Genetic signature of CTLA-4, BTLA, TIM-3 and LAG-3 molecular expression in colorectal cancer patients: Implications in diagnosis and survival outcomes.

OBJECTIVE: Accumulating evidences suggest that immune checkpoints (ICs) inhibit immune response against cancerous cells and promote tumor cell survival. Up-regulation of ICs in tumor microenvironment is reported in patients with colorectal cancer (CRC). Thus, evaluating the peripheral blood expression of ICs may be used as non-invasive biomarkers for diagnosis and prognosis of CRC. METHODS: This study included 60 primary and treatment naïve CRC patients along with 15 age and sex matched healthy volunteers as a control group. Total RNA was extracted from peripheral blood samples and gene expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and Lymphocyte activation gene-3 (LAG-3) was measured by quantitative real time polymerase chain reaction (qRT-PCR). All patients were followed for 12 months to correlate the measured ICs to patients' survival. RESULTS: The gene expression of CTLA-4, BTLA, TIM-3 and LAG-3 was significantly up-regulated in CRC patients compared to the control group (p < 0.001). Individually, CTLA-4 and BTLA showed 85% sensitivity in discriminating CRC patients from control group (p < 0.001). On the other hand, TIM-3 and LAG-3 expression showed higher sensitivity (93%) for diagnosis of CRC (p < 0.001). Conversely, CTLA-4 or BTLA strongly predicted CRC patients' survival (p < 0.001) compared to TIM-3 (p = 0.018) or LAG-3 (p = 0.035). CTLA-4, BTLA, TIM-3 and LAG-3 were independent prognostic factors of survival after adjustment for age and gender. CONCLUSION: The current study provided evidence that blood gene expression of ICs was up-regulated in CRC patients and associated with cancer stage and patients' survival, which highlights the diagnostic and prognostic values of ICs expression in CRC. Further investigations and validations in larger cohorts are required.

Aberrant p16INK4A methylation: Relation to viral related chronic liver disease and hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is currently the fifth most common solid tumor worldwide and the third leading cause of cancer related deaths. Several studies have shown that the tumor suppressor gene p16INK4A is frequently downregulated by aberrant methylation of the 5'-cytosine-phosphoguanine island within the promoter region. AIM: To find out the frequency of methylated p16INK4A in the peripheral blood of HCC and cirrhotic patients and to evaluate its role in hepatocarcinogenesis. PATIENTS AND METHODS: This study was performed on 58 subjects: 30 HCC patients, 20 cirrhotic patients, and eight healthy volunteers. Methylation of p16INK4A was examined using methylation specific polymerase chain reaction (PCR) (MSP). Comparison of quantitative variables between the study groups was done using Mann-Whitney U test for independent samples when not normally distributed. For comparing categorical data, Chi-square (χ(2)) test was performed. Exact test was used instead when the expected frequency was less than 5. RESULTS: Methylation of p16INK4A was found in 6.7% of HCC patients, 5% of liver cirrhosis (LC) patients, and none of the healthy volunteers; 66.67% of the p16INK4A-methylated cases (2/3) were positive for anti-hepatitis C virus (HCV) antibodies (one of them had HCC). All HCC cases with aberrant p16INK4A methylation show very high serum alpha fetoprotein (AFP) level (9,080; 30,000 µg/mL). There were no significant associations between the status of p16INK4A methylation and tumor size. CONCLUSION: Hypermethylation of p16INK4A was found to be infrequent among Egyptian patients with HCC.