Sherri Z. Millis

11599 Background: Immune checkpoint inhibitors have therapeutic benefit in the treatment of various cancers; however, an elucidation of the molecular determinants of response to such therapy is needed. A high nonsynonymous mutation burden in tumors is associated with improved response to checkpoint blockade. In this study we determined the relationship of several altered genes/pathways in solid tumor samples that appeared to be significantly mutated (SM). Methods: Comprehensive genomic profiling was performed on 374 advanced solid tumor samples. Targeted sequencing was performed on 562 cancer associated genes, and analysis of matched tumor-normal DNA was performed to filter out germline variants and identify true somatic mutations. Samples with greater than 17 mutations were considered SM based on extrapolation from other reports and considering the number of genes interrogated by our panel. Results: Our analysis revealed that 13% (48/374) of tumors were significantly mutated, exhibiting 17 or greater nonsynonymous mutations occurring in a wide variety of solid tumors including but not limited to lung, CRC and melanoma. Over 90% of mutations were either frameshift, missense or splice site alterations, which may result in tumor-specific neoantigens and increased amounts of tumor-infiltrating lymphocytes (TILs). Loss of function mutations in MMR genes indicative of MMR deficiency which has been associated with increased response to immune checkpoint blockade were present in 21% (10/48) of SM cases. Mutations in polymerase e (POLE), indicative of high neoantigen load, were identified in 17% (8/48) of SM cases. Genes in SWI/SNF pathway were mutated in 65% (31/48) of SM cases, which included loss of function mutations in ARID1A. Mutations in DNA repair genes, including homologous recombination, base excision and nucleotide excision repair pathway, were noted in over half of the cases with the hypermutant phenotype. Conclusions: Our findings suggest that 13% of solid tumors have high nonsynonymous mutation burden which may be associated with benefit from checkpoint inhibitor therapy. Targeted gene panel sequencing may identify significantly mutated cases that are potential candidates for clinical trials with immune-therapeutics.

603 Background: Anal squamous cell carcinoma (Anal SCC) is a rare, HPV-associated malignancy accounting for 2.4% of digestive system cancers. In most cases, these malignancies are detected in the early stages and successfully managed with chemoradiation therapy. Uncommonly, these cancers recur or present with metastases. In this setting, cisplatin and 5-fluorouracil represent the only endorsed regimen. Once beyond standard therapy, few therapeutic options exist for those patients with aggressive disease. The purpose of this study is to identify other novel, potential targets and therapeutic options for this disease, utilizing a multiplatform approach. Methods: 206 anal SCC specimenswere tested via a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ) consisting of gene sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]) and gene amplification (CISH or FISH). 6 cases were documented as positive for HPV or HIV; status was not provided on the remaining 200. Results: Key results are shown in the table below, shown as percent change/total cases. Conclusions: Multiplatform tumor profiling identified a low incidence of gene mutations. Protein expression aberrations identified potential treatment options not routinely considered, such as topoisomerase inhibitors and taxanes. Mutations in PIK3CA, Akt1, and FBXW7 as well as PTEN loss indicate potential for targeting the PI3 kinase pathway. Additionally, immunomodulatory agents may be a therapeutic option, based on PD-1 expression levels. Targeting the ErbB-family receptors, namely with anti-EGFR agents or newer generation pan-HER - inhibitors, may represent another option, given EGFR and HER2 amplification as well as EGFR overexpression. It should be mentioned that differences in anal carcinomas whose etiology is of viral origin may present different treatment options based on the driver mutations. [Table: see text]

283 Background: Effective treatment strategies for hepatocellular carcinoma (HCC) remain limited. Identification of additional therapies remains paramount as currently available agents have resulted in marginal improvements in overall survival. Methods: 313 HCC sampleswere evaluated using a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ), including gene sequencing (Sanger, NGS [N=79]), protein expression (IHC) and gene amplification (ISH). Results: Biomarker changes of interest are shown. See Table. TP53 was mutated in 28%, CTNNB1 in 23%, and BRCA2 in 20%; other gene mutation rates were < 5%.TP53-mutated tumors show significantly higher TOPO2A (89% vs. 39%, p

Comprehensive tumor profiling identifies numerous biomarkers of drug response in cancers of unknown primary site: Analysis of 1806 cases Comprehensive tumor profiling identifies numerous biomarkers of drug response in cancers of unknown primary site: Analysis of 1806 cases

5570 Background: Non-epithelial malignancies of the ovary account for <2%–4% of all ovarian tumors. The classification of non-epithelial ovarian cancer can be broadly divided into two histological subtypes, ovarian germ cell tumors (OGCTs) and ovarian sex cord stromal tumors (OSCSTs). Molecular phenotypes of these groupings may identify genetic susceptibilities to existing therapies and thus improve patient care. Methods: 275 non-epithelial ovarian cancers (non-EOC) were profiled by Caris Life Sciences between 2009 and 2013 using a multiplatform approach. Within this cohort, patients could be further grouped according to histopathological subtypes, particularly OGCTs (n=42) and OSCSTs (n=217). Testing of FFPE tissues included a combination of sequencing (Sanger, NGS and pyrosequencing), protein expression (IHC), gene amplification (CISH or FISH), and/or RNA fragment analysis. Results: Of interest, the frequency of positive hormone receptor over-expression was higher in OSCSTs than OGCTs(AR 76%v 0%, PR 90% v 12%, ER 12% v 0%). Granulosa Cell tumors (n=173) were particularly strongly expressing for AR (81.7%), PR (95.9%) but not for ER (9.3%), with Alk, APC, and ATM mutations occurring rarely. Sertoli-leydig tumors (n=25) were characterized by high Topo2A (66.7%), TUBB3 (65%), BRAF (14.3%) and kras (20%) mutation, with lower AR (38%) and PR (60%) and higher ER (28%) expression than granulosa cell tumors. OGCT (n=42) cohorts exhibited RRM1 (51%), TLE3 (55%), TOP2A (87%), TUBB3 (51%), PTEN loss (67%) with mutations of P53 (56%) and PI3K (22%) also occurring. Kras mutation was observed in 1 of 9 OGCTs tested. Conclusions: Tumor profiling has identified molecular differences between non-EOC histological subtypes, by both expression and NGS mutational approaches in FFPE tissues. Identification of these changes can provide a rationale for treatment options not routinely considered or those associated with targeted therapies and warrant future clinical trials in this cancer type

E15229 Background: Adenosquamous (AS), mucinous adenocarcinoma (MA), anaplastic (AP), acinar cell (Ac), and squamous (Sq) are rare exocrine pancreas cancer subtypes. Very little molecular analysis has been done in these tumor subtypes. No standard therapy exists for patients with these malignancies. Methods: Of 2400 pancreatic cancers referred to Caris Life Sciences globally, 116 cases were found to be rare subtypes (shown below) based on pathology and clinical history. Specific testing was performed per physician request, including sequencing (Sanger or NGS), protein expression (immunohistochemistry), and gene amplification (CISH or FISH). Results: IHC, Sequencing, and ISH analysis are shown in the accompanying table. Conclusions: Thisanalysisshows heretofore unsuspected leads to be considered for treatments of these rare variants of exocrine pancreas cancer. It is surprising to see the spectrum of different profiles among these tumor subtypes, including amplification of EGFR in of four of the subtypes. Profiling also shows possible roles of more traditional chemotherapies such as temozolomide (due to low MGMT), doxorubicin and etoposide (TOP2A inhibitors), nab-paclitaxel (SPARC inhibition), and platinum therapies (agents that target ERCC1). While the sample size is small, the heterogeneity in profiling results within each exocrine pancreas cancer subtype indicates that molecular profiling may be appropriate to provide maximum information regarding patient’s tumor.

107 Background: Prostate cancer is the second leading cause of cancer-related death among men in the U.S. Forty percent of men diagnosed will develop metastatic disease, which has few treatment options. We aim to describe the molecular profile of prostate cancer tumors and potential for novel therapeutic options. Methods: We reviewed profiling data of over 330 patients from a large referral laboratory (Caris Life Sciences, Phoenix, AZ) for biomarkers of drug response. Multiple methodologies were employed: sequencing (NGS, Sanger, pyrosequencing), in-situ hybridization (fluorescent and chromogenic), and immunohistochemistry. Results: High expression was observed for AR, MRP1, TOPO1, TLE3 and EGFR, with positivity rates of 89%, 87%, 63%, 48%, and 47%, respectively. Low expression was observed for TS, PGP, TUBB3, RRM1, PTEN and MGMT, with negativity rates of 94%, 87%, 75%, 69%, 54%, and 45%, respectively. Gene copy number increases for EGFR and cMYC were observed in 13% of patients. Sequencing data showed a 48% mutation rate for TP53, 18% for PTEN, 9% for CTNNB1, 8% for PIK3CA, 5% for RB1, ATM, and cMET, and approximately 2% for K/HRAS, ERBB4, ALK, BRAF, and cKIT. Targeted therapy options include imatinib for patients with high cKIT or PDGFRA (9 to 10%) and cetuximab for patients with EGFR positivity (13 to 47%). Promising agents may be considered, including cabozantinib, based on 4% of cohort with cMET aberrations or PAM pathway inhibitors (BEZ234, everolimus) based on approximately 30% of cohort with PIK3CA pathway activation. Lastly, HDAC inhibitors have recently been linked to cMYC driven cancers (13% amplified). 5-FU, gemcitabine, and temozolomide chemotherapies may be options, as approximately 70% of cohort with low TS, RRM1, or MGMT. Biomarker guidance for common prostate cancer drugs such as cabazitaxel is also provided, based on approximately 70% of cohort with low TUBB3 or PGP, or high TLE3. Also, continued dependence on androgen signaling is exhibited by 89% of cohort with high AR, indicating potential utility of anti-androgen agents like enzalutamide. Conclusions: Tumor profiling identified small subsets of patients that may benefit from targeted agents approved for other solid tumors (imatinib, cetuximab), promising therapies in clinical trials (cabozantinib) or agents not routinely used for prostate cancer (gemcitabine).

469 Background: Appendiceal cancers are rare and there are no established guidelines for adjuvant treatment and metastatic disease. Methods: Samples were identified from > 60,000 global tumors analyzed at a referral molecular profiling CLIA certified laboratory (Caris Life Sciences) for biomarkers of drug sensitivity, under an approved protocol. 285 samples with primary tumor sites of appendix were identified (M/F ratio of 38%/62%; mean age of 55). Approximately 80% of samples were adenocarcinomas, 10% carcinoids, and 10% of indeterminate histology. IHC assays (n=273) were performed with up to 30 biomarkers; FISH (n=39) for c-Myc, cMET, EGFR, HER2 and/or TOPO2A gene copy amplifications; and sequencing done ( n= 223) for KRAS, EGFR, PIK3CA, and BRAF. Results: Targets on IHC: 97% high BCRP; 16% high KIT; 80% high COX-2; 50%high EGFR; 30% low MGMT, 81% high MRP1, 35% high PDGFR, 82% low PTEN, 27% high RRM1, 39% high SPARC; 67% high TLE3, 27% high TOPO2A ; 63% high TOPOI, and 99% negative TS. Targets on FISH: 5% amplified EGFR and 0% amplified Her2/Neu. Sequencing results: 53% mutated KRAS and 2% mutated BRAF. Conclusions: The high levels of drug resistance proteins (BCRP and MRP1) highlights the difficulty in treating these tumors. The incidence of KRAS mutations (53%) and low expression of TS (99%) is noteworthy, and points towards using 5FU/capecitabine as a backbone of therapy. Therapeutic options are varied and options include TOPO1 inhibitors (irinotecan/topotecan), EGFR inhibitors (erlotinib, cetuximab), PDGFR antagonists (regorafenib, axitinib), MGMT (temazolamide), BRAF inhibitors (vemurafenib, dabrafenib) and SPARC (nab-paclitaxel). These findings indicate the need to evaluate patient samples for patterns in marker expression and alteration, in order to better understand the molecular biology and formulate a personalized therapy approach in these difficult to treat cancers, and to pursue whole genomic sequencing. Supported by a grant from Caris Life Sciences.

399 Background: Colorectal cancer (CRC) is the third most common cancer worldwide, with metastatic disease accounting for 40 to 50% of newly diagnosed patients. EGFR monoclonal antibodies (Mab), cetuximab and panitumumab, are effective treatment for KRAS wild type CRC. Although mutations in KRAS predict resistance to EGFR Mab therapy, only 80% of CRC patients with KRAS wild-type (WT) status respond to treatment. This study is a retrospective evaluation of genomic alterations in the EGFR pathway such as alterations in KRAS, BRAF, NRAS, PIK3CA and PTEN that may predict lack of response to EGFR Mab therapy in CRC patients. Methods: A large database of 5,900 consecutive CRC patients was analyzed from 2011 onwards for demographics (sex, age, geography, site of tumor) and biomarkers that might correlate with response to EGFR Mab therapy. Comprehensive testing included gene sequencing for KRAS, BRAF, NRAS, PTEN, and PIK3CA (next-generation sequencing, Sanger, pyrosequencing) and immunohistochemistry for PTEN protein expression. Results: Analysis revealed the incidence of KRAS mutation of 42% which is consistent with published literature. Analysis of the KRAS WT cohort revealed a mutation rate of 13.8 % for BRAF, 6.8% for NRAS, and 40% for patients with either PTEN loss of expression/mutation or a PIK3CA mutation. Earlier studies support that mutations in NRAS, BRAF and activation of the PI3K pathway by PTEN/PIK3CA analysis result in lower response rates to EGFR Mab therapy in CRC. Furthermore, multiplex biomarker testing revealed the rare occurrence of concurrent mutations: 1/5,900 subject harboring a BRAF and NRAS mutations, 7/5,900 others with BRAF and PIK3CA mutations, and 8/5,900 subjects with NRAS and PIK3CA mutations. Conclusions: This is a comprehensive analysis of a large international cohort evaluating the prevalence of predictive molecular aberrations suspected of lack of response to EGFR Mab therapy in patients with WT KRAS. Prospective controlled studies are in progress to validate the role of BRAF, NRAS, PIK3CA, and PTEN in clinical management of CRC. It is imperative to further explore the molecular pathology of CRC beyond KRAS in patient selection for EGFR Mab therapy.