Rajesh Patidar

MultiDimensional ClinOmics for Precision Therapy of Children and Adolescent Young Adults with Relapsed and Refractory Cancer: A Report from the Center for Cancer Research

Purpose: We undertook a multidimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome-guided precision therapy. Experimental Design: Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single-nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with FDA-approved drugs. Results: Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7 months to 25 years old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation. Conclusions: We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next-generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors. Clin Cancer Res; 22(15); 3810–20. ©2016 AACR.

Massively Parallel Sequencing Reveals an Accumulation of De Novo Mutations and an Activating Mutation of LPAR1 in a Patient with Metastatic Neuroblastoma

Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy). Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

High-Throughput Molecular and Histopathologic Profiling of Tumor Tissue in a Novel Transplantable Model of Murine Neuroblastoma: New Tools for Pediatric Drug Discovery

Using two MYCN transgenic mouse strains, we established 10 transplantable neuroblastoma cell lines via serial orthotopic passage in the adrenal gland. Tissue arrays demonstrate that by histochemistry, vascularity, immunohistochemical staining for neuroblastoma markers, catecholamine analysis, and concurrent cDNA microarray analysis, there is a close correspondence between the transplantable lines and the spontaneous tumors. Several genes closely associated with the pathobiology and immune evasion of neuroblastoma, novel targets that warrant evaluation, and decreased expression of tumor suppressor genes are demonstrated. These studies describe a unique and generalizable approach to expand the utility of transgenic models of spontaneous tumor, providing new tools for preclinical investigation.

Frequent inactivating germline mutations in DNA repair genes in patients with Ewing sarcoma

Purpose:Ewing sarcoma is a small round blue cell tumor that is highly malignant and predominantly affects the adolescent and young adult population. It has long been suspected that a genetic predisposition exists for this cancer, but the germ-line genetic underpinnings of this disease have not been well established.Methods:We performed germline variant analysis of whole-genome or whole-exome sequencing of samples from 175 patients affected by Ewing sarcoma.Results:We discovered pathogenic or likely pathogenic germline mutations in 13.1% of our cohort. Pathogenic mutations were highly enriched for genes involved with DNA damage repair and for genes associated with cancer predisposition syndromes.Conclusion:Our findings reported here have important clinical implications for patients and families affected by Ewing sarcoma. Genetic counseling should be considered for patients and families affected by this disease to take advantage of existing risk management strategies. Our study also highlights the importance of germline sequencing for patients enrolled in precision-medicine protocols.Genet Med advance online publication 26 January 2017

A mutational comparison of adult and adolescent and young adult (AYA) colon cancer: Comparison of Adult & AYA Colon Cancer

It is possible that the relative lack of progress in treatment outcomes among adolescent and young adult (AYA) patients with cancer is caused by a difference in disease biology compared with the corresponding diseases in younger and older individuals. There is evidence that colon cancer is more aggressive and has a poorer prognosis in AYA patients than in older adult patients.

Integrated proteogenomic analysis of metastatic thoracic tumors identifies APOBEC mutagenesis and copy number alterations as drivers of proteogenomic tumor evolution and heterogeneity

Elucidation of the proteogenomic evolution of metastatic tumors may offer insight into the poor prognosis of patients harboring metastatic disease. We performed whole-exome and transcriptome sequencing, copy number alterations (CNA) and mass spectrometry-based quantitative proteomics of 37 lung adenocarcinoma (LUAD) and thymic carcinoma (TC) metastases obtained by rapid autopsy and found evidence of patient-specific, multi-dimensional heterogeneity. Extreme mutational heterogeneity was evident in a subset of patients whose tumors showed increased APOBEC-signature mutations and expression of APOBEC3 region transcripts compared to patients with lesser mutational heterogeneity. TP53 mutation status was associated with APOBEC hypermutators in our cohort and in three independent LUAD datasets. In a thymic carcinoma patient, extreme heterogeneity and increased APOBEC3AB expression was associated with a high-risk germline APOBEC3AB variant allele. Patients with CNA occurring late in tumor evolution had corresponding changes in gene expression and protein abundance indicating genomic instability as a mechanism of downstream transcriptomic and proteomic heterogeneity between metastases. Across all tumors, proteomic heterogeneity was greater than copy number and transcriptomic heterogeneity. Enrichment of interferon pathways was evident both in the transcriptome and proteome of the tumors enriched for APOBEC mutagenesis despite a heterogeneous immune microenvironment across metastases suggesting a role for the immune microenvironment in the expression of APOBEC transcripts and generation of mutational heterogeneity. The evolving, heterogeneous nature of LUAD and TC, through APOBEC-mutagenesis and CNA illustrate the challenges facing treatment outcomes.

Abstract 3840: The National Cancer Institute’s patient-derived models repository (PDMR)

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community9s highest priority need was preclinical models that more faithfully reflect the patient9s tumor and are associated with the patient9s treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute9s patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 986.

Abstract 3003: Exome analysis of known hereditary cancer genes in 122 children with rhabdomyosarcoma

Introduction. Rhabdomyosarcoma (RMS) accounts for 5% of all pediatric cancer and is the most prevalent soft tissue tumor in childhood and adolescents. RMS is thought to arise from primitive mesenchymal stem cells directed towards myogenesis. Between 7-33% of RMS cases arise from a hereditary cancer syndrome, like LFS or NF1. We analyzed germline genetic variants in hereditary cancer genes in 122 children with RMS. Methodology. In 122 children with RMS and 1001 cancer-free adults, we examined germline exome data to determine the frequency of genetic variants in 51 cancer genes known to underlie syndromes associated with RMS. DNA was extracted from blood or buccal cells using standard methods. Exome enrichment was performed with NimbleGen SeqCap EZ Human Exome Library v3.0+UTR, on an Illumina HiSeq. Annotation of each exome variant was performed using a custom software pipeline. We evaluated all variants that passed quality controls with a population minor allele frequency (MAF) Results We compared the age, gender, histologic type and localization of the primary RMS of the patients with and without P/LP variants in the 51 genes. In the patients without P/LP variants, the mean age of diagnosis was 5 years and the most frequent site of diagnosis was head and neck. In the group with P/LP variants, the mean age of diagnosis was 10 years, and the most frequent site was pelvis. In the 51 genes that were analyzed we found 9 P and 12 LP variants in 15 genes: TP53, ATM, MSH6, PMS, DICER1, FANCA, RECQL4, PTEN, WRN, RB1, BUB1B, RET, APC, FANCM and TSC2; genes with 2 variants include WRN, PTEN, BUB1B, FANCA and RET. Most of the variations were stopgain, follow by missense, frameshift insertion and splicing genetic variations. Ten of this genes are associated with an autosomal dominant pattern of inheritance. In one 15-year-old female patient with an alveolar RMS in the paraspinal area we found P/LP variants in PTEN and PMS2. The frequency of P/LP variants in cases was 16% and 3% in controls. Conclusions. To our knowledge, this is the first study where multiple germline variation where analyses in children with RMS. We found P/LP variation in 16% of all the cases (pending orthogonal confirmation); the mean age of diagnosis was 10 years old and their primary tumor site was in the pelvis (50%). Identification of P/LP variation in genes underlying RMS-associated syndromes has implications for follow-up, screening and management of these patients and their families. We acknowledge the Children’s Oncology Group in helping to assemble the RMS cohort. Citation Format: Talia Wegman-Ostrosky, Rajesh Patidar, Sivasish Sindiri, Jack Shern, Douglas S. Hawkins, Daniel Catchpoole, Jun S. Wei, Stephen Skapek, Javed Khan, Douglas R. Stewart. Exome analysis of known hereditary cancer genes in 122 children with rhabdomyosarcoma [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 3003. doi:10.1158/1538-7445.AM2017-3003

Abstract 1456: Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas

BACKGROUND: Neurofibromatosis type 1 (NF1) is a genetic tumor predisposition disorder caused by germline mutations in tumor suppressor NF1. Plexiform neurofibromas (PN) are benign tumors that arise prenatally or early in childhood and affect 30-50% of NF1 population. Somatic inactivation of second copy of NF1 is believed to be primary genetic event leading to PN initiation. NF1 patients have 8-12% lifetime risk of developing malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma, often arising from pre-existing PN and atypical NF (ANF). ANF are pre-malignant tumors that often arise within PN and can transform into MPNST. They are distinct from both PN and MPNST clinically and histologically, thus representing an intermediate step in malignant transformation. Several studies identified deletion of the CDKN2A/2B locus as the most frequent genetic event in ANF, however it is not clear whether other genes or pathways play role in PN transformation into ANF and further into MPNST. In this study, we performed genomic analysis of 16 ANF and 4 MPNST matched with normal DNA obtained from 14 and 4 patients, respectively. METHODS: We performed whole exome sequencing and whole transcriptome RNASeq analyses on Illumina Hi-Seq 2500 platform and copy-number variant (CNV) analysis on Illumina HumanOmniExpressExome-8 SNP-arrays. In addition, we performed deep sequencing of NF1 and validation of select mutations on IonTorrent platform. For select tumors we estimated growth rate and metabolic activity by using volumetric MRI and FDG-PET. RESULTS: We identified inactivation of NF1 in the majority of ANF and all MPNST. We also detected CDKN2A/B locus deletion in the majority of ANF and MPNST (heterozygous in ANF and mostly homozygous in MPNST). We determined that PRC2 genes (EED and SUZ12) were mutated in multiple MPNST but never in ANF. We identified a low number of point mutations and small indels in the genomes of ANF (median 1, range 0-4) and somewhat elevated mutation burden in MPNST (median 23, range 18-31), however none of these mutations were recurrent and none of the mutant genes (other than NF1 and CDKN2A) were present in multiple samples. We found 93 CNV per tumor (median) in ANF that constituted ~2% of their genomes. In comparison, we observed 2,249 CNV (median) in MPNST that comprised ~75% of their genomes. We didn’t detect significant correlation between growth rate or metabolic activity and the degree of genomic instability or mutation burden in the tumors, however the size of the sample set was modest. RNAseq data analysis is pending. CONCLUSIONS: It appears that PN-ANF transition is predominantly if not exclusively driven by heterozygous deletion of the CDKN2A/2B locus. Further progression to MPNST likely involves homozygous loss of CDKN2A/B and complete inactivation of the PRC2 complex. Widespread LOH in MPNST may accelerate inactivation of key gatekeepers. Citation Format: Alexander Pemov, Nancy F. Hansen, Rajesh Patidar, Christine Higham, Eva Dombi, Joseph F. Boland, Settara C. Chandrasekharappa, NIH Intramural Sequencing Center, James C. Mullikin, Margaret Wallace, Javed Khan, Eric Legius, Brigitte Widemann, Douglas R. Stewart. Identifying early genetic steps in malignant transformation of neurofibromatosis type 1- associated plexiform neurofibromas [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 1456. doi:10.1158/1538-7445.AM2017-1456

Abstract PR16: Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma

Master transcription factors establish enhancers to regulate cell identity genes by recruiting epigenetic machinery, and are sequentially exchanged during changes in cell identity (ie, differentiation). Commonly, the fusion of transcription factors profoundly alters proper progression of cell identity, serving as the signature oncogenic event in many malignancies. The most common soft tissue cancer of childhood, rhabdomyosarcoma (RMS), is characterized by an inability to exit the proliferative myoblast-like state, presumably by blocking myogenic transcription factors from advancing the active enhancer landscape. This is achieved by either chromosomal translocation resulting in the oncogenic fusion transcription factor PAX3/7-FOXO1 (Fusion-Positive alveolar subtype, FP-RMS) or mutations in the tyrosine kinase/RAS/PIK3C axis (Fusion-Negative embryonal subtype, FN-RMS). Patients who harbor a PAX-fusion typically relapse despite aggressive therapy and have very poor survival. Here we hypothesized that the PAX3-FOXO1 fusion gene causes epigenetic reprogramming resulting in increased proliferation and a failure to terminally differentiate. Furthermore we hypothesized that disrupting the epigenetic machinery recruited by this fusion gene would provide a tractable target for therapy. We mapped the landscape of epigenetic alterations caused by the PAX3-FOXO1 fusion gene using a combination of RNA-seq, DNase hypersensitivity, and ChIP-seq against histone marks and transcription factors in cell lines and models of FP-RMS. We found high expression of several master transcription factors (including MYOD1, MYOG, MYCN, and SOX8) in FP-RMS primary tumors and cell lines, resembling human skeletal muscle myoblasts. ChIP-seq revealed that PAX3-FOXO1 is exclusively bound to distal, active enhancers and the histone modification most enriched surrounding PAX3-FOXO1 was acetylated H3K27. Furthermore we found that the introduction of the fusion gene into fibroblast cells opened up the chromatin at these same sites, completely rewiring the active enhancer landscape, recapitulating a transcriptome locked in a myoblast-like state. Genome-wide profiling of MYOD1, MYOG and MYCN reveals that all three master regulators collaborative bind at nearly every PAX3-FOXO1 driven super enhancer (SE), while typical enhancers (TEs) rarely have more than two of these four. PAX3-FOXO1 has a 7-fold preference for SEs over TEs. We also find that PAX3-FOXO1 bound, myogenic enhancers are decommissioned throughout normal skeletal muscle differentiation. To identify small molecules that would inhibit the PAX3-FOXO1 induced epigenetic machinery we treated a panel of FP-RMS cell lines with 1912 targeted agents and chemical probes at multiple concentrations and measured cell viability. Classes of molecules selectively potent for PAX3-FOXO1 driven cells (as compared to normal fibroblasts) hit connected biologically relevant targets including SE controlled receptor tyrosine kinases (including FGFR4, IGF1R, ALK), and transcriptional cofactors involved in SE complexes (including HDACs and BRD). In an expanded panel of RMS cell lines we confirmed that FP-RMS is selectively sensitive to the BET bromodomain inhibitors with the most potent being JQ1. These inhibitors selectively suppress PAX3-FOXO1 dependent transcription as measured by reporter assays and RNA-seq analysis. Indeed, coactivators of looped chromatin p300, MED1 and BRD4 excessively co-localize with PAX3-FOXO1 genome wide. In vivo , JQ1 selectively ablated PAX3-FOXO1 dependent SE driven transcription, and significantly delayed tumor progression in xenografts of PAX3-FOXO1 driven cell lines. In conclusion we found that PAX3-FOXO1 establishes myogenic super enhancers that are sensitive to BET bromodomain inhibition which constitutes a novel therapeutic strategy for children with PAX-fusion driven rhabdomyosarcoma. This abstract is also presented as Poster A16. Citation Format: Berkley E. Gryder, Marielle E. Yohe, Jack Shern, Hsien-Chao Chou, Young Song, Rajesh Patidar, Sam Li, Sivasish Sindiri, Abigail Cleveland, Hongling Liao, Xinyu Wen, Xiaohu Zhang, Lesley Mathews-Griner, Rajarshi Guha, Paul Shinn, Marc Ferrer, Scott Martin, Madhu Lal, Craig Thomas, Javed Khan. Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR16.