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PDF file - 470KB, All supplementary figures including tumor histology, RAD50 immunohistochemistry, candidate mutations and their clonality, and supporting yeast and MEF experimental data.

PDF file - 2461KB, A complete listing of somatic mutations and rearrangements detected as well as targeted genes, sequences, MRN complex mutation frequency, and strains utilized.

PDF file - 321KB, A detailed description of additional methods utilized (and associated references) for sequencing and analysis as well as an explanatory note regarding the TM pathway in yeast.

PDF file - 6545K, Detection of a K57N MEK1 mutation in DNA from formalin-fixed paraffin-embedded tumor and matched normal tissue from one patient. Multiple reads from tumor-derived DNA (panel on left) show a GC transversion in 21% of reads. This indicates a lysineaspargine mutation in MEK1. No such mutation is detected in germline DNA (panel at right).

PDF file - 3248K, Supplementary Table S1. Histopathologic findings for multi-region analysis. Supplementary Table S2.IMPACT gene list and their respective chromosomal positions. Supplementary Table S5. WEC run statistics on patients #4 and #5. Supplementary Table S6.List of all non-variant mutations detected by IMPACT assays in individual patient samples. Supplementary Table S7. Non-variant mutations identified by WEC in patients #4 and #5. Supplementary Figure 1. Flow chart depicts the IMPACT assay mutation identification and filtering algorithm. Supplementary Figure 2. Flow chart depicts the WEC assay mutation identification and filtering algorithm. Supplementary Figure 3. Sanger validations of mutations in the targeted pathway indentified by IMPACT assay for R1 of patients #1,#2, and #3. Supplementary Figure 4. Sanger validations of mutations in the targeted pathway indentified by IMPACT assay for additional regions analyzed for patients #1, and #3. Supplementary Figure 5. The Q2223K mTOR mutation provokes hyperactivation of mTORC1 in serum free growth conditions. Supplementary Figure 6. Hyperactivation of mTORC1 induced by Q2223K is sensitive to rapalog-based mTOR inhibition. Supplementary Figure 7. Immunoblots detecting phosphorylated mTOR at serine residues 2448 (S2448) and 2481 (S2481). Supplementary Figure 8. Copy number plots for multiple tumor regions in patient #3 showing the loss of chromosome 9 only in tumor regions carrying the TSC1 nonsense mutation (R3, R4). Supplementary Figure 9. Copy number plots for patient with tuberous sclerosis complex and metastatic unclassified RCC with extended benefit from everolimus therapy (44+ months). Supplementary Figure 10. PI3K pathway alterations as reported across 418 cases of clear cell RCC in TCGA. Alterations are detected in 42% of cases. Supplementary Figure 11: Hematoxylin and eosin (H&E) stains and immunohistochemistry for 4EBP1 phosphorylation (Thr 37/46), an mTORC1 downstream effector, in primary kidney tumors and adjacent normal kidney tissue for patients 1-5.

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Purpose: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity.Experimental Design: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list.Results: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions.Conclusions: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents. Clin Cancer Res; 20(7); 1955–64. ©2014 AACR.

PDF file - 2798K, Immunohistochemical (IHC) staining of high pAKT tumors

Purpose: Test the hypothesis that in BRAF-mutated melanomas, clinical responses to selumetinib, a MEK inhibitor, will be restricted to tumors in which the PI3K/AKT pathway is not activated.Experimental Design: We conducted a phase II trial in patients with melanoma whose tumors harbored a BRAF mutation. Patients were stratified by phosphorylated-AKT (pAKT) expression (high vs. low) and treated with selumetinib 75 mg per os twice daily. Pretreatment tumors were also analyzed for genetic changes in 230 genes of interest using an exon-capture approach.Results: The high pAKT cohort was closed after no responses were seen in the first 10 patients. The incidence of low pAKT melanoma tumors was low (∼25% of melanomas tested) and this cohort was eventually closed because of poor accrual. However, among the five patients with melanoma accrued in the low pAKT cohort, there was one partial response (PR). Two other patients had near PRs before undergoing surgical resection of residual disease (one patient) or discontinuation of treatment due to toxicity (one patient). Among the two nonresponding, low pAKT patients with melanoma, co-mutations in MAP2K1, NF1, and/or EGFR were detected.Conclusions: Tumor regression was seen in three of five patients with BRAF-mutated, low pAKT melanomas; no responses were seen in the high pAKT cohort. These results provide rationale for co-targeting MEK and PI3K/AKT in patients with BRAF mutant melanoma whose tumors express high pAKT. However, the complexity of genetic changes in melanoma indicates that additional genetic information will be needed for optimal selection of patients likely to respond to MEK inhibitors. Clin Cancer Res; 19(8); 2257–64. ©2013 AACR.

XLSX file - 13K, Supplementary Table S3. Impact assay run statistics on all samples.

PDF file - 132K

PDF file - 5171K, Schematic representation of gain or deletions across the set of genes on each chromosome in a tumor with MITF amplification. Each dot corresponds to an exon of a specific gene. Every gene expressed higher than 1.5 is considered amplified, while every gene expressed lower than 0.5 is considered deleted. Exons corresponding to MITF are circled in red.

Background The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway frequently is activated in patients with urothelial carcinoma (UC). In the current study, the authors performed a phase 2 study evaluating the efficacy of the pan-isoform class I PI3K inhibitor buparlisib in patients with platinum-refractory metastatic UC. Methods Two cohorts were recruited: an initial genetically unselected cohort and a subsequent expansion cohort of patients with PI3K/Akt/mTOR pathway-altered tumors. The primary endpoint was the 2-month progression-free survival rate. A rate of ≥80% was considered promising using a Simon 2-stage minimax design. Secondary endpoints included safety and correlation of markers of PI3K pathway activation with outcome. Results Six of 13 evaluable patients within the initial cohort demonstrated stable disease and 1 demonstrated a partial response, which was below the cutoff of 9 patients required to proceed to stage 2. Three of the patients with stable disease and the patient with a partial response harbored somatic TSC1 alterations. Four patients subsequently were recruited onto an expansion cohort: 3 patients with TSC1 alterations and 1 patient with a PIK3CA-activating mutation. No patient achieved disease control at 8 weeks and accrual was halted. Of the 19 patients evaluable for toxicity, 17 demonstrated treatment-related toxicities, 2 of whom had to discontinue therapy. Conclusions Buparlisib was found to demonstrate modest activity in patients with metastatic UC whose tumors harbored TSC1 loss of function alterations; however, this was not a robust predictor of response to buparlisib. The pattern of genetic coalterations likely influences drug sensitivity. Given the modest clinical activity and substantial toxicity of buparlisib, future trials of PI3K inhibitors in patients with UC should focus on isoform-selective PI3K inhibitors in genomically selected patients. Lay summary The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway frequently is upregulated in patients with metastatic urothelial carcinoma (UC). This trial explored buparlisib, an inhibitor of the pathway, in patients with heavily pretreated metastatic UC. Although the drug was found to have modest efficacy, with 6 patients experiencing stable disease and 1 patient achieving a partial response at 8 weeks on therapy, significant side effects also were observed. Patients with specific genetic alterations responded to treatment. Further studies of PI3K pathway inhibition are warranted using newer agents that have superior toxicity profiles and are more selective inhibitors of the pathway.

Clear cell carcinoma of the endometrium is a rare type of endometrial cancer that is generally associated with an aggressive clinical behaviour. Here, we sought to define the repertoire of somatic genetic alterations in endometrial clear cell carcinomas (ECCs), and whether ECCs could be classified into the molecular subtypes described for endometrial endometrioid and serous carcinomas. We performed a rigorous histopathological review, immunohistochemical analysis and massively parallel sequencing targeting 300 cancer-related genes of 32 pure ECCs. Eleven (34%), seven (22%) and six (19%) ECCs showed abnormal expression patterns for p53, ARID1A, and at least one DNA mismatch repair (MMR) protein, respectively. Targeted sequencing data were obtained from 30 of the 32 ECCs included in this study, and these revealed that two ECCs (7%) were ultramutated and harboured mutations affecting the exonuclease domain of POLE. In POLE wild-type ECCs, TP53 (46%), PIK3CA (36%), PPP2R1A (36%), FBXW7 (25%), ARID1A (21%), PIK3R1 (18%) and SPOP (18%) were the genes most commonly affected by mutations; 18% and 11% harboured CCNE1 and ERBB2 amplifications, respectively, and 11% showed DAXX homozygous deletions. ECCs less frequently harboured mutations affecting CTNNB1 and PTEN but more frequently harboured PPP2R1A and TP53 mutations than non-POLE endometrioid carcinomas from The Cancer Genome Atlas (TCGA). Compared to endometrial serous carcinomas (TCGA), ECCs less frequently harboured TP53 mutations. When a surrogate model for the molecular-based TCGA classification was used, all molecular subtypes previously identified in endometrial endometrioid and serous carcinomas were present in the ECCs studied, including POLE, MMR-deficient, copy-number high (serous-like)/p53 abnormal, and copy-number low (endometrioid)/p53 wild-type, which were significantly associated with disease-free survival in univariate analysis. These findings demonstrate that ECCs constitute a histologically and genetically heterogeneous group of tumours with varying outcomes. Furthermore, our data suggest that the classification of ECCs as being generally 'high-grade' or 'type II' tumours may not be warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BACKGROUND Biopsies from patients with high-risk (HR) non–muscle-invasive urothelial carcinoma (NMIUC), especially flat urothelial carcinoma in situ, frequently contain scant diagnostic material or denuded mucosa only, and this precludes further extensive genomic analysis. This study evaluated the use of next-generation sequencing (NGS) analysis of urine cytology material from patients with HR NMIUC in an attempt to identify genetic alterations that might correlate with clinical features and responses to bacille Calmette-Guerin (BCG) treatment. METHODS Forty-one cytology slides from patients with HR NMIUC treated with intravesical BCG were selected for this study. Histological confirmation was available for all cases. The specimens were subjected to NGS analysis with a customized targeted exome capture assay composed of 341 genes. RESULTS In this cohort, genomic alterations were successfully identified in all cytology samples. Mutations were detected down to a 2% allele frequency and chromosomal rearrangements including copy number alterations and gene fusions were identified. The most frequently altered genes included telomerase reverse transcriptase (TERT), tumor protein 53 (TP53), Erb-B2 receptor tyrosine kinase 2 (ERBB2), and chromatin remodeling genes such as lysine demethylase 6A (KDM6A) and AT-rich interaction domain 1A (ARID1A). For patients with matched tumor tissue, cytology specimens revealed all mutations detected in tissue as well as additional mutations, and this suggested that urine might more effectively capture the full genetic heterogeneity of disease than an individual cystectomy. Alterations in multiple genes correlated with clinical and histopathological features, including responses to BCG treatment, flat architecture versus papillary architecture, and smoking history. CONCLUSIONS Urine specimens can replace tissue as a substrate for NGS analysis of HR NMIUC. Several genomic alterations identified in urine specimens might be associated with histological features and clinical characteristics. Cancer Cytopathol 2017;125:416–26. © 2017 American Cancer Society.

BACKGROUND The authors characterized the genetic landscape of chemoradiation-treated urothelial carcinoma of the bladder (UCB) with the objective of identifying potential correlates of response. METHODS Primary tumors with (n = 8) or without (n = 40) matched recurrent tumors from 48 patients who had non-metastatic, high-grade UCB and received treatment primarily with chemoradiation were analyzed using a next-generation sequencing assay enriched for cancer-related and canonical DNA damage response (DDR) genes. Protein expression of meiotic recombination 11 homolog (MRE11), a previously suggested biomarker, was assessed in 44 patients. Recurrent tumors were compared with primary tumors, and the clinical impact of likely deleterious DDR gene alterations was evaluated. RESULTS The profile of alterations approximated that of prior UCB cohorts. Within 5 pairs of matched primary-recurrent tumors, a median of 92% of somatic mutations were shared. A median 33% of mutations were shared between 3 matched bladder-metastasis pairs. Of 26 patients (54%) who had DDR gene alterations, 12 (25%) harbored likely deleterious alterations. In multivariable analysis, these patients displayed a trend toward reduced bladder recurrence (hazard ratio, 0.32; P = .070) or any recurrence (hazard ratio, 0.37; P = .070). The most common of these alterations, ERCC2 (excision repair cross-complementation group 2) mutations, were associated with significantly lower 2-year metastatic recurrence (0% vs 43%; log-rank P = .044). No impact of MRE11 protein expression on outcome was detected. CONCLUSIONS A similar mutation profile between primary and recurrent tumors suggests that pre-existing, resistant clonal populations represent the primary mechanism of chemoradiation treatment failure. Deleterious DDR genetic alterations, particularly recurrent alterations in ERCC2, may be associated with improved chemoradiation outcomes in patients with UCB. A small sample size necessitates independent validation of these observations. Cancer 2016;122:3715-23. © 2016 American Cancer Society.

Summary Salivary duct carcinoma (SDC) is a rare, aggressive malignancy with limited treatment options and poor outcome. Twenty-nine primary resected SDC, including 15 SDC de novo (SDCDN), and 14 SDC ex pleomorphic adenoma (SDCXPA) were subjected to the massive parallel sequencing assay (MSK-IMPACT) targeting 287 to 468 cancer-related genes. TP53 was the most frequently altered gene (69%). TP53 mutations and ERBB2 amplification were more frequent in SDCXPA than in SDCDN (P = .0007 and P = .01, respectively). Potentially targetable mutations were detected in 79% (23/29) of SDC involving ERBB2 (31%), PIK3CA (28%), HRAS (21%), ALK (7%) and BRAF (3%), and 22% (5/23) of those cases harbored possible primary resistance mutations involving CCNE1, NF1 and PTEN. A novel HNRNPH3-ALK rearrangement was found in one SDCDN. In another case, EML4-ALK fusion detected in the primary tumor was associated with ALK G1202R secondary resistance mutation in the post-treatment metastasis. A germline analysis of the DNA repair genes revealed a case with a pathogenic BRCA1 E23fs germline variant. SDCDN and SDCXPA are genetically distinct. Although the majority of SDC may be amenable to molecular targeted therapy, concurrent possible resistance mutations may be found in a significant minority of cases. A broad genomic profiling is necessary to ensure detection of rare but clinically actionable somatic alterations in SDC.

Despite a similar histologic appearance, upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) tumors have distinct epidemiologic and clinicopathologic differences.To investigate whether the differences between UTUC and UCB result from intrinsic biological diversity.Tumor and germline DNA from patients with UTUC (n=83) and UCB (n=102) were analyzed using a custom next-generation sequencing assay to identify somatic mutations and copy number alterations in 300 cancer-associated genes.We described co-mutation patterns and copy number alterations in UTUC. We also compared mutation frequencies in high-grade UTUC (n=59) and high-grade UCB (n=102).Comparison of high-grade UTUC and UCB revealed significant differences in the prevalence of somatic alterations. Genes altered more commonly in high-grade UTUC included FGFR3 (35.6% vs 21.6%; p=0.065), HRAS (13.6% vs 1.0%; p=0.001), and CDKN2B (15.3% vs 3.9%; p=0.016). Genes less frequently mutated in high-grade UTUC included TP53 (25.4% vs 57.8%; p0.001), RB1 (0.0% vs 18.6%; p0.001), and ARID1A (13.6% vs 27.5%; p=0.050). Because our assay was restricted to genomic alterations in a targeted panel, rare mutations and epigenetic changes were not analyzed.High-grade UTUC tumors display a spectrum of genetic alterations similar to high-grade UCB. However, there were significant differences in the prevalence of several recurrently mutated genes including HRAS, TP53, and RB1. As relevant targeted inhibitors are being developed and tested, these results may have important implications for the site-specific management of patients with urothelial carcinoma.Comparison of next-generation sequencing of upper tract urothelial carcinoma (UTUC) with urothelial bladder cancer identified that similar mutations were present in both cancer types but at different frequencies, indicating a potential need for unique management strategies. UTUC tumors were found to have a high rate of mutations that could be targeted with novel therapies.

Cutaneous neuroendocrine (Merkel cell) carcinoma most often arises de novo in the background of a clonally integrated virus, the Merkel cell polyomavirus, and is notable for positive expression of retinoblastoma 1 (RB1) protein and low expression of p53 compared with the rare Merkel cell polyomavirus-negative Merkel cell carcinomas. Combined squamous and Merkel cell tumors are consistently negative for Merkel cell polyomavirus. Little is known about their immunophenotypic or molecular profile. Herein, we studied 10 combined cutaneous squamous cell and neuroendocrine carcinomas for immunohistochemical expression of p53, retinoblastoma 1 protein, neurofilament, p63, and cytokeratin 20 (CK20). We compared mutation profiles of five combined Merkel cell carcinomas and seven ‘pure’ Merkel cell carcinomas using targeted next-generation sequencing. Combined tumors were from the head, trunk, and leg of Caucasian males and one female aged 52–89. All cases were highly p53- and p63-positive and neurofilament-negative in the squamous component, whereas RB1-negative in both components. Eight out of 10 were p53-positive, 3/10 p63-positive, and 3/10 focally neurofilament-positive in the neuroendocrine component. Six out of 10 were CK20-positive in any part. By next-generation sequencing, combined tumors were highly mutated, with an average of 48 mutations per megabase compared with pure tumors, which showed 1.25 mutations per megabase. RB1 and p53 mutations were identified in all five combined tumors. Combined tumors represent an immunophenotypically and genetically distinct variant of primary cutaneous neuroendocrine carcinomas, notable for a highly mutated genetic profile, significant p53 expression and/or mutation, absent RB1 expression in the context of increased RB1 mutation, and minimal neurofilament expression.

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

BACKGROUND Mutation analysis for personalized treatment has become increasingly important in the management of different types of cancer. The advent of new DNA extraction protocols and sequencing platforms with reduced DNA input requirements might allow the use of cytology specimens for high-throughput mutation analysis. In this study, the authors evaluated the use of effusion fluid for next-generation sequencing-based, multigene mutation profiling. METHODS Four specimens from each of 5 patients who had at least stage III, high-grade serous ovarian carcinoma were selected: effusion fluid; frozen tumor; formalin-fixed, paraffin embedded tumor; and matched normal blood. Frozen tumors from each patient were previously characterized by The Cancer Genomic Atlas (TCGA). DNA was extracted from all specimens and was sequenced using a custom hybridization capture-based assay. Genomic alterations were compared among all specimens from each patient as well as with mutations reported in TCGA for the same tumors. RESULTS In total, 17 distinct somatic mutations were identified in the cohort. Ten of 17 mutations were reported in TCGA and were called in all 3 malignant specimens procured from the patients. Of the remaining 7 mutations, 2 were called in all 3 specimens, and the other 5 were sample-specific. Two mutations were detected only in the cytology specimens. Copy number profiles were concordant among the tumors analyzed. CONCLUSIONS Cytology specimens represent suitable material for high-throughput sequencing, because all mutations described by TCGA were independently identified in the effusion fluid. Differences in mutations detected in samples procured from the same patient may reflect tumor heterogeneity. Cancer (Cancer Cytopathol) 2015;123:289–97. © 2015 American Cancer Society.

Urothelial carcinoma of the bladder (UCB) is genomically heterogeneous, with frequent alterations in genes regulating chromatin state, cell cycle control, and receptor kinase signaling. To identify prognostic genomic markers in high-grade UCB, we used capture-based massively parallel sequencing to analyze 109 tumors. Mutations were detected in 240 genes, with 23 genes mutated in ≥5% of cases. The presence of a recurrent phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha ( PIK3CA ) mutation was associated with improved recurrence-free survival (RFS) (hazard ratio [HR]: 0.35; p =0.014) and improved cancer-specific survival (CSS) (HR: 0.35; p =0.040) in patients treated with radical cystectomy (RC). In multivariable analyses controlling for pT and pN stages, PIK3CA mutation remained associated with RFS (HR: 0.39; p =0.032). The most frequent alteration, TP53 mutation (57%), was more common in extravesical disease (69% vs 32%, p =0.005) and lymph node–positive disease (77% vs 56%, p =0.025). Patients with cyclin-dependent kinase inhibitor 2A ( CDKN2A )–altered tumors experienced worse RFS (HR: 5.76; p p =0.029) in multivariable analyses. Mutations in chromatin-modifying genes were highly prevalent but not associated with outcomes. In UCB patients treated with RC, PIK3CA mutations are associated with favorable outcomes, whereas TP53 and CDKN2A alterations are associated with poor outcomes. Genomic profiling may aid in the identification of UCB patients at highest risk following RC. Patient summary Using next-generation sequencing, we identified genomic subsets of high-grade urothelial bladder cancer associated with favorable and unfavorable outcomes. These findings may aid in the selection of patients most likely to benefit from novel combined modality approaches.

Plasmacytoid bladder cancer is an aggressive histologic variant with a high risk of disease-specific mortality. Using whole-exome and targeted sequencing, we find that truncating somatic alterations in the CDH1 gene occur in 84% of plasmacytoid carcinomas and are specific to this histologic variant. Consistent with the aggressive clinical behavior of plasmacytoid carcinomas, which frequently recur locally, CRISPR/Cas9-mediated knockout of CDH1 in bladder cancer cells enhanced cell migration.

Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function. Significance: Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer. Cancer Discov; 4(9); 1014–21. ©2014 AACR. See related commentary by Peng et al., p. 988 This article is highlighted in the In This Issue feature, p. 973

Purpose: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity. Experimental Design: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list. Results: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions. Conclusions: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents. Clin Cancer Res; 20(7); 1955–64. ©2014 AACR.

In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS, and RNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms. Nine pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin-embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median = 4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS, and RNF43 mutations. In conclusion, this study elucidates that ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm warrants being recognized separately.

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Desmoplastic melanoma (DM) is a rare variant of melanoma with distinct clinical, histopathologic, and immunohistochemical features. Clinically, DM differs from conventional melanoma by a higher propensity for local recurrence and less frequent metastatic spread to regional lymph nodes. In its pure form, DM has a distinct appearance displaying a low density of fusiform melanocytes in a collagen-rich matrix. While a number of mutations have been identified in primary melanoma, including BRAF, NRAS, GNAQ, GNA11 and KIT, and the occurrence of these mutations has been found to correlate to some extent with the histopathologic features, anatomic site and/or mode of sun exposure, no distinct set of mutations has so far been reported for DM. To study the potential association of neurofibromin (NF1) mutations with DM, we examined 15 desmoplastic and 20 non-desmoplastic melanomas by next-generation sequencing. Mutations of the NF1 gene were found in 14 of 15 (93%) desmoplastic and 4 of 20 (20%) non-desmoplastic melanomas. The high frequency of NF1 mutations in desmoplastic melanomas suggests an important role for NF1 in the biology of this type of melanoma.

Leiomyosarcoma (LMS) belongs to the class of genetically complex sarcomas and shows numerous, often non-recurrent chromosomal imbalances and aberrations. We investigated a group of LMS using NGS platform to identify recurrent genetic abnormalities and possible therapeutic targets. Targeted exome sequencing of 230 cancer-associated genes was performed on 35 primary soft tissue and visceral (extra-uterine) LMS. Sequence data were analyzed to identify single nucleotide variants, small insertions/deletions (indels), and copy number alterations. Key alterations were further investigated using FISH assay. The study group included patients with median age of 64 years and median tumor size of 7 cm. The primary sites included retroperitoneal/intra-abdominal, extremity, truncal, and visceral. Thirty-one tumors were high grade LMS, while four were low grade. Losses of chromosomal regions involving key tumor suppressor genes PTEN (10q), RB1 (13q), CDH1 (16q), and TP53 (17p) were the most frequent genetic events. Gains mainly involved chromosome regions 17p11.2 (MYOCD) and 15q25-26 (IGF1R). The most frequent mutations were identified in the TP53 gene in 13 of 35 (37%) cases. FISH analysis showed amplification of the myocardin (MYOCD) gene in 5 of 25 (20%) cases analyzed. None of the four low grade LMS showed losses or mutations of PTEN or TP53 genes. Genetic complexity is the hallmark of LMS with losses of important tumor suppressor genes being a common feature. MYOCD, a key gene associated with smooth muscle differentiation, is amplified in a subset of both retroperitoneal and extremity LMS. Further studies are necessary to investigate the significance of gains/amplifications in the development of these tumors.

INTRODUCTION AND OBJECTIVES: Macroautophagy, also known as type II programmed cell death, is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here, we investigated the activated basal autophagy in bladder cancer cells and its roles in maintaining cancer cell growth. METHODS: Human immortalized uroepithelium (SV-Huc-1) and bladder cancer cells including RT-4 (grade I), 5637 (grade II), HT1376 and T24 (grade III) were used. We also included human prostate cancer cells (Rv1 and PC3), breast cancer cell (MCF-7) and embryonic kidney cancer cell (293T) as reference cells. The cell viability was accessed by WST-8. Autophagy was detected by the expression level of LC3-II protein. Human bladder tumor tissue array (TMA; 12 cases, paired with normal tissue) was used to detect the LC3 expression level. Detection of apoptosis level was measured by caspase 3/7 activation, caspase 3 cleavage and TUNEL assays. The ROS generation, mitochondria membrane potential in cells treated with autophagy inhibitors was assayed by staining with H2DCFCA and JC-1 dyes, respectively, and subsequently subjected to flow cytometry analysis. Disruption of lysomome membrane (LMP) by was detected by immunofluorescent of cathepsin-D in cells treated with CQ. RESULTS: We found bladder cancer cells exhibited high basal level of autophagy compared to prostate, breast or embryonic kidney cancer cells. Immunohistochemistry (IHC) detection of LC3 in human bladder TMA showed increased level of LC3 in bladder tumor tissues compared to their paired normal tissues. In the cell-based experiment, we found that autophagy inhibitors alone inhibited bladder cancer cell growth and increased apoptosis. The ROS generation and MMP disruption were not detected in cells treated with CQ or HCQ. Immunofluorescent detection of cathepsin-D in cells treated with CQ showed dispread pattern in the cytosol, suggesting the disruption of LMP in CQ-treated bladder cancer cells. CONCLUSIONS: Our results showed that human bladder cancer exhibits high basal level of autophagy. Autophagy inhibitors showed significant effects on the growth of bladder cancer cells by disruption of lysosome function and elevation of apoptotic cell death.

BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We assessed the safety and activity of the oral BRAF inhibitor vemurafenib in patients with hairy-cell leukemia that had relapsed after treatment with a purine analogue or who had disease that was refractory to purine analogues.We conducted two phase 2, single-group, multicenter studies of vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the United States. The therapy was administered for a median of 16 weeks in the Italian study and 18 weeks in the U.S. study. Primary end points were the complete response rate (in the Italian trial) and the overall response rate (in the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in April 2013 and is still open (26 of 36 planned patients) in the U.S. trial.The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively. In the Italian trial, after a median follow-up of 23 months, the median relapse-free survival was 19 months among patients with a complete response and 6 months among those with a partial response; the median treatment-free survival was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the progression-free survival rate was 73% and the overall survival rate was 91%. Drug-related adverse events were usually of grade 1 or 2, and the events most frequently leading to dose reductions were rash and arthralgia or arthritis. Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50 patients. The frequent persistence of phosphorylated ERK-positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism.A short oral course of vemurafenib was highly effective in patients with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13; ClinicalTrials.gov number NCT01711632.).

Background Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors. Results We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations. Conclusions Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0454-7) contains supplementary material, which is available to authorized users.

*Time-dependent covariate analysis of the effect of first on-study SRE (starting 28 days before the event onset) on time to >1⁄4 2-point increase from baseline in worst pain score in patients with a baseline score of

INTRODUCTION AND OBJECTIVES: Genomic characterization of urothelial carcinoma of the bladder (UCB) has begun to reveal significant intertumor heterogeneity when comparing samples from different subjects. As in other malignancies, intratumor heterogeneity, which may allow for tumor evolution and adaption, poses a significant challenge to personalized-medicine strategies. METHODS: To examine UCB tumor evolution and heterogeneity, we performed next-generation targeted sequencing on multiple temporally and spatially separated bladder tumors obtained at time of transurethral resection (TUR) and radical cystectomy (RC). Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 300 cancer-associated genes. RESULTS: Phylogenetic reconstruction revealed evidence of branched evolutionary growth. Evaluation of multiple tumors from individual subjects identified both shared and unique potential driver mutations. Evidence of convergent phenotypic evolution was detected through analysis of multiple distinct tumors from several subjects. For example, three separate tumors in one subject (see Figure) shared a common PIK3CA mutation (E453K) and had unique second mutations in PIK3CA (E542V, E545K, and E545Q, respectively). In another subject, distinct inactivatingmutations of EP300were identified in two temporally separated tumor samples. Macrodissection of single tumors into non-invasive and invasive components revealed significant intratumor heterogeneity; one case illustrates howanalysis of amuscleinvasive TURspecimen could result in undersampling and therebymiss the tumor clone that persisted at time of RC. CONCLUSIONS: We demonstrate branched evolution of UCB through genomic analyses of multiple temporally and spatially distinct bladder tumors from individual subjects. Macrodissection of individual tumor samples identified significant intratumor heterogeneity. These concepts may present major challenges to personalized-medicine approaches that rely on sampling of a single tumor at a specific timepoint in the evolution of a patient’s UCB.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, highly aggressive form of ovarian cancer primarily diagnosed in young women. We identified inactivating biallelic SMARCA4 mutations in 100% of the 12 SCCOHT tumors examined. Protein studies confirmed loss of SMARCA4 expression, suggesting a key role for the SWI/SNF chromatin-remodeling complex in SCCOHT.

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

INTRODUCTION AND OBJECTIVES: High grade urothelial carcinoma (UC) is an aggressive malignancy with limited therapeutic options in the advanced, metastatic, or recurrent disease setting. Targeted genetic analysis of high grade tumors was performed to determine the prevalence of known cancer genes and to identify potential targets for therapy. METHODS: Frozen high grade bladder tumors and matched germline blood DNA were obtained from 60 patients undergoing transurethral resection (TUR) or cystectomy. Specimens were analyzed using a targeted, deep-sequencing assay designed to identify point mutations, indels, and copy number alterations in 275 cancer-associated genes. RESULTS: The cohort was predominantly male (N 49, 81%) with a median age of 71 (IQR 61, 76). Frozen tumor was obtained from radical cystectomy(RC) in 53 patients (88%), TUR in 6 (10%), and partial cystectomy in 1 (2%). 11 patients (18%) had a history of intravesical therapy. Tumor pathologic stage was Ta in 5 (8%), T1 in 5 (8 %), T2 in 11 (18%), T3 in 30 (50%), and T4 in 9 (15%). Of the 53 patients undergoing RC and lymph node dissection, 17 received neoadjuvant chemotherapy (32%) and 24 (45%) had lymph node metastases. Mean target coverage for all sequenced exons was 512X. The most common genomic event was somatic mutation of TP53, identified in 33 patients (55%). RB mutations were identified in 11 (18%). Alterations in the PI3K/AKT/mTOR signaling pathway were also common; 12 patients had mutations in PIK3CA (20%), and non-overlapping mutations were identified in other genes within the pathway including AKT1, PTEN, and MTOR. Frequent mutations in chromatin remodeling genes were also identified. KDM6A mutations were identified in 21 tumors (35%), MLL2 in 18 (30%), and ARID1A in 17 (28%). Overall, 60% of all analyzed tumors harbored potentially targetable genomic alterations, including those with alterations in ERBB2, BRAF, and FGFR3 (Figure). CONCLUSIONS: Genetic alterations are frequent in high grade bladder UC. Over half of the tumors analyzed harbor alterations in genes that have been targeted for therapeutic benefit in other solid tumors, thus providing a compelling case for clinical trials using novel agents. Analysis for correlations with clinical outcomes is ongoing to assess for the prognostic significance of these genetic events. Source of Funding: Sidney Kimmel Center for Prostate and Urologic Cancers, Michael and Zena Wiener Research and Therapeutics Program in Bladder Cancer

Purpose: Test the hypothesis that in BRAF-mutated melanomas, clinical responses to selumetinib, a MEK inhibitor, will be restricted to tumors in which the PI3K/AKT pathway is not activated. Experimental Design: We conducted a phase II trial in patients with melanoma whose tumors harbored a BRAF mutation. Patients were stratified by phosphorylated-AKT (pAKT) expression (high vs. low) and treated with selumetinib 75 mg per os twice daily. Pretreatment tumors were also analyzed for genetic changes in 230 genes of interest using an exon-capture approach. Results: The high pAKT cohort was closed after no responses were seen in the first 10 patients. The incidence of low pAKT melanoma tumors was low (∼25% of melanomas tested) and this cohort was eventually closed because of poor accrual. However, among the five patients with melanoma accrued in the low pAKT cohort, there was one partial response (PR). Two other patients had near PRs before undergoing surgical resection of residual disease (one patient) or discontinuation of treatment due to toxicity (one patient). Among the two nonresponding, low pAKT patients with melanoma, co-mutations in MAP2K1, NF1, and/or EGFR were detected. Conclusions: Tumor regression was seen in three of five patients with BRAF-mutated, low pAKT melanomas; no responses were seen in the high pAKT cohort. These results provide rationale for co-targeting MEK and PI3K/AKT in patients with BRAF mutant melanoma whose tumors express high pAKT. However, the complexity of genetic changes in melanoma indicates that additional genetic information will be needed for optimal selection of patients likely to respond to MEK inhibitors. Clin Cancer Res; 19(8); 2257–64. ©2013 AACR.

11608Background: Up to 30% of EG adenocarcinomas harbor ERBB2 (HER2) gene amplification or oncoprotein over-expression. While trastuzumab prolongs survival in patients with Stage IV HER2+ EG tumors...

Background: We previously reported potent anti-tumor activity of the oral BRAF inhibitor vemurafenib in patients with relapsed or refractory BRAF mutant hairy cell leukemia (HCL) (Park et al. ASH 2014). According to the study design, patients whose disease relapsed following the initial treatment were allowed to be re-treated with vemurafenib. Here we report the clinical outcome of patients who were retreated with vemurafenib at relapse following initial treatment, as well as the result of genomic analysis that provided an insight into mechanisms of resistance to BRAF inhibition in HCL. Patients and Methods: Patients with BRAF mutant HCL who were refractory or resistant to purine analogs, or who had ≥2 relapses with an indication for treatment (ANC ≤1.0, HGB ≤10, or PLT ≤100K) were enrolled. Eligible patients received vemurafenib 960mg twice daily for 3 months. Bone marrow (BM) evaluations were performed after 3 months to assess response. Patients with partial (PR) or complete response (CR) with detectable minimal residual disease were allowed to receive vemurafenib for up to 3 additional months. After a maximum of 6 months of therapy, patients were observed with monthly CBC. At disease relapse with peripheral blood (PB) counts low enough to meet the initial eligibility criteria, re-treatment with vemurafenib was allowed until disease progression or unacceptable toxicity. Serial PB and/or BM samples were collected for targeted next-generation sequencing analysis of a 300-gene panel to detect contributors to resistance and genes collaborating with BRAF mutations in HCL. Results: 26 patients have been enrolled. 2 patients discontinued treatment before response assessment: 1 patient due to primary refractory disease to vemurafenib and 1 patient due to grade 3 photosensitivity. 24 patients completed at least 3 months of treatment, and therefore are available for efficacy evaluations. Of the 24 evaluable patients, all patients achieved response (10 CR and 14 PR) with the overall response rate of 100% when assessed after 3 months of vemurafenib. With the median follow up of 11.7 months (range, 1.3 - 25.4 months), 7 patients experienced disease relapse (3 previous CR and 4 PR). Of the 7 relapse patients, 6 met re-treatment criteria and restarted vemurafenib. 4 of the 6 patients regained response (all PR) with complete hematologic recovery and remain on therapy. 2 patients discontinued re-treatment before response assessment: 1 patient due to grade 2 photosensitivity and fatigue, and 1 patient due to resistant disease with refractory cytopenia and a rapid increase in splenomegaly. Targeted genomic analysis in 20 patients pre-vemurafenib revealed at least 1 somatic alteration coexisting with the BRAF V600E mutation in every patient, including deletion of 7q in more than half of patients and recurrent mutations in MLL3 and MED12 (Figure). Genomic analysis of the patient with de novo resistance to vemurafenib identified a missense mutation in IRS1 (Insulin Receptor Substrate 1; IRS1 P1201S) in addition to the BRAF V600E mutation. Functional characterization of the IRS1 P1201S mutation in vitro revealed potent induction of MAP kinase and PI3K-AKT signaling by the IRS1 mutant relative to wildtype, consistent with prior knowledge that IRS1 activates both MAP kinase and PI3K-AKT signaling. These data suggest that bypass activation of ERK and parallel activation of the AKT pathway contributed to de novo vemurafenib resistance. In the patient with acquired resistance to vemurafenib, genetic analysis of pretreatment, remission and relapse PB mononuclear cells revealed emergence of 2 separate, activating subclonal KRAS mutations at relapse. The mutations in KRAS were not seen at pretreatment or at remission. Activating RAS mutations are well known mediators of vemurafenib resistance in BRAF V600E-mutant malignancies, and, in this case, the detection of KRAS mutations coincided with clinical relapse and insensitivity to vemurafenib. Conclusions: Despite high response rates after a short course of vemurafenib in most patients, we observed de novo and acquired resistance to vemurafenib. Serial genomic analysis revealed ERK-dependent and independent mechanisms of BRAF inhibitor resistance in HCL. Our data provide the first insights into genetic mechanisms of RAF inhibitor resistance in HCL and suggest combinatorial therapeutic strategies that may have a role in the therapy of HCL. Figure 1. Figure 1. Disclosures Park: Amgen: Consultancy; Juno Therapeutics: Other: Advisory Board, Research Funding; Genentech: Research Funding. Off Label Use: Vemurafenib in HCL. Stone:Agios: Consultancy; AROG: Consultancy; Juno: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Celator: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Roche/Genetech: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding. Rai:Nash Family Foundation: Research Funding; Karches Family Foundation: Research Funding; Nancy Marks Family Foundation: Research Funding; Leon Levy Foundation: Research Funding. Altman:Seattle Genetics: Other: Advisory board; Ariad: Other: Advisory board; Spectrum: Other: Advisory board; Novartis: Other: Advisory board; BMS: Other: Advisory board; Astellas: Other: Advisory board; assistance with abstract preparation. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Purpose: Radiotherapy (RT)-based bladder preservation is an alternative to perioperative chemotherapy and radical cystectomy (RC) for selected patients with muscle-invasive bladder cancer (MIBC). Recent data suggest that somatic DNA damage response (DDR) alterations are associated with improved outcome in MIBC patients receiving RC +/- perioperative chemotherapy. Improved understanding of the influence of DDR genetics on response to RT-based bladder preservation may aid patient selection. Methods: We performed deep, targeted capture sequencing of primary bladder tumors and paired/pooled normal specimens from 48 RT treated MIBC patients with a DDR gene-enriched panel. To test whether correlations of DDR alterations with outcomes were specific to a RT cohort, we also assessed a subset of these DDR genes that were sequenced in a previously published series of 89 patients who received RC +/- neoadjuvant platinum-based chemotherapy. Specimens were re-reviewed to confirm urothelial histology. Deleterious alterations were defined as somatic nonsense, frameshift, or splice site mutations, or missense mutations at or near functionally significant residues validated in the literature. Results: In the RT cohort, median RT dose was 66 Gy, and chemotherapy use was neoadjuvant and concurrent (46%), concurrent only (48%), or none (6%). Visibly complete TURBT was achieved in 71% of patients. Median surviving follow up was 28 months. There were 30 progression events (crude rate 63%), comprised of 22 metastases (crude 46%) and 24 local in-bladder recurrences (crude 50%). While 30 (63%) patients had alterations in DDR genes, only 13 (27%) patients had deleterious DDR alterations, specifically in ATM (2), BRCA1 (1), BRIP1 (1), ERCC2 (6), FANCD2 (1), and PALB2 (1). On multivariable Cox proportional hazards analysis, the presence of a deleterious DDR alteration was associated significantly with lower relapse risk (HR 0.28, 95% CI 0.08-0.95; p = 0.041), as well as with a trend towards lower risk for metastasis (HR 0.32, 95% CI 0.10-1.11; p = 0.07) and any disease progression (HR 0.35, 95% CI 0.12-1.03; p = 0.06). In the RC cohort, neoadjuvant chemotherapy was given in 39% of patients, and deleterious DDR gene alterations were noted only in 8% of the patients. On 2-sided log-rank testing, such alterations conferred a non-significant trend for improved recurrence-free survival (p = 0.20) and disease-specific survival (p = 0.10). Conclusion: Deleterious somatic alterations in DDR genes were associated with significantly improved outcomes in bladder cancer patients undergoing RT-based therapy and with a trend for improved outcomes in those treated with RC +/- platinum chemotherapy. Further research is warranted to validate these findings on an independent RT-treated dataset and to clarify the relationship in a larger chemotherapy-treated RC cohort. Citation Format: Neil B. Desai, Gopa Iyer, Eugene K. Cha, Sasinya N. Scott, Joseph Hreiki, John P. Sfakianos, Philip Kim, Aditya Bagroida, Bernard H. Bochner, Jonathan E. Rosenberg, Dean F. Bajorin, Michael F. Berger, Marisa A. Kollmeier, Hikmat Al-Ahmadie, David B. Solit. Deleterious alterations in DNA damage response genes are associated with improved outcome in muscle-invasive bladder cancer patients treated with radiation-based bladder preservation. [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 602. doi:10.1158/1538-7445.AM2015-602

Background Urothelial carcinomas are capable of arising at multiple sites within the urinary tract. About 90% of cases originate in the urinary bladder (lower tract) and about 10% of cases emerge in the pelvis or ureter (upper tract). While these sites have similar histologic appearances, there are differences in their epidemiologic, clinical and pathologic characteristics, suggesting they may represent two distinct diseases. Previous studies have observed a more aggressive disease from patients with upper tract urothelial carcinoma (UTUC) versus patients with urothelial carcinoma of the bladder (UCB). Our aim was to examine whether the clinicopathological differences between upper and lower tract urothelial tumors are the result of differences in their scope of somatic genetic alterations. Methods Tumors and matched germline DNA from 59 patients with high-grade UTUC and 102 patients with high-grade UCB were extracted. The genomic profiles of these patients were analyzed and compared using a custom hybridization capture-based sequencing assay to identify point mutations, small insertions, deletions and copy number alterations of 230 cancer-associated genes. Results Average next-generation sequencing coverage for high-grade UTUC (674x) and UCB (762x) tumors. The comparison between the high-grade UTUC and UCB tumors identified significant differences in the prevalence of somatic alterations between these cohorts. Alterations in oncogene HRAS (13.6% UTUC vs. 1.0% UCB, p = 0.001) were more common in high-grade UTUC. Another oncogene FGFR3 (35.6% UTUC vs. 21.6% UCB, p = 0.065) was not significantly different between the UTUC and UCB cohorts. Genes identified as significantly less frequently altered in UTUC compared to UCB tumors included tumor suppressor genes TP53 (25.4% vs. 57.8%, p Conclusions While the genes with somatic alterations in upper and lower tract urothelial tumors were similar, we did identify significant differences in the prevalence of several recurrently altered genes including TP53, RB1, HRAS and ARID1A between UTUC and UCB cohorts. These findings may account for the divergence in clinical outcomes observed between these two disease sites and the high prevalence of actionable genomic targets may assist in the development of novel therapeutic approaches for these diseases. Citation Format: Sasinya N. Scott, John P. Sfakianos, Eugene K. Cha, Gopa Iyer, Emily C. Zabor, Philip H. Kim, A. A. Hakimi, Irina Ostrovnaya, Ricardo Ramirez, Aphrothiti J. Hanrahan, Neil Desai, Qinghu Ren, Arony Sun, Jonathan E. Rosenberg, Guido Dalbagni, Dean F. Bajorin, Michael F. Berger, Bernard H. Bochner, Hikmat Al-Ahmadie, David B. Solit, Jonathan A. Coleman. Examining the genomic differences between upper and lower tract urothelial carcinomas. [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 LB-174. doi:10.1158/1538-7445.AM2015-LB-174

324 Background: PI3 kinase (PI3K) pathway alterations are found in 39% of urothelial carcinoma (UC) (TCGA). Prior reports have shown significant responses in metastatic UC patients (pts) whose tumors harbor PI3K pathway alterations treated with mTOR inhibitors, providing a rationale to investigate BKM120 in mUC. Methods: This phase II study enrolled mUC pts progressing on platinum-based chemotherapy (up to 4 prior agents). The primary and secondary endpoints were proportion of pts progression-free at 2 months and response rate (RR) by Response Criteria in Solid Tumors (RECIST) v1.1, respectively. A Simon 2-stage design was used to discriminate between a 2 month PFS rate of 80%. Pts received 100 mg drug once daily. To identify predictors of response/resistance to BKM120, targeted exon capture sequencing was performed to define the mutation status of PIK3CA, PTEN, AKT1, TSC1, and additional genes within tumors from all treated pts. Results: 13 of 15 enrolled pts were eligible for the primary endpoint. Median age was 65 (53-82). Pts had received an average of 3 agents (2-4) before enrollment. The median progression-free survival (PFS) was 2.77 months (95% CI: 1.83-3.71) with 6 pts displaying stable disease (SD) and 1 partial response (PR) at 2 months (PFS rate 54%). Sequencing identified 2 pts with PIK3CA mutations (E542K, H1047R) who experienced progression as best RECIST response. Tumor from the pt with a 16 month PR harbored a TSC1 R500* nonsense mutation. 1 pt with SD lasting 3.7 months had a TSC1 L330fs truncation. Conclusions: While BKM120 therapy did not display a significant improvement in 2-month PFS rate compared to standard chemotherapy in the second-line setting, 2 pts, one with a durable PR and one with SD, had tumors with inactivating mutations in TSC1. Based upon these results, an expansion cohort is accruing in which select mUC pts whose tumors harbor PI3K pathway alterations receive BKM120. Clinical trial information: NCT01551030.

301 Background: Urothelial carcinoma is characterized by multifocal and metachronous tumors. To explain this phenomenon, two hypotheses have been proposed: the ‘field defect’ hypothesis - urothelial cells are primed to undergo transformation by exposure to carcinogens, and the clonal, or ‘single progenitor cell,’ hypothesis - tumors arise from intraluminal seeding of transformed cells. Methods: To examine their clonal relationships, we compared the genomic profiles of primary upper tract urothelial carcinoma (UTUC) tumors and metachronous bladder tumors (intravesical recurrences) in patients treated with radical nephroureterectomy (RNU) and subsequent transurethral resection. Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 341 cancer-associated genes. Results: We analyzed 16 primary UTUC tumors and 41 intravesical recurrences in patients treated with RNU. The median number of intravesical recurrences per patient was 2 (range 1-7) and the interval from RNU to intravesical recurrence ranged from 3.5 months to 129 months. With an average sequencing coverage of 516x, we found strong evidence delineating the clonal relationship between primary UTUC tumors and subsequent bladder tumors. The majority of somatic mutations present in the primary UTUC tumors (median=7, range 4-39) were detected in all subsequent bladder tumors (128/146, 88%). In an illustrative case, one patient followed with periodic cystoscopy/cytology who had been NED for 5.5 years then developed 7 bladder tumors over the next 44 months, each with the identical mutation profile (8 mutations) as the primary tumor. Conclusions: We demonstrate that bladder tumors following RNU represent true intravesical recurrences, with almost all tumors sharing the same somatic mutation profile as the primary UTUC tumor. This has important implications for surgical techniques to minimize the risk of intraluminal seeding, the delivery of intravesical therapy following RNU, and the development of strategies employing systemic chemotherapy or targeted agents.

Deregulation of the HER family of receptor tyrosine kinases has been widely implicated in cancer initiation and progression. Activating mutations or gene amplification of EGFR and ERBB2 (HER2) are frequently found in non-small cell lung, breast, colorectal, glioblastoma and head and neck cancers. However, little is known regarding the prevalence or functionality of genomic alterations in the ERBB3 (HER3) isoform. Although ERBB3 has limited inherent kinase activity, ligand stimulation promotes its dimerization with active kinases like ERBB2 and EGFR that can phosphorylate ERBB3 which then promotes transformation. Thus genomic alterations in ERBB3 may be a key means of promoting oncogenic signaling despite the protein lacking robust enzymatic activity. We performed a meta-analysis of 40 cross-cancer next generation sequencing and copy number genomics data sets newly generated by our group (bladder) or found in recently published repositories (The Cancer Genome Atlas (TCGA), Broad, Genentech, Sanger, etc). Analogous to the singular 2013 publication on ERBB3 mutations to date, we found frequent mutations in ERBB3 in gastric tumors (∼11%). Preliminary analysis of our novel and expanding cohort of 109 high-grade bladder cancer tumors suggests that ERBB3 mutations are equally frequent in bladder cancer (∼10-12%). Collectively, EGFR, ERBB2 and ERBB3 alterations were found in 23-30% of all high-grade bladder cancers, typically in a mutually exclusive pattern suggesting that these may be driver events. ERBB3 mutations were distributed across all structural domains of the protein. We identified more than 15 novel ERBB3 mutations in our bladder cohort, including hotspot mutations in the extracellular domain (V104L/M, D297Y, others) that were recurrent across multiple cancers types. Stable retroviral overexpression of ERBB3 mutants into NIH-3T3 cells induced phosphorylation of ERBB3, activation of downstream signaling cascades and soft agar colony formation, all which were enhanced in the presence of neuregulin and inhibited by the dual EGFR/ERBB2 kinase inhibitor, neratinib, and a monoclonal antibody to ERBB3, AMG-888/U3-1287. These data highlight a large cohort of bladder cancer patients with novel, activating mutations in the tractable kinase ERBB3, that could potentially benefit from approved EGFR/ERBB2 targeted therapeutics in the clinic and novel ERBB3 therapeutics in early clinical trials. Citation Format: Aphrothiti J. Hanrahan, John P. Sfakianos, Ricardo Ramirez, Phillip H. Kim, Gopa Iyer, Hikmat A. Al-Ahmadie, Sasinya N. Scott, Dean F. Bajorin, Bernard H. Bochner, Jonathan A. Coleman, Jonathan E. Rosenberg, Michael F. Berger, Sarat Chandarlapaty, David B. Solit. Frequency and function of ERBB3 mutations in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3419. doi:10.1158/1538-7445.AM2014-3419