agent (Javier Cortés Castán, Alejandro Piris Giménez, Violeta Serra Elizalde. WO 2014/199294 A, Her2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic therapy. Aleix Prat, Antonio Llombart, Javier Cortés. US 2019/ 0338368 A1); and has received travel expenses from Roche, Pfizer, Eisai, Novartis, Daiichi-Sankyo, and Gilead Sciences. EC has received honoraria for advisory roles from Pfizer, AstraZeneca, Daiichi-Sankyo, Roche, Novartis, Lilly, and MSD; and has received a speaker’s fee from Roche and Lilly. MM reports research grants from Puma; consulting/advisory fees from Roche, Novartis, AstraZeneca, Daiichi-Sankyo, Seagen, Lilly, and Sanofi; speakers’ honoraria from Seagen, Lilly, AstraZeneca, Pfizer, Daiichi-Sankyo, and Roche; a leadership or fiduciary role as Chairman, GEICAM and as a member of the Board of Directors for TRIO. PC reports speakers’ bureaus with AstraZeneca, GlaxoSmithKline, Novartis, and Roche; travel, accommodation, and expenses from Celgene, GlaxoSmithKline, and Novartis; and research funding from Merck Serono, Novartis, and Roche. LAC reports participation on a Data Safety Monitoring Board or Advisory Board of Sanofi Aventis, Novartis, Genentech/Roche, GSK, AstraZeneca/Daiichi-Sankyo, and Aptitude Health; and has a spouse serving on the board of Falcon Therapeutics and spouse involvement in a neural stem cell therapy patent. NH received consulting fees from Pierre Fabre and Roche. AV has the DNADX patent filed (EP22382387.3). GC served as consultant or advisor for Roche, Lilly, and Bristol-Myers Squibb; served on the speaker’s bureau for Roche, Pfizer, and Lilly; received travel funding from Pfizer and Roche; and received honoraria from Roche, Pfizer, Lilly, Novartis, and Seagen, all outside the submitted work. PV is one of the stockholders of Reveal Genomics, and also reports personal fees from NanoString. JSP is an equity stockholder and consultant for Reveal Genomics and is listed as an inventor on patent applications for the Breast PAM50 assay. CMP is an equity stockholder and consultant of BioClassifier LLC and Reveal Genomics, and is listed as an inventor on patent applications for the Breast PAM50 assay. AP reports advisory and consulting fees from Roche, Pfizer, Novartis, Amgen, Bristol-Myers Squibb, Puma, Oncolytics Biotech, MSD, Guardant Health, Peptomyc, and Lilly; lecture fees from Roche, Pfizer, Novartis, Amgen, Bristol-Myers Squibb, NanoString Technologies, and Daiichi-Sankyo; institutional financial interests from Boehringer, Novartis, Roche, NanoString, Sysmex Europa GmbH, Medica Scientia Innovation Research, SL, Celgene, Astellas, and Pfizer; is a stockholder and consultant of Reveal Genomics and SL; and is listed as an inventor on patent applications for the HER2DX assay. SMT reports consulting or advisory roles for Novartis, Pfizer, Merck, Eli Lilly, AstraZeneca, Genentech/Roche, Eisai, Sanofi, Bristol-Myers Squibb, Seattle Genetics, CytomX Therapeutics, Daiichi-Sankyo, Gilead, OncXerna, Zymeworks, Zentalis, Blueprint Medicines, Reveal Genomics, ARC Therapeutics, Infinity Therapeutics, Sumitovant Biopharma, Zetagen, Umoja Biopharma, Artios Pharma, Menarini/Stemline, Aadi Biopharma, Bayer, Incyte Corp, Jazz Pharmaceuticals, Natera, Tango Therapeutics, Systimmune, eFFECTOR, and Hengrui USA; research funding from Genentech/Roche, Merck, Exelixis, Pfizer, Lilly, Novartis, Bristol-Myers Squibb, Eisai, AstraZeneca, Gilead, NanoString Technologies, Seattle Genetics, and OncoPep; and travel support from Eli Lilly, Sanofi, Gilead, and Pfizer. All other authors have declared no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Even with contemporary treatment strategies, more than 10% of HER2-positive early stage breast cancer patients may experience distant metastasis as first event during follow-up. Tools for predicting unique patterns of metastatic spread are needed to plan personalized surveillance. We evaluated how molecular heterogeneity affects the pattern of distant relapse in HER2-positive breast cancer., Methods: A total of 677 HER2-positive stage I-III breast cancer patients from ShortHER trial, Cher-LOB trial, and 2 institutional cohorts were included. PAM50 molecular subtypes and research-based HER2DX scores were evaluated. The cumulative incidence of distant relapse as the first event (any site and site specific) was evaluated using competing risk analysis. Median follow-up was 8.4 years. Tests of statistical significance are 2-sided., Results: Stage III and high HER2DX risk score identified patients at the highest risk of distant relapse as first event (10-year incidence 24.5% and 19.7%, respectively). Intrinsic molecular subtypes were associated with specific patterns of metastatic spread: compared with other subtypes, HER2-enriched tumors were more prone to develop brain metastases (10-year incidence 3.8% vs 0.6%, P = .005), basal-like tumors were associated with an increased risk of lung metastases (10-year incidence 11.1% vs 2.6%, P = .001), and luminal tumors developed more frequently bone-only metastases (10-year incidence 5.1% vs 2.0%, P = .042). When added to stage or HER2DX risk score in competing risk regression models, intrinsic subtype maintained an independent association with site-specific metastases., Conclusions: The integration of intrinsic molecular subtypes with stage or HER2DX risk score predicts site-specific metastatic risk in HER2-positive breast cancer, with potential implications for personalized surveillance and clinical trials aimed at preventing site-specific recurrence., (© The Author(s) 2023. Published by Oxford University Press.)

Purpose: In estrogen receptor-positive (ER+) breast cancer, single-nucleotide polymorphisms (SNP) in the aromatase gene might affect aromatase inhibitors (AI) metabolism and efficacy. Here, we assessed the impact of SNP on prognosis and toxicity of patients receiving adjuvant letrozole., Experimental Design: We enrolled 886 postmenopausal patients in the study. They were treated with letrozole for 2 to 5 years after taking tamoxifen for 2 to 6 years, continuing until they completed 5 to 10 years of therapy. Germline DNA was genotyped for SNP rs4646, rs10046, rs749292, and rs727479. Log-rank test and Cox model were used for disease-free survival (DFS) and overall survival (OS). Cumulative incidence (CI) of breast cancer metastasis was assessed through competing risk analysis, with contralateral breast cancer, second malignancies and non-breast cancer death as competing events. CI of skeletal and cardiovascular events were assessed using DFS events as competing events. Subdistribution HR (sHR) with 95% confidence intervals were calculated through Fine-Gray method., Results: No SNP was associated with DFS. Variants rs10046 [sHR 2.03, (1.04-2.94)], rs749292 [sHR 2.11, (1.12-3.94)], and rs727479 [sHR 2.62, (1.17-5.83)] were associated with breast cancer metastasis. Three groups were identified on the basis of the number of these variants (0, 1, >1). Variant-based groups were associated with breast cancer metastasis (10-year CI 2.5%, 7.6%, 10.7%, P = 0.035) and OS (10-year estimates 96.5%, 93.0%, 89.6%, P = 0.030). Co-occurrence of rs10046 and rs749292 was negatively associated with 10-year CI of skeletal events (3.2% vs. 10%, P = 0.033). A similar association emerged between rs727479 and cardiovascular events (0.3% vs. 2.1%, P = 0.026)., Conclusions: SNP of aromatase gene predict risk of metastasis and AI-related toxicity in ER+ early breast cancer, opening an opportunity for better treatment individualization., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: The HER2DX genomic test predicts pathological complete response (pCR) and survival outcome in early-stage HER2-positive (HER2+) breast cancer. Here, we evaluated the association of HER2DX scores with (i) pCR according to hormone receptor status and various treatment regimens, and (ii) survival outcome according to pCR status., Materials and Methods: Seven neoadjuvant cohorts with HER2DX and clinical individual patient data were evaluated (DAPHNe, GOM-HGUGM-2018-05, CALGB-40601, ISPY-2, BiOnHER, NEOHER and PAMELA). All patients were treated with neoadjuvant trastuzumab (n = 765) in combination with pertuzumab (n = 328), lapatinib (n = 187) or without a second anti-HER2 drug (n = 250). Event-free survival (EFS) and overall survival (OS) outcomes were available in a combined series of 268 patients (i.e. NEOHER and PAMELA) with a pCR (n = 118) and without a pCR (n = 150). Cox models were adjusted to evaluate whether HER2DX can identify patients with low or high risk beyond pCR status., Results: HER2DX pCR score was significantly associated with pCR in all patients [odds ratio (OR) per 10-unit increase = 1.59, 95% confidence interval 1.43-1.77; area under the ROC curve = 0.75], with or without dual HER2 blockade. A statistically significant increase in pCR rate due to dual HER2 blockade over trastuzumab-only was observed in HER2DX pCR-high tumors treated with chemotherapy (OR = 2.36 (1.09-5.42). A statistically significant increase in pCR rate due to multi-agent chemotherapy over a single taxane was observed in HER2DX pCR-medium tumors treated with dual HER2 blockade (OR = 3.11, 1.54-6.49). The pCR rates in HER2DX pCR-low tumors were ≤30.0% regardless of treatment administered. After adjusting by pCR status, patients identified as HER2DX low-risk had better EFS (P < 0.001) and OS (P = 0.006) compared with patients with HER2DX high-risk., Conclusions: HER2DX pCR score and risk score might help identify ideal candidates to receive neoadjuvant dual HER2 blockade in combination with a single taxane in early-stage HER2+ breast cancer., (Copyright © 2023 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

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 < .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 < .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 < .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.

Background: In hormone receptor-positive (HoR+) breast cancer (BC), gene expression analysis identifies luminal A (LumA), luminal B (LumB), human epidermal growth factor receptor 2 (HER2)-enriched (HER2-E), basal-like (BL) intrinsic subtypes and a normal-like group. This classification has an established prognostic value in early-stage HoR+ BC. Here, we carried out a trial-level meta-analysis to determine the prognostic ability of subtypes in metastatic BC (MBC)., Materials and Methods: We systematically reviewed all the available prospective phase II/III trials in HoR+ MBC where subtype was assessed. The primary endpoint was progression-free survival (PFS)/time to progression (TTP) of the LumA subtype compared to non-LumA. Secondary endpoints were PFS/TTP of each individual subtype, according to treatment, menopausal and HER2 status and overall survival (OS). The random-effect model was applied, and heterogeneity assessed through Cochran's Q and I 2 . Threshold for significance was set at P < 0.05. The study was registered in PROSPERO (ID: CRD42021255769)., Results: Seven studies were included (2536 patients). Non-LumA represented 55.2% and was associated with worse PFS/TTP than LumA [hazard ratio (HR) 1.77, P < 0.001, I 2  = 61%], independently of clinical HER2 status [P subgroup difference (P sub ) = 0.16], systemic treatment (P sub  = 0.96) and menopausal status (P sub  = 0.12). Non-LumA tumors also showed worse OS (HR 2.00, P < 0.001, I 2  = 65%), with significantly different outcomes for LumB (PFS/TTP HR 1.46; OS HR 1.41), HER2-E (PFS/TTP HR 2.39; OS HR 2.08) and BL (PFS/TTP HR 2.67; OS HR 3.26), separately (PFS/TTP P sub  = 0.01; OS P sub  = 0.005). Sensitivity analyses supported the main result. No publication bias was observed., Conclusions: In HoR+ MBC, non-LumA disease is associated with poorer PFS/TTP and OS than LumA, independently of HER2, treatment and menopausal status. Future trials in HoR+ MBC should consider this clinically relevant biological classification., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

Importance: Both tumor-infiltrating lymphocytes (TILs) assessment and immune-related gene expression signatures by RNA profiling predict higher pathologic complete response (pCR) and improved event-free survival (EFS) in patients with early-stage ERBB2/HER2-positive breast cancer. However, whether these 2 measures of immune activation provide similar or additive prognostic value is not known., Objective: To examine the prognostic ability of TILs and immune-related gene expression signatures, alone and in combination, to predict pCR and EFS in patients with early-stage ERBB2/HER2-positive breast cancer treated in 2 clinical trials., Design, Setting, and Participants: In this prognostic study, a correlative analysis was performed on the Cancer and Leukemia Group B (CALGB) 40601 trial and the PAMELA trial. In the CALGB 40601 trial, 305 patients were randomly assigned to weekly paclitaxel with trastuzumab, lapatinib, or both for 16 weeks. The primary end point was pCR, with a secondary end point of EFS. In the PAMELA trial, 151 patients received neoadjuvant treatment with trastuzumab and lapatinib for 18 weeks. The primary end point was the ability of the HER2-enriched subtype to predict pCR. The studies were conducted from October 2013 to November 2015 (PAMELA) and from December 2008 to February 2012 (CALGB 40601). Data analyses were performed from June 1, 2020, to January 1, 2022., Main Outcomes and Measures: Immune-related gene expression profiling by RNA sequencing and TILs were assessed on 230 CALGB 40601 trial pretreatment tumors and 138 PAMELA trial pretreatment tumors. The association of these biomarkers with pCR (CALGB 40601 and PAMELA) and EFS (CALGB 40601) was studied by logistic regression and Cox analyses., Results: The median age of the patients was 50 years (IQR, 42-50 years), and 305 (100%) were women. Of 202 immune signatures tested, 166 (82.2%) were significantly correlated with TILs. In both trials combined, TILs were significantly associated with pCR (odds ratio, 1.01; 95% CI, 1.01-1.02; P = .02). In addition to TILs, 36 immune signatures were significantly associated with higher pCR rates. Seven of these signatures outperformed TILs for predicting pCR, 6 of which were B-cell related. In a multivariable Cox model adjusted for clinicopathologic factors, including PAM50 intrinsic tumor subtype, the immunoglobulin G signature, but not TILs, was independently associated with EFS (immunoglobulin G signature-adjusted hazard ratio, 0.63; 95% CI, 0.42-0.93; P = .02; TIL-adjusted hazard ratio, 1.00; 95% CI, 0.98-1.02; P = .99)., Conclusions and Relevance: Results of this study suggest that multiple B-cell-related signatures were more strongly associated with pCR and EFS than TILs, which largely represent T cells. When both TILs and gene expression are available, the prognostic value of immune-related signatures appears to be superior.

In advanced HER2-positive (HER2+) breast cancer, the new antibody-drug conjugate trastuzumab deruxtecan is more effective compared with trastuzumab emtansine (T-DM1). However, trastuzumab deruxtecan can have considerable toxicities, and the right treatment sequence is unknown. Biomarkers to guide the use of anti-HER2 therapies beyond HER2 status are needed. Here, we evaluated if preestablished levels of ERBB2 mRNA expression according to the HER2DX standardized assay are associated with response and survival following T-DM1. In ERBB2 low, medium, and high groups, the overall response rate was 0%, 29%, and 56%, respectively (P < .001). ERBB2 mRNA was statistically significantly associated with better progression-free survival (P = .002) and overall survival (OS; P = .02). These findings were independent of HER2 immunohistochemistry (IHC) levels, hormone receptor, age, brain metastasis, and line of therapy. The HER2DX risk score (P = .04) and immunoglobulin signature (P = .04) were statistically significantly associated with overall survival since diagnosis. HER2DX provides prognostic and predictive information following T-DM1 in advanced HER2+ breast cancer., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Liquid biopsy has proven valuable in identifying individual genetic alterations; however, the ability of plasma ctDNA to capture complex tumor phenotypes with clinical value is unknown. To address this question, we have performed 0.5X shallow whole-genome sequencing in plasma from 459 patients with metastatic breast cancer, including 245 patients treated with endocrine therapy and a CDK4/6 inhibitor (ET + CDK4/6i) from 2 independent cohorts. We demonstrate that machine learning multi-gene signatures, obtained from ctDNA, identify complex biological features, including measures of tumor proliferation and estrogen receptor signaling, similar to what is accomplished using direct tumor tissue DNA or RNA profiling. More importantly, 4 DNA-based subtypes, and a ctDNA-based genomic signature tracking retinoblastoma loss-of-heterozygosity, are significantly associated with poor response and survival outcome following ET + CDK4/6i, independently of plasma tumor fraction. Our approach opens opportunities for the discovery of additional multi-feature genomic predictors coming from ctDNA in breast cancer and other cancer-types., (© 2023. The Author(s).)

Traditionally, the classification of breast cancer relies on the expression of immunohistochemical (IHC) biomarkers readily available in clinical practice. Using highly standardized and reproducible assays across patient cohorts, intrinsic molecular subtypes of breast cancer - also called "intrinsic subtypes" (IS) - have been identified based on the expression of 50 genes. Although IHC-based subgroups and IS moderately correlate to each other, they are not superimposable. In fact, non-luminal biology has been detected in a substantial proportion (5-20%) of hormone receptor-positive (HoR+) tumors, has prognostic value, and identifies reduced and increased sensitivity to endocrine therapy and chemotherapy, respectively. During tumor progression, a shift toward a non-luminal estrogen-independent and more aggressive phenotype has been demonstrated. Intrinsic genomic instability and cell plasticity, alone or combined with external constraints deriving from treatment selective pressure or interplay with the tumor microenvironment, may represent the determinants of such biological diversity between primary and metastatic disease, and during metastatic tumor evolution. In this review, we describe the distribution and the clinical behavior of IS as the disease progresses, focusing on HoR+/HER2-negative advanced breast cancer. In addition, we provide an overview of the ongoing clinical trials aiming to validate the predictive and prognostic value of IS towards their incorporation into routine care., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CF declares no competing financial and non-financial interests; FS declares no competing financial and non-financial interests but reports personal fees from Novartis for educational activities; TP declares no competing financial and non-financial interests; FBM declares no competing financial and non-financial interests but reports patent application EP21383165 and patent application on DNA-based predictors of breast tumor phenotypes filed; AP declares no competing non-financial interests but reports advisory and consulting fees from Roche, Pfizer, Novartis, Amgen, BMS, Puma, Oncolytics Biotech, MSD, Guardant Health, Peptomyc, and Lilly, lecture fees from Roche, Pfizer, Novartis, Amgen, BMS, Nanostring Technologies and Daiichi Sankyo, institutional financial interests from Boehringer, Novartis, Roche, Nanostring, Sysmex Europa GmbH, Medica Scientia inno. Research, SL, Celgene, Astellas, and Pfizer; a leadership role in Reveal Genomics, SL; and a patent PCT/EP2016/080056., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies., (© 2022. The Author(s).)