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e17025 Background: Bladder cancers (BC) are frequently highly mutated. Next generation sequencing (NGS) can both shed light on mutational burden and the specific alterations that provide insights into the underlying biology of individual tumors. Methods: We retrospectively reviewed BC cases assessed with UCSF500, an institutional NGS assay that uses hybrid capture enrichment of target DNA to interrogate approximately 500 frequently mutated cancer genes. Hypermutated tumors were defined as having TMB > 10 mutations/Mb. Fisher’s exact test was used to compare patients (pts) with hypermutated (HM) and non-hypermutated (NHM) tumors. Results: From 2015 to 2019, 74 pts with BC underwent UCSF500 testing; 48 pts were evaluable for TMB, of which 19 pts (40%) had HM tumors. 17/19 pts were evaluable for mutational signatures; all 17 had APOBEC signatures. Signatures were not assessed in NHM tumors due to low TMB. Clinicopathologic characteristics and most common alterations in the two groups are listed in the table. More HM pts had responses to immunotherapy (IO) treatment (86% vs 40%, p = 0.13). Conclusions: In this single-institution BC cohort, HM tumors were common and APOBEC mutational signature was the common underlying biology in HM tumors. There were relevant differences in common alterations between HM and NHM tumors, including more FGFR3 mutations in NHM tumors. HM status and APOBEC signature were suggested as relevant predictive biomarkers of response to IO, which should be investigated further in larger BC cohorts. [Table: see text]

Purpose Melanomas on mucosal membranes, acral skin (soles, palms, and nail bed), and skin with chronic sun-induced damage have infrequent mutations in BRAF and NRAS, genes within the mitogen-activated protein (MAP) kinase pathway commonly mutated in melanomas on intermittently sun-exposed skin. This raises the question of whether other aberrations are occurring in the MAP kinase cascade in the melanoma types with infrequent mutations of BRAF and NRAS. Patients and Methods We analyzed array comparative genomic hybridization data from 102 primary melanomas (38 from mucosa, 28 from acral skin, and 18 from skin with and 18 from skin without chronic sun-induced damage) for DNA copy number aberrations specific to melanoma subtypes where mutations in BRAF and NRAS are infrequent. A narrow amplification on 4q12 was found, and candidate genes within it were analyzed. Results Oncogenic mutations in KIT were found in three of seven tumors with amplifications. Examination of all 102 primary melanomas found mutations and/or copy number increases of KIT in 39% of mucosal, 36% of acral, and 28% of melanomas on chronically sun-damaged skin, but not in any (0%) melanomas on skin without chronic sun damage. Seventy-nine percent of tumors with mutations and 53% of tumors with multiple copies of KIT demonstrated increased KIT protein levels. Conclusion KIT is an important oncogene in melanoma. Because the majority of the KIT mutations we found in melanoma also occur in imatinib-responsive cancers of other types, imatinib may offer an immediate therapeutic benefit for a significant proportion of the global melanoma burden.

11575 Background: Targeted next-generation DNA sequencing of paired tumor and normal DNA samples allows for detection of biologically relevant variants in the tumor with significantly greater accuracy than tumor-only sequencing. In addition, this approach presents with the opportunity of unveiling previously unknown cancer predisposition traits in a patient’s germline DNA. Methods: We sought to determine the rate of pathogenic germline alterations in 546 consecutive pediatric and adult patients who underwent molecular profiling using the UCSF 500 assay, a hybrid capture-based DNA sequencing assay targeting the coding regions of ~500 cancer-related genes, TERT promoter, select introns from 40 genes (for detection of gene fusions and other structural variants), and intergenic regions at regular intervals along each chromosome (for chromosomal copy number and LOH assessment). Results: Pathogenic germline alterations were found in 89/546 patients (16.3%), including 25 pediatric and 64 adult cases. Germline variants were identified in 37 genes with MUTYH (n = 15, 17%), CHEK2 (n = 10, 11%), BRCA2 (n = 9, 10%), BRCA1 (n = 5, 6%), TP53 (n = 5, 5%), and APC (n = 4, 5%) being altered most frequently. Loss of heterozygosity of genes affected in the germline was seen in tumor DNA 37 (42%) cases, highlighting their likely role as drivers of tumorigenesis. Clinically relevant germline findings not associated with increased cancer risk were identified in 6 (7%) of the cases, for example a COL1A1 mutation associated with Ehlers-Danlos Syndrome. In In 73 (82%) of the cases, pathogenic germline alterations were new findings and genetic counseling was recommended. A possible, previously unknown, role of germline mutations was found in some instances, including a TSC2 germline mutation in a patient with hybrid oncocytoma/chromophobe tumor (HOCT) with loss of the normal TSC2 allele in the tumor. Conclusions: Our data suggest that paired tumor/normal DNA analysis uncovers actionable heritable traits in a substantial fraction of patients and represents the preferred approach to analyzing malignancies in children and adults.

9002 Background: Spitz tumors are melanocytic neoplasms with characteristic morphologic features that can overlap with melanoma. Predicting biologic behavior, which can range from an indolent disease to metastasis confined to regional lymph nodes and infrequent incidences of widespread metastatic disease with lethal outcome, is unreliable based on histopathological criteria and the genetic underpinnings of the disease as a whole are poorly understood. Methods: Genomic DNA and total RNA was isolated from 40 microns of FFPE sections from 20 benign Spitz nevi and 8 atypical Spitz tumors (with morphological features inconsistent with HRAS or BRAF/BAP1 mutations) in a CLIA-certified lab (Foundation Medicine). DNA sequencing was performed for 3230 exons of 182 cancer-related genes plus 37 introns of 14 genes commonly fused on indexed hybridization-captured libraries to an average unique coverage of 997x, with 99.96% of exons being sequenced at ≥100x coverage. RNA sequencing was performed on indexed libraries captured using the cDNA Kinome hybridization kit (Agilent) generating >50,000,000 unique pairs per specimen. Results: Only a single case harbored a point mutation in a gene known to be recurrently mutated in melanocytic neoplasms, HRAS Q61L and no known alterations were found in BRAF, NRAS, KIT, GNAQ or GNA11. Remarkably, genomic rearrangements were observed in 19/28 (68%) of cases. The rearrangements fused the intact kinase domains of ROS1 (36%), ALK (14%), RET (7%), NTRK1 (7%) and BRAF (4%) to a wide range of predominantly novel 5’ partners including PWWP2A, PPFIBP1, ERC1, MYO5A, CLIP1, HLA-A, ZCCHC8, DCNT1, LMNA and CEP89. These gene rearrangements, which were all expressed, formed constitutively activated chimeric oncogenes. All fusions occurred in a mutually exclusive pattern and were more common in younger patients compared to patients whose tumors did not harbor fusions (median age 14 versus 24 years, p=0.02). Conclusions: Next generation sequencing identified gene fusions in two thirds of Spitz tumors which are likely to be useful as diagnostic markers that may also serve as therapeutic targets for the rare subset of these tumors that metastasize.

11004 Background: NSCLC p with EGFR mutations respond initially to EGFR tyrosine kinase inhibitors (TKIs) but invariably develop acquired EGFR TKI resistance. Prior studies identified the EGFR T790M mutation and activation of MET, PI3K, AXL, HER2 and the MAPK pathway as drivers of acquired EGFR TKI resistance. To date, comprehensive molecular profiling to identify actionable modifiers of EGFR TKI response has not been conducted in NSCLC p on therapy. Methods: We performed next generation sequencing (NGS) using a 263-gene Nimblegen custom cancer panel on DNA isolated from primary patient lung adenocarcinoma FFPE specimens prior to initiating standard erlotinib treatment and upon the development of acquired erlotinib resistance after only 3 months of therapy. Results: In the pretreatment sample, we confirmed the presence of the EGFRL858R mutation in 95% of the sequencing reads and discovered a concurrent BRAF V600E mutation with a frequency of ~ 6%. NGS performed on the acquired erlotinib resistance sample revealed acquisition of the EGFR T790M mutation with a frequency of ~ 14%. Notably, the frequency of the BRAF V600E mutation increased 10-fold upon acquired erlotinib resistance from ~ 6% in the pretreatment tumor to ~ 60% in the recurrent tumor. We found that overexpression of BRAF V600E in H3255 human NSCLC, which harbor EGFR L858R (but not BRAF V600E) and are erlotinib sensitive, caused resistance to erlotinib treatment (10-fold increase in erlotinib IC50). BRAF V600E-mediated erlotinib resistance was reversed by treatment with the BRAF inhibitor vemurafenib. Additional functional studies are ongoing and the complete dataset will be presented. Conclusions: These results indicate that EGFR-mutant NSCLC can harbor additional oncogenic driver mutations in BRAF at low frequencies prior to therapy. EGFR TKI treatment can lead to expansion of BRAF V600E expressing tumor cells, resulting in acquired EGFR TKI resistance that can be reversed by BRAF inhibitor treatment. The data demonstrate the utility of routine molecular profiling of NSCLC p on targeted therapy and offer unprecedented insight into the genetic basis of therapeutic resistance.

10524 Background: As more therapies targeting genomic alterations become available, genotyping (e.g., by Sequenom) is increasingly performed in tumor types where mutational status may drive treatment choice. Next-generation sequencing (NGS) technology can expand on genotyping of individual base pairs because it can detect mutations across entire exons, copy changes and fusion genes. However, for NGS to be clinically viable, it must be made compatible with FFPE tissues and shown concordant with best current diagnostic methods. Methods: To explore a potential clinical role for NGS, we selected 120 FFPE specimens (68 NSCLC, 32 CRC, 20 melanoma) previously tested for 97 oncogenic mutations in 8 oncogenes by Sequenom, and sequenced all exons of 182 cancer genes. Genotyping and sequencing were both performed in CLIA compliant labs. DNA was extracted from 4x 10μ unstained sections from the diagnostic FFPE block, followed by library construction and hybridization capture of 3230 exons and 37 commonly rearranged introns. Average coverage of >900X uniquely-mapping reads was obtained. Sequence data were analyzed for all genomic alterations and examined for potentially actionable mutations. Results: High concordance was noted between Sequenom and NGS: 103 and 105 mutations were called by the two technologies, respectively, at mutually tested sites, with 97 mutation calls in common. Notably, mutant allele frequencies in concordant calls ranged as low as 2% by NGS, highlighting the sensitivity of detection enabled by both approaches. Furthermore, in 45/120 (38%) specimens, NGS revealed additional alterations that may confer sensitivity or resistance to approved or experimental targeted therapies and thus plausibly influence treatment decisions. These included 39 copy changes or loss-of-function variants best assayed by an NGS approach. Conclusions: Our results demonstrate technical feasibility and highlight potential benefits of comprehensive cancer gene characterization through next-generation sequencing of clinical FFPE specimens. As NGS is the most practical means to detect all classes of somatic alteration in a small, clinically relevant sample, we suggest that this type of testing will become an essential component of patient care.

8532 Background: Activation of the SRC-Stat pathway is present in melanoma and Stat3 activation is present in a large proportion of melanoma cell lines and human metastatic melanoma tumors. In prec...

8545 Background: Recent studies have shown that KIT gene aberrations can be found in melanomas originating from mucosal or acral sites, or skin with chronic sun-damage (CSD), and may respond to tre...

9001 Background: Three prior phase II studies of Imatinib mesylate (IM) in 62 pts with advanced melanoma reported only 1 response in a pt with acral melanoma. A proportion of melanomas arising from acral, mucosal, and chronic sun damaged (CSD) sites are characterized by KIT mutations (mut) or amplifications (amp) and we hypothesized that this subset of tumors would be sensitive to IM. We thus designed this phase II study of IM restricted to pts with melanoma harboring such alterations in KIT. Methods: Pts with unresectable melanoma arising from acral, mucosal, and CSD sites whose tumor harbored a 4q12 amp by FISH or mut in KIT and who had measureable disease by RECIST were eligible. Pts received IM 400 mg BID continually. Response was assessed every cycle (6 wks). A Simon 2-stage design was employed where initially 16 pts would be treated; if ≥ 2 responses were observed, a total of 25 pts would be enrolled. If ≥5 responses were seen in 25 pts, the study was to be considered positive. Results: Of 81 pt tumors screened, 17 (21%) had a KITmut or amp: 5/22 (23%) acral, 12/45 (27%) mucosal, 0/13 (0%) CSD, 0/1 (0%) unknown primary. 12 (15%) had a mut only; 4 (5%) had an amplification only; 1 (2%) had both. Thus far, 7 have been treated, with 5 currently evaluable for response. Median age: 64 yrs (range, 61–86); 2 male/5 female; median KPS: 90 (range, 80- 90); median # of prior therapies: 1 (range, 0–4). 3 pts (43%) achieved a PR (18 wks - exon 13 mut; 21 wks, ongoing - exon 11 mut; 18 wks, ongoing - exon 11 mut & amp); 2 pts (28%) achieved SD (12 wks - exon 11 mut; 11 wks - amp). 3 pts required a dose reduction to 400 mg QD for rash, GI toxicity and fatigue. 1 pt required a second dose reduction to 300 mg QD for visual changes. Conclusions: In this pt population, 21% of tumors are characterized by mut or amp of KIT. The 3 responses observed have allowed expansion to the second stage of enrollment which is currently on-going. While IM has limited activity in a non-selected melanoma pt population, a substantial proportion of melanomas harboring KIT mut or amp appear to respond. It may be possible to identify appropriate pts prospectively for treatment with IM. (Supported by R01FD003445–01, ASCO YIA, N01CM62206, and the Live4Life Foundation.) No significant financial relationships to disclose.