Anne-Katrin Emde

Detection of a Recurrent DNAJB1-PRKACA Chimeric Transcript in Fibrolamellar Hepatocellular Carcinoma

Oncogenic Suspect Exposed It can be difficult logistically to study the genomics of rare variants of common cancers. Nevertheless, Honeyman et al. (p. 1010) studied fibrolamellar hepatocellular carcinoma (FL-HCC), a rare and poorly understood liver tumor that affects adolescents and young adults and for which there is no effective treatment. FL-HCCs from 15 patients all expressed a chimeric RNA transcript and protein containing sequences from a molecular chaperone fused in frame with sequences from the catalytic domain of protein kinase A. The chimeric protein retained kinase activity in vitro. Such recurrent gene fusions in cancer may signal a role in pathogenesis and provide an opportunity for therapeutic intervention. A rare form of liver cancer affecting young adults expresses a chimeric kinase that may contribute to pathogenesis. Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

BackgroundColorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.ResultsWe performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.ConclusionsColorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response

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.

Disease variants in genomes of 44 centenarians

To identify previously reported disease mutations that are compatible with extraordinary longevity, we screened the coding regions of the genomes of 44 Ashkenazi Jewish centenarians. Individual genome sequences were generated with 30× coverage on the Illumina HiSeq 2000 and single‐nucleotide variants were called with the genome analysis toolkit (GATK). We identified 130 coding variants that were annotated as “pathogenic” or “likely pathogenic” based on the ClinVar database and that are infrequent in the general population. These variants were previously reported to cause a wide range of degenerative, neoplastic, and cardiac diseases with autosomal dominant, autosomal recessive, and X‐linked inheritance. Several of these variants are located in genes that harbor actionable incidental findings, according to the recommendations of the American College of Medical Genetics. In addition, we found risk variants for late‐onset neurodegenerative diseases, such as the APOE ε4 allele that was even present in a homozygous state in one centenarian who did not develop Alzheimers disease. Our data demonstrate that the incidental finding of certain reported disease variants in an individual genome may not preclude an extraordinarily long life. When the observed variants are encountered in the context of clinical sequencing, it is thus important to exercise caution in justifying clinical decisions.

Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

Objective: To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Methods: Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. Results: More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. Conclusions: The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. ClinicalTrials.gov identifier: NCT02725684.

Erratum: PGBD5 promotes site-specific oncogenic mutations in human tumors

Nat. Genet.; doi:10.1038/ng.3866; corrected online 24 May 2017 In the version of this article initially published online, the affiliations for Jiali Zhuang listed an incorrect present address instead of an equal contribution. The error has been corrected in the print, PDF and HTML versions of this article.

Whole Genome Sequencing-Based Discovery of Structural Variants in Glioblastoma

Next-generation DNA sequencing (NGS) technologies are currently being applied in both research and clinical settings for the understanding and management of disease. The goal is to use high-throughput sequencing to identify specific variants that drive tumorigenesis within each individuals tumor genomic profile. The significance of copy number and structural variants in glioblastoma makes it essential to broaden the search beyond oncogenic single nucleotide variants toward whole genome profiles of genetic aberrations that may contribute to disease progression. The heterogeneity of glioblastoma and its variability of cancer driver mutations necessitate a more robust examination of a patients tumor genome. Here, we present patient whole genome sequencing (WGS) information to identify oncogenic structural variants that may contribute to glioblastoma pathogenesis. We provide WGS protocols and bioinformatics approaches to identify copy number and structural variations in 41 glioblastoma patient samples. We present how WGS can identify structural diversity within glioblastoma samples. We specifically show how to apply current bioinformatics tools to detect EGFR variants and other structural aberrations from DNA whole genome sequencing and how to validate those variants within the laboratory. These comprehensive WGS protocols can provide additional information directing more precise therapeutic options in the treatment of glioblastoma.

Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors: Clinical Application of Whole-Genome Sequencing for Reporting Targetable Variants in Cancer

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patients tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York States Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.

Next-Generation Rapid Autopsies Enable Tumor Evolution Tracking and Generation of Preclinical Models

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 pre-mortem 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.

Abstract 2714: Analytical validation of clinical whole genome and transcriptome sequencing of patient derived tumors: clinical application of whole genome sequencing for reporting targetable variants in cancer

Next Generation DNA Sequencing (NGS) technologies are currently being applied in the clinical setting for the treatment of disease. The goal is to use high-throughput sequencing to identify specific variants within each tumor and recommend personalized treatment approaches or clinical trials tailored to the individual’s disease and genomic profile. These assays are comprised of either predefined sequencing panels, where a handpicked set of clinically significant genes are examined within each patient, or are cancer type specific targeted sequencing protocols or whole exome platforms covering only the coding region of the patient’s genome. Whole genome sequencing allows hypothesis-free interrogation of both coding and non-coding regions of the genome revealing more potential therapeutic options than examining a small set of genes or genomic loci. The protocol eliminates sequence capture related bias observed in whole exome or panel sequencing. The New York Genome Center therefore has performed analytical validation of whole genome and transcriptome sequencing (WGTS) of patient derived tumors and matched normals for the purposes of clinical testing and have devised a clinical reporting strategy of significant driver and therapeutic associated mutations. Many clinical NGS guidelines are directed toward targeted panel or exome sequencing validation. Here, we expanded on New York State’s Department of Health NGS guidelines developing them into novel standards applicable to WGTS for the purposes of clinical test validation. We first sequenced a virtual tumor at very high coverage (300x) and downsampled to determine the optimum depth of sequencing necessary for high confidence somatic variant calling across the entire genome. We then validated whole genome sequencing laboratory protocols for DNA and RNA sequencing on a total of 50 specimens derived from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumor samples. We performed a series of experiments to assess the accuracy and reliability of the results based on our laboratory and bioinformatics protocols. We performed our validation on the 50 tumor normal pairs, a subset of which had known genomic profiles. Comparisons were also made for variant calling concordance and reproducibility between matched FF and FFPE tumors. Here, we present our validation results and clinical WGTS standards for depth of sequencing, reproducibility, sensitivity, and present limit of detection analysis for SNV calling, copy number identification and structural variants. RNA sequencing is performed to call fusion or exon skipping events and to confirm the DNA variants. The New York Genome Center WGTS clinical assay is intended to provide a more comprehensive patient variant discovery approach suitable for directed oncological therapeutic applications. Citation Format: Kazimierz O. Wrzeszczynski, Avinash Abhyankar, Vanessa Felice, Esra Dikoglu, Lukasz Kozon, Nicolas Robine, Anne-Katrin Emde, Olca Basturk, Umesh K. Bhanot, Alex Kentsis, Mahesh Mansukhani, Govind Bhagat, Vaidehi Jobanputra. Analytical validation of clinical whole genome and transcriptome sequencing of patient derived tumors: clinical application of whole genome sequencing for reporting targetable variants in cancer [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 2714. doi:10.1158/1538-7445.AM2017-2714