Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

Clinical resistance mechanisms to CDK4/6 inhibitors in HR+ breast cancer have not been clearly defined. Whole exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of ER expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Besides inactivation of RB1, which accounts for ~5% of resistance, seven of these mechanisms have not been previously identified as clinical mediators of resistance to CDK4/6 inhibitors in patients. Three of these-RAS activation, AKT activation, and AURKA activation-have not to our knowledge been previously demonstrated preclinically. Together, these eight mechanisms were present in 80% of resistant tumors profiled and may define therapeutic opportunities in patients. SignificanceWe identified eight distinct mechanisms of resistance to CDK4/6 inhibitors present in 80% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precisionbased approaches to overcome resistance in many patients with HR+ MBC..

Preclinical data support a role for the IL-6/JAK2/STAT3 signaling pathway in breast cancer. Ruxolitinib is an orally bioavailable receptor tyrosine inhibitor targeting JAK1 and JAK2. We evaluated the safety and efficacy of ruxolitinib in patients with metastatic breast cancer. This was a non-randomized phase II study enrolling patients with refractory, metastatic triple-negative breast cancer. The primary endpoint was objective response by RECIST 1.1. The study was designed to enroll patients whose archival tumor tissue was pSTAT3-positive (T-score >5) by central immunohistochemistry. pSTAT3 staining was available from 171 of 217 consented patients and pSTAT3 T-score was positive in 67/171 (39.2%) tumors, suggesting that JAK–STAT activation is frequent. Twenty-three patients including one patient with inflammatory breast cancer were enrolled. Ruxolitinib was well-tolerated with infrequent grade 3 or higher toxicities with fatigue as the most common toxicity. Among 21 patients who received at least one dose of protocol therapy, no objective responses were observed and the study was closed to further accrual. Pharmacodynamic analyses of baseline vs. cycle 2 biopsies suggest on-target activity, including a significant decrease in the proportion of pSTAT3+ cells in three patients with paired biopsies and downregulation of JAK–STAT target genes and signatures via transcriptional analyses of 11 total baseline and four metastatic biopsies. Immuno-FISH analyses demonstrate intratumoral heterogeneity of pSTAT3 and JAK2 amplification. Ruxolitinib, as a single agent, did not meet the primary efficacy endpoint in this refractory patient population despite evidence of on-target activity.