Ho, Thai H.

424 Background: Programmed death-1 (PD-1) receptor negatively regulates T cell-mediated responses.PD-1 ligand (PD-L1) is aberrantly expressed in clear cell renal cell carcinoma (ccRCC) and is associated with worse prognosis. Levels of PD-L1 expressions in non-ccRCC and its association with clinicopathological features and survival are unknown. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 97 patients with chromophobe (CHR), papillary (PAP), translocation Xp11.2 (TrL) RCC and oncoytoma (ONC) and were included in the analysis. PD-L1 expression was evaluated by immunohistochemistry using a mouse monoclonal anti-PD-L1 antibody (405.9A11). The assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry. PD-L1 tumor positivity (PD-L1+) was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in immune cells, a combined score based on the intensity of infiltrate and percentage of positive cells was used. Baseline characteristics including stage/grade, and survival data were collected. Comparisons between PD-L1 expression and clinicopathological features were evaluated using chisq or wilcoxon rank sum tests. Cox model tests for association of PD-L1 expression with OS in univariate and multivariable analysis. Results: Among 97 patients, 12 (12.4%) were considered PD-L1+ in tumor cells: 2/36 (5%) of CHR RCC, 5/50 (10%) of PAP RCC, 3/7 (43%) of TrL RCC, and 2/4 (50%) of ONC. PD-L1 positivity in tumor cells was significantly associated with higher stage (p=0.026) and grade (p=0.046), as well as lower OS on univariate (p=0.02) but not multivariate analysis (p=0.29). On the other hand, PD-L1 positivity by immune cells was observed in 50 (51.5%) patients: 13/36 (36%) of CHR RCC, 30/50 (60%) of PAP RCC, 6/7 (86%) of TrL RCC, and 1/4 (25%) of ONC. PD-L1 positivity in immune cells did not correlate with stage (p=0.7), grade (p=0.1) or OS (p=0.8). Conclusions: PD-L1 expression is variable in non-ccRCC and depends on histology and tumor vs. immune cells scoring. Only PD-L1 positivity in tumors cells was associated with aggressive features. Patients with non-ccRCC should not be automatically excluded from trials of agents that target the PD-1/PD-L1 pathway.

414 Background: While mutations in PBRM1 (~40%) and BAP1(~10%) are associated with clinical outcomes and pathologic features in clear cell renal cell carcinoma (ccRCC), the impact of protein expression of these genes remains unknown. Herein, we quantify PBRM1/BAP1 protein expression in a large cohort of patients with localized ccRCC and associate expression with cancer-specific survival (CSS) and pathologic features. Methods: We utilized the Mayo Clinic Renal Registry and identified 1,416 patients who underwent nephrectomy to treat clinically localized ccRCC between 1/3/1990 and 4/14/2009. We used immunohistochemistry (IHC) to detect PBRM1/BAP1 expression, and a central pathologist blinded to the outcomes scored tumors as either positive or negative. Tumors with heterogeneous or equivocal staining were excluded from this analysis. We generated Cox proportional hazard regression models for associations with ccRCC-SS, and we employed Mann-Whitney U tests for associations with pathologic features. Results: Of the 1,416 samples, 1,232 (87%) were PBRM1/BAP1 positive or negative, 163 (11%) had heterogeneous staining, and 21 (1%) could not be assessed. The distribution and association of PBRM1/BAP1 phenotypes with clinical outcomes are listed in the table below. PBRM1+/BAP1+ tumors have the best CSS, and PBRM1-/BAP1- have the worst. In addition, PBRM1/BAP1 expression strongly associated with the tumor size, stage, grade, and tumor necrosis (p<0.0001). Conclusions: This study is the first and largest to quantify PRBM1/BAP1 protein expression in ccRCC tumors. We were able to quantify PBRM1/BAP1 through IHC in the vast majority of tumors (87%), and PRBM1/BAP1 expression strongly associates with both CSS and pathologic tumor characteristics. Our data confirms our previous findings of the importance of PRBM1/BAP1 in the molecular pathogenesis of ccRCC. [Table: see text]

386 Background: Angiogenesis plays a role in tumor growth and is partly mediated by factors in both the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) pathways. Brivanib inhibits both VEGF receptor 2 and FGF receptors (FGFR)-1/2. Methods: Nephrectomy material from 40 patients with metastatic renal cell carcinoma (RCC) receiving sorafenib ± interferon was assessed by immunohistochemical (IHC) staining. The Ariol imaging platform was used to stratify the specimens based on intensity of staining for FGF biomarkers: FGFR-1 and fibroblast growth factor receptor substrate 2 (pFRS2). The relationship between fibroblast growth factor pathway marker levels and progression-free survival (PFS) was analyzed using Kaplan-Meier and Cox proportional hazards regression methods. To assess the efficacy of dual targeting of VEGF and FGF receptor signaling by brivanib, we treated a xenograft RCC mouse model with sunitinib, brivanib, or sunitinib followed by brivanib. Results: Our analysis indicated that more intense FGFR-1 staining was associated with shorter PFS (p = 0.0452), and that pFRS2 staining were not significantly associated with PFS (p = 0.2610). FGF pathway activation was associated with decreased PFS in patients with metastatic RCC treated with sorafenib. Xenograft mice were treated with 20 mg/kg sunitinib and after an initial stabilization phase, resistance developed by day 20. A switch to 30 mg/kg brivanib prolonged the stabilization phase after progression on sunitinib (p = 0.0033). Conclusions: The PFS of patients treated with sorafenib is associated with FGF pathway activation. In a xenograft RCC mouse model, brivanib overcame the tumor escape after initial treatment with sunitinib. Switching from sunitinib to brivanib at the time of tumor escape prolonged the stabilization phase and overcame a sunitinib-induced resistance phenotype. The results suggest that FGF pathway activation mediates both innate and acquired tumor resistance to VEGF receptor inhibition. The efficacy of dual inhibition of FGF and VEGF in the subset of patients with increased FGF activation prior to treatment requires further study.

507 Background: Clear cell renal cell carcinoma (ccRCC) is a well-described molecularly heterogeneous tumor. Herein, we assessed the concordance of two of the most commonly mutated genes in ccRCC, PBRM1 (~50%), and BAP1 (~15%), in patient-matched primary and metastatic tumors. Methods: One pathologist (PK) assessed PBRM1 and BAP1 protein expression using immunohistochemistry (IHC) in 99 patients with a primary and at least one metastatic ccRCC tumor available for analysis. All available metastatic tumors were analyzed. Results: A total of 99 patients (48 M0 and 51 M1) had both a primary tumor and at least one metastatic tumor available for analysis. There were a total of 158 metastases with one patient having up to 7 metastases available for analysis. The concordance between primary and patient-matched metastasis was 87% for PBRM1 and 99% for BAP1. We observed a similar concordance between patients with M0 versus M1 disease. Conclusions: While ccRCC is molecularly heterogeneous, PRBM1, and BAP1 are largely concordant between primary and metastatic lesions suggesting that PBRM1 and BAP1 are genetically truncal events in the molecular pathogenesis of ccRCC.

501 Background: Predictive biomarkers of response to targeted therapy are lacking in renal cell carcinoma (RCC). We evaluated a cohort of RCC patients referred for molecular profiling to identify potentially actionable recurrent molecular aberrations. Methods: 166 consecutive renal cases referred to Caris Life Sciences over 2 yrs were evaluated with central pathology review. Cases were subtyped into clear cell (ccRCC), n=100, papillary (PRCC), n=20, sarcomatoid, n=10, translocation, n=6, medullary, n=4 or unclassified, n=26. 63% of cases were metastatic. Testing included a combination of sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]), and/or gene amplification (CISH or FISH). Results: ccRCC had a 52% loss of PTEN, while PRCC had a 21% loss (p value=0.02). 100% of ccRCC with sarcomatoid features (4) showed aberrant expression of PD-L1 and were infiltrated with PD-1+ tumor infiltrating lymphocytes; 100% of 10 non-ccRCC sarcomatoids also had aberrant expression of PD-L1. Loss of PBRM1 expression was observed in 60% of ccRCC. Loss of histone 3 lysine 36 trimethylation (H3K36me3), which is associated with SETD2 mutations, was observed in 30% of ccRCC. TOP2A was overexpressed in ccRCC at 30% and in non-ccRCC at 50%. 100% of ccRCC and PRCC overexpressed EGFR; a single unclassified renal cell carcinoma did not overexpress EGFR. 50% of cases had cMET overexpression, amplification, or mutation. HER2 was not amplified or overexpressed in any RCC; a single rare HER2 mutation (R816H) was found in a collecting duct carcinoma. VHL mutations were identified in 50% of ccRCC tumors. We observed lower rates of TP53 (14%), ATM (6%), and PIK3CA(4% ccRCC, 7% PRCC) mutations compared to other cancers. Conclusions: Multiplatform molecular profiling of renal cell carcinoma identifies potential predictive biomarkers in ccRCC. The documented activity of everolimus in ccRCC (RECORD-1 trial) may be attributable to PIK3CA pathway alterations. RCC with sarcomatoid features may respond to PD1/PD-L1 targeted immunotherapies. The impact of molecular profiling in ccRCC to predict responses to currently available targeted therapy has important implications for trial design and patient selection.

447 Background: Components of the peripheral blood counts (PBC) are associated with poor prognosis in patients diagnosed with non-metastatic clear cell renal cell carcinoma (ccRCC). Related to this, higher tumor expression of programmed death ligand one (PDL1) is also associated with poor ccRCC prognosis. Herein, we utilize a large cohort of patients undergoing nephrectomy for localized ccRCC to evaluate the association of PBC with tumor expression of PDL1. Secondarily, we examine the association of PBC and relapse free survival (RFS) after adjusting for age and PDL1 expression. Methods: Through chart review, we obtained PBC values at time of nephrectomy for patients enrolled in the Mayo Clinic Renal Registry from 1990-2009 who were treated for localized (M0) ccRCC including the following: hemoglobin (HGB), white blood cell (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute eosinophil count (AEC), absolute monocyte count (AMC), and platelets (PLT). We determined tumor PDL1 expression as a continuous variable (% of positive PDL1 tumor cells) using the 5H1 antibody on archived tissues. We analyzed the correlation of PBC values with PDL1 expression as a continuous variable using a linear estimate. To evaluate the association of PBC values with RFS after adjusting for PDL1 expression, we employed multivariate Cox regression models. Results: A total of 706 ccRCC patients had available PDL1 expression and PBC. PDL1 expression was inversely associated with HGB (linear estimate of -0.02, p<0.001) and positively associated with platelets (linear estimate 1.73, p<0.001) whereas there was no association with PDL1 with WBC, ANC, AEC, or AMC. After adjusting for age and PDL1 expression, a higher HGB (HR=0.87, p<0.001), higher ALC (HR 0.68, p=0.003), and higher AEC (HR 0.12, p=0.002) were all associated with improved relapse free survival. Conversely, higher PLT was associated with decreased RFS (HR 1.002, p<0.001). Conclusions: PDL1 expression is associated with decreased HGB and increased PLT at the time of nephrectomy. After adjusting for PDL1 expression and age, higher HGB, ALC, and AEC are associated with improved RFS, while higher platelets are associated with decreased RFS.

422 Background: There is significant variability in the response to VEGF-TT in mRCC with no validated predictive biomarkers. We hypothesized that whole exome analysis might identify markers of response and resistance to VEGF-TT in mRCC. Methods: mRCC patients who received first-line sunitinib or pazopanib and were in two extreme phenotypes of response were identified. Extreme responders (ER) were defined as PR or CR for ≥3 years (n=10) and primary refractory patients (PRP) were defined as PD within the first 3 months of therapy (n=10). WES was performed in pre-treatment specimens and established Broad Institute analytical pipelines were utilized to identify point mutations and copy number alterations across the exome. ER (n=4) and PRP (n=4) who were part of TCGA project for clear-cell RCC were included in this analysis. Nonsense, missense and indel mutations in established or novel RCC genes were investigated. Results: IMDC prognostic scores were not significantly different between the two groups. VHL mutations were observed at similar frequency in ER and PRP, overall 57%. Mutations in PBRM1 were identified in 7 ER (50%) vs. 1 PRP (7%) (p=0.03). In addition, mutations in TP53 were only found in PRP (p=0.09). No other gene had mutations that were associated with either response or primary refractory disease (Table). Conclusions: In this pilot study, PBRM1 mutations were associated with extreme response to VEGF-TT. However, this was exploratory, and multivariable analysis was not performed. Analysis of additional patient samples is ongoing to confirm or refute this association. If true it suggests that epigenetic effects of PBRM1 mutation may contribute to response to VEGF-TT in mRCC. [Table: see text]

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.

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]