Impact of gene polymorphism of TNF-α rs 1800629 and TNF-ß rs 909253 on plasma levels of South Indian breast cancer patients.

AIM: Inflammation plays a lead role in the tumor microenvironment and promotes metastasis. Single-nucleotide polymorphism (SNP) in the tumor necrosis factor (TNF) gene locus may alter the expression of genes and proteins. The objective of the study is to find the distribution of genetic polymorphism in the sites of TNF-α -308G>A and TNF- ß +252A>G in breast cancer and evaluate polymorphism effects on plasma levels. MATERIALS AND METHODS: The study group consisted of 109 invasive ductal primary breast cancer patients and 75 age-matched healthy female controls. Plasma cytokine concentrations were measured by the MILLIPLEX® MAP Human Cytokine/Chemokine Panel magnetic bead kits. The genotyping procedure for SNP included allele-specific polymerase chain reaction for TNFα and restriction fragment length polymorphism for TNFß. RESULTS: Odds ratio with 95% confidence interval showed that these polymorphisms were not a causative risk factor, and both polymorphisms were consistent with Hardy-Weinberg equilibrium. Plasma TNFα and TNFß median concentrations were significantly higher in cases when compared to controls (P < 0.01). When plasma TNFα levels were grouped under polymorphic subtypes, patients with mutant TNF- α -308A allele showed significantly higher values (P < 0.001). In addition, plasma TNFα values were significantly elevated in mutant TNF-ß +252G allele (P < 0.01). CONCLUSION: This study demonstrated that there is no significant association between SNPs and breast cancer susceptibility in South Indian population. However, plasma TNFα level is significantly elevated with mutant-recessive TNF-α -308 A and TNF-ß +252 G alleles of patients.

Identification of differentially expressed serum proteins in gastric adenocarcinoma.

Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu-Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. BIOLOGICAL SIGNIFICANCE: Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well classified patient samples would prove useful in identifying novel blood based biomarkers for gastric cancers. This article is part of a Special Issue entitled: Proteomics in India.

Calcium calmodulin dependent kinase kinase 2 - a novel therapeutic target for gastric adenocarcinoma.

Gastric cancer is one of the most common gastrointestinal malignancies and is associated with poor prognosis. Exploring alterations in the proteomic landscape of gastric cancer is likely to provide potential biomarkers for early detection and molecules for targeted therapeutic intervention. Using iTRAQ-based quantitative proteomic analysis, we identified 22 proteins that were overexpressed and 17 proteins that were downregulated in gastric tumor tissues as compared to the adjacent normal tissue. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was found to be 7-fold overexpressed in gastric tumor tissues. Immunohistochemical labeling of tumor tissue microarrays for validation of CAMKK2 overexpression revealed that it was indeed overexpressed in 94% (92 of 98) of gastric cancer cases. Silencing of CAMKK2 using siRNA significantly reduced cell proliferation, colony formation and invasion of gastric cancer cells. Our results demonstrate that CAMKK2 signals in gastric cancer through AMPK activation and suggest that CAMKK2 could be a novel therapeutic target in gastric cancer.

Prevalence of +405G>C,-1154G>A Vascular Endothelial Growth Factor Polymorphism in Breast Cancer.

Vascular endothelial growth factor (VEGF) plays an important role in the development of Breast Cancer. The aim of this study was to investigate the association of polymorphisms in the VEGF gene on prognosis of Breast Cancer patients. This study comprised 200 patients with histologically confirmed cases of Breast cancer and 200 controls. Genotyping of the VEGF gene polymorphisms at +405G>C,-1154G>A, were performed by PCR-RFLP analysis. Preoperative plasma VEGF levels were determined by ELISA. Amongst both cases and controls, the genotypic distribution of the individual SNPs were all in Hardy-Weinberg equilibrium. Mean VEGF level was significantly elevated in cases compared to controls (t = 8.248; P < 0.001). No significant association was found between +405G>C,-1154G>A VEGF polymorphism and Breast Cancer. Logistic regression analysis revealed that 405GG & 1154GG were associated with higher levels of VEGF.

SILAC-based quantitative proteomic analysis of gastric cancer secretome.

PURPOSE: Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood-based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer. EXPERIMENTAL DESIGN: A SILAC-based quantitative proteomic approach was employed to identify secreted proteins that were differentially expressed between neoplastic and non-neoplastic gastric epithelial cells. Proteins from the secretome were subjected to SDS-PAGE and SCX-based fractionation, followed by mass spectrometric analysis on an LTQ-Orbitrap Velos mass spectrometer. Immunohistochemical labeling was employed to validate a subset of candidates using tissue microarrays. RESULTS: We identified 2205 proteins in the gastric cancer secretome of which 263 proteins were overexpressed greater than fourfold in gastric cancer-derived cell lines as compared to non-neoplastic gastric epithelial cells. Three candidate proteins, proprotein convertase subtilisin/kexin type 9 (PCSK9), lectin mannose binding 2 (LMAN2), and PDGFA-associated protein 1 (PDAP1) were validated by immunohistochemical labeling. CONCLUSIONS AND CLINICAL RELEVANCE: We report here the largest cancer secretome described to date. The novel biomarkers identified in the current study are excellent candidates for further testing as early detection biomarkers for gastric adenocarcinoma.

Diagnostic Significance of CA15-3 with Combination of HER-2/neu Values at 85th Percentiles in Breast Cancer.

The human epidermal receptor-2/neu (HER-2/neu) oncogene encodes a transmembrane tyrosine kinase receptor. This molecule could have a diagnostic value since the extracellular domain of c-erbB-2 (HER-2) transmembrane is shed into the blood as a circulating antigen. The diagnostic value of serum HER-2/neu was calculated along with the conventional marker carbohydrate antigen 15-3 (CA15-3) and carcinoembryonic antigen (CEA) at 85th percentiles. Serum levels of breast carcinoma antigens HER-2/neu, CEA and CA15-3 were determined in 175 normal individuals and 268 malignant patients. The soluble form of serum HER-2/neu, CEA and CA15-3 was assayed by enzyme linked immunosorbent assay in control and breast cancer patients prior to treatment. Serum levels of the tested tumor markers HER-2/neu and CA15-3 and CEA were significantly higher in cancer patients compared to controls. At 85th percentile the sensitivity of HER-2/neu was 51.12 %; the specificity was 86.29 % and the overall accuracy was 64.56 %. The sensitivity of CA15-3 was 73.13 %; the specificity was 85.14 % and the overall accuracy was 77.88 %. The sensitivity of the combined testing was 82.84 %; the specificity was 73.71 % and the overall accuracy was 80.01 %. The sensitivity and the overall accuracy of combined testing were higher than those of HER-2/neu and CA15-3 testing single. The combined testing of HER-2/neu and CA15-3 can increase the sensitivity and overall accuracy of breast cancer diagnosis. The results of this study suggest that the use of multiple tumor markers may be employed as combination and at 85th percentiles to assess the prognosis.

Clinical outcome of adjuvant endocrine treatment according to Her-2/neu status in breast cancer.

BACKGROUND & OBJECTIVES: An association between over-expression of proto-oncogene Her-2/neu and resistance to tamoxifen in estrogen receptor (ER) positive, primary and metastatic breast cancer has been suggested. HR+/Her-2/neu+ patients have a poor response to endocrine therapy, making this group a matter of debate. The present study was carried out to examine whether Her-2/neu expression in breast cancer patients predicted tamoxifen effectiveness. METHODS: An enzyme-linked immunosorbent assay (ELISA) specific for the extracellular domain of the Her-2/neu oncoprotein product was used to detect serum Her-2/neu levels in 207 patients with histological confirmed breast cancer. Tissue Her-2 /neu expression was studied in 100 breast cancer patients by immunohistochemistry (IHC) and compared with serum Her-2/neu levels by ELISA. RESULTS: Among 207 histologically confirmed breast cancer patients, 53 were serum Her-2/neu positive. Patients who were treated with surgery, chemotherapy, and radiotherapy showed significantly (P<0.05) reduced serum Her-2/neu levels, showing good response to treatment. Patients who were treated with tamoxifen in addition to the above regimen did not show any significant reduction in serum Her-2/neu levels showing resistance to treatment. INTERPRETATION & CONCLUSIONS: The present findings study support the hypothesis that Her-2/neu overexpression contributes to tamoxifen resistance. Trastuzumab or other growth factor inhibitors should be used in combination with tamoxifen, since monotherapy is not likely to be optimal in HR+/Her-2/neu+ tumours.

Association of methylenetetrahydrofolate reductase gene polymorphisms & colorectal cancer in India.

BACKGROUND & OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and involved in DNA synthesis, DNA repair and DNA methylation. The two common functional polymorphisms of MTHFR, 677 C-->T and 1298 A-->C have shown to impact several diseases including cancer. This case-control study was undertaken to analyse the association of the MTHFR gene polymorphisms 677 C-->T and 1298 A-->C and risk of colorectal cancer (CRC). METHODS: One hundred patients with a confirmed histopathologic diagnosis of CRC and 86 age and gender matched controls with no history of cancer were taken for this study. DNA was isolated from peripheral blood samples and the genotypes were determined by PCR-RFLP. The risk association was estimated by compounding odds ratio (OR) with 95 per cent confidence interval (CI). RESULTS: Genotype frequency of MTHFR 677 CC, CT and TT were 76.7, 22.1 and 1.16 per cent in controls, and 74, 25 and 1.0 per cent among patients. The 'T' allele frequency was 12.21 and 13.5 per cent in controls and patients respectively. The genotype frequency of MTHFR 1298 AA, AC, and CC were 25.6, 58.1 and 16.3 per cent for controls and 22, 70 and 8 per cent for patents respectively. The 'C' allele frequency for 1298 A-->C was 43.0 and 45.3 per cent respectively for controls and patients. The OR for 677 CT was 1.18 (95% CI 0.59-2.32, P = 0.642), OR for 1298 AC was 1.68 (95% CI 0.92-3.08, P = 0.092) and OR for 1298 CC was 0.45 (95% CI 0.18-1.12, P = 0.081). The OR for the combined heterozygous state (677 CT and 1298 AC) was 1.18 (95% CI 0.52-2.64, P =0.697). INTERPRETATION & CONCLUSION: The frequency of the MTHFR 677 TT genotype is rare as compared to 1298 CC genotype in the population studied. There was no association between 677 C-->T and 1298 A-->C polymorphisms and risk of CRC either individually or in combination. The homozygous state for 1298 A-->C polymorphism appears to slightly lower risk of CRC. This needs to be confirmed with a larger sample size.

Association between plasma homocysteine and riboflavin status in Acute Lymphoblastic Leukemia in children.

Remethylation of homocysteine to methionine is dependent on an adequate supply of one or more of the B vitamins like folate, vitamin B(12) and vitamin B(6). Plasma total homocysteine (tHcy) is also influenced by genetic factors such as polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. MTHFR is a flavo enzyme and a key player in folate metabolism and changes in its activity could modify the susceptibility to Acute Lymphoblastic Leukemia (ALL). In this case - control study we have examined the effect of riboflavin status as measured by erythrocyte glutathione reductase activation coefficient (EGRAC) on homocysteine levels along with vitamin B(12) and folate in pediatric ALL. Folate and B(12) levels were significantly lower among cases as compared to controls while EGRAC and tHcy did not differ significantly among the groups. The multivariate regression analysis revealed that in the ALL group EGRAC significantly influences tHcy levels suggesting that riboflavin availability may be a predictor of tHcy levels in patients with ALL. This finding may have implications for tHcy lowering therapy.

Homocysteine, vitamin B12 and folate status in pediatric acute lymphoblastic leukemia.

OBJECTIVE: The cause of majority of acute leukemias is unknown, but likely to involve interaction of environment, hematopoitic development and weak susceptibility loci within an individual's genetic constitution. The present study evaluates the association between plasma levels of homocysteine, folate and vitamin B12 and acute lymphoblastic leukemia. METHODS: Plasma levels of homocysteine, folate and vitamin B12 were compared between cases of acute lymphoblastic leukemia and age and sex matched normal controls. Homocysteine levels were measured by solid immunoassay, while folate and vitamin B12 levels were determined by radioassay. RESULTS: Folate levels were significantly among cases as compared to control group (8.56 +/- 4.35) vs (14.04 +/- 2.62) ng/ml, P< 0.001). Although individually vitamin B12 and homocysteine were not significant different between cases and controls, the combined effect of all three parameters was significantly different (P< 0.001), with 83.3% of correct classification of cases and controls was obtained by discriminate function analysis. CONCLUSION: The data provide evidence for the role of folate, vitamin B12 and homocysteine levels in acute lymphoblastic leukemia, suggesting that gene-environment interaction may be an important factor in the development of acute lymphoblastic leukemia.

Effect of Vitamin B(12) and Folate on Homocysteine levels in colorectal cancer.

Folate and cobalamin (Vitamin B(12)) are two essential micronutrients involved in one-carbon metabolism, which affects heart disease, neural tube defects and cancer. Methylenetetrahydrofolate reductase, the key enzyme involved in one carbon metabolism produces methyl tetrahydrofolate from methylene tetrahydrofolate, which in turn donates methyl group to homocysteine to generate methionine. There exist two common low function polymorphic variants of the methylenetetrahydrofolate reductase gene involving nucleotides 677 C→T and 1298 A→C, which are associated with hyperhomocysteinemia. These polymorphisms are also linked with increased risk for certain cancers such as breast cancer and at the same time providing a protective effect on colorectal cancer. In this case control study, we have evaluated levels of folic acid, vitamin B(12) and homocysteine in patients with colorectal cancer. Folate and homocysteine levels did not differ significantly between the two groups; however an increasing trend was noticed with increase in homocysteine levels. Vitamin B(12) levels were increased in cases compared to control group.

Evaluation of immunohistochemistry and enzyme linked immunosorbent assay for HER-2/neu expression in breast carcinoma.

HER-2 is overexpressed in approximately 20-30% of invasive Breast Cancer. ECD of the HER-2 protein is frequently cleaved and released into the circulation, where it can be detected by ELISA in up to 45% of patients with metastatic breast cancer. The objective of our study was to compare the current methods for the detection of HER-2 protein. Tissue HER-2 levels were studied in 100 breast cancer patients by IHC and compared with serum HER-2 levels by ELISA. IHC frequency was 29%. Serum HER-2 ECD was positive in 42% of patients. A statistically significant correlation was observed. HER-2 detected by IHC correlates significantly with serum HER-2 levels detected by ELISA. Thus, ELISA is a reliable and economical tool to assess the HER-2 status in tumors, when breast tissue sample is not available.

Clinical utility of serum human epidermal receptor-2/neu detection in breast cancer patients.

BACKGROUND & OBJECTIVE: In breast cancer, the HER-2/neu gene is amplified in 20-30 per cent of cases. The mechanism by which the amplification/overexpression occurs is not known. Elevated serum HER-2/neu levels have been shown to be associated with a poor clinical prognosis and decreased survival in early stage breast cancer patients, and thus might help in management of the disease. The present study was therefore to estimate the serum HER-2/neu levels in breast cancer patients and associate with other prognostic factors. METHODS: Serum HER-2/neu levels were studied in 207 patients with cancer breast, 15 benign breast diseases (BBD) and 175 age-matched healthy controls. Patients' age, menopausal status, node and hormone receptor status were compared with serum HER-2/neu levels. RESULTS: Serum HER-2/neu overexpression was associated with age, disease stage and positive nodal status but not with menopausal status. Serum HER-2/neu levels were negatively related with hormone receptor positivity. INTERPRETATION & CONCLUSION: HER-2/neu serum test could be done more frequently in women with breast cancer irrespective of the hormone receptor status, to suggest modifications in systemic adjuvant therapy, including possibly the use of Herceptin.

Correlation study of Carcino Embryonic Antigen & Cancer Antigen 15.3 in pretreated female breast cancer patients.

Carcino Embryonic Antigen (CEA) and Cancer Antigen 15.3 (CA15.3) are the most common tumor markers in breast cancer patients. Measurement of circulating tumor markers is a non-invasive quantitative method. Serum levels of CEA and CA 15.3 were studied in female breast cancer patients prior to treatment. To evaluate the utility of these markers, 207 Breast carcinoma patients belonging to all the stages were considered. Healthy age matched 75 female individuals formed the control group. The serum levels of CEA and CA 15.3 were analyzed by Enzyme Linked Immunosorbent Assay (ELISA). Results were taken and compared with stages, tumor size, node and grade. The serum CA 15.3 levels were significant in all the study parameters whereas serum CEA levels showed no significant changes with any of the parameters. Measurement of serum CA 15.3 levels showed significant correlation (24.8%) with advanced stages and larger tumor sizes, whereas serum CEA levels did not show any significant correlation in breast cancer patients prior to treatment.