Karla Helvie

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors

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.Identifying the mutational landscape of tumours from cell-free DNA in the blood could help diagnostics in cancer. Here, the authors present ichorCNA, software that quantifies tumour content in cell free DNA, and they demonstrate that cell-free DNA whole-exome sequencing is concordant with metastatic tumour whole-exome sequencing.

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Abstract S1-01: Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer

Background: While great strides have been made in the treatment of estrogen receptor-positive (ER+) metastatic breast cancer (MBC), therapeutic resistance invariably occurs. A better understanding of the underlying resistance mechanisms is critical to enable durable control of this disease. Methods: We performed whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) on metastatic tumor biopsies from 88 patients with ER+ MBC who had developed resistance to one or more ER-directed therapies. For 27 of these patients, we sequenced the treatment-naive primary tumors for comparison to the resistant specimens. Tumors were analyzed for point mutations, insertions/deletions, copy number alterations, translocations, and gene expression. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of all metastatic samples demonstrated several recurrently altered genes whose incidence differed significantly from primary, treatment-naive ER+ breast cancers sequenced in the TCGA study (TCGA). These include ESR1 mutations (n=17, 19.3%; 32.86 fold enrichment, q.value Comparing to matched primary samples from the same patient, many alterations were found to be acquired in several cases, including for ESR1, ERBB2, PIK3CA, PTEN, RB1, AKT1, and others. Initial analysis of RNA-seq data from metastatic samples (n=59) allowed classification of individual resistance mechanisms into broader resistance modes based on the observed transcriptional state. Conclusions: We present a genomic landscape of resistant ER+ MBC using WES and RNA-seq. Multiple genes were recurrently altered in these tumors at significantly higher rates than in ER+ primary breast cancer. When compared with matched primary tumors from the same patient, alterations in these and other genes were often found to be acquired after treatment, suggesting a role in resistance to ER-directed therapies and/or metastasis. Potential resistance mechanisms appear to fall into several categories; integrating RNA-seq data may enhance the ability to identify these categories even when genomic alterations are not identified. Multiple clinically relevant genomic and molecular alterations are identified in metastatic biopsies– with implications for choice of next therapy, clinical trial eligibility, and novel drug targets. Citation Format: Cohen O, Kim D, Oh C, Waks A, Oliver N, Helvie K, Marini L, Rotem A, Lloyd M, Stover D, Adalsteinsson V, Freeman S, Ha G, Cibulskis C, Anderka K, Tamayo P, Johannessen C, Krop I, Garraway L, Winer E, Lin N, Wagle N. Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-01.

Abstract LB-136: High concordance of whole-exome sequencing of cell-free DNA and matched biopsies enables genomic discovery in metastatic cancer

Background: Circulating cell-free DNA (cfDNA) has largely been used to monitor blood for specific tumor mutations, but genome-wide discovery from cfDNA has not been well established. Here, we establish a scalable approach for whole-exome sequencing (WES) of cfDNA, making it possible to perform comprehensive genomic characterization of metastatic cancer in a routine and minimally-invasive manner. Comprehensive genomic characterization of metastatic cancer stands to uncover novel alterations of clinical significance. A major challenge is that metastatic tumors are infrequently biopsied. Cell-free DNA is shed abundantly into the bloodstream from metastatic tumors, presenting an opportunity for genomic discovery in advanced cancers that are rarely biopsied in routine clinical care. We report an efficient process to qualify and sequence whole-exomes from cfDNA at scale and systematically compare the somatic mutations, indels, and copy number alterations detected in WES of cfDNA to WES of matched tumor biopsies. Methods: We consented 86 patients with metastatic breast or prostate cancers for blood collection. We isolated cfDNA and germline DNA from blood and performed low coverage sequencing to estimate tumor content based on genome-wide copy number. We screened patient blood samples and prioritized those with higher tumor fractions for WES. In parallel, we analyzed cfDNA and germline DNA from healthy donors to calibrate our methods and assess false positive rate for genomic alterations. Results: We found the vast majority of patients with metastatic prostate or breast cancer to have detectable tumor-derived cfDNA. WES of cfDNA from healthy donors revealed very low false positive rates for somatic mutations, indels and copy number alterations (SCNAs). By analyzing WES of cfDNA and tumor biopsies from dozens of patients with metastatic breast or prostate cancers, we established guidelines for the coverage and tumor fraction required for mutation discovery in WES of cfDNA. We found WES of cfDNA to uncover 91% of the clonal mutations, 59% of the subclonal mutations, and 75% of the SCNAs detected in WES of matched tumor biopsies. In several cases, we observed mutations exclusive to cfDNA that were confirmed in later blood draws, suggesting that cfDNA-exclusive mutations may be derived from unsampled metastases. In some cases, cfDNA revealed clinically actionable mutations that were not detected in matched tumor biopsies. Conclusions: WES of cfDNA uncovers the majority of somatic mutations, indels, and SCNAs found in matched tumor biopsies of metastatic cancer. The high degree of concordance suggests that comprehensive sequencing of cfDNA can be leveraged for genomic discovery in settings where conventional biopsies are difficult to access. Furthermore, the detection of mutations in cfDNA that are not detected in concurrent biopsies suggests that cfDNA may be complementary to tumor biopsies for both translational studies and precision cancer medicine. Citation Format: Viktor A. Adalsteinsson, Gavin Ha, Sam Freeman, Atish D. Choudhury, Daniel G. Stover, Heather A. Parsons, Gregory Gydush, Sarah Reed, Denis Loginov, Dimitri Livitz, Daniel Rosebrock, Ignat Leshchiner, Ofir Cohen, Coyin Oh, Jaegil Kim, Chip Stewart, Mara Rosenberg, Huiming Ding, Maxwell R. Lloyd, Sairah Mahmud, Karla E. Helvie, Margaret S. Merrill, Rebecca A. Santiago, Edward P. O’Connor, Seong H. Jeong, Joseph F. Kramkowski, Jens G. Lohr, Laura Polacek, Nelly Oliver, Lori Marini, Joshua Francis, Lauren C. Harshman, Eliezer M. Van Allen, Eric P. Winer, Nancy U. Lin, Mari Nakabayashi, Mary-Ellen Taplin, Levi A. Garraway, Todd R. Golub, Jesse S. Boehm, Nikhil Wagle, Gad Getz, Matthew Meyerson, Christopher J. Love. High concordance of whole-exome sequencing of cell-free DNA and matched biopsies enables genomic discovery in metastatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-136.