Atish D. Choudhury

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

The Role of Genetic Markers in the Management of Prostate Cancer

CONTEXT Despite widespread screening for prostate cancer (PCa) and major advances in the treatment of metastatic disease, PCa remains the second most common cause of cancer death for men in the Western world. In addition, the use of prostate-specific antigen testing has led to the diagnosis of many potentially indolent cancers, and aggressive treatment of these cancers has caused significant morbidity without clinical benefit in many cases. The recent discoveries of inherited and acquired genetic markers associated with PCa initiation and progression provide an opportunity to apply these findings to guide clinical decision making. OBJECTIVE In this review, we discuss the potential use of genetic markers to better define groups of men at high risk of developing PCa, to improve screening techniques, to discriminate indolent versus aggressive disease, and to improve therapeutic strategies in patients with advanced disease. EVIDENCE ACQUISITION PubMed-based literature searches and abstracts through January 2012 provided the basis for this literature review. We also examined secondary sources from reference lists of retrieved articles and data presented at recent congresses. Cited review articles are only from the years 2007-2012, favoring more recent discussions because of the rapidly changing field. Original research articles were curated based on favoring large sample sizes, independent validation, frequent citations, and basic science directly related to potentially clinically relevant prognostic or predictive markers. In addition, all authors on the manuscript evaluated and interpreted the data acquired. EVIDENCE SYNTHESIS We address the use of inherited genetic variants to assess risk of PCa development, risk of advanced disease, and duration of response to hormonal therapies. The potential for using urine measurements such as prostate cancer antigen 3 (PCA3) RNA and the transmembrane protease, serine 2 v-ets erythroblastosis virus E26 oncogene homolog (avian) (TMPRSS2-ERG) gene fusion to aid screening is discussed. Multiple groups have developed gene expression signatures from primary prostate tumors correlating with poor prognosis, and attempts to improve and standardize these signatures as diagnostic tests are presented. Massive sequencing efforts are underway to define important somatic genetic alterations (amplifications, deletions, point mutations, translocations) in PCa, and these alterations hold great promise as prognostic markers and for predicting response to therapy. We provide a rationale for assessing genetic markers in metastatic disease for guiding choice of therapy and for stratifying patients in clinical trials, and discuss challenges in clinical trial design incorporating the use of these markers. CONCLUSIONS The use of genetic markers has the potential to aid disease screening, improve prognostic discrimination, and prediction of response to treatment. However, most markers have not been prospectively validated for providing useful prognostic or predictive information or improvement upon clinicopathologic parameters already in use. Significant efforts are underway to develop these research findings into clinically useful diagnostic tests in order to improve clinical decision making.

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.

Structural Alterations Driving Castration-Resistant Prostate Cancer Revealed by Linked-Read Genome Sequencing

Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%-87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.

Castration resistance in prostate cancer is mediated by the kinase NEK6

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753-65. ©2016 AACR.

Abstract 993: Whole exome sequencing of CTCs as a window into metastatic cancer

Comprehensive analysis of cancer genomes in clinical settings holds the promise to inform prognoses and guide the deployment of precise cancer treatments. A major barrier, however, is the inaccessibility of adequate metastatic tissue for accurate genomic analysis. The recognition that circulating tumor cells (CTCs) are present in many advanced cancer patients suggests an exciting opportunity to overcome this challenge. For instance, if CTCs could be comprehensively sequenced, it would be possible to obtain an orthogonal sample of the tumor burden_including subsets of transiting cells bound for metastatic colonization_potentially yielding new insights to complement the static sampling of resected or biopsied lesions. We report an integrated process to isolate, qualify, and sequence whole exomes of CTCs with high fidelity, using a census-based sequencing strategy. We isolated CTCs by magnetic bead purification (Illumina MagSweeper) from the blood of patients with prostate cancer, and integrated a nanowell platform to automatically image and recover candidate single CTCs. We then developed a strategy to qualify individual CTC-derived libraries for DNA sequencing after whole genome amplification, and established an analytical framework for accurate calling of mutations using census-based sequencing and MuTect. Whole exome sequencing was performed on 20 single CTCs, obtained from a patient with advanced prostate cancer. We validated our sequencing process by comparing CTC-derived mutations to mutations found in a lymph node metastasis and nine separate cores of the primary tumor. 51 of 73 CTC mutations (70%) were observed in the metastasis or the primary tumor. Moreover, we identified 9 early trunk mutations and 56 metastatic trunk mutations in the non-CTC tumor samples and found 100% and 73% of these, respectively, in CTC exomes. Our work demonstrates the feasibility of CTC sequencing and the ability to confidently call somatic mutations. CTCs may therefore represent a non-invasive window into the mutational landscape of metastatic cancer, and may have utility for genomics in clinical practice. Citation Format: Viktor A. Adalsteinsson, Jens G. Lohr, Kristian Cibulskis, Atish D. Choudhury, Mara Rosenberg, Peter Cruz-Gordillo, Joshua Francis, ChengZhong Zhang, Alexander K. Shalek, Rahul Satija, John T. Trombetta, Diana Lu, Naren Tallapragada, Narmin T. Tahirova, Sora Kim, Brendan Blumenstiel, Carrie Sougnez, Daniel Auclair, Eliezer M. Van Allen, Mari Nakabayashi, Rosina T. Lis, Gwo-Shu M. Lee, Tiantian Li, Matthew S. Chabot, Mary-Ellen Taplin, Thomas E. Clancy, Massimo Loda, Aviv Regev, Matthew Meyerson, William C. Hahn, Philip W. Kantoff, Todd R. Golub, Gad Getz, Jesse S. Boehm, J Christopher Love. Whole exome sequencing of CTCs as a window into metastatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2014-993

A dose finding clinical trial of cabozantinib (XL184) administered in combination with abiraterone acetate in metastatic castration-resistant prostate cancer

Cabozantinib can enhance the effect of abiraterone in preclinical prostate cancer models. This study aimed to define the recommended phase 2 dose (RP2D) and preliminary efficacy of abiraterone + cabozantinib in mCRPC.

Enrichment of FGFR3-TACC3 Fusions in Patients With Bladder Cancer Who Are Young, Asian, or Have Never Smoked

PurposeFGFR3-TACC3 (fibroblast growth factor receptor 3–transforming acidic coiled coil-containing protein 3) fusions have recently been identified as driver mutations that lead to the activation of FGFR3 in bladder cancer and other tumor types and are associated with sensitivity to tyrosine kinase inhibitors. We examined the clinical and molecular characteristics of patients with FGFR3-TACC3 fusions and hypothesized that they are enriched in a subset of patients with bladder cancer.Materials and MethodsWe correlated somatic FGFR3-TACC3 fusions with clinical and molecular features in two cohorts of patients with bladder cancer. The first cohort consisted of the muscle-invasive bladder cancer (MIBC) data set (n = 412) from The Cancer Genome Atlas. The second cohort consisted of patients with MIBC or high-grade non-MIBC at the Dana-Farber Cancer Institute that had targeted capture sequencing of a selected panel of cancer genes (n = 356). All statistical tests were two sided. The clinical response of one pat...

Abstract 5689: Identify tissue-of-origin in cancer cfDNA by whole genome sequencing

Cell free DNA (cfDNA) has been shown to be an emerging non-invasive biomarker to monitor tumor progression in cancer patients. Elevated cfDNA has been found not only from tumors, but also from normal tissues. Thus, the identification of cfDNA’s tissue-of-origin is critical to understand the mechanism of cfDNA release and tumor progression. Recent efforts to identify cfDNA’s tissue-of-origin begin to utilize cfDNA’s epigenomic status, such as DNA methylation and nucleosome spacing. However, both of these methods have limitations: (1) For nucleosome positioning, lack of reference nucleosome maps in different tumor and normal tissues has limited its application to tissue-of-origin deconvolution; (2) For DNA methylation, large DNA degradation during whole genome bisulfite sequencing (WGBS) library preparation, even with current low-input DNA technology, is still the major hurdle for its clinical application, although extensive DNA methylation studies by WGBS in tumor and normal tissues during the last decade have provided many reference maps. Very recently, a pioneer study showed significant differences between DNA fragment lengths of methylated and unmethylated cfDNA. Taking advantage of this experimental observation, we developed a machine learning approach to infer the base pair resolution DNA methylation level from fragment size information in whole genome sequencing (WGS). The predicted DNA methylation, from not only high coverage but also dozens of ultra-low-pass WGS (ULP-WGS), showed high concordance with the ground truth DNA methylation level from WGBS in the same cancer patients. Furthermore, by using hundreds of WGBS datasets from different tumor and normal tissues/cells as the reference map, we deconvoluted cfDNA’s tissue-of-origin status by inferred DNA methylation level at ULP-WGS from thousands of breast/prostate cancer samples and healthy individuals. The cfDNA’s tissue-of-origin status in cancer patients showed high concordance with confirmed metastasis tissues from physicians. Interestingly, some clinical information, such as cancer grades/stages, seemed to be correlated with cfDNA’s tissue-of-origin status. Overall, our methods here pave the road for cfDNA’s application in clinical diagnosis and monitoring. Citation Format: Yaping Liu, Sarah Reed, Atish D. Choudhury, Heather A. Parsons, Daniel G. Stover, Gavin Ha, Gregory Gydush, Justin Rhoades, Denisse Rotem, Samuel Freeman, Viktor Adalsteinsson, Manolis Kellis. Identify tissue-of-origin in cancer cfDNA by whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5689. doi:10.1158/1538-7445.AM2017-5689

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.