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The DNA damage response (DDR) promotes genome integrity and serves as a cancer barrier in precancerous lesions but paradoxically may promote cancer survival. Genes that activate the DDR when dysregulated could function as useful biomarkers for outcome in cancer patients. Using a siRNA screen in human pancreatic cancer cells, we identified the CHD5 tumor suppressor as a gene, which, when silenced, activates the DDR. We evaluated the relationship of CHD5 expression with DDR activation in human pancreatic cancer cells and the association of CHD5 expression in 80 patients with resected pancreatic adenocarcinoma (PAC) by immunohistochemical analysis with clinical outcome. CHD5 depletion and low CHD5 expression in human pancreatic cancer cells lead to increased H2AX-Ser139 and CHK2-Thr68 phosphorylation and accumulation into nuclear foci. On Kaplan-Meier log-rank survival analysis, patients with low CHD5 expression had a median recurrence-free survival (RFS) of 5.3 vs 15.4 months for patients with high CHD5 expression (P=0.03). In 59 patients receiving adjuvant chemotherapy, low CHD5 expression was associated with decreased RFS (4.5 vs 16.3 months; P=0.001) and overall survival (OS) (7.2 vs 21.6 months; P=0.003). On multivariate Cox regression analysis, low CHD5 expression remained associated with worse OS (HR: 3.187 (95% CI: 1.49-6.81); P=0.003) in patients undergoing adjuvant chemotherapy. Thus, low CHD5 expression activates the DDR and predicts for worse OS in patients with resected PAC receiving adjuvant chemotherapy. Our findings support a model in which dysregulated expression of tumor suppressor genes that induce DDR activation can be utilized as biomarkers for poor outcome.

Background Mixed lineage kinase domain-like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC).

BACKGROUND Adjuvant radiotherapy (A-RT) for patients with resected pancreatic adenocarcinoma (PAC) is controversial. In the current study, the authors aim to determine whether there is an association between overall survival (OS) and A-RT dose. METHODS National Cancer Data Base (NCDB) data were obtained for all patients who received A-RT for resected PAC from 1998 through 2002. Univariate and multivariate survival analyses were performed along with Kaplan-Meier estimates for A-RT levels

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to use a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes, including 55 genes linked to DNA damage responses (DDR), that demonstrated gemcitabine sensitization when silenced, including CHD7, which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 patients with resected PDAC receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment. Cancer Res; 74(10); 2677–87. ©2014 AACR.

Background: To dosimetrically evaluate the effect of reduced margin radiotherapy on hippocampal dose for glioblastoma multiforme (GBM) patients. Methods: GBM patients enrolled on the Radiation Therapy Oncology Group (RTOG) 0825 trial at our institution were identified. Standard RTOG 0825 expansions were 2 cm + 3-5 mm from the gross tumor volume (GTV) to the clinical tumor volume (CTV) and from the CTV to the planning tumor volume (PTV), respectively. These same patients also had reduced margin tumor volumes generated with 8 mm (GTV to CTV) + 3 mm (CTV to PTV) expansions. Individual plans were created for both standard and reduced margin structures. The dose-volume histograms were statistically compared with a paired, two-tailed Student’s t-test with a significance level of p

Purpose/Objectives: Pancreatic cancer is notorious for its devastating disease course and prognosis. Gemcitabine is a widely used regimen for pancreatic cancer treatment but lack of response and resistance often limits its effectiveness. Thus, a better understanding of which patients are likely to respond to gemcitabine treatment would allow the personalization of therapies that are most effective for a patient while potentially reducing toxicity. The objective of this study was to identify the genes and mechanisms involved in determining gemcitabine sensitivity in pancreatic cancer. Materials/Methods: We completed a loss of function genetic screen to identify genes, which when silenced cause sensitization of resistance to a low dose of gemcitabine in human pancreatic cancer cells. We optimized a high-throughput assay using ATR or CHK1 siRNA as positive controls and ATM or nontargeting (NT) siRNA as negative controls. Our siRNA library included 4,474 siRNAs corresponding to 1,006 unique human genes arrayed in a one-gene:one-well format in 96-well plates. Genes chosen for our library consisted predominantly of nuclear enzymes, which we reasoned were more likely to function directly in DNA repair processes and be targetable. Mia PaCa-2 cells were transfected with 25 nM siRNA and treated 48 hours later with or without 13 nM gemcitabine for 72 hours prior to assaying for cell proliferation using WST-1 reagent. Candidate genes were deconvoluted with individual siRNAs to eliminate off-target effects and validated by secondary screens for cell cycle recovery after a challenge of hydroxyurea (HU) and γH2AX phosphorylation in the absence of exogenous damage following gene silencing. A rigorous statistical algorithm was used to determine positive hits. Genes with variable expression in pancreatic cancer tissue samples were identified by mining through published data sets. Results: We identified 49 genes in which at least 2 unique siRNAs yielded gemcitabine sensitization and 17 genes in which at least 2 unique siRNAs yielded gemcitabine resistance. Positive hits included 25 known genome maintenance genes, including well characterized ATR signaling pathway genes CHK1, RAD9, HUS1 , and CDC25A , 27 putative ATM/ATR substrates, and 26 genes identified in previously published DNA damage sensitivity screens. Eight of our genes are above the 90th percentile in variability of expression amongst a panel of pancreatic cancer tissue samples. Conclusion: We identified gemcitabine sensitization and resistance genes that are variably expressed in pancreatic cancer, which may function as novel targets or biomarkers for individualizing treatment for patients with pancreatic cancer. Citation Format: Claire W. Hardy, Brooke G. Pantazides, Khanjan Gandhi, Jerome C. Landry, Joseph W. Shelton, Shishir K. Maithel, Bassel El-Rayes, Jeanne Kowalski, David S. Yu. A loss of function genetic screen identifies determinants of gemcitabine sensitivity in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A33.

investigated the role of DAB2IP and/or EZH2 as prognostic biomarkers following radiation therapy (RT) in high-risk PCa patients. Materials/Methods: Immunohistochemistry was performed and scored by an expert genitourinary pathologist. Freedom from biochemical failure (FFBF) was determined using the Phoenix definition. Castrate resistancefree survival (CRFS) was determined when 2 episodes of rising prostatespecific antigen (PSA) occurred while on standard hormone therapy. Distant metastasis-free survival (DMFS) was determined from clinical data review. Log-rank test and Cox regression were used to determine significance of DAB2IP and EZH2 levels with clinical outcome. Results: Fifty-four patients with high-risk PCa (stage T3a, or Gleason score 8, or PSA 20) treated with RT from 2005-2012 at UT Southwestern were evaluated. 28.3% (13/46) of patients revealed DAB2IPreduction while 71.7% (33/46) retained DAB2IP. Nearly all patients expressed EZH2 (97.9%), supporting prior reports that EZH2 expression precedes DAB2IP reduction. For EZH2, the intensity level was grade (G) 0 in 1 (2.1%), G1 in 9 (18.8%), G2 in 29 (60.4%), and G3 in 9 (18.8%) patients. Median follow-up was 34.0 months (mos) (range, 6.7-76.1 mos) for DAB2IP-reduced patients, 29.9 mos (range, 6.1-84.6 mos) for DAB2IP-retained patients, and 32.6 m (range, 2.8-84.6 mos) for the EZH2 study. Patients with reduced DAB2IP demonstrated worse outcome compared to patients retaining DAB2IP, including FFBF (2-year Z 73% vs 93%; 4-yearZ 37% vs 89%; p Z 0.04; HR Z 3.65), DMFS (2-yearZ 90% vs 97%; 4-year Z 36% vs 97%; p Z 0.05), and CRFS (4-year Z 50% vs 90%; p Z 0.02). Intensity of EZH2 expression trended toward significance for worse FFBF and CRFS (p Z 0.07). Patients with reduced DAB2IP or highest intensity (G3) EZH2 expression exhibited even worse FFBF (p Z 0.03; HR Z 5.65). Six of the 7 patients that developed CRPC had reduced DAB2IP or G3 EZH2. Even with modest follow-up and after controlling for duration of hormone therapy, DAB2IP remains significant. Conclusions: This study suggests that DAB2IP loss is a potent biomarker for identifying high-risk PCa patients that portends worse outcome despite definitive RT. EZH2 is expressed in most high-risk PCa tumors evaluated, and is a less potent discriminator of outcome in this group of patients. DAB2IP status in combination with degree of EZH2 expression may be useful for determining patients with worse outcome within the high-risk patient population. Author Disclosure: C. Jacobs: None. P. Kapur: None. J. Yan: None. V. Tumati: None. X. Xie: None. D. Pistenmaa: None. M. Bhuiyan: None. J. Hsieh: None. D. Saha: None. D. Kim: None.

Purpose/Objectives: Pancreatic cancer is notorious for its devastating disease course and prognosis. Gemcitabine is a widely used regimen for pancreatic cancer treatment but lack of response and resistance often limits its effectiveness. Thus, a better understanding of which patients are likely to respond to gemcitabine treatment would allow the personalization of therapies that are most effective for a patient while potentially reducing toxicity. The objective of this study was to identify the genes and mechanisms involved in determining gemcitabine sensitivity in pancreatic cancer. Materials/Methods: We completed a loss of function genetic screen to identify genes, which when silenced cause sensitization of resistance to a low dose of gemcitabine in human pancreatic cancer cells. We optimized a high-throughput assay using ATR or CHK1 siRNA as positive controls and ATM or non-targeting (NT) siRNA as negative controls. Our siRNA library included 4,474 siRNAs corresponding to 1,006 unique human genes arrayed in a one-gene:one-well format in 96-well plates. Genes chosen for our library consisted predominantly of nuclear enzymes, which we reasoned were more likely to function directly in DNA repair processes and be targetable. Mia PaCa-2 cells were transfected with 25 nM siRNA and treated 48 hours later with or without 13 nM gemcitabine for 72 hours prior to assaying for cell proliferation using WST-1 reagent. Candidate genes were deconvoluted with individual siRNAs to eliminate off-target effects and validated by secondary screens for cell cycle recovery after a challenge of hydroxyurea (HU) and γH2AX phosphorylation in the absence of exogenous damage following gene silencing. A rigorous statistical algorithm was used to determine positive hits. Genes with variable expression in pancreatic cancer tissue samples were identified by mining through published data sets. Results: We identified 49 genes in which at least 2 unique siRNAs yielded gemcitabine sensitization and 17 genes in which at least 2 unique siRNAs yielded gemcitabine resistance. Positive hits included 25 known genome maintenance genes, including well characterized ATR signaling pathway genes CHK1, RAD9, HUS1, and CDC25A, 27 putative ATM/ATR substrates, and 26 genes identified in previously published DNA damage sensitivity screens. Eight of our genes are above the 90th percentile in variability of expression amongst a panel of pancreatic cancer tissue samples. Conclusion: We identified gemcitabine sensitization and resistance genes that are variably expressed in pancreatic cancer, which may function as novel targets or biomarkers for individualizing treatment for patients with pancreatic cancer. Citation Format: Matthew D. Warren, Claire W. Hardy, Brooke G. Pantazides, Khanjan Gandhi, Jerome C. Landry, Joseph W. Shelton, Shishir K. Maithel, Bassel El-Rayes, Jeanne Kowalski, David S. Yu. A synthetic lethal screen identifies determinants of gemcitabine sensitivity in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1767. doi:10.1158/1538-7445.AM2013-1767

Background/ Objectives Chromodomain helicase DNA Binding protein 7 (CHD7) modulates chromatin remodeling during genome maintenance. Its role in pancreatic cancer (PAC) is unestablished, but preliminary data for Gemcitabine (GEM) sensitivity in pancreatic cell lines and pilot clinical data is promising. This study will evaluate CHD7 as a prognostic and predictive factor in resected PAC using 1) immunohistochemistry (IHC) of patient samples from RTOG 97-04, 2) prospective evaluation of CHD7 over-expression and treatment response in patients receiving FOLFIRINOX and GEM-based chemoradiotherapy (CRT) for locally advanced PAC at our institution and 3) a Phase II molecular-driven randomized trial of personalized therapy based on CHD7 expression. Methodology A loss of function siRNA screen identified genes, including CHD7, that conferred GEM sensitivity when silenced using GEM-treated Mia PaCa-2 cells. CHD7 expression was assessed by IHC scoring in 80 patients who underwent curative intent resection of PAC between 1/2000 and 10/2008. Kaplan-meier survival analysis was performed for recurrence-free survival (RFS) and overall survival (OS). Univariate (UV) and Multivariate (MV) Cox regression analyses using clinically relevant covariates were used to correlate CHD7 expression levels with RFS and OS in patients receiving GEM therapy (n=42). This analysis will be validated in 200 patient samples from RTOG 97-04. CHD7 expression will be correlated with RFS and OS in GEM and 5-Fluorouracil (5-FU) treatment arms. CHD7 expression will also be measured in pre-chemotherapy core biopsies from locally advanced PAC patients treated at our institution. Patients will receive either two cycles of FOLFIRINOX (FOL) followed by GEM-based CRT at a dose of 1000mg/m2 once weekly or two cycles of FOL followed by Capecitabine (CAP)-based CRT. CHD7 expression will again be correlated with survival. These results will be followed by an ECOG- sponsored multi-institutional Phase II randomized trial of FOL and GEM-based CRT or FOL and CAP-based CRT based on CHD7 gene expression. Preliminary Data/ Anticipated Results In patients receiving GEM (n=42), high CHD7 was associated with poor RFS (7 months vs 15 months; p=.025) and poor OS (10 months vs 18 months; p=.015). On MV analysis, high CHD7 expression remained a predictor of poor RFS (HR 8.2 [95% CI 2.4-28]; p=.001) and poor OS (HR 11.6 [95% CI 3.4-40); p We expect our RTOG 97-04 validation study to demonstrate stronger correlation of low CHD7 expression with improved RFS and OS in GEM-treated patients. Similarly, we expect our institutional trial to demonstrate improved RFS and OS with low CHD7 in the GEM-treated arm. Citation Format: Lauren E. Colbert, William A. Hall, Claire W. Hardy, Sarah B. Fisher, David S. Yu, Shishir K. Maithel, Bassel El-Rayes, Burcu Saka, N Volkan Adsay, Aleksandra Petrova, Brooke Pantazides, Jeanne Kowalski, Khanjan Gandhi, David Kooby, Charles Staley, Jerome C. Landry. A pilot clinical trial of chromodomain-helicase-DNA-binding protein 7 (CHD7) expression as a prognostic and predictive biomarker in patients with early-stage pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-168. doi:10.1158/1538-7445.AM2013-LB-168