Stanton, Melissa

Tissue microarrays (TMAs) are important tools to conserve precious tissue resources from increasingly smaller biopsies and to control experimental costs and variation across sample sets. The quality assurance assessment of TMA materials created at centralized biobanks has not been standardized. Herein, we outline 2 processes for the construction of TMAs (“recipient block” and “tape” methods) and the associated preconstruction quality control measures (pathology review, protein and RNA assessment, map creation, and storage conditions) developed by the AIDS Cancer Specimen Resource (ACSR) Network’s Science and Technology Core. These steps provide a suggested framework for quality assessment that allows end-users, receiving materials from tissue banks, confidence in their experimental results.

e15567 Background: Esophageal adenocarcinoma (EAC) prognosis is poor and there is a need to identify patients that benefit most from neoadjuvant therapy. To examine the association of various biomarkers with clinical outcomes in neoadjuvant treatment of EAC, we retrospectively evaluated the biomarker expression (TS, ERCC1, TOPO1, PD-L1, PD-1) in patient matched formalin-fixed paraffin-embedded (FFPE) tumor samples. Methods: Immunohistochemistry of TS (TS106/4H4B1) , ERCC1 (Ab. 8F1), TOPO1 (1D6), PD-L1 (both 22c3 and SP142), PD-1 (NAT105), and chromogenic in-situ hybridization (CISH) of Her2 were performed on FFPE samples from 35 patients across 2 institutions at time of EAC diagnosis and after treatment when available. Retrospective clinical data and survival (5/2006-1/2016) was analyzed with a mean follow up of 110 months (range 22-306). Results: Overexpression (pre/post-treatment) of TS (60%/54%), ERCC1 (69%/16%), TOPO1 (74%/50%), PD-1 (54%/63%), PD-L1 (SP142) (2.9%/4%), PD-L1 (22c3) (0%/4%) and amplification of Her2 (18%/23%) were observed. Pretreatment observed PD-L1 levels were lower in our study (3%) when compared to other studies in EAC specimens (35%). Immunohistochemistry and changes observed after chemoradiation are reviewed in Table. No markers had significant correlation with prognosis however TS negative expression showed a non-significant (p=0.15) trend towards improved survival. Conclusions: Analyzing biomarkers in our neoadjuvant EAC cohort demonstrated a lower than expected PD-L1 positivity. In the largest cohort, to our knowledge, of patient matched FFPE tumor samples, we did not observe a statistically significant association between TS, ERCC1, TOPO1, PD-L1, or PD-1 with improved clinical outcomes. [Table: see text]

e15563 Background: Expression of thymidylate synthase (TS), a target of fluoropyrimidine-based (5-FU) chemotherapy, has prognostic value in colorectal adenocarcinoma. Its value as a biomarker in treatment of esophageal adenocarcinoma (EAC) is unknown. With widespread adoption of CROSS trial protocol for treatment of EAC, 5-FU is frequently omitted from regimens. We retrospectively analyzed the association between TS protein expression and clinical outcomes in EAC. Methods: TS expression levels were measured by immunohistochemistry in formalin-fixed paraffin-embedded specimens from 35 primary EAC patients from 2 institutions (range 5/2006-1/2016); at time of diagnosis and surgical resection (33 neoadjuvant chemoradiation and 2 surgery alone). TS expression higher than 1+, 5% was read as positive. Mean follow up was 1323 days (range 263-3677). TS expression correlated with treatment response and survival in patients treated with and without 5-FU therapy (log-rank test, Kaplan-Meier method). Results: In the cohort of 35 patients, 21 patients (60%) had pretreatment positive TS expression. Overall survival (OS) for TS negative patients was 1216 days versus 974 days for TS positive (p = 0.15). In the 23 patients treated with 5-FU, TS-negative patients had a trend towards longer overall survival compared to TS-positive patients, mean OS (1272 days versus 743 days, p = 0.08). In the group of 12 patients not treated with 5-FU, 2 patients showed negative TS staining. Neoadjuvant chemoradiation was performed in 33/35 patients. Downstaging was observed in 69% (9/13) of patients with negative pre-treatment TS levels versus 55% (11/20) in pre-treatment TS positive patients (p = 0.48). In 23 patients treated with 5FU, 9/12 (75%) were downstaged in the TS-negative cohort versus 6/11 (45%) in the TS-positive cohort (p = 0.21). In the 5FU treatment group, complete response was seen in 2 patients with negative TS staining. Conclusions: Low TS expression in EAC patients trended towards improved survival and tumor downstaging, particularly in patients treated with neoadjuvant 5-FU and platinum based therapy. Prospective data and greater patient numbers may be necessary to demonstrate statistical significance.

Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) is a rare but potentially life-threatening cutaneous hypersensitivity reaction characterized by extensive mucocutaneous eruption, fever, hematologic abnormalities, and extensive organ involvement. Here, we present a case of a young woman with DRESS syndrome following exposure to vancomycin with renal, cutaneous, and gastrointestinal involvement. To the best of our knowledge, this is the first case description in the literature of DRESS of the gastrointestinal tract with autoimmune enteropathy.

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.

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.

287 Background: Our preliminary immunohistochemical studies of bladder cancer patients have shown that the inflammatory/immune responses associated with bladder cancer appear to be Th2-polarized as judged by the preponderance of tumor infiltrating GATA-3+ (Th2) vs. T-bet+ (Th1) T cells, as well as by evidence of tumor-associated eosinophil infiltration and activation. The standard-of-care treatment for Tis bladder cancer is intravesical administration of BCG, a Th1-polarizing immunomodulator. The aim of the present study was to assess the Th2/Th1 ratio correlation with response to BCG therapy in patients with Tis bladder cancer. Methods: Two groups of patients were studied. Group A included 20 patients that responded to BCG, and Group B included 20 patients that did not respond to therapy. Response to BCG was determined as presence/absence of disease at 6 months post-BCG therapy. The initial diagnostic biopsies of these patients were subjected to immunohistochemical analysis using commercially available antibodies specific for Th2 (GATA-3+) and Th1 (T-bet+) lymphocytes, and our unique monoclonal antibody specific for eosinophil peroxidase (EPX-mAb). Ten random high powered (400X) fields at the maximum focus of mononuclear infiltration were examined and the number of GATA 3+ and T-bet+ tumor infiltrating T cells were counted to define the G/T ratio for each patient sample. Eosinophils and their degranulation activity were counted in the same fields. Results: Patients with response to BCG therapy had a G/T ratio of ≥ 3 and patients with non-response to BCG therapy had a G/T ratio of ≤ 1.5. Correlation of these scores with subsequent patient outcomes following BCG immune therapy demonstrated that patient responsiveness (i.e., tumor elimination at 6 months) trended with the higher G/T ratio patients. In addition, initial biopsies of BCG responsive patients display a higher number of eosinophils and degranulation relative to BCG non-responsive patients. Conclusions: Evaluations of initial biopsies revealed a strong correlation between Th1 and Th2 immune response with BCG therapy outcomes. The development of specific and novel tools to identify/measure Th2 immune responses in Tis bladder cancer provides a unique opportunity to predict outcomes at the time of diagnosis.

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]