Unknown,Transcriptomics,Genomics,Proteomics

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Study of gene expression markers for predictive significance for bevacizumab benefit in patients with metastatic colon cancer: A translational research study of the Hellenic Cooperative Oncology Group (HeCOG)

ABSTRACT: Background. Bevacizumab, an antibody neutralizing Vascular Endothelial Growth Factor (VEGF), is licensed for the management of patients with advanced colon cancer. However, tumor biomarkers identifying the molecular tumor subsets most amenable to angiogenesis modulation are lacking. Patients and Methods. We profiled expession of 24526 genes by means of whole genome 24K DASL (c-DNA-mediated, Annealing, Selection and Ligation) arrays, (Illumina, CA) in 16 bevacizumab-treated patients with advanced colon cancer (Test set). Genes with correlation to 8-month Progression-free status were studied by means of qPCR in two independent colon cancer cohorts: 49 patients treated with bevacizumab+chemotherapy (Bevacizumab qPCR set) and 72 patients treated with chemotherapy only (Control qPCR set). Endpoints were best tumor response before metastasectomy (ORR) and progression-free survival (PFS). Results. Five genes were significantly correlated to 8-month progression-free status in the Test set: overexpression of KLF12 and downregulation of AGR2, ALDH6A1, MCM5, TFF2. In the two independent datasets, irinotecan- or oxaliplatin-based chemotherapy was administered as first-line treatment and metastasectomies were subsequently applied in 8-14% of patients. No prognostically significant gene classifier encompassing all five genes could be validated in the Bevacizumab or Control qPCR sets. The complex gene expression profile of all-low tumor (ALDH6A1+TFF2+MCM5) was strongly associated with ORR in the Bevacizumab qPCR set (ORR 85.7%, p=0.007), but not in the Control set (ORR 36.4%, p=0.747). The Odds Ratio for response for the all-low tumor (ALDH6A1+TFF2+MCM5) profile versus any other ALDH6A1+TFF2+MCM5 profile was 15 (p=0.018) in the Bevacizumab qPCR set but only 0.72 (p=0.63) in the Control set. The tumor expression profile of (KLF12-high+TFF2-low) was significantly associated with PFS only in the Bevacizumab qPCR set: bevacizumab-treated patients with (KLF12-high + TFF2-low) tumors had superior PFS (median 14 months, 95% CI 2-21) compared to patients with any other (KLF12+TFF2) expression profile (median PFS 7 months, 95% CI 5-10, p=0.021). The Hazard Ratio for disease progression for (KLF12-high + TFF2-low) versus any other KLF12+TFF2 expression profile was 2.92 (p=0.03) in the Validation and 1.29 (p=0.39) in the Control set. Conclusions. Our hypothesis-generating study failed to validate the prognostic significance of a five-gene classifier in mCRC patients. Exploratory analyses suggest two gene signatures that are potentially associated with bevazicumab benefit in patients with advanced colon cancer. 18 tumor tissue samples were analysed, no replicates

ORGANISM(S): Homo sapiens

SUBMITTER: elena fountzilas

PROVIDER: E-GEOD-53127 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background. Bevacizumab, an antibody neutralizing Vascular Endothelial Growth Factor (VEGF), is licensed for the management of patients with advanced colon cancer. However, tumor biomarkers identifying the molecular tumor subsets most amenable to angiogenesis modulation are lacking. Patients and Methods. We profiled expession of 24526 genes by means of whole genome 24K DASL (c-DNA-mediated, Annealing, Selection and Ligation) arrays, (Illumina, CA) in 16 bevacizumab-treated patients with advanced colon cancer (Test set). Genes with correlation to 8-month Progression-free status were studied by means of qPCR in two independent colon cancer cohorts: 49 patients treated with bevacizumab+chemotherapy (Bevacizumab qPCR set) and 72 patients treated with chemotherapy only (Control qPCR set). Endpoints were best tumor response before metastasectomy (ORR) and progression-free survival (PFS). Results. Five genes were significantly correlated to 8-month progression-free status in the Test set: overexpression of KLF12 and downregulation of AGR2, ALDH6A1, MCM5, TFF2. In the two independent datasets, irinotecan- or oxaliplatin-based chemotherapy was administered as first-line treatment and metastasectomies were subsequently applied in 8-14% of patients. No prognostically significant gene classifier encompassing all five genes could be validated in the Bevacizumab or Control qPCR sets. The complex gene expression profile of all-low tumor (ALDH6A1+TFF2+MCM5) was strongly associated with ORR in the Bevacizumab qPCR set (ORR 85.7%, p=0.007), but not in the Control set (ORR 36.4%, p=0.747). The Odds Ratio for response for the all-low tumor (ALDH6A1+TFF2+MCM5) profile versus any other ALDH6A1+TFF2+MCM5 profile was 15 (p=0.018) in the Bevacizumab qPCR set but only 0.72 (p=0.63) in the Control set. The tumor expression profile of (KLF12-high+TFF2-low) was significantly associated with PFS only in the Bevacizumab qPCR set: bevacizumab-treated patients with (KLF12-high + TFF2-low) tumors had superior PFS (median 14 months, 95% CI 2-21) compared to patients with any other (KLF12+TFF2) expression profile (median PFS 7 months, 95% CI 5-10, p=0.021). The Hazard Ratio for disease progression for (KLF12-high + TFF2-low) versus any other KLF12+TFF2 expression profile was 2.92 (p=0.03) in the Validation and 1.29 (p=0.39) in the Control set. Conclusions. Our <three-stage> hypothesis-generating study failed to validate the prognostic significance of a five-gene classifier in mCRC patients. Exploratory analyses suggest two gene signatures that are potentially associated with bevazicumab benefit in patients with advanced colon cancer.

Project description:The mechanisms by which tumors develop resistance to angiogenesis inhibitors, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. We developed three human lung adenocarcinoma murine models of resistance to the VEGF inhibitor bevacizumab and, using species-specific profiling, separately investigated tumor cell and stromal molecules associated with resistance. Gene expression changes associated with acquired resistance occurred predominantly in stromal (mouse) and not tumor (human) cells. Components of the EGFR and FGFR2 pathways were significantly upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Dual targeting of VEGF and EGFR pathways with bevacizumab and erlotinib, or the VEGFR/EGFR inhibitor vandetanib, reduced pericyte coverage and increased progression-free survival. These findings demonstrate that alterations in tumor stromal pathways, including EGFR and FGFR2, are associated with, and may contribute to VEGF inhibitor resistance and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens. To identify changes in stromal and tumor gene expression associated with acquired resistance to anti-VEGF therapy, we performed RNA microarray analyses comparing H1975 control (vehicle-treated) and BV-resistant (bevacizumab-treated until progression) xenografts (n=3 biologic replicates per group of treatment) using Illumina mouse (WG-6 v2) and human (WG-6 v3)-specific expression arrays. Probes in these arrays have been designed to minimize cross-species reactivity and consistent with this essentially no cross reactivity was observed in experiments mixing human and mouse cell lines.Total RNA was extracted from snap-frozen tissues using the mirVanaTM miRNA Isolation Kit (Ambion, Austin, TX) according to manufacturer’s protocol. Biotin labeled cRNA samples for hybridization were prepared by using Illumina Total Prep RNA Amplification Kit (Ambion Inc., Austin, TX). One microgram of total RNA was used for the synthesis of cDNA and followed by an amplification and biotin labeling. Each of 1.5 μg of biotinylated cRNAs was hybridized to both mouseWG-6 v2 and human WG-6v3 Expression BeadChips (Illumina®, San Diego, CA) at the same time, for analysis of murine and human transcriptomes. Signals were developed by Amersham fluorolink streptavidin-Cy3 (GE Health care Bio-Sciences, Little Chalfont, UK). Gene expression data were collected by using Illumina bead Array Reader confocal scanner (BeadStation 500GXDW; Illumina Inc.). Data were analyzed using the BRB-ArrayTools Version 3.7.0 Beta platform developed by Dr. Richard Simon and the BRB-Array Tools development team. A log base 2 transformation was applied to the data set prior to data normalization. A median array was selected as the reference array for normalization and statistical significance was set using a p<0.01. Genes differentially expressed between groups were determined applying univariate t-test with estimation of the false discovery rate (FDR). Genes were determined using selection criteria of a p<0.005 and a fold-change ≥1.5.

Project description:The function of human circulating proangiogenic cells (PACs) has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines for 3 days and then exposed to proinflammatory cytokines or oxLDL for 2 days. Under these conditions, PACs converted into APCs (APCcy and APCox, respectively), expressed maturation markers CD80 and CD83, and showed an up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters", showing reciprocal modulation in APCcy vs. APCox justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotype and function into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions. PBMC-derived PACs were committed with GM-CSF and IL-4 to a dendritic phenotype, and then exposed to proinflammatory cytokines (IL-1β and TNF-α) or to the oxidized form of LDL (oxLDL). Functional assays and whole genome gene expression profiling were performed both on PACs (n=6) and or resulting cytokine- (APCcy, n=6) and oxLDL-induced APCs (APCox, n=6). RNA was reverse transcribed, labeled, and linearly amplified, and then hybridized to HumanHT-12 v.4 Expression BeadChip microarrays. To estimate technical variability, ≈20% of the samples (n=4) were hybridized in duplicate.

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Study of gene expression markers for predictive significance for bevacizumab benefit in patients with metastatic colon cancer: A translational research study of the Hellenic Cooperative Oncology Group (HeCOG)

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