Nasser, Sara A.

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 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.

Abstract Background: Genomic assessment of cancer has been revolutionized by Next-Generation sequencing and is increasingly being applied to guide clinical prognostic and therapeutic decision-making. Initial clinical applications have been limited to gene panels and whole exome based strategies due to challenges with time to reporting of results, specimen quantity, analyte quality, and ethical-legal-social issues (ELSI). Methods: Excisional or core tumor biopsies or bone marrow biopsies were obtained from consenting participants. Nucleic acid was extracted from fresh or frozen samples followed by WGS/WES/RNASeq on the Illumina HiSeq2000 or HiSeq2500 and bioinformatics analysis. Therapeutic targets were then prioritized by a multi-disciplinary Genomic Tumor Board (GTB) and CLIA validated using Sanger sequencing, RT-qPCR, FISH and/or IHC. Treatment was delivered using on/off-label FDA approved drugs, available clinical trials and single patient INDs. Results: We consented 64 and enrolled 35 patients with advanced, treatment-refractory cancers. Median age was 59 (range 27-91) with 62% male. The majority had ECOG scores of 1 (94%). A median of 79 (range 28-8891) potentially functional somatic point mutations were identified in each case. One to two mutations/case were further identified as therapeutically targetable in 60% of the cases. The median time from tissue acquisition to CLIA validated results was 116 days (range 42-282) with CLIA validation of targets achieved in 21 of 22 patients. Genomic, target directed treatment was ultimately instituted in 13 patients utilizing: on/off label FDA approved drugs (n = 9), clinical trial (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in 5 patients (2 PR, 3 SD). Integration of WES, long-insert WGS and RNA-Seq identified a case with an ERRFI1 mutant allele present in only 11% of the DNA reads while 82% of the RNA-Seq reads had the mutant transcript. The patient was treated with erlotinib and achieved a partial response by RECIST criteria. In another case, a fusion between FGFR2-MGEA5 was observed in WES and RNA-Seq data leading to prioritization as a drug target. Reasons for patients not receiving targeted therapy included: inability to access treatment (n = 1), death prior to intended treatment (n = 3), results returned after death (n = 3), no targets identified (n = 2) and decision to pursue alternate therapy (n = 5). Conclusions: Integrating whole genome analysis in a CLIA setting is not only feasible, but also valuable, in the prioritization and selection of potential targeted therapies for patients with advanced tumors. Continued barriers to broad application include the need for shorter time to reporting as well as broad availability of therapies through basket studies. Citation Format: Mitesh J. Borad, Jan Egan, Mia Champion, Katherine Hunt, Robert McWilliams, Ann McCullough, Jessica Aldrich, Sara Nasser, Winnie Liang, Michael Barrett, David Craig, Ramesh Ramanathan, John Carpten, A. Keith Stewart, Alan Bryce. Implementation of CLIA enabled integrated whole genome (WGS)/exome (WES)/transcriptome (RNAseq) next-gen sequencing to identify therapeutically relevant targets in advanced cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT112. doi:10.1158/1538-7445.AM2015-CT112

Abstract Glioblastoma is the most common and lethal type of primary brain tumor. Despite recent therapeutic advances in other cancers, the treatment of glioblastomas remains ineffective and essentially palliative due the highly invasive nature of the disease. To discern molecular mechanisms that drive GBM migration, we investigated the potential role of differential expression of microRNAs (miRNA) by comparing the matched large-scale, genome-wide miRNA expression profiles of migrating and migration-restricted human glioma cells. Using a radial migration assay, migrating and non-migrating populations from seven well-established glioma cell lines were isolated and utilized for miRNA expression analysis. Statistical analyses revealed 22 miRNAs common to all seven glioma cell lines were down-regulated in the migrating cell population (p < 0.0005) relative to cells in the non-migratory population. Specifically, we report here that the expression level of miR-23b was markedly down-regulated in migrating glioma cells as compared to migration-restricted cells. Over-expression of miR-23b significantly inhibited glioma cell migration and invasion. A bioinformatics search revealed a conserved target site within the 3’untranslated region (UTR) of Pyk2, a non-receptor tyrosine kinase that has been previously implicated in the regulation of glioma cell migration and invasion. Increased expression of miR-23b reduced the expression level of Pyk2 in glioma cells indicating that miR-23b targets Pyk2. Notably, increased expression of miR-23b did not alter the expression level of the related focal adhesion kinase FAK indicating that miR-23b specifically inhibits the expression Pyk2. Taken together, our study demonstrates that reduced expression of miR-23b facilitates the enhancement of glioma cell migration and invasion via modulation of Pyk2 expression. Thus, these data suggest that certain miRNAs may regulate glioma migration and invasion to influence the progression of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3994. doi:10.1158/1538-7445.AM2011-3994