Common variation in a long non-coding RNA gene modulates variation of circulating TGF-ß2 levels in metastatic colorectal cancer patients (Alliance).

BACKGROUND: Herein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC). The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings. RESULTS: The final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-ß2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73. CONCLUSIONS: This study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-ß2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.

Common variation in a long non-coding RNA gene modulates variation of circulating TGF-ß2 levels in metastatic colorectal cancer patients (Alliance).

Background: Herein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC).The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings. Results: The final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-ß2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73. Conclusions: This study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-ß2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.

High-fat diet and obesity are associated with differential angiogenic gene expression in epithelial ovarian cancer.

OBJECTIVE: We sought to evaluate the association between diet and angiogenic biomarkers in KpB mice, and the association between these markers, body mass index (BMI), and overall survival (OS) in high-grade serous cancers (HGSC). METHODS: Tumors previously obtained from KpB mice subjected to high-fat diets (HFD, n = 10) or low-fat diets (LFD, n = 10) were evaluated for angiogenesis based on CD-31 microvessel density (MVD). Data from prior microarray analysis (Agilent 244 K arrays) conducted in 10 mice were utilized to assess associations between diet and angiogenetic biomarkers. Agilent (mouse) and Affymetrix Human Genome U133a probes were linked to 162 angiogenic-related genes. The associations between biomarkers, BMI, and OS were evaluated in an HGSC internal database (IDB) (n = 40). Genes with unadjusted p < 0.05 were evaluated for association with OS in the TCGA-OV database (n = 339). RESULTS: There was no association between CD-31 and diet in mice (p = 0.66). Sixteen angiogenic-related genes passed the p < 0.05 threshold for association with HFD vs. LFD. Transforming growth factor-alpha (TGFA) demonstrated 72% higher expression in HFD vs. LFD mice (p = 0.04). Similar to the mouse study, in our HGSC IDB, higher TGFA expression correlated with higher BMI (p = 0.01) and shorter survival (p = 0.001). In the TCGA-OV dataset, BMI data was not available and there was no association between TGFA and OS (p = 0.48). CONCLUSIONS: HFD and obesity may promote tumor progression via differential modulation of TGFA. We were unable to confirm this finding in the TCGA dataset. Further evaluation of TGFA is needed to determine if this is a target unique to obesity-driven HGSC.

Bevacizumab-induced hypertension and proteinuria: a genome-wide study of more than 1000 patients.

BACKGROUND: Hypertension and proteinuria are common bevacizumab-induced toxicities. No validated biomarkers are available for identifying patients at risk of these toxicities. METHODS: A genome-wide association study (GWAS) meta-analysis was performed in 1039 bevacizumab-treated patients of European ancestry in four clinical trials (CALGB 40502, 40503, 80303, 90401). Grade ≥2 hypertension and proteinuria were recorded (CTCAE v.3.0). Single-nucleotide polymorphism (SNP)-toxicity associations were determined using a cause-specific Cox model adjusting for age and sex. RESULTS: The most significant SNP associated with hypertension with concordant effect in three out of the four studies (p-value <0.05 for each study) was rs6770663 (A > G) in KCNAB1, with the G allele increasing the risk of hypertension (p-value = 4.16 × 10-6). The effect of the G allele was replicated in ECOG-ACRIN E5103 in 582 patients (p-value = 0.005). The meta-analysis of all five studies for rs6770663 led to p-value = 7.73 × 10-8, close to genome-wide significance. The most significant SNP associated with proteinuria was rs339947 (C > A, between DNAH5 and TRIO), with the A allele increasing the risk of proteinuria (p-value = 1.58 × 10-7). CONCLUSIONS: The results from the largest study of bevacizumab toxicity provide new markers of drug safety for further evaluations. SNP in KCNAB1 validated in an independent dataset provides evidence toward its clinical applicability to predict bevacizumab-induced hypertension. ClinicalTrials.gov Identifier: NCT00785291 (CALGB 40502); NCT00601900 (CALGB 40503); NCT00088894 (CALGB 80303) and NCT00110214 (CALGB 90401).

Genome-wide association studies of survival in 1520 cancer patients treated with bevacizumab-containing regimens.

Germline variants might predict cancer progression. Bevacizumab improves overall survival (OS) in patients with advanced cancers. No biomarkers are available to identify patients that benefit from bevacizumab. A meta-analysis of genome-wide association studies (GWAS) was conducted in 1,520 patients from Phase III trials (CALGB 80303, 40503, 80405 and ICON7), where bevacizumab was randomized to treatment without bevacizumab. We aimed to identify genes and single nucleotide polymorphisms (SNPs) associated with survival independently of bevacizumab treatment or through interaction with bevacizumab. A cause-specific Cox model was used to test the SNP-OS association in both arms combined (prognostic), and the effect of SNPs-bevacizumab interaction on OS (predictive) in each study. The SNP effects across studies were combined using inverse variance. Findings were tested for replication in advanced colorectal and ovarian cancer patients from The Cancer Genome Atlas (TGCA). In the GWAS meta-analysis, patients with rs680949 in PRUNE2 experienced shorter OS compared to patients without it (P = 1.02 × 10-7 , hazard ratio [HR] = 1.57, 95% confidence interval [CI] 1.33-1.86), as well as in TCGA (P = .0219, HR = 1.58, 95% CI 1.07-2.35). In the GWAS meta-analysis, patients with rs16852804 in BARD1 experienced shorter OS compared to patients without it (P = 1.40 × 10-5 , HR = 1.51, 95% CI 1.25-1.82) as well as in TCGA (P = 1.39 × 10-4 , HR = 3.09, 95% CI 1.73-5.51). Patients with rs3795897 in AGAP1 experienced shorter OS in the bevacizumab arm compared to the nonbevacizumab arm (P = 1.43 × 10-5 ). The largest GWAS meta-analysis of bevacizumab treated patients identified PRUNE2 and BARD1 (tumor suppressor genes) as prognostic genes of colorectal and ovarian cancer, respectively, and AGAP1 as a potentially predictive gene that interacts with bevacizumab with respect to patient survival.

Plasma Protein Biomarkers in Advanced or Metastatic Colorectal Cancer Patients Receiving Chemotherapy With Bevacizumab or Cetuximab: Results from CALGB 80405 (Alliance).

PURPOSE: CALGB 80405 compared the combination of first-line chemotherapy with cetuximab or bevacizumab in the treatment of advanced or metastatic colorectal cancer (mCRC). Although similar clinical outcomes were observed in the cetuximab-chemotherapy group and the bevacizumab-chemotherapy group, biomarkers could identify patients deriving more benefit from either biologic agent. PATIENTS AND METHODS: In this exploratory analysis, the Angiome, a panel of 24 soluble protein biomarkers were measured in baseline plasma samples in CALGB 80405. Prognostic biomarkers were determined using univariate Cox proportional hazards models. Predictive biomarkers were identified using multivariable Cox regression models including interaction between biomarker level and treatment. RESULTS: In the total population, high plasma levels of Ang-2, CD73, HGF, ICAM-1, IL6, OPN, TIMP-1, TSP-2, VCAM-1, and VEGF-R3 were identified as prognostic of worse progression-free survival (PFS) and overall survival (OS). PlGF was identified as predictive of lack of PFS benefit from bevacizumab [bevacizumab HR, 1.51; 95% confidence interval (CI), 1.10-2.06; cetuximab HR, 0.94; 95% CI, 0.71-1.25; Pinteraction = 0.0298] in the combined FOLFIRI/FOLFOX regimens. High levels of VEGF-D were predictive of lack of PFS benefit from bevacizumab in patients receiving FOLFOX regimen only (FOLFOX/bevacizumab HR, 1.70; 95% CI, 1.19-2.42; FOLFOX/cetuximab HR, 0.92; 95% CI, 0.68-1.24; Pinteraction = 0.0097). CONCLUSIONS: In this exploratory, hypothesis-generating analysis, the Angiome identified multiple prognostic biomarkers and two potential predictive biomarkers for patients with mCRC enrolled in CALGB 80405. PlGF and VEGF-D predicted lack of benefit from bevacizumab in a chemo-dependent manner. See related commentaries by Mishkin and Kohn, p. 2722 and George and Bertagnolli, p. 2725.

A prospective trial of abiraterone acetate plus prednisone in Black and White men with metastatic castrate-resistant prostate cancer.

BACKGROUND: Retrospective analyses of randomized trials suggest that Black men with metastatic castration-resistant prostate cancer (mCRPC) have longer survival than White men. The authors conducted a prospective study of abiraterone acetate plus prednisone to explore outcomes by race. METHODS: This race-stratified, multicenter study estimated radiographic progression-free survival (rPFS) in Black and White men with mCRPC. Secondary end points included prostate-specific antigen (PSA) kinetics, overall survival (OS), and safety. Exploratory analysis included genome-wide genotyping to identify single nucleotide polymorphisms associated with progression in a model incorporating genetic ancestry. One hundred patients self-identified as White (n = 50) or Black (n = 50) were enrolled. Eligibility criteria were modified to facilitate the enrollment of individual Black patients. RESULTS: The median rPFS for Black and White patients was 16.6 and 16.8 months, respectively; their times to PSA progression (TTP) were 16.6 and 11.5 months, respectively; and their OS was 35.9 and 35.7 months, respectively. Estimated rates of PSA decline by ≥50% in Black and White patients were 74% and 66%, respectively; and PSA declines to <0.2 ng/mL were 26% and 10%, respectively. Rates of grade 3 and 4 hypertension, hypokalemia, and hyperglycemia were higher in Black men. CONCLUSIONS: Multicenter prospective studies by race are feasible in men with mCRPC but require less restrictive eligibility. Despite higher comorbidity rates, Black patients demonstrated rPFS and OS similar to those of White patients and trended toward greater TTP and PSA declines, consistent with retrospective reports. Importantly, Black men may have higher side-effect rates than White men. This exploratory genome-wide analysis of TTP identified a possible candidate marker of ancestry-dependent treatment outcomes.

Whole-Exome Sequencing of Radiation-Induced Thymic Lymphoma in Mouse Models Identifies Notch1 Activation as a Driver of p53 Wild-Type Lymphoma.

Mouse models of radiation-induced thymic lymphoma are widely used to study the development of radiation-induced blood cancers and to gain insights into the biology of human T-cell lymphoblastic leukemia/lymphoma. Here we aimed to identify key oncogenic drivers for the development of radiation-induced thymic lymphoma by performing whole-exome sequencing using tumors and paired normal tissues from mice with and without irradiation. Thymic lymphomas from irradiated wild-type (WT), p53+/-, and KrasLA1 mice were not observed to harbor significantly higher numbers of nonsynonymous somatic mutations compared with thymic lymphomas from unirradiated p53-/- mice. However, distinct patterns of recurrent mutations arose in genes that control the Notch1 signaling pathway based on the mutational status of p53. Preferential activation of Notch1 signaling in p53 WT lymphomas was also observed at the RNA and protein level. Reporter mice for activation of Notch1 signaling revealed that total-body irradiation (TBI) enriched Notch1hi CD44+ thymocytes that could propagate in vivo after thymocyte transplantation. Mechanistically, genetic inhibition of Notch1 signaling in immature thymocytes prevented formation of radiation-induced thymic lymphoma in p53 WT mice. Taken together, these results demonstrate a critical role of activated Notch1 signaling in driving multistep carcinogenesis of thymic lymphoma following TBI in p53 WT mice. SIGNIFICANCE: These findings reveal the mutational landscape and key drivers in murine radiation-induced thymic lymphoma, a classic animal model that has been used to study radiation carcinogenesis for over 70 years.

Genomic Analysis of Germline Variation Associated with Survival of Patients with Colorectal Cancer Treated with Chemotherapy Plus Biologics in CALGB/SWOG 80405 (Alliance).

PURPOSE: Irinotecan/5-fluorouracil (5-FU; FOLFIRI) or oxaliplatin/5-FU (FOLFOX), combined with bevacizumab or cetuximab, are approved, first-line treatments for metastatic colorectal cancer (mCRC). We aimed at identifying germline variants associated with survival in patients with mCRC treated with these regimens in Cancer and Leukemia Group B/SWOG 80405. EXPERIMENTAL DESIGN: Patients with mCRC receiving either FOLFOX or FOLFIRI were randomized to either cetuximab or bevacizumab. DNA from peripheral blood was genotyped for approximately 700,000 SNPs. The association between SNPs and overall survival (OS) was tested in 613 patients of genetically estimated European ancestry using Cox proportional hazards models. RESULTS: The four most significant SNPs associated with OS were three haplotypic SNPs between microsomal glutathione S-transferase 1 (MGST1) and LIM domain only 3 (LMO3, representative HR, 1.56; P = 1.30 × 10-6), and rs11644916 in AXIN1 (HR, 1.39, P = 4.26 × 10-6). AXIN1 is a well-established tumor suppressor gene in colorectal cancer, and rs11644916 (G>A) conferred shorter OS. Median OS for patients with the AA, AG, or GG genotypes was 18.4, 25.6, or 36.4 months, respectively. In 90 patients with stage IV colorectal cancer from The Cancer Genome Atlas (TCGA), rs11649255 in AXIN1 [in almost complete linkage disequilibrium (LD) with rs11644916], was associated with shorter OS (HR, 2.24, P = 0.0096). Using rs11648673 in AXIN1 (in very high LD with rs11644916 and with functional evidence), luciferase activity in three colorectal cancer cell lines was reduced. CONCLUSIONS: This is the first large genome-wide association study ever conducted in patients with mCRC treated with first-line standard treatment in a randomized phase III trial. A common SNP in AXIN1 conferred worse OS and the effect was replicated in TCGA. Further studies in colorectal cancer experimental models are required.

Adaptation and selection shape clonal evolution of tumors during residual disease and recurrence.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

Prognostic and Predictive Biomarkers in Patients with Metastatic Colorectal Cancer Receiving Regorafenib.

Regorafenib is a tyrosine kinase inhibitor approved by the FDA for the treatment of patients with chemotherapy refractory metastatic colorectal cancer (mCRC). Regorafenib inhibits signaling through multiple receptors associated with angiogenesis, metastasis, and tumor immunity. Here, we report biomarker results from LCCC1029, a randomized, placebo-controlled, phase II trial of chemotherapy ± regorafenib in patients with second-line mCRC. A panel of 20 soluble protein biomarkers (termed the Angiome) was assessed in the plasma of 149 patients from the LCCC1029 trial both at baseline and along the treatment continuum. Baseline protein levels were analyzed for prognostic and predictive value for progression-free survival (PFS) and overall survival (OS). Changes in protein levels during treatment were analyzed for potential pharmacodynamic effects. Six markers (HGF, IL6, PlGF, VEGF-R1, OPN, and IL6R) were found to be prognostic for PFS. Nine markers (IL6, TIMP-1, PlGF, VCAM-1, ICAM-1, OPN, TSP-2, HGF, and VEGF-R1) were prognostic for OS. Higher baseline levels of OPN (P intx = 0.0167), VCAM-1 (P intx = 0.0216), and PDGF-AA (P intx = 0.0435) appeared to predict for PFS benefit from regorafenib compared with placebo. VCAM-1 was also potentially predictive of OS benefit from regorafenib compared with placebo (P intx = 0.0124). On-treatment changes of six markers reflected potential on-target effect of regorafenib. Consistent results were observed in an Italian cohort where 105 patients with late-stage mCRC received regorafenib monotherapy. The key findings of this study suggest that VCAM-1 may be a predictive biomarker for regorafenib benefit, while multiple protein markers may be prognostic of outcome in patients with mCRC.

Predictive Blood-Based Biomarkers in Patients with Epithelial Ovarian Cancer Treated with Carboplatin and Paclitaxel with or without Bevacizumab: Results from GOG-0218.

PURPOSE: GOG-0218, a double-blind placebo-controlled phase III trial, compared carboplatin and paclitaxel with placebo, bevacizumab followed by placebo, or bevacizumab followed by bevacizumab in advanced epithelial ovarian cancer (EOC). Results demonstrated significantly improved progression-free survival (PFS), but no overall survival (OS) benefit with bevacizumab. Blood samples were collected for biomarker analyses. EXPERIMENTAL DESIGN: Plasma samples were analyzed via multiplex ELISA technology for seven prespecified biomarkers [IL6, Ang-2, osteopontin (OPN), stromal cell-derived factor-1 (SDF-1), VEGF-D, IL6 receptor (IL6R), and GP130]. The predictive value of each biomarker with respect to PFS and OS was assessed using a protein marker by treatment interaction term within the framework of a Cox proportional hazards model. Prognostic markers were identified using Cox models adjusted for baseline covariates. RESULTS: Baseline samples were available from 751 patients. According to our prespecified analysis plan, IL6 was predictive of a therapeutic advantage with bevacizumab for PFS (P = 0.007) and OS (P = 0.003). IL6 and OPN were found to be negative prognostic markers for both PFS and OS (P < 0.001). Patients with high median IL6 levels (dichotomized at the median) treated with bevacizumab had longer PFS (14.2 vs. 8.7 months) and OS (39.6 vs. 33.1 months) compared with placebo. CONCLUSIONS: The inflammatory cytokine IL6 may be predictive of therapeutic benefit from bevacizumab when combined with carboplatin and paclitaxel. Aligning with results observed in patients with renal cancer treated with antiangiogenic therapies, it appears plasma IL6 may also define those patients with EOC more or less likely to benefit from the addition of bevacizumab to standard chemotherapy.

Efficient estimation of grouped survival models.

BACKGROUND: Time- and dose-to-event phenotypes used in basic science and translational studies are commonly measured imprecisely or incompletely due to limitations of the experimental design or data collection schema. For example, drug-induced toxicities are not reported by the actual time or dose triggering the event, but rather are inferred from the cycle or dose to which the event is attributed. This exemplifies a prevalent type of imprecise measurement called grouped failure time, where times or doses are restricted to discrete increments. Failure to appropriately account for the grouped nature of the data, when present, may lead to biased analyses. RESULTS: We present groupedSurv, an R package which implements a statistically rigorous and computationally efficient approach for conducting genome-wide analyses based on grouped failure time phenotypes. Our approach accommodates adjustments for baseline covariates, and analysis at the variant or gene level. We illustrate the statistical properties of the approach and computational performance of the package by simulation. We present the results of a reanalysis of a published genome-wide study to identify common germline variants associated with the risk of taxane-induced peripheral neuropathy in breast cancer patients. CONCLUSIONS: groupedSurv enables fast and rigorous genome-wide analysis on the basis of grouped failure time phenotypes at the variant, gene or pathway level. The package is freely available under a public license through the Comprehensive R Archive Network.

Mutational landscape in genetically engineered, carcinogen-induced, and radiation-induced mouse sarcoma.

Cancer development is influenced by hereditary mutations, somatic mutations due to random errors in DNA replication, or external factors. It remains unclear how distinct cell-intrinsic and -extrinsic factors impact oncogenesis within the same tissue type. We investigated murine soft tissue sarcomas generated by oncogenic alterations (KrasG12D activation and p53 deletion), carcinogens (3-methylcholanthrene [MCA] or ionizing radiation), and in a novel model combining both factors (MCA plus p53 deletion). Whole-exome sequencing demonstrated distinct mutational signatures in individual sarcoma cohorts. MCA-induced sarcomas exhibited high mutational burden and predominantly G-to-T transversions, while radiation-induced sarcomas exhibited low mutational burden and a distinct genetic signature characterized by C-to-T transitions. The indel to substitution ratio and amount of gene copy number variations were high for radiation-induced sarcomas. MCA-induced tumors generated on a p53-deficient background showed the highest genomic instability. MCA-induced sarcomas harbored mutations in putative cancer-driver genes that regulate MAPK signaling (Kras and Nf1) and the Hippo pathway (Fat1 and Fat4). In contrast, radiation-induced sarcomas and KrasG12Dp53-/- sarcomas did not harbor recurrent oncogenic mutations, rather they exhibited amplifications of specific oncogenes: Kras and Myc in KrasG12Dp53-/- sarcomas, and Met and Yap1 for radiation-induced sarcomas. These results reveal that different initiating events drive oncogenesis through distinct mechanisms.

An initial genetic analysis of gemcitabine-induced high-grade neutropenia in pancreatic cancer patients in CALGB 80303 (Alliance).

OBJECTIVES: One of the standard of care regimens for advanced pancreatic cancer is gemcitabine-based chemotherapy. The efficacy of gemcitabine is limited by dose-limiting hematologic toxicities especially neutropenia. Uncovering the variability of these toxicities attributed to germline DNA variation is of great importance. PATIENTS AND METHODS: CALGB 80303 was a randomized study in advanced pancreatic cancer patients treated with gemcitabine with or without bevacizumab. The study protocol included genotyping of genes of gemcitabine disposition (CDA, DCTD, SLC29A1, SLC28A1, and SLC29A2), as well as a genome-wide analysis. The clinical phenotype was time to early high-grade neutropenia event accounting for progression or death or other treatment-terminating adverse events as competing for informative events. The inference was carried out on the basis of the association between genotype and cause-specific hazard of a neutropenic event. RESULTS: The primary analyses were carried out on the basis of 294 genetically estimated European pancreatic cancer patients. For CDA rs2072671 (A>C), AC and CC patients had a lower risk of neutropenia than AA patients (P=0.01, hazard ratio: 0.61, 95% confidence interval: 0.41-0.89). For SLC28A1 rs3825876 (G>A), AA patients have a higher risk of neutropenia than GA and GG patients (P=0.02, hazard ratio: 1.51, 95% confidence interval: 1.06-2.16). CDA rs2072671 was associated with increased mRNA expression in whole blood in three studies (P=2.7e-14, 6.61e-62, and 9.70e-65). In the genome-wide analysis, variants in TGFB2 were among the top hits (lowest P=1.62e-06) but had no effect in luciferase assays. CONCLUSION: This is the first genetic analysis of gemcitabine-induced neutropenia using a competing risk model in a prospective randomized clinical study has proposed a potentially novel mechanism of the protective effect of the CDA rs2072671 variant. Further confirmation is needed.

Genetic variation determines VEGF-A plasma levels in cancer patients.

Angiogenesis is essential in tumor biology and is regulated by vascular endothelial growth factor (VEGF) ligands and receptors. Here we aimed to discover genetic variants associated with levels of circulating angiogenic proteins in cancer patients. Plasma was collected at baseline in 216 pancreatic and 114 colorectal cancer patients. Thirty-one angiogenic proteins were measured by ELISA. 484,523 Single Nucleotide Polymorphisms (SNP) were tested for association with plasma levels for each protein in pancreatic cancer patients. Three top-ranked hits were then genotyped in colorectal cancer patients, where associations with the same proteins were measured. The results demonstrated rs2284284 and MCP1 (P-value = 6.7e-08), rs7504372 and VEGF-C (P-value = 9.8e-09), and rs7767396 and VEGF-A (P-value = 5.8e-09) were SNP-protein pairs identified in pancreatic cancer patients. In colorectal cancer patients, only rs7767396 (A > G) and VEGF-A was validated (P-value = 5.18e-05). The AA genotype of rs7767396 exhibited 2.04-2.3 and 2.7-3.4-fold higher VEGF-A levels than those with AG and GG genotypes. The G allele of rs7767396 reduces binding of the NF-AT1 transcription factor. In conclusion, a common genetic variant predicts the plasma levels of VEGF-A in cancer patients through altered binding of NF-AT1.

The vitamin D receptor gene as a determinant of survival in pancreatic cancer patients: Genomic analysis and experimental validation.

PURPOSE: Advanced pancreatic cancer is a highly refractory disease almost always associated with survival of little more than a year. New interventions based on novel targets are needed. We aim to identify new genetic determinants of overall survival (OS) in patients after treatment with gemcitabine using genome-wide screens of germline DNA. We aim also to support these findings with in vitro functional analysis. PATIENTS AND METHODS: Genome-wide screens of germline DNA in two independent cohorts of pancreatic cancer patients (from the Cancer and Leukemia Group B (CALGB) 80303 and the Mayo Clinic) were used to select new genes associated with OS. The vitamin D receptor gene (VDR) was selected, and the interactions of genetic variation in VDR with circulating vitamin D levels and gemcitabine treatment were evaluated. Functional effects of common VDR variants were also evaluated in experimental assays in human cell lines. RESULTS: The rs2853564 variant in VDR was associated with OS in patients from both the Mayo Clinic (HR 0.81, 95% CI 0.70-0.94, p = 0.0059) and CALGB 80303 (HR 0.74, 0.63-0.87, p = 0.0002). rs2853564 interacted with high pre-treatment levels of 25-hydroxyvitamin D (25(OH)D, a measure of endogenous vitamin D) (p = 0.0079 for interaction) and with gemcitabine treatment (p = 0.024 for interaction) to confer increased OS. rs2853564 increased transcriptional activity in luciferase assays and reduced the binding of the IRF4 transcription factor. CONCLUSION: Our findings propose VDR as a novel determinant of survival in advanced pancreatic cancer patients. Common functional variation in this gene might interact with endogenous vitamin D and gemcitabine treatment to determine improved patient survival. These results support evidence for a modulatory role of the vitamin D pathway for the survival of advanced pancreatic cancer patients.

Leveraging population information in family-based rare variant association analyses of quantitative traits.

Confounding due to population substructure is always a concern in genetic association studies. Although methods have been proposed to adjust for population stratification in the context of common variation, it is unclear how well these approaches will work when interrogating rare variation. Family-based association tests can be constructed that are robust to population stratification. For example, when considering a quantitative trait, a linear model can be used that decomposes genetic effects into between- and within-family components and a test of the within-family component is robust to population stratification. However, this within-family test ignores between-family information potentially leading to a loss of power. Here, we propose a family-based two-stage rare-variant test for quantitative traits. We first construct a weight for each variant within a gene, or other genetic unit, based on score tests of between-family effect parameters. These weights are then used to combine variants using score tests of within-family effect parameters. Because the between-family and within-family tests are orthogonal under the null hypothesis, this two-stage approach can increase power while still maintaining validity. Using simulation, we show that this two-stage test can significantly improve power while correctly maintaining type I error. We further show that the two-stage approach maintains the robustness to population stratification of the within-family test and we illustrate this using simulations reflecting samples composed of continental and closely related subpopulations.

Blood-based markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: results from CALGB 80203 (Alliance).

Circulating protein markers were assessed in patients with colorectal cancer (CRC) treated with cetuximab in CALGB 80203 to identify prognostic and predictive biomarkers. Patients with locally advanced or metastatic CRC received FOLFOX or FOLFIRI chemotherapy (chemo) or chemo in combination with cetuximab. Baseline plasma samples from 152 patients were analyzed for six candidate markers [epidermal growth factor (EGF), heparin-binding EGF (HBEGF), epidermal growth factor receptor (EGFR), HER2, HER3, and CD73]. Analyte levels were associated with survival endpoints using univariate Cox proportional hazards models. Predictive markers were identified using a treatment-by-marker interaction term in the Cox model. Plasma levels of EGF, HBEGF, HER3, and CD73 were prognostic for overall survival (OS) across all patients (KRAS mutant and wild-type). High levels of EGF predicted for lack of OS benefit from cetuximab in KRAS wild-type (WT) patients (chemo HR = 0.98, 95% CI = 0.74-1.29; chemo+cetuximab HR = 1.54, 95% CI = 1.05-2.25; interaction P = 0.045) and benefit from cetuximab in KRAS mutant patients (chemo HR = 1.72, 95% CI = 1.02-2.92; chemo+cetuximab HR = 0.90, 95% CI = 0.67-1.21; interaction P = 0.026). Across all patients, higher HER3 levels were associated with significant OS benefit from cetuximab treatment (chemo HR = 4.82, 95% CI = 1.68-13.84; chemo+cetuximab HR = 0.95, 95% CI = 0.31-2.95; interaction P = 0.046). CD73 was also identified as predictive of OS benefit in KRAS WT patients (chemo HR = 1.28, 95% CI = 0.88-1.84; chemo+cetuximab HR = 0.60, 95% CI = 0.32-1.13; interaction P = 0.049). Although these results are preliminary, and confirmatory studies are necessary before clinical application, the data suggest that HER3 and CD73 may play important roles in the biological response to cetuximab.