Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression.

Antibody targeting of the immune checkpoint receptor PD1 produces therapeutic activity in a variety of solid tumors, but most patients exhibit partial or complete resistance to treatment for reasons that are unclear. In this study, we evaluated tumor specimens from 65 patients with melanoma, lung nonsquamous, squamous cell lung or head and neck cancers who were treated with the approved PD1-targeting antibodies pembrolizumab or nivolumab. Tumor RNA before anti-PD1 therapy was analyzed on the nCounter system using the PanCancer 730-Immune Panel, and we identified 23 immune-related genes or signatures linked to response and progression-free survival (PFS). In addition, we evaluated intra- and interbiopsy variability of PD1, PD-L1, CD8A, and CD4 mRNAs and their relationship with tumor-infiltrating lymphocytes (TIL) and PD-L1 IHC expression. Among the biomarkers examined, PD1 gene expression along with 12 signatures tracking CD8 and CD4 T-cell activation, natural killer cells, and IFN activation associated significantly with nonprogressive disease and PFS. These associations were independent of sample timing, drug used, or cancer type. TIL correlated moderately (∼0.50) with PD1 and CD8A mRNA levels and weakly (∼0.35) with CD4 and PD-L1. IHC expression of PD-L1 correlated strongly with PD-L1 (0.90), moderately with CD4 and CD8A, and weakly with PD1. Reproducibility of gene expression in intra- and interbiopsy specimens was very high (total SD <3%). Overall, our results support the hypothesis that identification of a preexisting and stable adaptive immune response as defined by mRNA expression pattern is reproducible and sufficient to predict clinical outcome, regardless of the type of cancer or the PD1 therapeutic antibody administered to patients. Cancer Res; 77(13); 3540–50. ©2017 AACR.

Purpose: The therascreenPIK3CAmutation assay and the alpha-specific PI3K inhibitor alpelisib are FDA-approved for identifying and treating patients with advancedPIK3CA-mutated (PIK3CAmut) breast cancer (BC). However, it is currently unknown to what extend this assay detects mostPIK3CAmutations in BC. This information is critical as patients and clinicians are using this and other genomic assays to indicate alpelisib.Methods: Data from 6338 patients with BC was explored across 10 publicly available studies. The primary objective was to evaluate the proportion and distribution ofPIK3CAmutations in BC. Secondary objectives were (1) to evaluate in silico the spectrum ofPIK3CAmutations in BC that would be captured by the therascreen panel; (2) to evaluate the proportion and distribution ofPIK3CAmutations in hormone receptor-positive/HER2-negative (HR+/HER2−), HER2+, and triple-negative BC (TNBC); and (3) to explore the identification ofPIK3CAmutations in a cohort of 48 HR+/HER2− advanced BC patients by the Guardant B360 circulating tumor DNA (ctDNA) assay.Results: Patients withPIK3CAmut tumors represented 35.7% (2261/6338). FivePIK3CAmutations comprised 73% of allPIK3CAmutations: H1047R (35%), E545K (17%), E542K (11%), N345K (6%), and H1047L (4%). Therascreen gene list would capture 72% of allPIK3CAmutations and 80% of patients with a knownPIK3CAmut BC. Among patients with doublePIK3CAmut tumors (12% of allPIK3CAmut), the therascreen panel would capture 78% as harboring 1 singlePIK3CAmutation, 17% asPIK3CAmut undetected, and 5% asPIK3CAdouble-mut.PIK3CAmutation rates were lower in TNBC (16%) compared to HR+/HER2 (42%) and HER2+ (31%) BC; however, the distribution of the 4 mainPIK3CAmutations across subtypes was similar. Finally, 28% ofPIK3CAmutations identified in ctDNA in 48 patients with advanced HR+/HER2− BC were not part of the therascreen panel.Conclusion: PIK3CAmutations in BC are heterogenous and ~ 20% of patients with a knownPIK3CAmutation, and 95% with a known doublePIK3CAmut tumor, would not be captured by the therascreen panel. Finally, the clinical utility ofPIK3CAmutations not present in the therascreen companion diagnostic assay or identified by other sequencing-based assays needs further investigation.

Both clinical and genomic data independently predict survival and treatment response in early-stage HER2-positive breast cancer. Here we present the development and validation of a new HER2DX risk score, and a new HER2DX pathological complete response (pCR) score, both based on a 27-gene expression plus clinical feature-based classifier. HER2DX is a supervised learning algorithm incorporating tumour size, nodal staging, and 4 gene expression signatures tracking immune infiltration, tumour cell proliferation, luminal differentiation, and the expression of the HER2 amplicon, into a single score. 434 HER2-positive tumours from the Short-HER trial were used to train a prognostic risk model; 268 cases from an independent cohort were used to verify the accuracy of the HER2DX risk score. In addition, 116 cases treated with neoadjuvant anti-HER2-based chemotherapy were used to train a predictive model of pathological complete response (pCR); two independent cohorts of 91 and 67 cases were used to verify the accuracy of the HER2DX pCR likelihood score. Five publicly available independent datasets with >1,000 patients with early-stage HER2-positive disease were also analysed. In Short-HER, HER2DX variables were associated with good risk outcomes (i.e., immune, and luminal) and poor risk outcomes (i.e., proliferation, and tumour and nodal staging). In an independent cohort, continuous HER2DX risk score was significantly associated with disease-free survival (DFS) (p=0·002); the 5-year DFS in the low-risk group was 97·4% (94·4-100·0%). For the neoadjuvant pCR predictor training cohort, HER2DX variables were associated with pCR (i.e., immune, proliferation and HER2 amplicon) and non-pCR (i.e., luminal, and tumour and nodal staging). In both independent test set cohorts, continuous HER2DX pCR likelihood score was significantly associated with pCR (p<0·0001). A weak negative correlation was found between the HER2DX risk score versus the pCR score (correlation coefficient -0·19). The two HER2DX tests provide accurate estimates of the risk of recurrence, and the likelihood to achieve a pCR, in early-stage HER2-positive breast cancer. This study received funding from Reveal Genomics, IDIBAPS and the University of Padova.