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Supplemental Figures 1-6. Supplemental Figure 1. A typical KRAS Sanger dye termination sequencing trace for a pancreatic cancer sample. The arrow indicates the mutation site; the black peak represents a wild-type allele and the red peak represents the mutant allele. Supplemental Figure 2. (A) Copy numbers per sample of methylated KCNK12 assayed form pancreatic juice of patients with normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated KCNK12 for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 3. (A) Copy numbers per sample of methylated CLEC11A assayed form pancreatic juice of patients with normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated CLEC11A for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 4. (A) Copy numbers per sample of methylated NDRG4 assayed form pancreatic juice of patients with normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated NDRG4 for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 5. (A) Genomic copy numbers of methylated IKZF1 in normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated IKZF1 for the detection of PC in comparison to NP (black) and CP (grey). Supplemental Figure 6. (A) Genomic copy numbers of methylated PKRCB in normal pancreas (NP), chronic pancreatitis (CP), high grade dysplastic main-duct intraductal pancreatic mucinous neoplasm (IPMN), and pancreatic cancer (PC) were used to calculate (B) receiver operating characteristics curves of methylated PKRCB for the detection of PC in comparison to NP (black) and CP (grey).

Supplementary Figure 1. Gut microbial composition and diversity in patients with and without adenomas. A) Microbial composition was similar between groups across all taxonomic levels. The groups did not differ in terms of α-diversity: B) observed number of operational taxonomic units (OTUs) or C) the Shannon diversity index. The groups did not cluster by β-diversity: D) unweighted and E) weighted UniFrac distance metrics.

Supplementary Table 1. Pharmacological Inhibitors of Endocytic Pathways

Supplementary Fig. 5. Adrenomedullin increases reactive oxygen species production. Reactive oxygen/nitrogen species (ROS/RNS) were detected in INS-1 cells pre-treated with either 1 or 20 pM adrenomedullin for 24 hours. The orange filter detects superoxide and nitric oxide generation, while the green filter detects all reactive oxygen species. Positive control cells were treated with Pyocyanin (Pyo) to induce ROS production and negative control cells were treated with Pyo + N-acetyl-L-cysteine (NAC) to inhibit ROS production. Scale bar represents 10 µm.

Purpose: To determine the frequency and prognostic association of molecular markers by anatomic tumor site in patients with stage III colon carcinomas.Experimental Design: In a randomized trial of adjuvant FOLFOX ± cetuximab, BRAFV600E and KRAS (exon 2) mutations and DNA mismatch repair (MMR) proteins were analyzed in tumors (N = 3,018) in relationship to tumor location, including subsite. Cox models were used to assess clinical outcome, including overall survival (OS).Results: KRAS codon 12 mutations were most frequent at the splenic flexure and cecum; codon 13 mutations were evenly distributed. BRAF mutation frequency sharply increased from transverse colon to cecum in parallel with deficient (d) MMR. Nonmutated BRAF and KRAS tumors progressively decreased from sigmoid to transverse (all P < 0.0001). Significantly, poorer OS was found for mutant KRAS in distal [HR, 1.98; 95% confidence interval (CI), 1.49–2.63; P < 0.0001] versus proximal (1.25; 95% CI, 0.97–1.60; P = 0.079) cancers. BRAF status and outcome were not significantly associated with tumor site. Proximal versus distal dMMR tumors had significantly better outcome. An interaction test was significant for tumor site by KRAS (Padjusted = 0.043) and MMR (Padjusted = 0.010) for OS. Significant prognostic differences for biomarkers by tumor site were maintained in the FOLFOX arm. Tumor site was independently prognostic with a stepwise improvement from cecum to sigmoid (OS: Padjusted = 0.001).Conclusions: Mutation in BRAF or KRAS codon 12 was enriched in proximal cancers whereas nonmutated BRAF/KRAS was increased in distal tumors. Significant differences in outcome for KRAS mutations and dMMR were found by tumor site, indicating that their interpretation should occur in the context of tumor location. Clin Cancer Res; 21(23); 5294–304. ©2015 AACR.

Supplemental Table 2. Comparison to ICGC array data

Supplemental Table 1. Univariate Cox Proportional Hazards Regression Models for Clinical Factors and Biomarkers (TTR, DFS, and OS)

Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls.Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls.Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity.Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Supplemental Figure 1: Quantitative allele-specific real-time target and signal amplification (QuARTS) assay.

Supplemental Table 3. Function and cancer biology associations of candidates

Supplemental Figure 2. Box plots of methylated DNA marker levels in various types of cancers were made from publically available hybridization array data from The Cancer Genome Atlas.

Supplementary Table 1. MiRKAT association P values for various adenoma characteristics in individuals with adenomas

Supplementary Figure 2. Heat map of significantly predictive taxa. Hierarchical clustering is based on the Euclidean distance of log proportion, complete linkage. The left cluster is enriched in adenoma samples (P=0.1, Fisher's exact test).

Background: Host immune response may predict the course of colorectal cancer. We examined the survival of 468 colorectal cancer patients associated with two tumor-infiltrating immune biomarkers, the number of cytotoxic T lymphocytes (CTLs), and the activated CTLs, as reflected by the number of cells expressing granzyme B (GZMB) in the prospective Iowa Women's Health Study.Methods: Using paraffin-embedded tissue samples, we constructed and immunostained tumor microarrays with CD8 (for CTL) and GZMB antibodies. We scored CTL and GZMB densities in tumor epithelial and stromal tissues and also created a composite score for each biomarker (sum of the scores across tissue compartments). Cox regression estimated the HR and 95% confidence intervals (CI) for all-cause and colorectal cancer–specific death associated with each composite score.Results: CTL and GZMB composite scores were positively correlated (r = 0.65) and each biomarker was inversely correlated with stage at diagnosis. Both composite scores were higher in proximal colon tumors and tumors characterized by MSI-high, CIMP-high, or BRAF mutation status. HRs (95% CI) were 0.53 (0.38–0.75; Ptrend = 0.0004) and 0.66 (0.51–0.86; Ptrend = 0.002) for all-cause death, respectively, and 0.30 (0.18–0.51; Ptrend < 0.0001) and 0.41 (0.27–0.63; Ptrend < 0.0001) for colorectal cancer–related death, respectively. Including CTL and GZMB scores simultaneously in the model significantly improved the predictive performance of the models for all-cause and colorectal cancer–related death.Conclusions: Higher tumor infiltration with CTL and GZMB cells is associated with improved all-cause and cancer-specific survival of colorectal cancer patients.Impact: Both the number of CTLs and GZMB appear to be useful prognostic factors in colorectal cancer, irrespective of stage. Cancer Epidemiol Biomarkers Prev; 26(4); 622–31. ©2016 AACR.

Purpose:The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma.Experimental Design:Whole-methylome sequencing was performed on DNA extracted from 37 tissues (28 EC; 9 normal esophagus) and 8 buffy coat samples. Top MDMs were validated by methylation specific PCR on tissue from 76 EC (41 adeno, 35 squamous cell) and 17 normal esophagus. Quantitative allele-specific real-time target and signal amplification was used to assay MDMs in plasma from 183 patients (85 EC, 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Validation was performed in silico by bootstrapping.Results:From discovery, 23 candidate MDMs were selected for independent tissue validation; median area under the receiver operating curve (AUC) for individual MDMs was 0.93. Among 12 MDMs advanced to plasma testing, rPART modeling selected a 5 MDM panel (FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4) which achieved an AUC of 0.93 (95% CI, 0.89–0.96) on best-fit and 0.81 (95% CI, 0.75–0.88) on cross-validation. At 91% specificity, the panel detected 74% of esophageal cancer overall, and 43%, 64%, 77%, and 92% of stages I, II, III, and IV, respectively. Discrimination was not affected by age, sex, smoking, or body mass index.Conclusions:Novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy. Further optimization and clinical testing are warranted.

Background: Adenomatous polyps are the most common precursor to colorectal cancer, the second leading cause of cancer-related death in the United States. We sought to learn more about early events of carcinogenesis by investigating shifts in the gut microbiota of patients with adenomas.Methods: We analyzed 16S rRNA gene sequences from the fecal microbiota of patients with adenomas (n = 233) and without (n = 547).Results: Multiple taxa were significantly more abundant in patients with adenomas, including Bilophila, Desulfovibrio, proinflammatory bacteria in the genus Mogibacterium, and multiple Bacteroidetes species. Patients without adenomas had greater abundances of Veillonella, Firmicutes (Order Clostridia), and Actinobacteria (family Bifidobacteriales). Our findings were consistent with previously reported shifts in the gut microbiota of colorectal cancer patients. Importantly, the altered adenoma profile is predicted to increase primary and secondary bile acid production, as well as starch, sucrose, lipid, and phenylpropanoid metabolism.Conclusions: These data hint that increased sugar, protein, and lipid metabolism along with increased bile acid production could promote a colonic environment that supports the growth of bile-tolerant microbes such as Bilophilia and Desulfovibrio. In turn, these microbes may produce genotoxic or inflammatory metabolites such as H2S and secondary bile acids, which could play a role in catalyzing adenoma development and eventually colorectal cancer.Impact: This study suggests a plausible biological mechanism to explain the links between shifts in the microbiota and colorectal cancer. This represents a first step toward resolving the complex interactions that shape the adenoma–carcinoma sequence of colorectal cancer and may facilitate personalized therapeutics focused on the microbiota. Cancer Epidemiol Biomarkers Prev; 26(1); 85–94. ©2016 AACR.

Supplementary Figure 3. A receiver operating characteristic curve generated using the four significant taxa (Streptococcus, Veillonella, Mogibacterium, and Sutterella) identified by the random forests algorithm demonstrates a poor ability to predict adenoma status.

Supplementary Fig. 4. AM and PC-Exosomes modestly upregulate insulin gene expression. All studies were done using INS-1, a rat β-cell line. (A) Relative expression of insulin genes INS1 and INS2 in response to varying concentrations (1, 20, or 40 pM) of AM and AM inhibitor AM 22-52. (B) Western blot analysis of insulin protein output in response to 1, 20, or 40 pM of AM. 20 µg of protein lysate was loaded. (C) Relative expression of insulin genes INS1 and INS2 in response to the addition of 50 µg of PC-Exo. * P

Supplemental Table 1. AUC and 95% confidence intervals for the technical validation study

Supplementary Fig. 1. NanoTracker results of PC-Exosome isolated from PC patient-derived cell lines and patient plasma. (A-B) PC-Exo isolated from a PC patient-derived cell line show the majority of microparticles are of exosomal size ([A] mode, 105{plus minus}4.2 nm). In (B), microparticles are more of microvesicle size (mode, 294{plus minus}13.5 nm). Thus, depending on the cell line, a 100,000 x g isolation can yield both exosomes and microvesicles. (C-F) PC-Exo isolated from the plasma of different PC patients show that the majority of microparticles in plasma are exosomes (modes, 156{plus minus}8.6 nm; 88{plus minus}5.0 nm; 92{plus minus}3.1 nm; 100{plus minus}7.0 nm, respectively). Analysis was taken by averaging 5, 60-second movies.

Supplementary Figure Legends

Purpose: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer–derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion.Experimental Methods: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown.Results: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species.Conclusions: Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. Clin Cancer Res; 21(7); 1722–33. ©2014 AACR.See related commentary by Korc, p. 1508

Supplemental Table 1: Discrimination for gastric adenocarcinoma (GAC) from normal gastric mucosa by the top 22 methylated DNA marker candidates in Discovery and Technical Validation Sets. Supplemental Table 2: Function of Genes Containing Differentially Methylated Regions Identified as Targets for Candidate Markers Supplemental Table 3: Covariate Effects on Tissue Levels of Methylated DNA Marker Candidates (Data shown represent p-values).

Supplementary Fig. 2. PC-derived exosomes from additional PC cell lines and PC patient plasma can decrease insulin secretion. (A-B) PC-Exo isolated from 2 PC patient-derived cell lines decreased insulin secretion in INS-1 cells. (C-D) PC-Exo isolated from 2 different PC patient blood plasma samples can decrease insulin secretion at varying levels. * P

Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets.Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated.Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS).Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

Supplementary Figure 1. Receiver operator curve (ROC) plots in the logistic regression model (Model 2) for all-cause (B) and CRC-death (A) within 5 years after diagnosis (adjusted for age of diagnosis, BMI, smoking status, stage and grade at diagnosis). AUC=area under the ROC curve. Supplementary Table 1. Characteristics of women diagnosed with CRC across tumor composite scores for cytotoxic T lymphocytes (CTL) and granzymeB (GZMB)a. Supplementary Table 2. Tumor stromal and epithelial cytotoxic T lymphocytes (CTL) scoresa and risk of all-cause and CRC-specific death in the CRC patients, IWHS, 1986-2011. Supplementary Table 3. Tumor stromal and epithelial granzyme B (GZMB) scores and risk of all-cause and CRC-specific death the CRC patients, IWHS, 1986-2011. Supplementary Table 4. Tumor composite scores for cytotoxic T lymphocytes (CTL) and granzyme B (GZMB) for all-cause and CRC-specific death in the CRC patients followed for 5 years, IWHS, 1986-2011.

Supplementary Fig. 3. Chlorpromazine and Nocodazole do not inhibit exosome internalization. INS-1 cells were treated with varying concentrations of chlorpromazine (A) or Nocodazole (C) for 30 minutes and 10 minutes, respectively. PKH67-dyed PANC-1 exosomes were then added to the cells for 5 hours to allow internalization. Scale bars represent 10 µm. (B) and (D) show the quantification of internalized green fluorescence per cell for chlorpromazine (B) and Nocodazole (D) treated cells.

Supplementary Table 1 from Expression and Regulatory Role of GAIP-Interacting Protein GIPC in Pancreatic Adenocarcinoma

Supplementary Figure 2 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 1 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 5 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Regulator of G-protein signaling–GAIP-interacting protein COOH terminus (GIPC) is involved in protein trafficking, endocytosis, and receptor clustering and is associated with insulin-like growth factor I receptor (IGF-IR), a receptor important for proliferation and anchorage-independent growth. Here, we described GIPC expression in different human pancreatic adenocarcinoma (PCA) cell lines and we examined the role of GIPC in the regulation of IGF-IR protein levels in PCA. Interestingly, inhibition of GIPC expression by RNA interference led to reduced IGF-IR protein levels and a subsequent decrease in proliferation of PCA cells. We also determined that the PDZ domain of GIPC is essential for the post-translational regulation and the binding of IGF-IR. The importance of GIPC in pancreatic cancer development and progression is supported by tissue microarray data of 300 pancreatic cancer specimens where GIPC is highly expressed in PCA. Taken together, our data suggest that GIPC is a central molecule for the stability of IGF-IR and could be a target for future therapy. (Cancer Res 2006; 66(21): 10264-8)

Supplementary Figure 3 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Tables 1-2 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 6 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure Legends 1-6 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Supplementary Figure 4 from Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls.Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls.Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity.Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Purpose: To determine the frequency and prognostic association of molecular markers by anatomic tumor site in patients with stage III colon carcinomas. Experimental Design: In a randomized trial of adjuvant FOLFOX ± cetuximab, BRAFV600E and KRAS (exon 2) mutations and DNA mismatch repair (MMR) proteins were analyzed in tumors (N = 3,018) in relationship to tumor location, including subsite. Cox models were used to assess clinical outcome, including overall survival (OS). Results: KRAS codon 12 mutations were most frequent at the splenic flexure and cecum; codon 13 mutations were evenly distributed. BRAF mutation frequency sharply increased from transverse colon to cecum in parallel with deficient (d) MMR. Nonmutated BRAF and KRAS tumors progressively decreased from sigmoid to transverse (all P < 0.0001). Significantly, poorer OS was found for mutant KRAS in distal [HR, 1.98; 95% confidence interval (CI), 1.49–2.63; P < 0.0001] versus proximal (1.25; 95% CI, 0.97–1.60; P = 0.079) cancers. BRAF status and outcome were not significantly associated with tumor site. Proximal versus distal dMMR tumors had significantly better outcome. An interaction test was significant for tumor site by KRAS (Padjusted = 0.043) and MMR (Padjusted = 0.010) for OS. Significant prognostic differences for biomarkers by tumor site were maintained in the FOLFOX arm. Tumor site was independently prognostic with a stepwise improvement from cecum to sigmoid (OS: Padjusted = 0.001). Conclusions: Mutation in BRAF or KRAS codon 12 was enriched in proximal cancers whereas nonmutated BRAF/KRAS was increased in distal tumors. Significant differences in outcome for KRAS mutations and dMMR were found by tumor site, indicating that their interpretation should occur in the context of tumor location. Clin Cancer Res; 21(23); 5294–304. ©2015 AACR.

Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

Background: Cigarette smoking (smoking), hormone therapy (MHT), and folate intake (folate) are each thought to influence colorectal cancer risk, but the underlying molecular mechanisms remain incompletely defined. Expression of estrogen receptor β (ESR2) has been associated with colorectal cancer stage and survival. Methods: In this prospective cohort study, we examined smoking, MHT, and folate-associated colorectal cancer risks by ESR2 protein expression level among participants in the Iowa Women's Health Study (IWHS). Self-reported exposure variables were assessed at baseline. Archived, paraffin-embedded colorectal cancer tissue specimens were collected and evaluated for ESR2 protein expression by IHC. Multivariate Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI) for associations between smoking, MHT, or folate and ESR2-defined colorectal cancer subtypes. Results: Informative environmental exposure and protein expression data were available for 491 incident colorectal cancer cases. Positive associations between ESR2-low and -high tumors and several smoking-related variables were noted, most prominently with average number of cigarettes per day (RR, 4.24; 95% CI, 1.81–9.91 for ESR2-low and RR, 2.15; 95% CI, 1.05–4.41 for ESR2-high for ≥40 cigarettes compared with nonsmokers). For MHT, a statistically significant association with ESR2-low tumors was observed with longer duration of exposure (RR, 0.54; 95% CI, 0.26–1.13 for >5 years compared with never use). No associations were found for folate. Conclusions: In this study, smoking and MHT were associated with ESR2 expression patterns. Impact: These data support possible heterogeneous effects from smoking and MHT on ERβ-related pathways of colorectal carcinogenesis in older women. Cancer Epidemiol Biomarkers Prev; 24(4); 713–9. ©2015 AACR.

Cigarette smoking (CS), hormone therapy (HT), and folate intake (FI) are each thought to influence colorectal cancer risk, but the underlying molecular mechanisms remain incompletely defined. The TP53 (p53) protein, encoded by the TP53 tumor-suppressor gene that is commonly mutated in colorectal cancer, can be readily assessed to differentiate biologically distinct colorectal cancer subtypes. In this prospective cohort study, we examined CS-, HT-, and FI-associated colorectal cancer risks by TP53 protein expression level among Iowa Women's Health Study (IWHS) participants. The IWHS recruited 41,836 randomly selected Iowa women, ages 55 to 69 years, with a valid driver's license at study entry in 1986. Self-reported exposure variables were assessed at baseline. Incident colorectal cancer cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected and evaluated for TP53 protein expression by immunohistochemistry. Multivariate Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI) for associations between CS, HT, or FI and TP53-defined colorectal cancer subtypes. Informative environmental exposure and protein expression data were available for 492 incident colorectal cancer cases: 222 (45.1%) TP53 negative, 72 (14.6%) TP53 low, and 198 (40.2%) TP53 high. Longer duration (>5 years) of HT was inversely associated with TP53 high colorectal cancers (RR, 0.50; 95% CI, 0.27–0.94). No other statistically significant associations were observed. These data support possible heterogeneous effects from HT on TP53-related pathways of colorectal carcinogenesis in older women. Cancer Epidemiol Biomarkers Prev; 23(2); 350–5. ©2013 AACR.

Background: Postmenopausal hormone (PMH) therapy represents a controversial colorectal cancer (CRC) preventive intervention. Because colorectal carcinogenesis is a heterogeneous process, we evaluated associations between PMH therapy and incident CRC in relation to KRAS mutation status in a population-based cohort of older women [Iowa Women's Health Study (IWHS)]. Methods: The IWHS enrolled 41,836 randomly selected women, ages 55 to 69 years, in 1986. PMH therapy and other exposure data were recorded at baseline. Tissue samples from prospectively identified CRC cases (n = 507) were analyzed for somatic KRAS mutations (exon 2, codons 12 and 13). Multivariable Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI). Results: PMH therapy (ever vs. never) was inversely associated with KRAS mutation–negative (RR = 0.83; 95% CI, 0.66–1.06; P = 0.14) and KRAS mutation–positive (RR = 0.82; 95% CI, 0.58–1.16; P = 0.27) tumors, although the observed risk estimates were not statistically significant. When anatomic subsite was additionally considered, the strongest association was found for KRAS mutation–negative, distal colorectal tumors (RR = 0.64; 95% CI, 0.43–0.96; P = 0.03). Conclusions: To our knowledge, we provide the first report of KRAS-defined CRC risks associated with PMH therapy. These data suggest that PMH therapy may reduce CRC risk through mechanisms beyond KRAS mutation status but might provide greater benefits for KRAS mutation–negative than mutation-positive tumors (at least in the distal colorectum). Impact: Findings from this prospective cohort study provide novel insights about the molecular biology of PMH therapy–related CRC risk reduction. Cancer Epidemiol Biomarkers Prev; 21(4); 681–4. ©2012 AACR.

Increased alcohol consumption is a putative colorectal cancer (CRC) risk factor. However, existing data are less conclusive for women than men. Also, to date, relatively few studies have reported alcohol-related CRC risks based on molecularly defined tumor subtypes. We evaluated associations between alcohol intake and incident CRC, overall and by microsatellite instability [MSI high (MSI-H) or MSI low/microsatellite stable (MSI-L/MSS)], CpG island methylator phenotype (CIMP positive or CIMP negative), and BRAF mutation (mutated or wild-type) status in the prospective, population-based Iowa Women's Health Study (IWHS; n = 41,836). Subjects were 55 to 69 years at baseline (1986), and exposure data were obtained by self-report. Incident CRCs were prospectively identified and archived, paraffin-embedded tissue specimens were collected from 732 representative cases, diagnosed through December 31, 2002. Multivariate Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI). Among alcohol consumers, the median intake (range) was 3.4 (0.9–292.8) g/d. Compared with nonconsumers, alcohol intake levels of 3.4 g/d or less (RR = 1.00; 95% CI, 0.86–1.15) and more than 3.4 g/d (RR = 1.06; 95% CI, 0.91–1.24) were not significantly associated with overall CRC risk. Analyses based on alcohol intake levels of 30 g/d or less and more than 30 g/d or quartile distributions yielded similar risk estimates. Null associations were also observed between each alcohol intake level and the MSI-, CIMP- or, BRAF-defined CRC subtypes (P > 0.05 for each comparison). These data do not support an adverse effect from alcohol intake on CRC risk, overall or by specific molecularly defined subtypes, among older women. Cancer Prev Res; 4(12); 2035–43. ©2011 AACR.

Purpose: Colorectal carcinoma (CRC) can be divided into two nonoverlapping groups: those that are chromosomally unstable but microsatellite stable (MSS CIN+) and those that are chromosomally stable but microsatellite unstable (MSI CIN−). However, a third group with neither chromosome nor microsatellite instability (MSS CIN−) makes a substantial contribution to the total CRC burden. The clinicopathologic features of MSS CIN− CRC are not well delineated. We assessed the relationship between age and chromosomal instability (CIN) status as measured by ploidy and allelic imbalance in a series of MSS tumors.Experimental Design: We studied a prospectively collected series of CRC patients at Mayo Clinic Rochester. A total of 84 samples of MSS CRC in patients ≤50 years old were identified between 1994 and 1997. A consecutive series of 90 MSS CRC in patients ≥65 years old served as a comparison group. CIN status was assessed using two techniques: ploidy analysis by flow cytometry and small chromosome changes as measured by genomewide fractional allelic imbalance.Results: CRC in the young-onset group was more likely to involve the rectum and to be high stage. MSS tumors in the young-onset group were more often diploid (46%) than those in older patients (26%; P = 0.006). This difference was maintained in the subset of MSS CRC that were high stage (42% versus 18%; P = 0.02) and in rectal cancers (50% versus 23%; P = 0.04).Conclusion: A greater proportion of young patients with MSS CRC has diploid tumors than patients who develop MSS CRC over age 65.

Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) β regulates nuclear factor-κB (NF-κB)–mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3β in pancreatic cancer cells and assess the antitumor effect of GSK-3 inhibition in vivo to improve our understanding of the mechanism by which GSK-3β affects NF-κB activity in pancreatic cancer. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3β in human pancreatic tumors. We studied the effect of GSK-3 inhibition on tumor growth, cancer cell proliferation, and survival in established CAPAN2 tumor xenografts using a tumor regrowth delay assay, Western blotting, bromodeoxyuridine incorporation, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Results: We found nuclear accumulation of GSK-3β in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3β nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3β can accumulate in the nucleus of pancreatic cancer cells and that inhibition of GSK-3 kinase activity represses its nuclear accumulation via proteasomal degradation within the nucleus. Lastly, we have found that inhibition of GSK-3 arrests pancreatic tumor growth in vivo and decreases NF-κB-mediated pancreatic cancer cell survival and proliferation in established tumor xenografts. Conclusions: Our results show the antitumor effect of GSK-3 inhibition in vivo, identify GSK-3β nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3β regulates NF-κB activity in pancreatic cancer.

Background: The increased number of T-lymphocytes residing in the tumors of colorectal cancer (CRC) patients is consistently shown to predict better survival of CRC patients independent of stage. In addition to tumor itself, T-lymphocytes also infiltrate normal tissues adjacent to tumors; however, little is known about how the inter-individual T-cell variability in tumor-adjacent tissues affects CRC prognosis. Our goal was to characterize total T-cells in colorectal tumor and tumor-adjacent tissues and study their associations with all-cause and CRC-specific survival in the Iowa Women’s Health Study. Methods: We constructed tissue microarrays and quantified CD3 antibody staining (Clone F.7.238; Dako), an established marker for total T-cells, in paraffin-embedded tissue samples from 463 women diagnosed with incident CRC from 1986-2002 (mean age at diagnosis was 74 years). Up to 3 tumor and 3 tumor-adjacent cores were immunostained for each person. An experienced pathologist quantified CD3+ T cells in each area using 4 categories: non-detected, mild (1-10 cells per 0.28 mm2); moderate (11-29 cells per 0.28 mm2); and strong infiltration (≥30 cells per 0.28 mm2). The obtained scores were averaged over all quantified cores for each person. Cox regression was used to estimate the hazard ratio (HR) and 95% CI for all-cause and CRC mortality in relation to (1) tumor score; (2) tumor-adjacent score; and (3) the ratio of tumor to tumor-adjacent score, hereafter called Score ratio. All scores were categorized in quartiles. The final model adjusted for age at diagnosis, SEER stage, tumor grade, body mass and smoking history. Results: During follow-up for a maximum of 25 years, 67% of participants diagnosed with CRC died (~30% died from CRC). There was a weak but significant correlation between CD3 tumor and tumor-adjacent scores (Spearman coefficient: r=0.15, p=0.02). CD3 tumor score was inversely associated with stage and was higher in those with proximal colon cancer, and for MSI-high, CIMP-high and BRAF-mutated tumors. The HRs (95%CI) for the highest versus lowest category of CD3 tumor score were 0.61 (0.44-0.84) (p-trend=0.003) for all-cause mortality and 0.27 (0.15-0.48) (p-trend Conclusions. Based on data from prospectively identified CRC cases in the IWHS cohort, colorectal tumor T-cell infiltration is associated with improved survival. Further investigation is needed to determine the T-cell subtypes that are most predictive of CRC survival outcomes. Citation Format: Anna E. Prizment, Robert A. Vierkant, Thomas C. Smyrk, Lori S. Tillmans, Heather H. Nelson, Charles F. Lynch, Stephen N. Thibodeau, Timothy R. Church, James R. Cerhan, Kristin E. Anderson, Paul J. Limburg. Associations between total T-lymphocytes and colorectal cancer survival in a prospective cohort study of older women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2017-2247

Background: The immune response has been shown to impact the course of colorectal cancer (CRC). Our goal was to examine the survival of CRC patients in relation to tumor-infiltrating cytotoxic (CD8+) T lymphocytes (CTL). We quantified CTLs in tumor epithelial and stromal tissues and correlated them with clinicopathological characteristics and survival of CRC patients in the Iowa Women's Health Study. Methods: Paraffin-embedded tissue samples were available from 465 post-menopausal women diagnosed with incident CRC in 1986-2002. Tumor microarrays were constructed and immunostained with a CD8 antibody (Clone 144B; Dako). CTLs in tumor epithelial and stromal tissues were categorized by an experienced pathologist into non-detected; mild (1-10 cells per core); moderate (11-29 cells); and strong infiltration (≥30 cells per core), and averaged over cores per each person. We used Cox regression to estimate the hazard ratio (HR) and 95% CI for total and CRC death in relation to each CTL score after adjusting for age of diagnosis, SEER stage, grade, body mass, smoking history and integrated molecular pathway for CRC. The tumors were categorized by integrated pathway (traditional, alternate, serrated, unassigned) based on the combination of phenotypes: microsatellite instability (MSI) status, CpG island methylator (CIMP) phenotype; and mutations in BRAF and/or KRAS genes (described in Samadder, 2013). Results: During follow-up until 2011, 31% of participants died from CRC, 36% from all other causes, and 33% were alive (median follow-up: 8.4 y). The Spearman correlation coefficient between epithelial and stromal CTL scores was 0.52. Both scores were inversely correlated with stage at diagnosis and were higher in tumors characterized by MSI-high, CIMP-high and BRAF mutation-positive status. The highest categories in the epithelial and stromal scores were associated with statistically significant decreases in total death (by 44% and 40%, respectively) and CRC death (by 68% and 56%, respectively) compared to lowest categories. After summing epithelial and stromal CTL scores, the combined HRs (95% CI) in the highest versus lowest category were 0.55 (0.38-0.78) for total death (p-trend = 0.0002) and 0.36 (0.20-0.63) for CRC death (p-trend Conclusions. Our study further supports infiltration of tumors with CTLs as an important prognostic factor in CRC. Citation Format: Anna E. Prizment, Robert A. Vierkant, Thomas C. Smyrk, Lori S. Tillmans, Heather H. Nelson, Charles F. Lynch, Stephen N. Thibodeau, Timothy R. Church, James R. Cerhan, Kristin E. Anderson, Paul J. Limburg. Tumor-infiltrating cytotoxic T-cells are associated with improved survival of colorectal cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2636.

Purpose: Methylated DNA markers can discriminate upper from lower GI neoplasms (Gastroenterology 2013;144(5):S84). It is unknown if organ-specific molecular prediction of GI tumor site can be achieved. Experimental design: An unbiased search for neoplasm markers at each major GI organ site was conducted by reduced representation bisulfite sequencing. Top marker candidates were then blindly validated by methylation specific PCR on independent tissue DNA samples from normal mucosa [17 esophagus (E), 13 stomach (S), 33 pancreas (P), 35 bile duct/liver (BD), 21 colorectal (CR)] and neoplasia [42 E, 43 S, 36 P, 48 BD, 97 CR]. Recursive partitioning (rPart) trees modeled all stepwise combinations of single markers to classify controls vs neoplasms and then neoplasms by site (CR, gastroesophageal or pancreaticobiliary). A 2nd model was designed from tissue data for blinded clinical piloting on 42 independent plasma DNA samples (14 normal and 28 cancers (14 P, 14 CR). Results: From the top 100 markers, 95 were validated and used for rPart modeling. A 3- marker panel (chr12.133484978-5047, BMP3, chr11.123301058-255) was selected for the universal detection of GI neoplasms, as it classified neoplasms and control tissues with 95% accuracy. Two markers (chr7.25896389-501, QKI) then assigned lower vs upper GI neoplasms with 94% accuracy. Finally, 3 markers (PDGFD, ELOVL2, PCBP3) called pancreaticobiliary vs gastroesophageal neoplasms with 94% accuracy. All 8 markers applied in a single model (Table) accurately predicted control vs tumor by site [88% (p Predicted by ModelTissue SourceControlsColorectal neoplasiaPancreatico-biliary cancerGastro-esophageal neoplasiaControls1169102Colorectal neoplasia18617Pancreatico-biliary cancer10654Gastro-esophageal neoplasia12872 Conclusion: Panels of methylated DNA markers were identified for both universal detection of GI neoplasia and accurate prediction of tumor site. Pilot testing of these markers in plasma supports potential clinical feasibility. Citation Format: John B. Kisiel, William R. Taylor, Tracy C. Yab, Hassan M. Ghoz, Patrick H. Foote, Mary E. Devens, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Gloria M. Petersen, Navtej S. Buttar, Lewis R. Roberts, Graham P. Lidgard, David A. Ahlquist. Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation. [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 4252. doi:10.1158/1538-7445.AM2015-4252

Oncogenic K-RAS activation is a major menace that drives aggressive tumor progression and metastasis in 30% of all human cancer. Currently, there are no effective therapies to treat stage III and IV metastatic human cancers with oncogenic K-RAS hyperactivation that often confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS pathway activation is an urgent goal and the major challenge in cancer therapy and anti-K-RAS-based drug development. Instead of targeting an upstream signaling module such as EGFR/HER2/K-RAS/B-RAF, we targeted the most downstream signaling module in the oncogenic K-RAS signaling pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of Seven-In-Absentia (SINA), an evolutionarily conserved RING E3 ligase - the most downstream signaling module and a critical “gatekeeper” required for proper RAS signal transduction. Guided by the insights and fundamental principles learned from the Drosophila RAS signal transduction, we conducted preclinical studies to dissect SIAH function in promoting the oncogenic K-RAS-driven tumorigenesis and metastasis in human cancer. We found that (1) SIAH is a new biomarker reflective of oncogenic K-RAS activation in human cancer, and (2) SIAH loss-of-function is highly effective to block tumorigenesis and metastasis against the well-established, end-stage and metastatic pancreatic cancer and triple negative breast cancer (TNBC). These findings demonstrate that SIAH is an attractive and logical new therapeutic target for developing novel and effective anti-K-RAS and anticancer therapy against metastatic human cancer. Through our work, SIAH has emerged as a promising new drug target against oncogenic K-RAS hyperactivation in metastatic human cancer cells. Using anti-SIAH molecules to block oncogenic K-RAS signaling in human cancer is an excellent example of science going “from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials)”. As a highly evolutionarily conserved E3 ligase that is the most downstream and the most conserved “signaling gatekeeper” in the oncogenic K-RAS signaling network, SIAH is uniquely and strategically positioned to become a great and logical anti-K-RAS drug target. Our preclinical studies have demonstrated that “SIAH-dependent proteolysis” is indeed an Achilles' heel in metastatic human cancer cells. Knowledge gained from our preclinical study has promising translational values. Anti-SIAH-based small molecule inhibitors are likely to aid in expanding our limited arsenal of novel anti-K-RAS-based anticancer therapies. By attacking the oncogenic K-RAS pathway using multi-pronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a position to control the late-stage, relapsed and metastatic human cancers by shutting down the hyperactivated K-RAS signaling transduction cascades in cancer cells in the future. Citation Format: Minglei Bian, Yang Liao, Vasilena Zheleva, Zena Urban, Monicah Njogu, Justin J. Odanga, Andrew J. Isbell, Roger R. Perry, Richard A. Hoefer, Thomas C. Smyrk, Gloria M. Petersen, Amy H. Tang. Attacking the most downstream “gatekeeper,” the SIAH-dependent proteolytic machinery, in the oncogenic ERBB/K-RAS signaling pathway to block tumorigenesis and control metastasis in human cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A26. doi: 10.1158/1557-3125.RASONC14-A26