Prasant Yadav

Erratum to : Expression of serum miR-200a, miR-200b and miR-200c as candidate biomarkers in epithelial ovarian cancer and their association with clinicopathological features

Background MicroRNAs (miRs) have been implicated in the etiology of various human cancers. The aim of this study was to investigate the association of the expression of three members—miR 200a, miR 200b, and miR 200c belonging to the miR-200 family with clinicopathological characteristics and their impact on the progression of epithelial ovarian cancer (EOC).

Serum microRNA-21 expression as a prognostic and therapeutic biomarker for breast cancer patients

MiRNA-21 is recognized as the main active candidate and high expression in many solid tumors consequential cell proliferation, differentiation, apoptosis, and closely related to metastasis of disease. The study aimed to evaluate the serum miRNA-21 expression and therapy outcome in breast cancer patients and cell lines. Seventy-five histopathologically confirmed newly diagnosed breast cancer patients were included in the study; before and after therapy, patient’s blood sample were collected and analyzed for serum microRNA-21 expression by quantitative real-time PCR. In patients, 8.9 mean fold increased microRNA-21 expression was observed compared to controls. Increased expression was found to be associated with advanced stage (11.72-fold), lymph node involvement (11.12-fold), and distant metastases (20.17-fold). After treatment significant decrease in miRNA-21 expression was observed and found to be significant (p < 0.0001). Patients treated with neoadjuvant therapy had significant impact on miRNA-21 suppression and found to be significantly associated with different clinicopathological features of patients. Increased miRNA-21 expression was also found to be significantly associated with poor survival of breast cancer patients (p = 0.002). MicroRNA-21 expression could be used as promising predictive indicators for breast cancer prognosis. MicroRNA-21 over-expression was associated with response to neoadjuvant therapy and may perhaps be considered as primary treatment choice.

Epigenetic Silencing of DAPK1 Gene is Associated with Faster Disease Progression in India Populations with Chronic Myeloid Leukemia

Background: One of the major epigenetic changes in human cancer is DNA methylation of tumour suppressor genes which leads to silencing of gene leading to disease progression. Therefore, DNA methylation status of such genes may serve as the epigenetic biomarker for prognosis of human Chronic Myeloid Leukemia. Material and methods: We used MSP-PCR technique for the analysis of aberrant promoter DAPK1 methylation on 200 CML venous blood samples. Stastical analysis was done for evaluating differences between different parameters using SPSS 16.0 version. Results: We could detect 91/200 promoter methylation (45.5%) in CML patients. Percentage of methylation detected was seen higher in blast phase (63.07%) and in accelerated phase (48.1%) than in chronic phase (29.6%). A significant correlation was seen between CML stages and DAPK1 aberrant methylation. We also found a significant association of DAPK1 methylation in gender and in haematological resistance CML patients. However no correlation was found between DAPK1 promoter methylation and other clinical parameters like age, BCR-ABL type and Thrombocytopenia. Conclusion: In summary we concluded that methylation status of DAPK1 gene is associated with advanced phase of CML and may be related to disease progression in chronic myeloid leukemia. Further study on a more number of patients is needed to explore the role of DAPK1 methylation in the prognosis of CML.

Simple multiplex RT-PCR for identifying common fusion BCR-ABL transcript types and evaluation of molecular response of the a2b2 and a2b3 transcripts to Imatinib resistance in north Indian chronic myeloid leukemia patients.

INTRODUCTION Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, an abnormally shortened chromosome 22. It is the result of a reciprocal translocation of chromosomes 9 and 22, creating BCR-ABL fusion transcripts, b3a2, b2a2, and e1a2. The aim of our study was to determine the type of BCR-ABL fusion transcripts for molecular diagnosis and investigate the frequency of BCR-ABL fusion transcripts in CML patients by multiplex RT-PCR in CML. MATERIALS AND METHODS A single reaction with multiple primers multiplex PCR was used to detect and investigate the type and frequency in 200 CML patients among which 116, 33, and 51 were in CP, AP, and BC phase, respectively. RESULTS The study included 200 CML patients, among whom breakpoints in b3a2, b2a2 transcripts were detected in 68% and 24%, respectively, while 8% of the patients showed both b3a2/b2a2. A statistically significant difference was seen between frequency of BCR-ABL fusion transcripts and gender (P = 0.03), molecular response (P = 0.04), and hematological response (P = 0.05). However, there was no correlation found between frequencies of BCR-/ABL fusion transcripts and other clinicopathological parameters like age, type of therapy, thrombocytopenia, and white blood cell count. CONCLUSION Multiplex reverse transcriptase-polymerase chain reaction is useful and saves time in the detection of BCR-ABL variants; the occurrence of these transcripts associated with CML can assist in prognosis and treatment of disease.

Inactivation of RIZ1 Gene by Promoter Hypermethylation is Associated with Disease Progression and Resistance to Imatinib in Indian Chronic Myelogenous Leukemia Patients, First Study from India

Background: The epigenetic impact of DNA methylation in chronic myelogenous leukemia (CML) is not completely understood. RIZ1 expression and activity are reduced in many cancers. In CML, blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML. Methods: To examine whether promoter methylation is involved in the disease development and progression of CML, we investigated promoter methylation status of RIZ1 gene in 100 chronic myeloid leukemia’s (CML) patients and 50 controls by MSP method. Results: The RIZ1 methylation was studied in 100 CML patients, 9 were cases were methylation positive cases, six of nine were in blastic phase, 2 in chronic phase and one patient in accelerated phase. It was seen that RIZ1 methylation was increased significantly from early to advanced phase. The higher frequency of RIZ1 methylation was reported in haematologically resistant cases (42% vs 2%) and molecularly resistant cases (16.77% vs 1.92%) than the responders. The higher frequency of RIZ1 methylation was found in CML patients who were treated with interferon initially followed by imatinib treatment. Also RIZ1 hypermethylation was associated with faster disease progression p<0.003 than the non methylated cases. No correlation was found between RIZ1 gene methylation with age, thrombocytopenia, types of bcr/abl transcripts of CML patients. Conclusion: We conclude that epigenetic silencing of RIZ1 gene is associated with CML progression and imatinib resistance. Early detection of RIZ1 methylation could be a predictive marker for imatinib resistance and disease progression in CML.

A deletion polymorphism in the RIZ gene is associated with increased progression of imatinib treated chronic myeloid leukemia patients

Abstract RIZ1 encodes a retinoblastoma (Rb)-interacting zinc finger protein, is commonly lost or expressed at reduced levels in cancer cells. The RIZ1 gene locus commonly undergoes LOH in many cancers. Here, we analyzed Proline insertion–deletion polymorphism at amino acid position 704 in the RIZ1 gene and its association with CML. The RIZ1 pro-704 LOH genotypes were determined by AS-PCR in 100 CML patients among which 50 were in CP-CML, 25 in AP-CML, and 25 in BC-CML. Pro704 ins/del polymorphism (LOH) was detected in 27% CML patients. Proline ins–ins homozygosity, del–del homozygosity and ins–del heterozygosity was detected in 9%, 18%, and 73% CML patients compared with 3%, 3%, and 94% in healthy controls, respectively (p < .0003). A four-fold increased risk was found to be associated del-del genotype. We found a statistically significant association between RIZ1 LOH and stage (p > .01) and hematological resistance (p > .001). However, there were no correlations found with other clinical parameters like age, gender, thrombocytopia, type of BCR–ABL, and molecular response. Our findings suggest that proline 704 del–del homozygosity phenotype can play an important role in progression of CML.

Biological and Clinical Implications of Exon 8 P53 (R282W) GeneMutation in Relation to Development and Progression of Chronic MyeloidLeukaemia Patients in India Population

Background: TP53, located on chromosome 17p13, is one of the most mutated genes affecting many types of human cancers .To establish an association between the incidence of exon 8 p53 (R282W) gene and progression of the disease in CML and also to correlate the presence of mutation with the clinicopathological features of the disease. Methods: p53 status was investigated by studying mutations in the p53 gene at exon 8 region after confirming the diagnosis by BCR-ABL. 100 CML samples were analyzed using the Allele-Specific Oligonucleotide PCR assay. Mutations occurred in 58% of the cases in exon 8 codon 282 region of the p53 gene. C : T transitions occurred at a high frequency with a statistically significant result (p=0.03). Results: Of the 100 clinically confirmed specimens, 58% tested positive for the mutation. Also, the mutation was found to be higher in the progressed stages (88.2% in accelerated phase and 60.0% in blast crisis) of CML compared to the chronic stage (35.2%). A statistically significant association (p=0.001) was found between the occurrence of p53 R282W mutation and the clinical phase of CML with chronic, accelerated and blast crisis phases. The mutation was detected in a vast majority (88.2%) of patients in the accelerated and the blast crisis phase (60.0%) indicating that this mutation might play a critical role in predicting the progression of disease in CML. Clinicopathological correlation with TLC, platelet count and the haematological response elicited a significant association with patients harboring the mutation with (p=0.01), (p=0.001) and (p=0.01) respectively. Conclusion: Our study suggests that p53 mutations in the exon 8 region might have a strong influence on disease progression and poor response of imatiib (Tyrosine kinase inhibitor) in CML patients.

Impact of MDM2 SNP309T>G Polymorphism: Increased Risk of Developing Non Small Cell Lung Cancer and Poor Prognosis in Indian Patients

Background: MDM2 is an important negative regulator of the TP53 pathway, over expressed in many cancers as oncoprotein. Polymorphisms in the promoter region of the MDM2 gene have been shown to alter protein expression and may, thus play an important role in carcinogenesis. Aim and methods: To test our hypothesis that the MDM2 promoter polymorphisms are associated with risk of non small cell lung cancer, we conducted a hospital-based, case–control study of 136 Indian patients diagnosed with NSCLC and 136 cancer-free controls and investigated the association between genetic variation in the promoter region of MDM2 (c.–51309G4T, rs2279744:g.G4T) and the risk of developing NSCLC by tetra-primer ARMS-PCR and ASO-PCR. Results: Compared with the MDM2-2580TT genotype, we found that the MDM2-309G variant genotypes were associated with an increased risk of NSCLC in Indian patients [OR 3.88 (1.82-8.27) RR 1.94 (1.27-2.96) RD 32.6 (15.7-49.6) p 0.0004 for GG and OR 2.60 (1.49-4.57) RR 1.52 (1.20-1.93) RD 23.16 (10.3-36.0) p 0.0009 for GT genotype]. GG genotype was found to be associated with poor survival outcome of NSCLC patients and in addition significant association was observed with stage (p 0.01) and metastasis status (p 0.002) of NSCLC patients. Conclusion: Genetic polymorphism in cell cycle regulatory genes MDM2 contribute to the risk of developing NSCLC in Indian Patients. In addition G allele was associated with an increased risk and poor survival outcome than T allele.

Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC) patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168) cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75%) in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08%) than squamous cell carcinoma (52.83%). Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%). Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease.

Biological and Clinical Implications of Functional Promoter Polymorphism of CASPASE 9 Gene in Non Small Cell Lung Cancer Patients

Purpose: The study was conducted to determine the impact of promoter polymorphism in CASP9 gene on susceptibility and prognosis of Non Small Cell Lung Cancer patients of Indian origin. Patients and methods: Present case control study includes newly diagnosed 160 NSCLC patients and 160 healthy controls. Promoter polymorphism (-712C>T, rs4645981) in CASP9 gene was investigated using PCR-RFLP technique. Results: Our findings reveal that a statistically significant increased risk of about 2.6 fold was associated with homozygous TT genotype of CASP9 (-712C>T) promoter polymorphism (OR 4.3, 95%CI 2.2-8.3, p=0.0000) in Indian population .In addition smokers were at high risk for NSCLC which was more predominant in heavy smokers with pack-year >40 and in cigarette or beedi smokers. Compared to males, females were at high risk; about 2.3 fold more in association with homozygous TT genotype [OR, 3.9(1.9-8.2) in males vs. 6.2(1.4-27.1) in females]. Significant association was observed between advanced TNM stage (p<0.0001) and distant metastasis status (p<0.0001) of NSCLC patients with the polymorphism. Patients homozygous for T allele exhibited a significant poor overall survival compared with patients displaying CT+TT or CT or CC genotype (Median survival 6.0 vs. 9.0, 11.0, and 30.0, months respectively, p<0.0001). Also advanced stage patients with TT genotype showed lower median survival time than early stage NSCLC patients (Median 5.0 vs. 9.0 months respectively). Conclusion: The functional polymorphism (-712C>T) in the promoter region of CASP9 gene is associated with risk and susceptibility to NSCLC in north Indian population, and also is an important factor for poor prognosis in patients with NSCLC.