Harris, Faye R.

AbstractA crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment‐naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome‐wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome‐wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.

7568 Background: The molecular events in the initiation and progression of invasive lung adenocarcinoma remain poorly characterized. These tumors are thought to develop through an adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), invasive adenocarcinoma (AD) sequence. The goal of our study was to assess lineage relationships between in situ and invasive components within adenocarcinomas with prominent lepidic growth patterns. Methods: Frozen tissue from fourteen lung adenocarcinomas with mixtures of AD together with an adjacent in situ component (AIS), varying in ratio between 40 to 80%, were selected from our Lung Specimen Registry. Laser capture microdissection (LCM) of each component was performed separately for each tumor. Genomic DNA was isolated using a direct in situ whole genome amplification (WGA) methodology, and Next Generation Sequencing performed using an Illumina Mate Pair (MP) library protocol. MP sequence reads were mapped to the human genome and primers spanning the fusion junctions were used in validation PCRs. Results: Genomic break points identical and unique to a specific patient were identified between the AIS and AD components in 13 (of 14) cases. The total number of events per case ranged from 4-215, and the number of identical events shared between the AIS and AD components ranged from 30 to 90%. Recurrent genomic breakpoints between cases were also observed, with the 2 most common involving 8q24.3 in 5 cases and FAM19A2 on chromosome 12 in 4 cases. PCR validation of selected genomic alterations confirmed the genomic break points identified from sequencing in both the AIS and AD in all cases. Interestingly, we also observed a limited number of genomic alterations present in a zonal distribution of surrounding normal lung around the tumor mass. Conclusions: Our study demonstrates unique chromosomal alterations present in both the AIS and AD components of individual lung tumors with features of lepidic growth. These data provide evidence for lineage relationship and clonal relatedness between the two components, and provide genomic evidence for a model of stepwise progression from AIS to invasive AD in this subset of lung adenocarcinomas.

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.

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.

Abstract Purpose: The treatment of ovarian cancer poses a challenge since 70% of patients will relapse with incurable platinum resistant disease. Although ovarian cancers lack frequently-occurring driver mutations or amplifications, the study presented herein examines the concept of individualized therapy based on mate pair sequencing and protein expression in ovarian cancer patient-derived xenografts. Experimental Procedure: Macrodissection of patient tumor was followed by genomic DNA isolation and next-generation sequencing using an Illumina Mate Pair protocol to identify structural genomic changes and copy number variations. Secondary analyses were carried out to select potential therapeutic targets among those. The tumors were also propagated intraperitoneally in immunocompromised mice and treated with standard chemotherapy or/and targeted therapy chosen based on genomic analyses. Results: Mate pair analysis revealed that, in general, copy number changes (amplifications, gains and losses) were common in ovarian tumors and included genes potentially targetable. Tumors from early generation patient- derived xenografts showed a landscape of genomic aberrations identical to that of original patient tumor. One PDX model in which high expression of HER2 and copy number gains at RICTOR and AKT genetic loci were revealed, was treated with either chemotherapy and Pertuzumab/trastuzumab (HER2 inhibitors), MK-8669 (mTOR inhibitor) or MK-2206 (AKT inhibitor) or chemotherapy alone. Tumor size was measured by serial abdominal ultrasound and after 28 days, the final tumor size ratio compared to baseline was 0.23, 0.36, 0.55 and 0.56, respectively, indicating that the best response was seen in the combined chemotherapy and Pertuzumab/trastuzumab cohort and that was better than in the chemotherapy only cohort. Conclusions: Patient-derived xenografts models in conjunction with genomic and protein analyses of patient tumors are valuable for testing of combination therapies including specific targeting drugs. This approach also provides actionable information for more tailored treatment of ovarian cancer patients. Citation Format: Konstantinos Leventakos, Faye R. Harris, Lin Yang, Xiaonan Hou, Saravut Weroha, Irina V. Kovtun. Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts [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 4814. doi:10.1158/1538-7445.AM2017-4814

Abstract Over the recent years significant knowledge has been accumulated on which drugs can be therapeutically beneficial for a given patient based on mutational profiling of their tumors using selected gene panels. Much less, however, is known under which conditions targeting of genes which are amplified could be beneficial. Apart from successfully targeting HER2-positive cancers, many of which frequently carry amplified ERBB2 gene, no other targeted therapy based on DNA amplifications or gains of the genes is offered to the patients due to lack of clinical response data. Using mate pair sequencing we have profiled 60 high grade serous ovarian tumors to compare genomic rearrangements and copy number changes. The analyses revealed that these tumors carry highly rearranged genome. Copy number changes amplifications, gains and losses were very common and included genes potentially therapeutically targetable. In contrast, only a few of targetable fusion genes were identified in a set of 60 cases. The tumors were propagated in immunocompromised mice (mouse avatars), sequenced and showed identical to original patient tumor landscape of rearrangements. Using these mouse avatar models we tested whether a therapy inhibiting selected based on genomic amplification targets provided a benefit compared to standard chemotherapy. Mice which harbored tumors showing amplifications of CDKs (CDK2, 4 and 9) and ERBB3 were treated with flavopiridol and pertuzumab respectively, and response was compared to that of standard for ovarian cancer chemotherapy (carboplatin and paclitaxel combination). We observed that application of either agent, significantly inhibited tumor growth in treated mice as compared to untreated. However, neither drug alone caused tumor regression. The treatment regimen then was modified to administer chemotherapy and targeted therapy together. A mouse avatars in which genomic analyses revealed amplification at loci involving AKT3 and RICTOR genes was treated with chemotherapy alone and in combination with MK2206 (AKT inhibitor) or MK8669 (mTOR inhibitor). The high level of both target proteins was confirmed by immunoblotting. Significant regression of tumor was observed after 6 weeks of treatment. Addition of either targeted drug provided an extra benefit in killing tumor cells compared to chemotherapy alone. The mice are followed to study tumor recurrence. These data indicated that genomic information obtained using next generation sequencing can aid therapy decisions by tailoring treatments to individual tumor makeup. Avatar models in conjunction with genomic analyses of the original tumors are invaluable for testing specific targeted therapies selected based on amplification/gains of putative genes-targets and for studying mechanisms of tumor recurrence and therapy resistance. Citation Format: Faye R. Harris, Lin Yang, Xiaonan Hou, Konstantinos Leventakos, Saravut J. Weroha, George Vasmatzis, Irina V. Kovtun. Targeting genes which are amplified in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A34.

Abstract Circulating tumor DNA (ctDNA) provides great potential for the non-invasive clinical assessment of tumor burden. We developed a monitoring protocol using next generation mate-pair sequencing to identify chromosomal rearrangement signatures in primary tumors to follow ctDNA in blood. Primary tumors of 10 patients with ovarian cancer were sequenced, and individualized tumor-specific panels were designed to detect the junctions in ctDNA. A novel algorithm was developed and applied to data generated by quantitative PCR to calculate the percent of ctDNA. Selected junctions, some with potential as therapeutic targets, were detected in pre-surgically drawn blood in eight out of ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA post-surgery, and five had undetectable levels, consistent with their documented presence or absence of disease. The detection of somatic junctions derived from ctDNA could be an effective way to monitor ovarian cancer patients for relapse and therapy response. Citation Format: Faye R. Harris, Irina Kovtun, James Smadbeck, Francesco Multinu, Aminah Jatoi, Kimberly R. Kalli, Stephen J. Murphy, Geoffrey C. Halling, Farhad Kosari, Sarah H. Johnson, Andrea Mariani, George Vasmatzis. Quantification of somatic chromosomal rearrangements in circulating cell-free DNA from ovarian cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 440.