Vasmatzis, George

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo‐resistant disease after initial response to standard‐of‐care treatment with platinum‐based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient‐derived xenografts (PDX) allow validation of personalized treatments. Using a whole‐genome mate‐pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS‐OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti‐HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2‐targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo‐resistant lines. When chemotherapy and HER2‐targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post‐treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS‐OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2‐targeted therapy.

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 The mechanistic target of rapamycin (mTOR) is a central regulator of cellular proliferation and metabolism. Depending on its binding partners, mTOR is at the core of 2 complexes that either promote protein biosynthesis (mTOR complex 1; mTORC1) or provide survival and proliferation signals (mTORC2). Protein biosynthesis downstream of mTORC1 plays an important role in MYC-driven oncogenesis with translation inhibitors garnering increasing therapeutic attention. The germinal center B-cell oncogene UCHL1 encodes a deubiquitinating enzyme that regulates the balance between mTOR complexes by disrupting mTORC1 and promoting mTORC2 assembly. While supporting mTORC2-dependent growth and survival signals may contribute to its role in cancer, the suppression of mTORC1 activity is enigmatic, as its phosphorylation of its substrate 4EBP1 promotes protein biosynthesis. To address this, we used proximity-based proteomics to identify molecular complexes with which UCH-L1 associates in malignant B cells. We identified a novel association of UCH-L1 with the translation initiation complex eIF4F, the target of 4EBP1. UCH-L1 associates with and promotes the assembly of eIF4F and stimulates protein synthesis through a mechanism that requires its catalytic activity. Because of the importance of mTOR in MYC-driven oncogenesis, we used novel mutant Uchl1 transgenic mice and found that catalytic activity is required for its acceleration of lymphoma in the Eμ-myc model. Further, we demonstrate that mice lacking UCH-L1 are resistant to MYC-induced lymphomas. We conclude that UCH-L1 bypasses the need for mTORC1-dependent protein synthesis by directly promoting translation initiation, and that this mechanism may be essential for MYC in B-cell malignancy.

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.

Abstract Ovarian cancer (OC) is most lethal gynecologic malignancy. About 75% of OC patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in OC are reported to vary and responses to HER2 inhibitors have been feeble. Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using whole-genome mate-pair next generation sequencing protocol we profiled a set of serous OC to identify copy number changes and gene fusions. Specific focus was on DNA alterations which were therapeutically targetable. The efficiency of anti-Her2 therapy was tested in three different PDX lines with alterations at genes of ERBB pathway and various levels of HER2 protein expression. Tumor in one line harbored a duplication of ERBB2 and high expression of HER2 protein. Second PDX model had a fusion at NRG1 gene, a ligand for HER4 and HER4 receptors, that was highly expressed on protein level. Third line harbored an amplification of NRG3 gene, a known ligand for HER4 receptor. Treatment responses to pertzumab/trastuzumab were compared in each PDX line to standard carboplatin and paclitaxel combination treatment. In all three PDX models HER2-targeted therapy resulted in significant inhibition of tumor growth compared to untreated control. However, the responses in each were inferior to chemotherapy, even in chemo-resistant case. There was an extra benefit when chemotherapy and HER-2 targeted therapy were administered together, as a significant regression of tumor was observed after 6 weeks of treatment using this regiment compared to chemotherapy alone. Post-treatment analysis of the tissues, nevertheless, revealed inhibition of ERBB2 pathway on the level of phosphorylation and expression of downstream targets. In conclusion, targeting of presumably activated ERBB2 pathway alone in OC results in a modest treatment benefit. A combination therapy including both chemo drugs and Her2 inhibitors provides far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to Her2 targeted therapy. Citation Format: Irina V. Kovtun, Zhang Piyan, Faye R. Harris, Xiaonan Hou, Saravut J. Weroha, George Vasmatzis. Targeting ERBB2 pathway in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A19.

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.

Abstract The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2–LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Mol Cancer Res; 16(10); 1556–67. ©2018 AACR.

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.