Smadbeck, James B.

ABSTRACTThe MLLT10 (formerly AF10) gene is the fourth most common KMT2A fusion partner across all acute leukemias and requires at least 3 breaks to form an in‐frame KMT2A/MLLT10 fusion due to the opposite orientation of each gene. A 10‐year retrospective review was performed to identify individuals from all age groups that harbor KMT2A/MLLT10 fusion obtained by our KMT2A/MLLT10 dual‐color dual‐fusion fluorescence in situ hybridization (D‐FISH) assay. Of the 60 unique individuals identified, 31 were male and 29 were female (M:F ratio, 1.1:1) with ages ranging from 3 days to 86 years (mean 21.5 years, median 5.5 years). The diagnoses included acute myeloid leukemia (AML) (49 patients, 82%), B‐ or T‐lymphoblastic leukemia/lymphoma (7 patients, 12%), myeloid sarcoma (3 patients, 5%), and a single case (2%) of undifferentiated leukemia. Twenty‐seven of 49 patients (55%) with AML were in the infant or pediatric age group. Fifty‐three of 60 patients (88%) had KMT2A/MLLT10 D‐FISH signal patterns mostly consisting of single fusions. In addition, 10 (26%) of 38 patients with conventional chromosome studies had “normal” (5 patients) or abnormal (5 patients) chromosome studies that lacked structural or numeric abnormalities involving chromosomes 10 or 11, implying cryptic cytogenetic mechanisms for KMT2A/MLLT10 fusion. Lastly, mate‐pair sequencing was performed on 4 AML cases, 2 of which had “normal” chromosome studies and cryptic KMT2A/MLLT10 fusion as detected by KMT2A/MLLT10 D‐FISH studies, and verified the multiple breaks required to generate KMT2A/MLLT10 fusion.

AbstractObjectiveAcute 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.MethodWe evaluated whether an NGS‐based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML.ResultsUsing 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML‐specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML‐fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML‐specific structural rearrangements in 10/88 (11%) of cases analyzed.ConclusionUtilization of MPseq represents a precise, molecular‐based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies.

Abstract 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.

Objectives: 1) Describe the genetic alterations discovered in a series of sporadic vestibular schwannomas (VS). 2) Identify if more clinically aggressive variants possess different genetic alterations compared to more indolent-behaving VS. Methods: Fresh frozen tumor and matched peripheral blood leukocytes from 23 individuals with sporadic VS were analyzed using whole-exome sequencing, tumor whole transcriptome expression profiling (mRNA-Seq), and tumor mate-pair analysis. Source cases included tumors with fast preoperative growth, giant tumors in young patients, tumors with macrocystic change, recurrent tumors following radiation or microsurgery, and indolent small tumors with minimal or no growth before surgery. Somatic and germ-line alterations of the NF2 gene and beyond the NF2 locus were identified and analyzed using complementing analyses. Results: Biallelic somatic events involving the NF2 gene were discovered in every analyzed tumor specimen with no concurrent NF2 variants identified in matching peripheral blood specimens. Thirteen tumors showed loss of one chromosome 22 (ch22), 4 tumors showed copy-neutral 22q loss of heterozygosity, and 31 unique small variants in the NF2 gene were discovered. Of the latter, 10 were essential splice site, 11 frame shift, 7 stop gain, 2 missense, and 1 in-frame mutation. No other common or recurring NF2 mutations were identified. However, several other notable large chromosomal aberrations were discovered including 2 tumors with loss of a chromosome 21, 3 with loss of an X or Y chromosome, 1 with copy-neutral loss of heterozygosity in chromosome 15, and 1 with loss of 18p and 16q. All of these other major chromosomal abnormalities only occurred in tumors demonstrating a more aggressive phenotype. Conclusions: To date, few studies have used whole-exome sequencing, mate-pair analysis, and RNA-seq to profile genome-wide alterations in sporadic VS. Using high-throughput deep sequencing, “two-hit” alterations in the NF2 gene were identified in every tumor and were not present in peripheral blood supporting that all events were somatic. Type of NF2 gene alteration and accessory mutations outside the NF2 locus may predict phenotypic expression and clinical course.

Background Cahan's 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 Cahan's criteria cannot be validly applied. Objective This article proposes an update to Cahan's 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.

AbstractCopy 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.

Abstract 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. Supplementary information Supplementary data are available at Bioinformatics online.

Abstract TMPRSS2-ERG gene fusions occur in over 50% of prostate cancers, but their impact on clinical outcomes is not well understood. Retention of interstitial genes between TMPRSS2 and ERG has been reported to influence tumor progression in an animal model. In this study, we analyzed the status of TMPRSS2-ERG fusion genes and interstitial genes in tumors from a large cohort of men treated surgically for prostate cancer, associating alterations with biochemical progression. Through whole-genome mate pair sequencing, we mapped and classified rearrangements driving ETS family gene fusions in 133 cases of very low-, low-, intermediate-, and high-risk prostate cancer from radical prostatectomy specimens. TMPRSS2-ERG gene fusions were observed in 44% of cases, and over 90% of these fusions occurred in ERG exons 3 or 4. ERG fusions retaining interstitial sequences occurred more frequently in very low-risk tumors. These tumors also frequently displayed ERG gene fusions involving alternative 5′-partners to TMPRSS2, specifically SLC45A3 and NDRG1 and other ETS family genes, which retained interstitial TMPRSS2/ERG sequences. Lastly, tumors displaying TMPRSS2-ERG fusions that retained interstitial genes were less likely to be associated with biochemical recurrence (P = 0.028). Our results point to more favorable clinical outcomes in patients with ETS family fusion-positive prostate cancers, which retain potential tumor-suppressor genes in the interstitial regions between TMPRSS2 and ERG. Identifying these patients at biopsy might improve patient management, particularly with regard to active surveillance. Cancer Res; 77(22); 6157–67. ©2017 AACR.