Search

Table S2: Odds ratio and 95% confidence limits for cancers associated with variants with pleiotropic associations. Figure S1: Flowchart showing analysis steps. Figure S2. Manhattan plots by chromosome for individual cancer sites. Figure S3. Results for rs11571833. Figure S4: Conditional QQ-plots.

Kaplan-Meier graphs in the validation cohort with number of subjects at risk showing colorectal cancer-specific survival according to SMAD4 status.

Supplementary Methods from Cathepsin B Expression and Survival in Colon Cancer: Implications for Molecular Detection of Neoplasia

This file contains Supplementary tables 1 - 7 and the text for Supplementary Figure 1A-D.

figure legends for supp. figures 1, 2, and 3

Kaplan-Meier graphs with number of subjects at risk comparing recurrence-free survival (RFS) by (a) TIL status, or (b) PLA status, with arrows indicating patients whose tumors were also SMAD4 loss.

Background: Higher levels of physical activity are associated with lower colorectal carcinoma incidence and mortality, perhaps through influencing energy balance, cellular prosta7 systemic inflammation. Although evidence suggests interactive effects of energetics, sedentary lifestyle, and tumor CTNNB1 (β-catenin) or CDKN1B (p27) status on colon cancer prognosis, interactive effects of physical activity and tumor PTGS2 (the official symbol for COX-2) status on clinical outcome remain unknown.Methods: Using molecular pathological epidemiology database of 605 stage I–III colon and rectal cancers in two prospective cohort studies (the Nurse's Health Study and the Health Professionals Follow-up Study), we examined patient survival according to postdiagnosis physical activity and tumor PTGS2 status (with 382 PTGS2-positive and 223 PTGS2-negative tumors by immunohistochemistry). Cox proportional hazards models were used to calculate colorectal cancer-specific mortality HR, adjusting for clinical and other tumor variables including microsatellite instability status.Results: Among PTGS2-positive cases, compared with the least active first quartile, the multivariate HRs (95% confidence interval) were 0.30 (0.14–0.62) for the second, 0.38 (0.20–0.71) for the third, and 0.18 (0.08–0.41) for the fourth quartile of physical activity level (Ptrend = 0.0002). In contrast, among PTGS2-negative cases, physical activity level was not significantly associated with survival (Ptrend = 0.84; Pinteraction = 0.024, between physical activity and tumor PTGS2 status).Conclusions: Postdiagnosis physical activity is associated with better survival among patients with PTGS2-positive tumors but not among patients with PTGS2-negative tumors.Impact: Immunohistochemical PTGS2 expression in colorectal carcinoma may serve as a predictive biomarker in pathology practice, which may predict stronger benefit from exercise. Cancer Epidemiol Biomarkers Prev; 22(6); 1142–52. ©2013 AACR.

PDF file - 59K, The description of studies included in this pooled analysis

Supplementary Tables S1-3. Supplementary Table S1 Description of the characteristics by study. Supplementary Table S2 Associations between confounders and body mass index and the weighted genetic risk score among controls. Supplementary Table S3 Odds ratios (OR) and 95% confidence intervals (CI) for the association between a 1-unit increase in the weighted genetic risk score (the instrumental variable [IV]) and risk of colorectal cancer.

Clinical, pathological, and molecular characteristics of colorectal cancer cases according to SMAD4 expression status in validation cohort

PDF file, 64K.

Background: Considerable evidence suggests that cigarette smoking is associated with a higher risk of colorectal cancer (CRC). What is unclear, however, is the impact of quitting smoking on risk attenuation and whether other risk factors for CRC modify this association.Methods: We conducted a pooled analysis of eight studies, including 6,796 CRC cases and 7,770 controls, to evaluate the association between cigarette smoking history and CRC risk and to investigate potential effect modification by other risk factors.Results: Current smokers [OR, 1.26; 95% confidence interval (CI), 1.11–1.43] and former smokers (OR, 1.18; 95% CI, 1.09–1.27), relative to never smokers, showed higher risks of CRC. Former smokers remained at higher CRC risk, relative to never smokers, for up to about 25 years after quitting. The impact of time since quitting varied by cancer subsite: The excess risk due to smoking decreased immediately after quitting for proximal colon and rectal cancer but not until about 20 years post-quitting for distal colon cancer. Furthermore, we observed borderline statistically significant additive interactions between smoking status and body mass index [BMI; relative excess risk due to interaction (RERI]), 0.15; 95% CI, −0.01 to 0.31; P = 0.06] and significant additive interaction between smoking status and fruit consumption (RERI, 0.16; 95% CI, 0.01–0.30; P = 0.04).Conclusion: CRC risk remained increased for about 25 years after quitting smoking, and the pattern of decline in risk varied by cancer subsite. BMI and fruit intake modified the risk associated with smoking.Impact: These results contribute to a better understanding of the mechanisms through which smoking impacts CRC etiology. Cancer Epidemiol Biomarkers Prev; 21(11); 1974–85. ©2012 AACR.

Descriptive table or cohorts and results from sensitivity analyses

Supplementary Table 1. Clinical and histologic characteristics of cutaneous melanocytic nevi, primary melanomas, and melanoma metastases and their relationship to ITK protein levels

Biliary tract cancers are rare but highly fatal with poorly understood etiology. Identifying potentially modifiable risk factors for these cancers is essential for prevention. Here we estimated the relationship between adiposity and cancer across the biliary tract, including cancers of the gallbladder (GBC), intrahepatic bile ducts (IHBDC), extrahepatic bile ducts (EHBDC), and the ampulla of Vater (AVC). We pooled data from 27 prospective cohorts with over 2.7 million adults. Adiposity was measured using baseline body mass index (BMI), waist circumference, hip circumference, waist-to-hip, and waist-to-height ratios. HRs and 95% confidence intervals (95% CI) were estimated using Cox proportional hazards models adjusted for sex, education, race, smoking, and alcohol consumption with age as the time metric and the baseline hazard stratified by study. During 37,883,648 person-years of follow-up, 1,343 GBC cases, 1,194 EHBDC cases, 784 IHBDC cases, and 623 AVC cases occurred. For each 5 kg/m2 increase in BMI, there were risk increases for GBC (HR = 1.27; 95% CI, 1.19–1.36), IHBDC (HR = 1.32; 95% CI, 1.21–1.45), and EHBDC (HR = 1.13; 95% CI, 1.03–1.23), but not AVC (HR = 0.99; 95% CI, 0.88–1.11). Increasing waist circumference, hip circumference, waist-to-hip ratio, and waist-to-height ratio were associated with GBC and IHBDC but not EHBDC or AVC. These results indicate that adult adiposity is associated with an increased risk of biliary tract cancer, particularly GBC and IHBDC. Moreover, they provide evidence for recommending weight maintenance programs to reduce the risk of developing these cancers.Significance:These findings identify a correlation between adiposity and biliary tract cancers, indicating that weight management programs may help minimize the risk of these diseases.

Ordinal logistic regression analyses to assess the association of SMAD4 expression status with lymphocytic reaction patterns in validation cohort

Legends for Supplementary Figures 1 and 2

Results from individual participant meta-analysis of WC. Footnote to Supplementary Figure 2. Multivariable models include age, sex, study, alcohol, smoking, race and body mass index.

Background: High body mass index (BMI) is consistently linked to increased risk of colorectal cancer for men, whereas the association is less clear for women. As risk estimates from observational studies may be biased and/or confounded, we conducted a Mendelian randomization study to estimate the causal association between BMI and colorectal cancer.Methods: We used data from 10,226 colorectal cancer cases and 10,286 controls of European ancestry. The Mendelian randomization analysis used a weighted genetic risk score, derived from 77 genome-wide association study–identified variants associated with higher BMI, as an instrumental variable (IV). We compared the IV odds ratio (IV-OR) with the OR obtained using a conventional covariate-adjusted analysis.Results: Individuals carrying greater numbers of BMI-increasing alleles had higher colorectal cancer risk [per weighted allele OR, 1.31; 95% confidence interval (CI), 1.10–1.57]. Our IV estimation results support the hypothesis that genetically influenced BMI is directly associated with risk for colorectal cancer (IV-OR per 5 kg/m2, 1.50; 95% CI, 1.13–2.01). In the sex-specific IV analyses higher BMI was associated with higher risk of colorectal cancer among women (IV-OR per 5 kg/m2, 1.82; 95% CI, 1.26–2.61). For men, genetically influenced BMI was not associated with colorectal cancer (IV-OR per 5 kg/m2, 1.18; 95% CI, 0.73–1.92).Conclusions: High BMI was associated with increased colorectal cancer risk for women. Whether abdominal obesity, rather than overall obesity, is a more important risk factor for men requires further investigation.Impact: Overall, conventional epidemiologic and Mendelian randomization studies suggest a strong association between obesity and the risk of colorectal cancer. Cancer Epidemiol Biomarkers Prev; 24(7); 1024–31. ©2015 AACR.

Purpose:SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death.Experimental Design: A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance.Results:The discovery cohort consisted of 364 patients with stage I–IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3–8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all P < 0.04). SMAD4 loss was associated with worse RFS (P = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS (P = 0.002 and P = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival.Conclusions:Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer.

Kaplan-Meier graph with number of subjects at risk comparing (a) SMAD4, (b) TIL, and (c) PLA status, separately, as well as (d) SMAD4 and TIL, and (e) SMAD4 and PLA, together, with recurrence-free survival (RFS) among only the MMR-proficient patients.

Table S2 shows no significant differences were observed between regular use of NSAIDs and CRC risk across cancer subsites or stages.

Identifying genetic variants with pleiotropic associations can uncover common pathways influencing multiple cancers. We took a two-stage approach to conduct genome-wide association studies for lung, ovary, breast, prostate, and colorectal cancer from the GAME-ON/GECCO Network (61,851 cases, 61,820 controls) to identify pleiotropic loci. Findings were replicated in independent association studies (55,789 cases, 330,490 controls). We identified a novel pleiotropic association at 1q22 involving breast and lung squamous cell carcinoma, with eQTL analysis showing an association with ADAM15/THBS3 gene expression in lung. We also identified a known breast cancer locus CASP8/ALS2CR12 associated with prostate cancer, a known cancer locus at CDKN2B-AS1 with different variants associated with lung adenocarcinoma and prostate cancer, and confirmed the associations of a breast BRCA2 locus with lung and serous ovarian cancer. This is the largest study to date examining pleiotropy across multiple cancer-associated loci, identifying common mechanisms of cancer development and progression. Cancer Res; 76(17); 5103–14. ©2016 AACR.

Contains more detailed information on the individual cohorts contributing to the study and their genotyping results; Supplemental Tables 1 and 2

Purpose:While evidence indicates that Fusobacterium nucleatum (F. nucleatum) may promote colorectal carcinogenesis through its suppressive effect on T-cell–mediated antitumor immunity, the specific T-cell subsets involved remain uncertain.Experimental Design:We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum–positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets.Results:The amount of F. nucleatum was inversely associated with tumor stromal CD3+ lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28–0.79, for F. nucleatum–high vs. -negative category; Ptrend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32–0.85; Ptrend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs.Conclusions:The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.

Supplementary Figure 1 shows [18F]F-AraG Structure and Schematic of AraG Uptake into T Cells Supplementary Figure 2 shows [18F]F-AraG and [3H]F-AraC Uptake and Retention Across Various Immune Cell Lines Supplementary Figure 3 shows AraG Competitive Inhibition of [18F]F-AraG Uptake Across Various Immune Cell Lines Supplementary Figure 4 shows Comparison of [18F]F-AraG and [3H]F-AraC Uptake and Retention Across Various Solid Tumor Cell Lines Supplementary Figure 5 shows Western Blot Analysis of dCK Expression in Wild-type and Mutant CCRF-CEM cells Supplementary Figure 6 shows dGK overexpression in CHO-K1 cells leads to increased [18F]F-AraG uptake and retention Supplementary Figure 7 shows [3H]F-AraC Accumulates in Cells via dCK Activity Supplementary Figure 8 shows Uptake of [18F]F-AraG in Activated Versus Resting Murine T Cells Supplementary Figure 9 shows Isolation and Activation of Primary Human T Cells Supplementary Figure 10 shows Uptake and Retention of [3H]AraG and [3H]F-AraC in Resting and Activated Human Primary T Cells Supplementary Figure 11 shows Evaluation of GFP+ Donor T cells in Spleens and Cervical Lymph Nodes (CLNs) at Day 3 and Day 10 of Acute GVHD Supplementary Figure 12 shows Evaluation of Proliferation of Donor T Cells in Cervical Lymph Nodes

CHO Cell Transfection Tracer Uptake and Efflux Assays for Adherent Cells Western Blot Immunofluorescence Staining of Lymphoid Organs Ex Vivo Donor T Cell Proliferation Analysis Automated preparation of [18F]FAraG for human imaging using the Neptis perform PET synthesizer Imaging Studies in Humans Statistics

Supplementary Table S1: Cohorts participating in analysis of coffee variables, Liver Cancer Pooling Project. Supplementary Table S2: Assessment of coffee consumption, Liver Cancer Pooling Project. Supplementary Table S3: Characteristics of participants in the Liver Cancer Pooling Project by coffee drinking. Supplementary Table S4: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Intrahepatic Cholangiocarcinoma Incidence by Sex, Smoking Status, Body Mass Index, and Diabetes, Liver Cancer Pooling Project. Supplementary Table S5: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma Incidence by Caffeine Content; Sensitivity Analysis Excluding WHI and Jointly Modeling Caffeine Content and Drinking Intensity, Liver Cancer Pooling Project. Supplementary Table S6: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Suspected Hepatocellular Carcinoma Incidence by Caffeine Content, Liver Cancer Pooling Project. Supplementary Table S7: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma Incidence by Caffeine Content in the AARP Study, Liver Cancer Pooling Project. Supplementary Table S8: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma Incidence by Sex in the AARP Study, Liver Cancer Pooling Project. Supplementary Table S9: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma Incidence by Caffeine Content excluding the AARP Study, Liver Cancer Pooling Project. Supplementary Table S10: Adjusted Hazard Ratios (HR) and 95% Confidence Intervals (CI) for Associations Between Coffee Consumption and Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma Incidence by Sex excluding the AARP Study, Liver Cancer Pooling Project. Supplementary Figure S1: Meta-influence Graph of the Influence of Individuals Studies for Associations between Coffee Consumption versus No Consumption and Hepatocellular Carcinoma, Liver Cancer Pooling Project.

PDF file, 1411K, Supplemental Figure 1. Determination of IC50 values for carfilzomib and ONX 0912 in HNSCC cell lines. UMSCC-22A, UMSCC-22B, 1483, UMSCC-1, Cal33, PCI-15A, PCI-15B, and OSC-19 cells were treated in triplicate wells for 48 hours with varying concentrations of carfilzomib (A) or ONX 0912 (B). Following treatment, floating and attached cells were collected and combined, and cell viabilities were determined by trypan blue exclusion assays. Each data point represents the average of values obtained in 3 independent experiments. Error bars represent the standard error of the means. IC50 values (nmol/L) were determined using GraphPad PRISM sofeware (GraphPad Software, Inc.) Supplemental Figure 2. Inhibition of caspase-3 partially attenuates carfilzomib- and ONX 0912-induced apoptosis of HNSCC cell lines. UMSCC-22A (A) and 1483 (B) cells were pre-incubated for 2 hours with 0.1% DMSO (vehicle), or 20 �mol/L z-DEVD or z-VAD, followed by treatment for 36 hours with 100 nmol/L carfilzomib or ONX 0912. Following treatment, cells were collected and analyzed by flow cytometry for Annexin V/PI staining. Numbers indicate the percentage of Annexin V-positive cells. Supplemental Figure 3. Carfilzomib- and ONX 0912-induced apoptosis is mediated by Bik upregulation, and antagonized by Mcl-1 upregulation. 1483 (A) and UMSCC-1 (B) cells were transfected with nonspecific siRNA, Bik siRNA, or Mcl-1 siRNA for 6 hours. Following transfection, 1483 cells were treated for 36 hours with 100 nmol/L carfilzomib or ONX 0912 and UMSCC-1 cells were treated for 24 hours with 100 nmol/L carfilzomib or for 36 hours with 100 nmol/L ONX 0912. Flow cytometry was used to assess Annexin V/PI staining. Numbers represent the percentage of Annexin V-positive cells. Supplemental Figure 4. Carfilzomib and ONX 0912 induction of LC3-II is partially dependent on upregulation of ATF4. 1483 (A) and UMSCC-1 (B) cells transfected with nonspecific siRNA or ATF4 siRNA were treated for 24 hours with 0.1% DMSO, or 100 nmol/L carfilzomib or ONX 0912, then subjected to immunoblotting for ATF4, LC3-II, or β-actin. Supplemental Figure 5. Impact of autophagy inhibition on ONX 0912-induced HNSCC cell death. UMSCC-22A, UMSCC-22B, 1483 and UMSCC-1 cells were treated with the indicated concentrations of ONX 0912 in the absence or presence of 10 �mol/L chloroquine. After 48 hours treatment, cell viabilities were determined by trypan blue exclusion assays. Data represent the mean + SEM from 3 independent experiments. Supplemental Figure 6. Suppression of ATF4 expression enhances apoptosis in carfilzomib- and ONX 0912-treated HNSCC cells. UMSCC-22A (A) or 1483 (B) cells transfected with nonspecific siRNA or ATF4 siRNA were treated for 24 hours with 100 nmol/L carfilzomib or ONX 0912. Following treatment, Annexin V/PI staining was assessed by flow cytometry. Numbers represent the percentage of Annexin V-positive cells. Supplemental Figure 7. Carfilzomib inhibits HNSCC xenograft tumor growth in vivo. HNSCC tumor-bearing mice were randomized into 3 groups on day 0. Treatment with vehicle (1% carboxymethyl cellulose) or carfilzomib (3 or 5 mg/kg) was initiated on day 0. Mice were treated once per day for two consecutive days and the treatment repeated weekly for 2 weeks (arrows represent treatment days). Mean tumor volumes + SEM are presented.

Protective associations of fruits, vegetables, and fiber intake with colorectal cancer risk have been shown in many, but not all epidemiologic studies. One possible reason for study heterogeneity is that dietary factors may have distinct effects by colorectal cancer molecular subtypes. Here, we investigate the association of fruit, vegetables, and fiber intake with four well-established colorectal cancer molecular subtypes separately and in combination. Nine observational studies including 9,592 cases with molecular subtypes for microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and somatic mutations in BRAF and KRAS genes, and 7,869 controls were analyzed. Both case-only logistic regression analyses and polytomous logistic regression analyses (with one control set and multiple case groups) were used. Higher fruit intake was associated with a trend toward decreased risk of BRAF-mutated tumors [OR 4th vs. 1st quartile = 0.82 (95% confidence interval, 0.65–1.04)] but not BRAF-wildtype tumors [1.09 (0.97–1.22); P difference as shown in case-only analysis = 0.02]. This difference was observed in case–control studies and not in cohort studies. Compared with controls, higher fiber intake showed negative association with colorectal cancer risk for cases with microsatellite stable/MSI-low, CIMP-negative, BRAF-wildtype, and KRAS-wildtype tumors (Ptrend range from 0.03 to 3.4e-03), which is consistent with the traditional adenoma-colorectal cancer pathway. These negative associations were stronger compared with MSI-high, CIMP-positive, BRAF-mutated, or KRAS-mutated tumors, but the differences were not statistically significant. These inverse associations for fruit and fiber intake may explain, in part, inconsistent findings between fruit or fiber intake and colorectal cancer risk that have previously been reported.Significance:These analyses by colorectal cancer molecular subtypes potentially explain the inconsistent findings between dietary fruit or fiber intake and overall colorectal cancer risk that have previously been reported.

Purpose: Prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2; a target of aspirin) produces inflammatory mediator prostaglandin E2 (PGE2), and contributes to colorectal neoplasia development. PTGS2-driven inflammatory responses can induce tumor expression of microRNA MIR21 (miR-21) that can increase local PGE2 level by downregulating PGE2-metabolizing enzymes. We hypothesized that the prognostic association of tumor MIR21 expression level in colorectal carcinoma might depend on inflammatory tumor microenvironment and be stronger in tumors expressing high-level PTGS2.Experimental Design: Utilizing 765 rectal and colon cancer specimens in the Nurses' Health Study and the Health Professionals Follow-up Study, we measured MIR21 expression by quantitative reverse transcription PCR, and PTGS2 expression by immunohistochemistry. Cox proportional hazards regression model was used to assess statistical interaction between MIR21 and PTGS2 in colorectal cancer–specific survival analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutations.Results: Tumor MIR21 expression level was associated with higher colorectal cancer–specific mortality (Ptrend = 0.029), and there was a statistically significant interaction between MIR21 and PTGS2 (Pinteraction = 0.0004). The association between MIR21 expression and colorectal cancer–specific mortality was statistically significant in PTGS2-high cancers (multivariable hazard ratio of the highest vs. lowest quartile of MIR21, 2.28; 95% confidence interval, 1.42–3.67; Ptrend = 0.0004) but not in PTGS2-absent/low cancers (Ptrend = 0.22).Conclusions:MIR21 expression level in colorectal carcinoma is associated with worse clinical outcome, and this association is stronger in carcinomas expressing high-level PTGS2, suggesting complex roles of immunity and inflammation in tumor progression. Clin Cancer Res; 22(15); 3841–8. ©2016 AACR.

Regular aspirin use after diagnosis and colorectal cancer mortality according to tumor MIR21 expression level among 579 patients with stage I to III colorectal cancer

Supplementary Tables S1-5 and Figure S1. Supplementary Table S1 - Multivariate model of overall survival including known prognostic factors; Supplementary Table S2 - Univariate analyses of survival in our validation phase; Supplementary Table S3: Gene-dose effect for rs9929218; Supplementary Table S4 - Distribution of rs9929218 genotype by disease stage; Supplementary Table S5 - Univariate analysis of rs9929218 on overall survival according to COIN trial arm; Supplementary Figure S1 - The effect of rs9929218 on survival from the time of diagnosis to death.

Supplementary Figures 1 - 3

Purpose: We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti–IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti–IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807.Experimental Design: An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti–IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy.Results: The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti–IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe.Conclusions: These studies provide a clear biological rationale to test anti–IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC. Clin Cancer Res; 17(8); 2314–27. ©2011 AACR.

Supplementary Figure 2. Ingenuity Pathway Analysis Results from RPPA assay and effects of ITK activity on specific cellular proteins by westerns and RPPA results.

Results from individual participant meta-analysis of diabetes. Multivariable models include age, sex, study, alcohol, smoking, race and body mass index.

Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation.Experimental Design: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined.Results: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0–G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Ptennull/BrafV600E) melanomas.Conclusions: We conclude that ITK, formerly considered an immune cell–specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanoma model supports this possibility. Clin Cancer Res; 21(9); 2167–76. ©2015 AACR.

Supplementary Table 6. Significant pathways identified by Ingenuity Pathway Analysis for Reverse Phase Protein Array (RPPA) results of RPMI 8322

Supplemental Figure legends

Supplemental Table 3. Environmental Factors and colorectal cancer: associations by study design

Purpose: Genome-wide association studies have identified numerous loci associated with colorectal cancer risk. Several of these have also been associated with patient survival, although none have been validated. Here, we used large independent training and validation cohorts to identify robust prognostic biomarkers for colorectal cancer.Experimental Design: In our training phase, we analyzed 20 colorectal cancer-risk SNPs from 14 genome-wide associated loci, for their effects on survival in 2,083 patients with advanced colorectal cancer. A Cox survival model was used, stratified for treatment, adjusted for known prognostic factors, and corrected for multiple testing. Three SNPs were subsequently analyzed in an independent validation cohort of 5,552 colorectal cancer patients. A validated SNP was analyzed by disease stage and response to treatment.Results: Three variants associated with survival in the training phase; however, only rs9929218 at 16q22 (intron 2 of CDH1, encoding E-cadherin) was significant in the validation phase. Patients homozygous for the minor allele (AA genotype) had worse survival (training phase HR, 1.43; 95% confidence intervals; CI, 1.20–1.71, P = 5.8 × 10−5; validation phase HR, 1.18; 95% CI, 1.01–1.37, P = 3.2 × 10−2; combined HR, 1.28; 95% CI, 1.14–1.43, P = 2.2 × 10−5). This effect was independent of known prognostic factors, and was significant amongst patients with stage IV disease (P = 2.7 × 10−5). rs9929218 was also associated with poor response to chemotherapy (P = 3.9 × 10−4).Conclusions: We demonstrate the potential of common inherited genetic variants to inform patient outcome and show that rs9929218 identifies approximately 8% of colorectal cancer patients with poor prognosis. rs9929218 may affect CDH1 expression and E-cadherin plays a role in epithelial-to-mesenchymal transition providing a mechanism underlying its prognostic potential. Clin Cancer Res; 21(15); 3453–61. ©2015 AACR.

Purpose:Immunotherapy is a promising approach for many oncological malignancies, including glioblastoma, however, there are currently no available tools or biomarkers to accurately assess whole-body immune responses in patients with glioblastoma treated with immunotherapy. Here, the utility of OX40, a costimulatory molecule mainly expressed on activated effector T cells known to play an important role in eliminating cancer cells, was evaluated as a PET imaging biomarker to quantify and track response to immunotherapy.Experimental Design:A subcutaneous vaccination approach of CpG oligodeoxynucleotide, OX40 mAb, and tumor lysate at a remote site in a murine orthotopic glioma model was developed to induce activation of T cells distantly while monitoring their distribution in stimulated lymphoid organs with respect to observed therapeutic effects. To detect OX40-positive T cells, we utilized our in-house–developed 89Zr-DFO-OX40 mAb and in vivo PET/CT imaging.Results:ImmunoPET with 89Zr-DFO-OX40 mAb revealed strong OX40-positive responses with high specificity, not only in the nearest lymph node from vaccinated area (mean, 20.8%ID/cc) but also in the spleen (16.7%ID/cc) and the tumor draining lymph node (11.4%ID/cc). When the tumor was small (6 p/sec/cm2/sr in bioluminescence imaging), a high number of responders and percentage shrinkage in tumor signal was indicated after only a single cycle of vaccination.Conclusions:The results highlight the promise of clinically translating cancer vaccination as a potential glioma therapy, as well as the benefits of monitoring efficacy of these treatments using immunoPET imaging of T-cell activation.

Table S1. Analyses of MSI, DNA methylation, KRAS, BRAF, and PIK3CA mutations, and neoantigen load. Table S2. Clinical and pathological characteristics of colorectal cancer cases according to tumor stromal immune cell densities in TCGA dataset. Table S3. Steps of immune cell detection and quantification and tissue category classification from images of H&E stained sections using QuPath. Table S4. Statistical methods for survival analysis. Table S5. Immune cell densities in tumor intraepithelial and stromal regions and colorectal cancer-specific survival with inverse probability weighting (IPW). Table S6. Densities of stromal lymphocytes, plasma cells, and eosinophils and patient survival in strata of MSI status with inverse probability weighting (IPW). Table S7. Comparison of the prognostic power of immune cell densities in colorectal cancer-specific survival analyses. Table S8. Combination of spatial G-cross measurement and density measurements of lymphocytes, plasma cells, neutrophils, and eosinophils in relation to colorectal cancer-specific survival. Table S9. Densities of intraepithelial and stromal immune cells and patient survival in TCGA dataset. Table S10. Tumor:Immune cell G-cross function area under the curve and patient survival in TCGA dataset. Figure S1. Reproducibility of the immune cell counting. Figure S2. Boxplots of the densities of intraepithelial and stromal lymphocytes, plasma cells, neutrophils, and eosinophils across 10 TMAs. Figure S3. UMAP analysis of colorectal cancer cases according to intraepithelial and stromal densities of lymphocytes, plasma cells, neutrophils, and eosinophils. Figure S4. Relationships of intraepithelial and stromal immune cell densities with LINE-1 methylation level, BRAF mutation, KRAS mutation, and PIK3CA mutation. Figure S5. Inverse probability weighting-adjusted Kaplan-Meier curves of colorectal cancer-specific survival according to ordinal categories (C1-C4) of intraepithelial lymphocyte (A), plasma cell (B), neutrophil (C), and eosinophil (D) densities.

PDF file - 68K, The description of smoking variables included in this analysis

Supplementary Tables 3 and 4. Supplementary Table 3. ITK protein expression levels in cell lines by single fluorescent immunocytochemistry Supplementary Table 4. Statistical analysis of shRNA and BI 10N effects on the proliferation and motility rates of melanoma cell lines

Background: Genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) that are associated with risk of colorectal cancer. Prior research has evaluated the presence of gene–environment interaction involving the first 10 identified susceptibility loci, but little work has been conducted on interaction involving SNPs at recently identified susceptibility loci, including: rs10911251, rs6691170, rs6687758, rs11903757, rs10936599, rs647161, rs1321311, rs719725, rs1665650, rs3824999, rs7136702, rs11169552, rs59336, rs3217810, rs4925386, and rs2423279.Methods: Data on 9,160 cases and 9,280 controls from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and Colon Cancer Family Registry (CCFR) were used to evaluate the presence of interaction involving the above-listed SNPs and sex, body mass index (BMI), alcohol consumption, smoking, aspirin use, postmenopausal hormone (PMH) use, as well as intake of dietary calcium, dietary fiber, dietary folate, red meat, processed meat, fruit, and vegetables. Interaction was evaluated using a fixed effects meta-analysis of an efficient Empirical Bayes estimator, and permutation was used to account for multiple comparisons.Results: None of the permutation-adjusted P values reached statistical significance.Conclusions: The associations between recently identified genetic susceptibility loci and colorectal cancer are not strongly modified by sex, BMI, alcohol, smoking, aspirin, PMH use, and various dietary factors.Impact: Results suggest no evidence of strong gene–environment interactions involving the recently identified 16 susceptibility loci for colorectal cancer taken one at a time. Cancer Epidemiol Biomarkers Prev; 23(9); 1824–33. ©2014 AACR.

Purpose: To determine whether optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this, we evaluated the known Her2 response to 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) treatment, an Hsp90 inhibitor.Experimental Design: After in vitro 17-DMAG treatment response evaluation of MCF7 parental cells and 2 HER2-transfected clones (clone A medium, B high Her2 expression), we established human breast cancer xenografts in nude mice (only parental and clone B) for in vivo evaluation. Mice received 120 mg/kg of 17-DMAG in 4 doses at 12-hour intervals intraperitonially (n = 14) or PBS as carrier control (n = 9). Optical images were obtained both pretreatment (day 0) and posttreatment (day 3, 6, and 9), always 5 hours postinjection of 500 pmol of anti-Her2 Affibody-AlexaFluor680 via tail vein (with preinjection background subtraction). Days 3 and 9 in vivo optical imaging signal was further correlated with ex vivo Her2 levels by Western blot after sacrifice.Results: Her2 expression decreased with 17-DMAG dose in vitro. In vivo optical imaging signal was reduced by 22.5% in clone B (P = 0.003) and by 9% in MCF7 parental tumors (P = 0.23) 3 days after 17-DMAG treatment; optical imaging signal recovered in both tumor types at days 6 to 9. In the carrier group, no signal reduction was observed. Pearson correlation of in vivo optical imaging signal with ex vivo Her2 levels ranged from 0.73 to 0.89.Conclusions: Optical imaging with an affibody can be used to noninvasively monitor changes in Her2 expression in vivo as a response to treatment with an Hsp90 inhibitor, with results similar to response measurements in positron emission tomography imaging studies. Clin Cancer Res; 18(4); 1073–81. ©2012 AACR.

Results from individual participant meta-analysis of BMI. Multivariable models include age, sex, study, alcohol, smoking, and race.

Supplementary Figure 1. IHC staining for co-localization of immune markers with cells expressing ITK and determining the presence of ITK in immune cell subtypes infiltrating the tumor and the effect of ITK ablation on the cell cycle of the melanoma cells