Anish Parekh

Association of variants in genes encoding for macrophage-related functions with clinical outcome in patients with locoregional gastric cancer

BACKGROUND Nuclear factor-kappaB (NF-κB) and CCL2/CCR2 chemokine axis play a central role in tumor progression such as stimulation of angiogenesis, acceleration of tumor invasion and migration, and suppression of innate immunosurveillance in the macrophage-related functions. There have been few reports regarding association of the macrophage function-related genes with the clinical outcome in gastric cancer. We hypothesized that variants in genes encoding for NF-κB and CCL2/CCR2 axis may predict prognosis in gastric cancer and tested whether the functional single-nucleotide polymorphisms (SNPs) will be associated with clinical outcome in patients with gastric cancer across two independent groups. PATIENTS AND METHODS This study enrolled two cohorts which consisted of 160 Japanese patients and 104 US patients with locoregional gastric cancer. Genomic DNA was analyzed for association of 11 SNPs in NFKB1, RELA, CCL2, and CCR2 with clinical outcome using PCR-based direct DNA sequencing. RESULTS The univariable analysis showed four SNPs had significant association with clinical outcome in the Japanese cohort, NFKB1 rs230510 remained significant upon multivariable analysis. The patients with the A allele of the NFKB1 rs230510 had significantly longer overall survival (OS) compared with those with the T/T genotype in both the Japanese and US cohort in the univariable analysis. In contrast, genotypes with the T allele of CCL2 rs4586 were significantly associated with shorter OS compared with the C/C genotype in the US cohort [hazard ratio (HR) 2.43; P = 0.015] but longer OS in the Japanese cohort (HR 0.58; P = 0.021), resulting in the statistically significant opposite impact on OS (P = 0.001). CONCLUSIONS Our study provides the first evidence that the NFKB1 rs230510 and CCL2 rs4586 are significantly associated with the clinical outcome in patients with locoregional gastric cancer. These results also suggest that the genetic predisposition of the host may dictate the immune-related component of the tumor for progression in gastric cancer.

Cytokeratin-20 and Survivin-Expressing Circulating Tumor Cells Predict Survival in Metastatic Colorectal Cancer Patients by a Combined Immunomagnetic qRT-PCR Approach

Circulating tumor cells (CTC) express epithelial and stem cell–like genes, though current approved detection methods mainly use epithelial markers. We optimized a CTC isolation method that could capture their molecular heterogeneity and predict overall survival (OS) in metastatic colorectal cancer (mCRC) patients receiving various chemotherapy regimens. We combined immunomagnetic enrichment of CD45-negative, EpCAM-positive circulating cancer cells with qRT-PCR amplification of CK20 and survivin expression in 88 mCRC patients and 20 healthy controls. We then evaluated the prognostic value of baseline CTC CK20 and survivin expression in mCRC patients. The presence of elevated CTC CK20 or survivin expression distinguished mCRC patients from controls with sufficient sensitivity (79.6%) and specificity (85%). In univariate analysis, patients with high CTC-CK20 expression (9 vs. 33.2+ months, log-rank P < 0.001) or high CTC-survivin expression (10 vs. 33.2+ months, log-rank P = 0.032) had a significantly worse median OS than those with low expression of either marker. In multivariable analysis, the high CTC-CK20 group had significantly shortened OS (HR, 3.11; adjusted P = 0.01), and there was a trend toward inferior OS in the high CTC-survivin group (HR, 1.76; adjusted P = 0.099). Patients with either high CTC CK20 or survivin expression had inferior OS compared with those with low expression of both markers (HR, 4.39; 95% confidence interval, 1.56–12.35; adjusted P = 0.005). Colorectal cancer CTCs can be reliably isolated using epithelial and stem cell markers. CTC CK20 and survivin expression may effectively predict OS in mCRC patients receiving chemotherapy. Mol Cancer Ther; 14(10); 2401–8. ©2015 AACR.

Variations in genes regulating tumor-associated macrophages (TAMs) to predict outcomes of bevacizumab-based treatment in patients with metastatic colorectal cancer: results from TRIBE and FIRE3 trials

BACKGROUND Tumor-associated macrophages (TAMs) with the M2-like phenotype are regulated by mainly NF-kB pathway including TBK1, which can influence tumor progression by secretion of proangiogenic factors such as vascular endothelial growth factor. The CCL2/CCR2 axis, histidine-rich glycoprotein (HRG), and placenta growth factor (PIGF) play a critical role in the polarization of M1/M2 phenotypes and the recruitment of TAMs to tumor microenvironment. We therefore hypothesized that variations in genes involved in regulating TAMs may predict clinical outcomes of bevacizumab treatment in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS We analyzed genomic DNA extracted from samples of patients receiving bevacizumab plus FOLFIRI as a first-line treatment using PCR-based direct sequencing. Twelve functional single-nucleotide polymorphisms in eight genes (CCL2, CCR2, HRG, PIGF, NFKB1, TBK1, CCL18, and IRF3) were tested for associations with clinical outcomes in a discovery cohort of 228 participants in TRIBE trial (NCT00719797), then validated in 248 KRAS exon2 (KRAS) wild-type participants in FIRE3 trial (NCT00433927). FIRE3-cetuximab cohort served as a negative control. RESULTS TBK1 rs7486100 was significantly associated with overall survival in 95 KRAS wild-type patients of TRIBE cohort in univariate analysis and had a strong trend in multivariable analysis; furthermore, the association of the T allele was observed for progression-free survival (PFS) in both univariate and multivariable analyses in FIRE3-bevacizumab but not cetuximab cohort. CCL2 rs4586, CCL18 rs14304, and IRF3 rs2304205 had univariate and multivariable correlations with PFS in KRAS mutant patients of the TRIBE cohort, whereas they had no correlations in KRAS wild-type patients of the TRIBE cohort. No association was seen in control cohort. CONCLUSIONS Our study demonstrates for the first time that variations in genes regulating TAMs-related functions are significantly associated with clinical outcomes in mCRC patients treated with bevacizumab-containing chemotherapy. These results also suggest that some TAM-related gene variations may predict outcomes of bevacizumab treatment in KRAS status-dependent manner.

Clinical significance of tlr1 i602s polymorphism for patients with metastatic colorectal cancer treated with folfiri plus bevacizumab

The purpose of this study was to evaluate the clinical significance of single-nucleotide polymorphisms in TLR1, TLR2, TLR6, and TAK1 in patients with metastatic colorectal cancer (mCRC). We genotyped 9 SNPs of TLR1, TLR2, TLR6, and TAK1 in mCRC patients treated with first-line FOLFIRI (combination therapy of irinotecan, 5-fluorouracil, and folinic acid) plus bevacizumab, using a discovery cohort (TRIBE trial, n = 228) and a validation cohort (FIRE-3 trial, n = 297), and analyzed for the association with response rate (RR), progression-free survival (PFS), and overall survival (OS). There was a significant association of TLR1 rs5743618 (T1805G) with the clinical outcome. In the TRIBE cohort, a homozygous wild-type genotype (T/T) associated with a significantly lower RR compared with variant T/G and G/G genotypes (43% vs. 62%, P = 0.025), and this observation was validated in the FIRE-3 cohort (46% vs. 65%, P = 0.021). In addition, those patients with the T/T genotype had significantly worse PFS (median, 8.2 vs. 10.5 months; HR, 1.57; 95% CI, 1.09–2.28, P = 0.014) and OS (median: 19.9 vs. 27.9 months; HR, 1.63; 95% CI, 1.14–2.35, P = 0.007), compared with those with other genotypes in the TRIBE cohort. These differences remained statistically significant in multivariate analysis. Our data suggest that TLR1 rs5743618 could serve as a predictor of clinical response to FOLFIRI plus bevacizumab in patients with mCRC. Mol Cancer Ther; 15(7); 1740–5. ©2016 AACR.

Variations in genes involved in dormancy associated with outcome in patients with resected colorectal liver metastases

BACKGROUND Tumor dormancy has been described as a state of hibernation. Dormancy can be switched to proliferation by different pathways, which may play a critical role in tumor recurrence. In this study, we investigated genetic variations within genes involved in tumor dormancy and their association with recurrence and outcome in patients with colorectal liver metastases (CLM) who underwent neoadjuvant bevacizumab-based chemotherapy. PATIENTS AND METHODS Genomic DNA was extracted from resected CLM (FFPE) from 149 patients. Single-nucleotide polymorphisms (SNPs) in 14 genes associated with dormancy were analyzed by direct Sanger DNA sequencing and evaluated for response, recurrence-free survival (RFS), overall survival (OS) and recurrence patterns. RESULTS NME1 rs34214448 C>A was significantly associated with RFS in univariable analysis (P = 0.039) and with intrahepatic recurrence (P = 0.014). NOTCH3 rs1044009 T>C and CD44 rs8193 C>T showed a significant difference in 3-year OS rates (P = 0.004 and P = 0.042, respectively). With respect to radiological response, CD44 rs8193 C>T variant genotypes were associated with a significantly higher response rate (P = 0.033). Recursive partitioning analyses revealed that Dll4 rs12441495 C>G, NME1 rs34214448 C>A and NOTCH3 rs1044009 T>C were the dominant SNPs predicting histological response, RFS and OS, respectively. CONCLUSION Our data suggest that gene variations within genes involved in tumor dormancy pathways are associated with response and outcome in patients with resected CLM. These data may lead to new and more effective treatment strategies targeting tumor dormancy.

TWIST1 Polymorphisms Predict Survival in Patients with Metastatic Colorectal Cancer Receiving First-Line Bevacizumab plus Oxaliplatin-Based Chemotherapy

The epithelial–mesenchymal transition (EMT) is an important mechanism of resistance to angiogenesis inhibition. The ability of EMT pathway genetic variants to predict the efficacy of antiangiogenic therapy is unknown. We analyzed associations between functional SNPs in EMT-related genes and outcomes in metastatic colorectal cancer (mCRC) patients undergoing first-line bevacizumab-based chemotherapy. A total of 220 mCRC patients were included in this study: 143 patients treated with first-line bevacizumab-based chemotherapy (bevacizumab cohort) and 77 patients treated with cetuximab-based chemotherapy (cetuximab cohort). SNPs in TWIST1 (rs2285682, rs2285681), ZEB1 (rs10826943, rs2839658), SNAIL (rs1543442, rs4647958), and E-cadherin (rs16260) genes were analyzed by PCR-based direct sequencing. Patients carrying a TWIST1 rs2285682 G allele had a significantly longer median progression-free survival (PFS) of 18.1 months and overall survival (OS) of 44.1 months compared with those with the T/T genotype, who had a median PFS of 13.3 months (HR, 0.57; P = 0.003) and OS of 29.2 months (HR, 0.53; P = 0.001) in the bevacizumab cohort. In multivariate analysis, associations between TWIST1 rs2285682 and PFS and OS remained significant. Among women, the G allele of TWIST1 rs2285682 (PFS HR, 0.39; P = 0.007; OS HR, 0.30; P = 0.001) and TWIST1 rs2285681 (PFS HR, 0.27; P < 0.001; OS HR, 0.25; P < 0.001) was associated with improved survival. No significant associations were found in the cetuximab cohort. Our findings suggest that TWIST1 polymorphisms are associated with survival in mCRC patients treated with first-line bevacizumab-based chemotherapy and may serve as clinically useful biomarkers for antiangiogenic therapy. Mol Cancer Ther; 15(6); 1405–11. ©2016 AACR.

Prognostic Impact of IL6 Genetic Variants in Patients with Metastatic Colorectal Cancer Treated with Bevacizumab-Based Chemotherapy

Purpose: The IL6/STAT3 axis promotes inflammation, angiogenesis, and cancer. The effect of genetic variants within this pathway on benefit from antiangiogenic cancer therapy is unknown. We tested whether SNPs in genes involved in IL6/STAT3 signaling can predict efficacy of bevacizumab-based chemotherapy in metastatic colorectal cancer (mCRC) patients. Experimental Design: Associations between potentially functional IL6 (rs2069837 and rs1800795) and STAT3 (rs744166 and rs4796793) SNPs and clinical outcomes [progression-free survival (PFS), overall survival, and tumor response rate] were evaluated in mCRC patients receiving first-line FOLFIRI plus bevacizumab in two randomized phase III trials: TRIBE (n = 223, training cohort) and FIRE-3 (n = 288, validation cohort). Patients receiving FOLFIRI plus cetuximab in FIRE-3 (n = 264) served as a control cohort. The interaction between genotype and primary tumor location with clinical outcomes was examined. Genomic DNA isolated from whole blood or tumor tissue was analyzed by PCR-based direct sequencing. Results: Patients with an IL6 rs2069837 G allele treated with FOLFIRI plus bevacizumab had an inferior PFS than those with the A/A genotype in TRIBE [9.4 vs. 11.1 months; HR = 1.53; 95% confidence interval (CI), 1.12–2.10; P = 0.004] and FIRE-3 (8.8 vs. 10.9 months; HR = 1.40; 95% CI, 1.06–1.85; P = 0.015). These associations were confirmed in multivariable analyses and were not seen in the control cohort. In subgroup analysis, the effect of IL6 rs2069837 on PFS was present only in patients with left-sided cancers, but the test for interaction was not significant. Conclusions: IL6 rs2069837 genotype is a clinically relevant prognostic factor in mCRC patients treated with first-line bevacizumab-based chemotherapy. Clin Cancer Res; 22(13); 3218–26. ©2016 AACR.

Expression of genes involved in vascular morphogenesis and maturation predicts efficacy of bevacizumab-based chemotherapy in patients undergoing liver resection

Angiogenesis-related gene expression is associated with the efficacy of anti-VEGF therapy. We tested whether intratumoral mRNA expression levels of genes involved in vascular morphogenesis and early vessel maturation predict response, recurrence-free survival (RFS), and overall survival (OS) in a unique cohort of patients with colorectal liver metastases (CLM) treated with bevacizumab-based chemotherapy followed by curative liver resection. Intratumoral mRNA was isolated from resected bevacizumab-pretreated CLM from 125 patients. In 42 patients, a matching primary tumor sample collected before bevacizumab treatment was available. Relative mRNA levels of 9 genes (ACVRL1, EGFL7, EPHB4, HIF1A, VEGFA, VEGFB, VEGFC, FLT1, and KDR) were analyzed by RT-PCR and evaluated for associations with response, RFS, and OS. P values for the associations between the individual dichotomized expression level and RFS were adjusted for choosing the optimal cut-off value. In CLM, high expression of VEGFB, VEGFC, HIF1A, and KDR and low expression of EGFL7 were associated with favorable RFS in multivariable analysis (P < 0.05). High ACVRL1 levels predicted favorable 3-year OS (P = 0.041) and radiologic response (PR = 1.093, SD = 0.539, P = 0.002). In primary tumors, low VEGFA and high EGFL7 were associated with radiologic and histologic response (P < 0.05). High VEGFA expression predicted shorter RFS (10.1 vs. 22.6 months; HR = 2.83, P = 0.038). High VEGFB (46% vs. 85%; HR = 5.75, P = 0.009) and low FLT1 (55% vs. 100%; P = 0.031) predicted lower 3-year OS rates. Our data suggest that intratumoral mRNA expression of genes involved in vascular morphogenesis and early vessel maturation may be promising predictive and/or prognostic biomarkers. Mol Cancer Ther; 15(11); 2814–21. ©2016 AACR.

Association between single nucleotide polymorphisms (SNPs) of genes involved in spindle assembly checkpoint (SAC) and clinical outcomes in advanced gastric cancer (AGC) patients (pts) treated with taxane-based chemotherapy.

79 Background: Taxanes which disrupt the microtubule function and inhibit the process of cell division have shown encouraging activity in the treatment of AGC. Resistance to these agents often becomes a problem in clinical settings and its mechanism hasn’t been dissolved conclusively. SAC is a safety device ensures the proper chromosome segregation in mitosis and is required for taxane-induced cell death. We hypothesized genetic variants in SAC genes may be associated with clinical outcomes in pts with AGC treated with taxane. Methods: Genomic DNA was isolated from blood or tissue samples of 39 U.S. pts (median age 57; median follow-up 6.4 months) for evaluation set and 39 Japanese (JPN) pts (median age 63; median follow-up 9.4 months) for validation set, with AGC treated with taxane. Twenty-five functionally significant SNPs in SAC genes (MAD1, MAD2, BUB3, BUBR1, RAN, TPX2, CDC20, AURKA, AURKB, RCC1, ANAPC10, ANAPC13) were analyzed by PCR-based direct sequencing and evaluated for association with outcome...

Polymorphism of the chemokine CXCR4 to predict treatment benefit of first-line bevacizumab-based chemotherapy in patients with metastatic colorectal cancer.

635 Background: The chemokine receptor CXCR4 and its ligand CXCL12 promote angiogenesis and the migration of tumor cells into the metastatic sites in many cancers. CXCR4 expression on tumor cells is upregulated by hypoxia and angiogenic factors, such as vascular endothelial growth factor. Therefore, we analyzed the association between CXCR4/CXCL12 polymorphisms and prognosis in metastatic colorectal cancer (mCRC) patients underwent bevacizumab-based chemotherapy. Methods: This study included 144 Japanese patients (pts) for evaluation set and 424 patients from two clinical trials (204 of TRIBE arm A and 220 of PROVETTA) for validation set, with mCRC treated with bevacizumab-based chemotherapy as first line. A total of 144 Japanese pts with (male/female; 81/63, median age 61 years, median follow-up 4.2 years) and 424 pts (male/female; 252/172, median age 62 years; median follow-up time; 2.8 years) were enrolled in a pharmacogenomics translational study. Genomic DNA was extracted from the pts’ blood or tissu...