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Optimal treatment for advanced cervical cancer after first line chemotherapy remains undefined. Immune checkpoint inhibition with pembrolizumab, a programmed cell death protein 1(PD-1) inhibitor, is under investigation. We analyzed the micro-environmental and molecular genetic profile of tumors from 4 patients with metastatic cervical cancer treated with off-label second-line pembrolizumab in an effort to identify predictive biomarkers. All patients received 2 mg/kg of pembrolizumab, 3-weekly until disease progression. Immunohistochemistry(IHC) for PD-1, PD-L1, CD3 and CD8, as well as next generation sequencing (NGS) for 50 cancer-related genes were performed on tumor samples. All patients tolerated treatment well with no discontinuation of treatment due to toxicity. One patient experienced dramatic and prolonged partial response, and remains stable on pembrolizumab with a progression free survival (PFS) of 21 months at the time of reporting of this series. Three patients experienced disease progression as best response. In the exceptional responder, there was no tumoral expression of PD-L1, however, combined positive score (CPS) for PD-L1 was 1 and we identified somatic mutations in ERBB4(R612W), PIK3CA(E542K) and RB1(E365K). In 2 patients, despite progressive disease defined by RECIST v1.1, symptom stabilization on pembrolizumab was observed. The tumors of both patients had PD-1 expression in ≥1% of stromal lymphocytes. All patients with response or clinical benefit had CPS for PD-L1 ≥ 1. NGS revealed PIK3CA mutations in 3 tumors. Pembrolizumab is a promising therapeutic option in advanced cervical cancer. Further evaluation of biomarkers may guide optimal patient selection., Highlights • 1 of 4 patients receiving second line pembrolizumab, experienced dramatic and durable response. • All patients with response or clinical benefit had CPS for PD-L1 ≥ 1. • Germline BRCA1 mutation was detected in 1 patient, but she was a non-responder. • All tumors had somatic mutation in PIK3CA or PTEN.

Summary: Therapies targeting epidermal growth factor receptor (EGFR) have variable and unpredictable responses in breast cancer. Screening triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), we identify a subset responsive to EGFR inhibition by gefitinib, which displays heterogeneous expression of wild-type EGFR. Deep single-cell RNA sequencing of 3,500 cells from an exceptional responder identified subpopulations displaying distinct biological features, where elevated EGFR expression was significantly enriched in a mesenchymal/stem-like cellular cluster. Sorted EGFRhi subpopulations exhibited enhanced stem-like features, including ALDH activity, sphere-forming efficiency, and tumorigenic and metastatic potential. EGFRhi cells gave rise to EGFRhi and EGFRlo cells in primary and metastatic tumors, demonstrating an EGFR-dependent expansion and hierarchical state transition. Similar tumorigenic EGFRhi subpopulations were identified in independent PDXs, where heterogeneous EGFR expression correlated with gefitinib sensitivity. This provides new understanding for an EGFR-dependent hierarchy in TNBC and for patient stratification for therapeutic intervention. : Savage et al. demonstrate that sensitivity to EGFR inhibitor, gefitinib, in triple-negative breast cancer is paradoxically associated with EGFR heterogeneity. Using single-cell RNA sequencing in conjunction with functional assays, they identify TNBC tumors in which EGFR expression identifies cells with tumor-initiating capacity whose proliferative expansion is sensitive to EGFR inhibition. Keywords: breast cancer, tumor heterogeneity, patient-derived xenograft, single-cell RNA sequencing, EGFR inhibition, therapeutic response, tumor-initiating cell, cell hierarchy, BRCA1 mutation