Bryce, Alan H.

348 Background: Patients (pts) with mUC have few treatment options beyond the second-line setting. HER2 gene amplification has been reported in a minority of pts with UC, but there have been anecdotal reports of the activity of HER2-targeted agents. MyPathway is a multi-basket study evaluating the efficacy and safety of targeted therapies in non-indicated tumor types harboring relevant molecular alterations. We present preliminary data for pts with HER2-positive mUC receiving HER2-targeted treatment with pertuzumab + trastuzumab. Methods: MyPathway (NCT02091141) is an open-label, multicenter, phase IIA study. Pts in this subset analysis had refractory mUC with HER2 amplification or putative activating mutations by gene sequencing, FISH, or IHC. Pts received standard doses of pertuzumab + trastuzumab without chemotherapy until disease progression or unacceptable toxicity. The primary endpoint is investigator-assessed overall response rate (RECIST v1.1). Results: As of July 31, 2016, 12 pts with platinum-resistant HER2-positive mUC (HER2-amplified, n=9; HER2-mutated, n=3) have been enrolled. At a median follow-up of 5.4 (range 0.9–14.5) mos, 1 pt had complete response (CR, ongoing at 12.5 mos), 2 had partial responses (PR; duration of response, 3.7 and 5.5 mos), and 2 had stable disease (SD) for >4 mos (Table). Safety was consistent with the product labels. Conclusions: Preliminary results indicate that the combination of pertuzumab + trastuzumab has activity in previously treated HER2-amplified mUC, including a durable CR in a pt with peritoneal metastases. Accrual to MyPathway is ongoing. Clinical trial information: NCT02091141. [Table: see text]

175 Background: Genome and transcriptome aberrations associated with primary resistance to abiraterone acetate/prednisone (AA/P) in mCRPC are not known. In a prospective trial (NCT#01953640) we assessed whole-exome and RNA-seq based aberrations in metastases of CRPC stage patients (pts) for identifying markers associated with primary resistance to AA/P. Methods: Whole-exome and RNA-seq of biopsies from metastases was performed followed by analyses for association between genomic aberrations & primary resistance. Primary resistance was defined by progression on AA/P after 12-weeks of therapy(non-responders) using PSA, RECIST, bone scan and symptom criteria (per PCWG2). Gene network analysis was performed in genes mutated more frequently in non-responders, and also in genes that were differentially expressed between non-responders and responders and a “risk ratio” (RR) was calculated thereof. Results: Between 6/2013 & 8/2015, 92 pts were enrolled of which 82 had complete whole-exome, RNA-seq and 12-week outcome data available for analysis. Median age was 72.5 yrs (IQR: 68.5-78); median PSA-18 ng/ml (IQR: 8.1- 46.6). At 12-weeks 33/82 had progressed. Using the RR of 2 as threshold, we identified 113 and 292 genes that were more frequently mutated in non-responders & responders respectively. Among the 113 genes, OBSCN, ADAM21, LPHN3, DOCK10 ( P = 0.08, RR= inf) & USP42 ( P = 0.16, RR = 5.71) were the top candidates.Gene network analysis revealed that in non-responders, genes involved in the Wnt/β-catenin pathway were frequently mutated and negative regulators of Wnt pathway ( DKK4, SFRP2 & LRP6) were also frequently deleted. Gene expression analyses revealed the expression levels of Wnt/β-catenin pathway inhibitors were significantly reduced while expression levels of cell cycle regulatory genes were significantly increased in non-responders. Conclusions: In this study we observed Wnt/β-catenin pathway activation to be associated with primary AA/P resistance. This finding offers a potential for the development of predictive biomarkers and modulation of targeted pathways to overcome AA/P resistance. Clinical trial information: NCT# 01953640.

464 Background: Although the von Hippel-Lindau (VHL) tumor suppressor gene is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. We hypothesize that loss of SETD2 methyltransferase activity alters the genome wide pattern of H3K36 trimethylation (H3K36me3) in ccRCC, and contributes to the cancer phenotype. Methods: To generate a genome-wide profile of H3K36me3 in frozen nephrectomy samples and RCC cell lines, we optimized a chromatin immunoprecipitation (ChIP) protocol for the isolation of DNA associated with H3K36me3. H3K36me3 is associated with open chromatin and an H3K36me3-specific antibody was used for immunoprecipitation of endogenous H3K36me3-bound DNA. ChIP PCR primers were optimized for active genes, such as actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a “gene desert” on chromosome 12 (negative control). ChIP libraries were then generated from 3 paired uninvolved kidney and RCC and 2 RCC cell lines. In order to identify H3K36Me3 upregulated regions in uninvolved kidney and RCC, reads from the ChIP sequencing were mapped to the human genome using Burrows-Wheeler Aligner and SICER algorithms. Results: Using ChIP PCR, we found that active genomic regions were enriched 15-30 fold over the negative controls indicating that the quality and yield of immunoprecipitated DNA/chromatin complexes from frozen tissue was sufficient for ChIP sequencing. A preliminary ChIP sequencing analysis of RCC cell lines and frozen ccRCC tissue indicates that H3K36me3 enriched DNA sequences were mapped to exons (31.3%) compared to introns (13.5%, p<0.001), consistent with the role of H3K36me3 in transcription. Conclusions: Genomic regions enriched for H3K36Me3 binding were identified from patient-derived tissue and RCC cell lines. Current efforts are focused on comparing the H3K36me3 profiles between matched tumor and uninvolved kidney ChIP libraries to generate a genome wide map of dysregulated H3K36me3 modifications.

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

243 Background: Cbz/pred extends survival in mCRPC patients (pts) following docetaxel and first-line studies comparing Cbz to docetaxel are ongoing. Mito/pred also has anti-tumor activity in mCRPC and a non-overlapping mechanism of action and toxicity profile with Cbz. A multicenter phase I trial was initiated through the DOD PCCTC to establish the maximally tolerated dose (MTD) of the combination of Cbz, Mito, and Pred. Methods: Chemotherapy (chemo)-naive pts with mCRPC were enrolled in an accelerated titration design. The primary objective was to determine the MTD of CAMP; secondary objectives included assessing PSA and objective response rates and duration of response. Starting dose of Cbz was 25 mg/m2 (the currently approved single-agent dose) and Mito 4 mg/m2, administered on day 1 of a 21-day cycle. Mito was escalated by 2 mg/m2 per dose level. Pred 5 mg BID and pegfilgrastim were administered with each cycle. Results: 9 pts were enrolled (dose level I, N = 1; dose level II, N = 1; dose level III, N = 3; dose level IV N = 4). The median age was 67 (range 55-81) and the median baseline PSA was 82.3 (range 20.4-791.2). There were 2 DLTs observed at dose level IV (Cbz 25 mg/m2 + mito 10 mg/m2), both grade 4 sepsis. The most common grade 3 adverse events were hematologic (neutropenia, n = 5; febrile neutropenia, n = 2, thrombocytopenia, n = 2). 6 of 9 pts remain on study (range 2.2 - 10.5+ months). Maximal PSA declines from baseline > 50% have been observed in 4/7 (57%) evaluable pts to date. 2 of 4 pts with measurable disease have experienced partial responses and a third has had a minor response after 4 cycles of chemo. The median progression-free survival and duration of response have not been reached. Conclusions: The approved single-agent dosage of Mito (12 mg/m2) could not be safely reached in combination with Cbz 25 mg/m2. Pre-planned evaluation of an alternate dosing strategy combining Mito with Cbz 20 mg/m2, which has demonstrated activity in mCRPC and potentially less hematologic toxicity, is underway. Preliminary efficacy data are encouraging but additional follow-up and further study of the treatment combination is required. Clinical trial information: NCT01594918.

249 Background: Evaluating tumor specific pathologic and genetic profiles in mCRPC stage is difficult due to the limited availability of mCRPC tissue. We describe findings from a prospective cohort study performed to obtain concomitant histopathology and genetic information in mCRPC. Methods: Patients with mCRPC initiating abiraterone acetate therapy underwent 2 serial (3 months apart) metastatic (met) site needle core biopsies (NCB1/2). Bone lesions were biopsied using 11-13G core needles and 18G core biopsy devices were used for non-osseous masses. Up to 4 cores were obtained at each biopsy with the 1st core (S1) sent for DNA sequencing, the 2nd (S2) for RNA sequencing, and the 3rd/4th (X3, X4) submitted for xenograft implantation. From each, 1-2 mm segments were separated and formalin-fixed for histopathologic examination (HPE). Results: A total of 54 patients, enrolled between June 2013 and July 2014, underwent 94 NCB (54 NCB1, 40 NCB2), rendering a total of 259 samples (94 S1; 75 S2; 59 X3 and 31 X4) of which 85 (32%) were positive (pos) for tumor by HPE. Positivity for tumor in S1, S2, X3, and X4 cores was 42%, 33%, 22%, and 19%, respectively. At least one core pos for met tumor was observed in 62% NCB1 and 52% NCB2 (overall 52/94; 56%). Met sites biopsied include bone (71), lymph nodes (18), liver (3), penile (1) and pelvic (1) soft tissues. HPE revealed met adenocarcinoma in 44/52, and poorly differentiated carcinoma in 8/52. Gleason grade applied to the met ranged from 3+4 to 5+5. 45/85 pos samples (53%) had > 50% tumor cellularity. Pos NCB in bone lesions was observed in 23/71 (32%), compared to 17/23 (73%) of non-bone sites (p=.0004). 85 S1 samples yielded DNA material, of which 66 (77%) had ≥10% tumoral DNA. 24 cases with negative HPE had ≥10% tumoral DNA; 7 cases with pos HPE had <10 tumoral DNA. In all, 76/94 (81%) NCB yielded tumor material either by HPE or DNA analysis. Conclusions: NCB of mCRPC is feasible and provides adequate tissue for pursuing HPE and sequencing studies. HPE of extracted material correlates with DNA sequencing data and provides complementary information on tumor features. Bone lesions yield significantly less tumoral material than non-osseous sites.

238 Background: Cbz/pred extends survival in mCRPC patients (pts) following docetaxel and first-line studies comparing Cbz to docetaxel are ongoing. Mito/pred also has anti-tumor activity in mCRPC and a non-overlapping mechanism of action and toxicity profile with Cbz. A multicenter phase 1 trial was initiated through the DOD PCCTC to establish the maximally tolerated dose (MTD) and recommended phase 2 dose (RP2D) of the combination of Cbz, Mito, and Pred (CAMP). Methods: Chemotherapy (chemo)-naïve pts with mCRPC were enrolled in an accelerated titration design. The primary objective was to determine the MTD and RP2D of CAMP; secondary objectives included PSA response rate and duration of response. Cbz 20 and 25 mg/m2 were each evaluated in combination with escalating doses of Mito (starting dose 4 mg/m2), both administered on day 1 of a 21-day cycle. Pred 5 mg BID and pegfilgrastim were given with each cycle. Results: 20 pts were enrolled. The median age was 66 (range 51-78) and the median baseline PSA was 62.5 (range 3.0-791.2). There were 2 DLTs observed at the dose level of Cbz 25 mg/m2 + Mito 10 mg/m2 (sepsis and febrile neutropenia). No DLTs were observed with Cbz 20 mg/m2 (N = 10), establishing Cbz 20 mg/m2 + Mito 12 mg/m2 as the MTD and RP2D. The most common grade ≥ 3 related adverse events were hematologic (neutropenia, n = 8; thrombocytopenia, n = 3; febrile neutropenia, n = 2). No cardiac adverse events were observed. The median number of treatment cycles was 9 (range 2-16), and 5 pts remain on study. Greater than 50% maximal PSA declines from baseline were observed in 8 of 18 evaluable pts (44%). Objective tumor responses have been observed in 2 of 4 pts (50%) with measurable disease, with evaluation ongoing. The median duration of response has not been reached (range 4.9-10.0+ months). Conclusions: The approved single-agent dose of Mito (12 mg/m2) was safely combined with Cbz 20 mg/m2, a dose with demonstrated activity in mCRPC and potentially less hematologic toxicity than 25 mg/m2. Preliminary efficacy data are encouraging with durable responses observed in a subset of pts. Further study of the treatment combination is warranted. Clinical trial information: NCT01594918.

190 Background: It is not known if biopsy of CRPC stage metastases alters CTC results. We evaluated pre & post biopsy CTC counts in a prospective cohort undergoing pre-chemotherapy abiraterone acetate/prednisone (AA/P). Methods: CTC enumeration was performed on 7.5mL blood using the FDA cleared immunomagnetic/immunofluorescence assay (CellSearch). Time of blood draw for CTC counts pre/post biopsy was recorded for each biopsy. Biopsies were performed using 11/13 gauge core biopsy device for bone lesions and 18 gauge core needle biopsy device for soft tissue/nodal metastases. All patients (pts) underwent two serial biopsies 3 months apart. Change in CTC counts pre/post biopsy for visits 1 and 2 were evaluated using Wilcoxon signed rank test (significance at p<0.05). Differences in CTC count change (visit1) pre/post biopsy based on metastatic site (skeletal vs. non-skeletal) was compared using Wilcoxon rank-sum test. Results: Median age of the cohort was 74 years (IQR: 68-78); median PSA at V1 was 16.4 ng/ml (IQR: 6.4-87.6); at V2 was 11.5 ng/ml (IQR: 1.8-23.7). Of 59 pts undergoing biopsies 42 were in bone and 17 in nodal/soft tissue masses. At V1, pre/post biopsy CTC counts were measured on 50 and 49 pts, respectively; at V2, pre/post CTC counts were measured on 43 and 36 pts. High volume metastatic disease at V1 was observed in 67.8 % of all pts. Table lists CTC counts and time of collection pre/post biopsy for both visits. Mean change in pre/post biopsy CTC counts for skeletal sites was 0.7 (range: -12 to 64) compared to 12.1 (range: -21 to 13) for non-skeletal sites (p=0.23). Conclusions: An increase in mean CTC counts after biopsy of CRPC metastases was observed. No significant differences in change of CTC counts based on site biopsied (skeletal versus non-skeletal) was noted. Correlation with clinical outcomes is on-going. Clinical trial information: NCT# 01953640. [Table: see text]

174 Background: AA/P is an FDA approved treatment for mCRPC. Since markers of early resistance to AA/P are unknown, we report initial findings of androgen receptor (AR) based associations with short term (12 week) progression on AA/P. Methods: mCRPC stage patients (pts) initiating pre-chemotherapy AA/P underwent metastatic site biopsies at baseline (pre AA/P) and after 12 weeks. Baseline somatic whole exome DNA, tumor gene expression for AR full length (ARFL), AR splice variant 7 (ARV7) and ARV7/ARFL ratios were compared in progessors versus non-progressors. Progression at or within 12 weeks of AA/P therapy was defined as death or disease progression by PCWG2 “composite progression (CP)” and/or “radiographic progression” endpoints. Wilcoxon rank-sum tests were used to test for differences in the two groups for comparing ARFL, ARV7 expressions and ARV7/ARFL ratios and chi square tests were used for differences in copy number variation. Results: Between 1/2013 and 6/2014, 59 pts were enrolled of which 44 have disease assessment data at the12-week time point. CP was observed in 17/44 patients. DNA seq and clinical data was available for 42/44 pts. Using radiographic progression at 12 weeks, AR Amplification/Gain was observed in 20/26 non-progressors (13 with Amplification) and in 9/16 progressors (P-value = 0.19; OR 2.5). ARFL and ARV7 gene expressions in both groups is provided in table. Conclusions: A trend towards higher ARV7/ARFL gene expression ratio in metastases was observed with early progression on AA/P. AR gain/amplification is observed less often in pts with early progression. Validation of these findings is on-going in this prospective trial. Clinical trial information: NCT# 01953640. [Table: see text]