Liang, Winnie S.

Abstract Background Tumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions. Methods Whole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens. Results Private mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions. Conclusions Subclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences.

Abstract Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT. Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT. Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%. Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Abstract Purpose: Glioblastoma is an aggressive and molecularly heterogeneous cancer with few effective treatment options. We hypothesized that next-generation sequencing can be used to guide treatment recommendations within a clinically acceptable time frame following surgery for patients with recurrent glioblastoma. Experimental Design: We conducted a prospective genomics-informed feasibility trial in adults with recurrent and progressive glioblastoma. Following surgical resection, genome-wide tumor/normal exome sequencing and tumor RNA sequencing were performed to identify molecular targets for potential matched therapy. A multidisciplinary molecular tumor board issued treatment recommendations based on the genomic results, blood–brain barrier penetration of the indicated therapies, drug–drug interactions, and drug safety profiles. Feasibility of generating genomics-informed treatment recommendations within 35 days of surgery was assessed. Results: Of the 20 patients enrolled in the study, 16 patients had sufficient tumor tissue for analysis. Exome sequencing was completed for all patients, and RNA sequencing was completed for 14 patients. Treatment recommendations were provided within the study's feasibility time frame for 15 of 16 (94%) patients. Seven patients received treatment based on the tumor board recommendations. Two patients reached 12-month progression-free survival, both adhering to treatments based on the molecular profiling results. One patient remained on treatment and progression free 21 months after surgery, 3 times longer than the patient's previous time to progression. Analysis of matched nonenhancing tissue from 12 patients revealed overlapping as well as novel putatively actionable genomic alterations. Conclusions: Use of genome-wide molecular profiling is feasible and can be informative for guiding real-time, central nervous system–penetrant, genomics-informed treatment recommendations for patients with recurrent glioblastoma. Clin Cancer Res; 24(2); 295–305. ©2017 AACR. See related commentary by Wick and Kessler, p. 256

Abstract Archival tumor samples present a rich resource of annotated specimens for genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available with archival tissue. Without a matched normal sample, it is very difficult to distinguish between true somatic variants and germline variants that are private to the individual. Archival FFPE sections often contain adjacent normal tissue, but this normal tissue is often contaminated with infiltrating tumor cells. Comparative somatic variant callers are designed to exclude variants present in the normal sample, so a novel approach is required to leverage sequencing of adjacent normal tissue. Here we present LumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient. The approach is based on the concept that the allelic fraction of somatic variants, but not germline variants, would be diluted by the presence of normal tissue. Given the allele specific copy number state and expected tumor fraction of each sample, one can model the expected allelic fractions of somatic and germline variants. LumosVar estimates allele specific copy number and tumor sample fractions from the data, and uses the model to call somatic and germline variants. LumosVar is also able to detect sub clonal events and spatial heterogeneity between samples. To evaluate this approach, we used a glioblastoma dataset, where enhancing (high tumor content) and non-enhancing (low tumor content) samples were available for each patient, as well as the matched normal sample to define the true somatic variants. We show that both sensitivity and positive predictive value are improved by analyzing the high tumor and low tumor jointly compared to analyzing the samples individually or in silico pooling of the two samples. Finally, we apply this approach to a set of breast and prostate archival FFPE tumor samples where normal samples were not available for germline sequencing. Citation Format: Rebecca F. Halperin, Winnie S. Liang, Erica Tassone, Jonathan Adkins, Megan Russell, Nhan Tran, Nicole Hank, James Newell, Sandeep Gupta, Michael Berens, Ronald Korn, David W. Craig, Sara A. Byron. Leveraging spatial heterogeneity in tumor purity for improved somatic variant calling of archival tumor-only samples [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 LB-007. doi:10.1158/1538-7445.AM2017-LB-007

Abstract Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. Contrasting with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis, and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM. Citation Format: Winnie S. Liang, William Hendricks, Jeffrey Kiefer, Jessica Schmidt, Shobana Sekar, John Carpten, David W. Craig, Jonathan Adkins, Lori Cuyugan, Zarko Manojlovic, Rebecca F. Halperin, Adrienne Helland, Sara Nasser, Christophe Legendre, Laurence H. Hurley, Karthigayini Sivaprakasam, Douglas B. Johnson, Holly Crandall, Klaus J. Busam, Victoria Zismann, Valerie De Luca, Jeeyun Lee, Aleksandar Sekulic, Charlotte E. Ariyan, Jeffrey Sosman, Jeffrey Trent. Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma [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 3389. doi:10.1158/1538-7445.AM2017-3389

463 Background: The genomic assessment of cancer has been revolutionized by next-generation sequencing and is increasingly being applied in the clinic to guide therapeutic decision-making. Time to reporting of results, specimen quantity, and analyte quality have constrained initial clinical applications to gene panels and whole exome based strategies. Methods: Patients underwent surgical resection, excisional or core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing and RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients with advanced, treatment-refractory cancers of whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in the CLIA enabled phase. Upon availability of the initial report, identified targets of putative therapeutic relevance were then prioritized by the CGB in 22/27 patients (81%) for subsequent CLIA validation. Eleven patients have been treated with genomically selected therapy with partial response in 3/10 assessed patients. A testicular cancer patient had aberrations in two testes specific genes, a copy number gain in TSSK6 and a novel gene fusion between thyroid hormone receptor associated protein 3 (THRAP3) and Tektin 2 (TEKT2). Additionally, a case of papillary renal cell carcinoma had an amplification of YAP1 and a previously unreported P287T mutation in CCND1, suggesting potential benefit with a CDK4/6 inhibitor. Treatment is ongoing and results will be reported. Conclusions: Integrated whole genome analysis in a CLIA setting is feasible. Integration of SNV, copy number and transcriptional data may allow for selection of putative driver genes to enhance targeted therapy decisions. Barriers for future broader implementation include the need for reduced time from biopsy to report and availability of therapies.