James B. Smadbeck

Quantification of Somatic Chromosomal Rearrangements in Circulating Cell-Free DNA from Ovarian Cancers.

Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA.

Integrated Genomic Analysis of Pancreatic Ductal Adenocarcinomas Reveals Genomic Rearrangement Events as Significant Drivers of Disease

Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFβ signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression.

YAP-associated chromosomal instability and cholangiocarcinoma in mice

Deregulated Hippo pathway signaling is associated with aberrant activation of the downstream effector yes-associated protein (YAP), an emerging key oncogenic mediator in cholangiocarcinoma (CCA). In our prior work, we have demonstrated that biliary transduction of YAP along with Akt as a permissive factor induces CCA in mice. To further delineate the mechanisms associated with YAP-associated biliary oncogenesis, we have established seven malignant murine cell lines from our YAP-driven murine CCA model. These cells express the CCA markers SRY (Sex Determining Region Y)-Box 9 (SOX9), cytokeratin (CK)-7 and 19 but lack hepatocyte nuclear factor 4 alpha and alpha-smooth muscle actin, markers of hepatocellular carcinoma and cancer-associated fibroblasts, respectively. Notably, the murine CCA cells can be readily implanted into mouse livers with resultant orthotopic tumor formation. In this unique syngeneic orthotopic murine model, tumors exhibit histopathologic features resembling human CCA. We analyzed transcriptome data from YAP-associated parent CCA tumor nodules and identified a gene expression pattern associated with chromosomal instability, known as CIN25. Similarly, mate-pair sequencing of the murine CCA cells revealed chromosomal missegregation with gains and losses of several whole chromosomes demonstrating aneuploidy. Of the CIN25 genes, forkhead box M1 (Foxm1), a key cell cycle regulator, was the most significantly upregulated CIN25 gene product. Accordingly, small interfering RNA (siRNA)-mediated silencing of YAP as well as FOXM1 inhibition with thiostrepton induced CCA cell death. These preclinical data imply a role for YAP-mediated chromosomal instability in cholangiocarcinoma, and suggest FOXM1 inhibition as a therapeutic target for CCA.

Copy number variant analysis using genome-wide mate-pair sequencing

Copy number variation (CNV) is a common form of structural variation detected in human genomes, occurring as both constitutional and somatic events. Cytogenetic techniques like chromosomal microarray (CMA) are widely used in analyzing CNVs. However, CMA techniques cannot resolve the full nature of these structural variations (i.e. the orientation and location of associated breakpoint junctions) and must be combined with other cytogenetic techniques, such as karyotyping or FISH, to do so. This makes the development of a next‐generation sequencing (NGS) approach capable of resolving both CNVs and breakpoint junctions desirable. Mate‐pair sequencing (MPseq) is a NGS technology designed to find large structural rearrangements across the entire genome. Here we present an algorithm capable of performing copy number analysis from mate‐pair sequencing data. The algorithm uses a step‐wise procedure involving normalization, segmentation, and classification of the sequencing data. The segmentation technique combines both read depth and discordant mate‐pair reads to increase the sensitivity and resolution of CNV calls. The method is particularly suited to MPseq, which is designed to detect breakpoint junctions at high resolution. This allows for the classification step to accurately calculate copy number levels at the relatively low read depth of MPseq. Here we compare results for a series of hematological cancer samples that were tested with CMA and MPseq. We demonstrate comparable sensitivity to the state‐of‐the‐art CMA technology, with the benefit of improved breakpoint resolution. The algorithm provides a powerful analytical tool for the analysis of MPseq results in cancer.

Genome U-Plot: a whole genome visualization

Motivation: The ability to produce and analyze whole genome sequencing (WGS) data from samples with structural variations (SV) generated the need to visualize such abnormalities in simplified plots. Conventional two‐dimensional representations of WGS data frequently use either circular or linear layouts. There are several diverse advantages regarding both these representations, but their major disadvantage is that they do not use the two‐dimensional space very efficiently. We propose a layout, termed the Genome U‐Plot, which spreads the chromosomes on a two‐dimensional surface and essentially quadruples the spatial resolution. We present the Genome U‐Plot for producing clear and intuitive graphs that allows researchers to generate novel insights and hypotheses by visualizing SVs such as deletions, amplifications, and chromoanagenesis events. The main features of the Genome U‐Plot are its layered layout, its high spatial resolution and its improved aesthetic qualities. We compare conventional visualization schemas with the Genome U‐Plot using visualization metrics such as number of line crossings and crossing angle resolution measures. Based on our metrics, we improve the readability of the resulting graph by at least 2‐fold, making apparent important features and making it easy to identify important genomic changes. Results: A whole genome visualization tool with high spatial resolution and improved aesthetic qualities. Availability and implementation: An implementation and documentation of the Genome U‐Plot is publicly available at https://github.com/gaitat/GenomeUPlot. Contact: vasmatzis.george@mayo.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Proposal for Modification of Cahan's Criteria Utilizing Molecular Genetic Analyses for Cases without Baseline Histopathology: A Unique Method Applicable to Primary Radiosurgery

Background Cahans criteria have been utilized since 1948 to establish causality between prior radiation treatment and the development of secondary malignancy. One major criterion specifies that histological and radiographic evidence collected before and after radiation treatment must confirm separate tumor types; however, pretreatment biopsy is rarely obtained prior to radiosurgery for vestibular schwannoma and many other skull base and cranial lesions. Therefore, in these cases Cahans criteria cannot be validly applied. Objective This article proposes an update to Cahans criteria using modern molecular genetic analysis for cases lacking baseline histopathology. Methods Mate‐pair sequencing and whole exome sequencing of a cerebellopontine angle undifferentiated high‐grade pleomorphic sarcoma (UHGPS) that developed after stereotactic radiosurgery of a presumed benign vestibular schwannoma. Results Mate‐pair sequencing and whole exome sequencing of the sarcoma revealed complex chromosomal aberrations. Notably, the tumor contained a deletion in the NF2 gene at 22q12 and an in‐frame deletion on exon 5 of the remaining copy of NF2. Biallelic events impacting NF2 are atypical for UHGPS but are characteristic for vestibular schwannoma. These findings help support the conclusion that the UHGPS arose from a benign vestibular schwannoma all along. Conclusions Next‐generation sequencing can be successfully applied to a radiation‐induced sarcoma when both the original and malignant tumors harbor separate signature genetic markers. As our understanding of the genetic profile of various tumors expand, we believe that next‐generation sequencing and other genomic tools will play an increasingly important role in establishing causality between radiation and the development of secondary malignancy.

Neoantigenic potential of complex chromosomal rearrangements in mesothelioma

Introduction: Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Whereas other carcinogen‐related tumors are associated with a high tumor mutation burden, mesothelioma is not. We sought to resolve this discrepancy. Methods: We used mate‐pair (n = 22), RNA (n = 28), and T cell receptor sequencing along with in silico predictions and immunologic assays to understand how structural variants of chromosomes affect the transcriptome. Results: We observed that inter‐ or intrachromosomal rearrangements were present in every specimen and were frequently in a pattern of chromoanagenesis such as chromoplexy or chromothripsis. Transcription of rearrangement‐related junctions was predicted to result in many potential neoantigens, some of which were proven to bind patient‐specific major histocompatibility complex molecules and to expand intratumoral T cell clones. T cells responsive to these predicted neoantigens were also present in a patients circulating T cell repertoire. Analysis of genomic array data from the mesothelioma cohort in The Cancer Genome Atlas suggested that multiple chromothriptic‐like events negatively impact survival. Conclusions: Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel immunotherapeutic strategies and the selection of patients to receive immunotherapies.

Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia

Acute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints.

Abstract 5726: Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma

Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [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 5726.

Chromoanasynthesis is a common mechanism that leads to ERBB2 amplifications in a cohort of early stage HER2+ breast cancer samples

BackgroundHER2 positive (HER2+) breast cancers involve chromosomal structural alterations that act as oncogenic driver events.MethodsWe interrogated the genomic structure of 18 clinically-defined HER2+ breast tumors through integrated analysis of whole genome and transcriptome sequencing, coupled with clinical information.ResultsERBB2 overexpression in 15 of these tumors was associated with ERBB2 amplification due to chromoanasynthesis with six of them containing single events and the other nine exhibiting multiple events. Two of the more complex cases had adverse clinical outcomes. Chromosomes 8 was commonly involved in the same chromoanasynthesis with 17. In ten cases where chromosome 8 was involved we observed NRG1 fusions (two cases), NRG1 amplification (one case), FGFR1 amplification and ADAM32 or ADAM5 fusions. ERBB3 over-expression was associated with NRG1 fusions and EGFR and ERBB3 expressions were anti-correlated. Of the remaining three cases, one had a small duplication fully encompassing ERBB2 and was accompanied with a pathogenic mutation.ConclusionChromoanasynthesis involving chromosome 17 can lead to ERBB2 amplifications in HER2+ breast cancer. However, additional large genomic alterations contribute to a high level of genomic complexity, generating the hypothesis that worse outcome could be associated with multiple chromoanasynthetic events.