Detection of IGF2BP3, HOXB7, and NEK2 mRNA expression in brush cytology specimens as a new diagnostic tool in patients with biliary strictures.

INTRODUCTION: It is a challenging task to distinguish between benign and malignant lesions in patients with biliary strictures. Here we analyze whether determination of target gene mRNA levels in intraductal brush cytology specimens may be used to improve the diagnosis of bile duct carcinoma. MATERIALS AND METHODS: Brush cytology specimens from 119 patients with biliary strictures (malignant: n = 72; benign: n = 47) were analyzed in a retrospective cohort study. mRNA of IGF-II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), Forkhead box M1 (FOXM1), kinesin family member 2C (KIF2C) and serine/threonine kinase NEK2 was determined by semi-quantitative RT-PCR using the ΔCt method. RESULTS: IGF2BP3 (p<0.0001), HOXB7 (p<0.0001), and NEK2 (p<0.0001) mRNA expression levels were significantly increased in patients with cholangiocarcinoma or pancreatic cancer. Median ΔCt values differed by 3.5 cycles (IGF2BP3), 2.8 cycles (HOXB7) and 1.3 cycles (NEK2) corresponding to 11-fold, 7-fold and 2.5-fold increased mRNA levels in malignant versus benign samples. Sensitivity to detect biliary cancer was 76.4% for IGF2BP3 (80.9% specificity); 72.2% for HOXB7 (78.7% specificity) and 65.3% for NEK2 (72.3% specificity), whereas routine cytology reached only 43.1% sensitivity (85.4% specificity). Diagnostic precision was further improved, when all three molecular markers were assessed in combination (77.8% sensitivity, 87.2% specificity) and achieved 87.5% sensitivity and 87.2% specificity when molecular markers were combined with routine cytology. CONCLUSIONS: Our data suggest that measuring IGF2BP3, HOXB7 and NEK2 mRNA levels by RT-PCR in addition to cytology has the potential to improve detection of malignant biliary disorders from brush cytology specimens.

The toll-like receptor 2 (TLR2) -196 to -174 del/ins polymorphism affects viral loads and susceptibility to hepatocellular carcinoma in chronic hepatitis C.

Chronic hepatitis C virus (HCV) infection is a major risk factor for hepatocellular carcinoma (HCC). HCV proteins core and NS3 can bind to toll-like receptor 2 (TLR2) and trigger inflammatory responses. Polymorphisms in the TLR2 gene predispose to various forms of malignancy but have not been studied in HCV-associated HCC. Here, we investigated whether single nucleotide polymorphisms (SNPs), rs4696480, rs5743708, rs5743704 and the -196 to -174 del/ins polymorphism of the TLR2 gene affect the risk for HCC in chronic hepatitis C. The study involved 189 and 192 HCV genotype 1 infected patients with and without HCC, respectively, as well as 347 healthy controls. TLR2 alleles were determined by hybridization probe assays and allele-specific short fragment polymerase chain reaction on a LightCycler system. All TLR2 polymorphisms matched the Hardy-Weinberg equilibrium in each study group. Although TLR2 SNPs showed no effect, the frequency of the TLR2 -196 to -174 del allele was significantly higher in patients with HCV-associated HCC (22.5%) than in HCV-infected patients without HCC (15.6%, p = 0.016) and healthy controls (15.3%, p = 0.003). HCV-infected carriers of a TLR2 -196 to -174 del allele had significantly higher HCV viral loads than TLR2 -196 to -174 ins/ins homozygous patients (p = 0.031). Finally, in carriers of the TLR2 -196 to -174 del allele, stimulation of monocytes resulted in significantly lower TLR2 expression levels and interleukin-8 (IL-8) induction than in individuals with the TLR2 -196 to -174 ins/ins genotype (p < 0.05). Our data suggest the TLR2 -196 to -174 del allele to affect HCV viral loads and to increase the risk for HCC in HCV genotype1-infected patients.