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BACKGROUND Metastatic biopsies are increasingly being performed in patients with advanced prostate cancer to search for actionable targets and/or to identify emerging resistance mechanisms. Due to a predominance of bone metastases and their sclerotic nature, obtaining sufficient tissue for clinical and genomic studies is challenging. METHODS Patients with prostate cancer bone metastases were enrolled between February 2013 and March 2017 on an institutional review board-approved protocol for prospective image-guided bone biopsy. Bone biopsies and blood clots were collected fresh. Compact bone was subjected to formalin with a decalcifying agent for diagnosis; bone marrow and blood clots were frozen in optimum cutting temperature formulation for next-generation sequencing. Frozen slides were cut from optimum cutting temperature cryomolds and evaluated for tumor histology and purity. Tissue was macrodissected for DNA and RNA extraction, and whole-exome sequencing and RNA sequencing were performed. RESULTS Seventy bone biopsies from 64 patients were performed. Diagnostic material confirming prostate cancer was successful in 60 of 70 cases (85.7%). The median DNA/RNA yield was 25.5 ng/μL and 16.2 ng/μL, respectively. Whole-exome sequencing was performed successfully in 49 of 60 cases (81.7%), with additional RNA sequencing performed in 20 of 60 cases (33.3%). Recurrent alterations were as expected, including those involving the AR, PTEN, TP53, BRCA2, and SPOP genes. CONCLUSIONS This prostate cancer bone biopsy protocol ensures a valuable source for high-quality DNA and RNA for tumor sequencing and may be used to detect actionable alterations and resistance mechanisms in patients with bone metastases. Cancer 2017. © 2017 American Cancer Society.

PURPOSE We developed a precision medicine program for patients with advanced cancer using integrative whole-exome sequencing and transcriptome analysis. PATIENTS AND METHODS Five hundred fifteen patients with locally advanced/metastatic solid tumors were prospectively enrolled, and paired tumor/normal sequencing was performed. Seven hundred fifty-nine tumors from 515 patients were evaluated. RESULTS Most frequent tumor types were prostate (19.4%), brain (16.5%), bladder (15.4%), and kidney cancer (9.2%). Most frequently altered genes were TP53 (33%), CDKN2A (11%), APC (10%), KTM2D (8%), PTEN (8%), and BRCA2 (8%). Pathogenic germline alterations were present in 10.7% of patients, most frequently CHEK2 (1.9%), BRCA1 (1.5%), BRCA2 (1.5%), and MSH6 (1.4%). Novel gene fusions were identified, including a RBM47-CDK12 fusion in a metastatic prostate cancer sample. The rate of clinically relevant alterations was 39% by whole-exome sequencing, which was improved by 16% by adding RNA sequencing. In patients with more than one sequenced tumor sample (n = 146), 84.62% of actionable mutations were concordant. CONCLUSION Integrative analysis may uncover informative alterations for an advanced pan-cancer patient population. These alterations are consistent in spatially and temporally heterogeneous samples.

Genomic testing with next-generation sequencing (NGS) has become a pillar of precision medicine, whose aim is to identify the genomic alterations of a patient’s tumor and provide guidelines to clinicians for optimal treatment. Clinical testing is typically performed with targeted panels interrogating a limited set of genes, selected based on our best scientific knowledge on their diagnostic or prognostic role. Despite more recent efforts to be more inclusive, most genomic databases have a limited representation of non-European populations, resulting in a biased selection of those genes, and the potential exclusion of under-represented groups from the benefit of precision medicine. At the Englander Institute for Precision Medicine (EIPM), we developed a whole-exome sequencing (WES) clinical test, EXaCT-1, which interrogates about 21,000 protein coding genes for single-nucleotide variants, indels, and copy number. EXaCT-1 enables an unbiased view of the genomic landscape of a patient’s tumor and allows for the collection of data to investigate genomic diversity. We also tackled one of the major barriers of precision medicine: the infrastructure to execute clinical sequencing. From ordering a test, collecting and processing samples, to the analysis and review of the data and generation of reports, several systems, procedures, and expertise are involved, and their effective coordination is a key component for the timely delivery of results. We have built a framework supporting the entire process of clinical genomic testing: a Laboratory Information Management System (LIMS) helps the clinical lab to receive orders, acquire and process specimens, and seamlessly communicate with the sequencers and the computational pipelines. Molecular pathologists use NGSReporter, a secure web application, to review the data and sign-out reports. NGSReporter integrates the results of a test with our Precision Medicine Knowledge Base (PMKB – https://pmkb.weill.cornell.edu), which classifies variants based on their relevance to clinical management and provides standardized interpretations. Reports are sent to the electronic health record (EHR) as PDFs as well as discrete entities, enabling queries such as: “Which Hispanic patients with KRAS mutations are diabetic?” Sharing de-identified data is also a key aspect of precision medicine. To this end, we provide our investigators and collaborators with a protected cBioPortal instance that, in addition to publicly available datasets, includes internal data, thus enabling the exploration of hypotheses about the role of alterations across different cohorts and clinical features. Being in the center of New York City has the added benefit of an ethnically diverse patient population. Finding the “right treatment for the right person and at the right time” requires a concerted effort of multiple partners. The EIPM infrastructure facilitates these efforts, with the goal of making precision medicine accessible to everyone. Citation Format: Andrea Sboner, Cora Sternberg, Juan Miguel Mosquera, Wei Song, Michael Kluk, Wayne Tam, Hanna Rennert, David Pisapia, Jeffrey Catalano, Gloria Cheang, David Wilkes, Danielle Bulaon, M. Laura Martin, Alexandros Sigaras, Kenneth Eng, Rohan Bareja, Rob Kim, Massimo Loda, Olivier Elemento. Precision medicine at Weill Cornell Medicine/New York Presbyterian: Breaking silos, integrating resources, being inclusive [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr IA33.

SummaryToward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%–60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.

Purpose Patients with cancer who graciously consent for autopsy represent an invaluable resource for the study of cancer biology. To advance the study of tumor evolution, metastases, and resistance to treatment, we developed a next-generation rapid autopsy program integrated within a broader precision medicine clinical trial that interrogates pre- and postmortem tissue samples for patients of all ages and cancer types. Materials and Methods One hundred twenty-three (22%) of 554 patients who consented to the clinical trial also consented for rapid autopsy. This report comprises the first 15 autopsies, including patients with metastatic carcinoma (n = 10), melanoma (n = 1), and glioma (n = 4). Whole-exome sequencing (WES) was performed on frozen autopsy tumor samples from multiple anatomic sites and on non-neoplastic tissue. RNA sequencing (RNA-Seq) was performed on a subset of frozen samples. Tissue was also used for the development of preclinical models, including tumor organoids and patient-derived xenografts. Results Three hundred forty-six frozen samples were procured in total. WES was performed on 113 samples and RNA-Seq on 72 samples. Successful cell strain, tumor organoid, and/or patient-derived xenograft development was achieved in four samples, including an inoperable pediatric glioma. WES data were used to assess clonal evolution and molecular heterogeneity of tumors in individual patients. Mutational profiles of primary tumors and metastases yielded candidate mediators of metastatic spread and organotropism including CUL9 and PIGM in metastatic ependymoma and ANKRD52 in metastatic melanoma to the lung. RNA-Seq data identified novel gene fusion candidates. Conclusion A next-generation sequencing–based autopsy program in conjunction with a premortem precision medicine pipeline for diverse tumors affords a valuable window into clonal evolution, metastasis, and alterations underlying treatment. Moreover, such an autopsy program yields robust preclinical models of disease.

Importance Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. Objective To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. Design, Setting, and Patients Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. Main Outcomes and Measures Feasibility, use of WES for decision making, and identification of novel biomarkers. Results A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. Conclusions and Relevance The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Background: Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low frequency mutations. We describe an evidence-based precision medicine program designed to bring whole exome sequencing into clinical practice and illustrate how this can be utilized to identify novel predictive biomarkers associated with exceptional systemic response. Methods: Metastatic and treatment-resistant cancer patients are prospectively enrolled under an IRB approved protocol for paired metastatic tumor and normal WES through our Precision Medicine Clinic. Using a comprehensive computational pipeline, point mutations, indels and copy number alterations are detected. Mutations are categorized as Category I-III based on actionability and a report is generated and discussed in tumor board. Patients are followed prospectively for correlation of molecular information with clinical response and patient outcomes. A rapid response functional team follows on key findings to expand actionability based on the WES results. Results: In the first year, 154 tumor-normal pairs from 97 patients were sequenced, with an average coverage of 95X of over 21,000 genes and 16 somatic alterations detected on average per patient. In total, 16 mutations were Category I, 98 were Category II, and 1474 were Category III. Tumor purity ranged from 14 to near 100%. Among unexpected findings, a prostate cancer exceptional responder was identified that harbored a somatic hemizygous deletion of the DNA repair gene FANCA and likely loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. Conclusions: The establishment of a clinical program for whole exome sequencing of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response. Citation Format: Himisha Beltran, Kenneth Eng, Juan Miguel Mosquera, Alexandros Sigaras, Alessandro Romanel, Hanna Rennart, Myriam Kossai, Chantal Pauli, Bishoy Faltas, Jacqueline Fontugne, Brian Robinson, David M. Nanus, Scott T. Tagawa, Jenny Z. Xiang, Francesca Demichelis, David Rickman, Andrea Sboner, Olivier Elemento, Mark A. Rubin. Precision cancer medicine program for whole-exome sequencing of metastatic tumors reveals biomarkers of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4745. doi:10.1158/1538-7445.AM2015-4745