Manjula Gorre

Methylation status of CEBPA gene promoter in chronic myeloid leukemia

Abstract CCAAT/enhancer binding protein alpha is one of the crucial transcription factors for myeloid cell development that has been found to be involved in hematopoietic differentiation and leukemiogenesis. Recently, epigenetic regulation of CEBPA expression through DNA methylation has been demonstrated in leukemia. The aim of this study was to investigate the methylation status of CEBPA gene in chronic myeloid leukemia (CML) patients. The methylation status of CEBPA promoter was studied in 100 patients with CML and 98 normal healthy individuals from Hyderabad, India, using methylation-specific polymerase chain reaction. The aberrant methylation of CEBPA gene promoter was found in 32 of the 100 CML cases. A highly significant association was found between the frequency of CEBPA gene promoter hypermethylation and the CML stages (P = 0.017), but association with respect to age and gender of the patient was not found. The results suggest that aberrant methylation in the CpG island of the promoter region of this gene might be a common event in CML, and systemic expression studies will be needed to unfold the role of CEBPA promoter methylation in the development, progression, and prognosis of CML.

Association of XRCC1 gene polymorphisms with chronic myeloid leukemia in the population of Andhra Pradesh, India.

Abstract Chronic myeloid leukemia (CML), a clonal myeloproliferative disorder, is characterized primarily by the presence of chimeric BCR-ABL oncogene, and its progression from chronic to blast phase is associated with the accumulation of additional molecular and chromosomal abnormalities. The molecular mechanisms underlying this genetic instability are poorly understood. The activity of BCR-ABL is known to be associated with the increased production of intracellular reactive oxygen species and spontaneous DNA damage, which when effected by impaired/inaccurate DNA repair systems result in increased susceptibility to CML progression. Using case-control study design, we explored possible association of the repair gene, XRCC1, particularly the codons 399, 280, and 194 polymorphisms screened through PCR-RFLP, with the CML in the sample of 350 cases (206 male and 144 female) and 350 controls from Hyderabad, the capital city of state of the Andhra Pradesh, India. The patient group constituted 301 early chronic phase cases followed by 28 accelerated and 21 blast phase cases. The median age of the patients was 42 years (range, 9–70 years). The genotype distribution revealed significant association of codons 399 (χ2 = 11.904, degree of freedom (d.f.) = 2; P = 0.002) and 194 (χ2 = 8.091, d.f. = 2, P = 0.017) with CML, not 280 (P = 0.29). Although these polymorphisms are known to affect the function of XRCC1, the nature and extent of their genetic association with CML does not indicate their direct role in its development. The results seem to suggest that XRCC1 gene might have an important role in CML progression but not in its causation.

HIF-1α (1772C>T) polymorphism as marker for breast cancer development

Hypoxia-inducible factor 1α (HIF-1α) is an important transcription factor that regulates different cellular responses to hypoxia. HIF-1α is rapidly degraded by von Hippel–Lindau (VHL) protein under normoxic conditions and stabilized under hypoxia. A common variant of HIF-1α (1772C>T) (rs 11549465) polymorphism, corresponding to an amino acid change from proline to serine at 582 position within the oxygen-dependent degradation domain, results in increased stability of the protein and altered transactivation of its target genes. The present study was aimed to find the association between HIF-1α (1772C>T) (rs 11549465) polymorphism and breast cancer development. For this purpose, 348 primary breast cancer patients and 320 healthy and age-matched controls were genotyped through PCR-RFLP method. The genotype frequencies were compared between patients and controls, and their influence on clinical characteristics of breast cancer patients was analyzed. Our study revealed a significant increase of TT genotype in breast cancer patients compared to controls (p = 0.038). Further, TT genotype and T allele were found to be associated with progesterone receptor (PR)-negative status (p < 0.09). None of the clinical variables revealed significant association with HIF-1α (1772C>T) (rs 11549465) polymorphism.

Significance of ATM Gene Polymorphisms in Chronic Myeloid Leukemia - a Case Control Study from India

BACKGROUND Development of chronic myeloid leukemia (CML) involves formation of double strand breaks (DSBs) which are initially sensed by the ataxia telangiectasia mutated (ATM) signal kinase to induce a DNA damage response (DDR). Mutations or single nucleotide polymorphisms in ATM gene are known to influence the signaling capacity resulting in susceptibility to certain genetic diseases such as cancers. MATERIALS AND METHODS In the present study, we have analyzed -5144A>T (rs228589) and C4138T (rs3092856) polymorphisms of theATM gene through polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 925 subjects (476 CML cases and 449 controls). RESULTS The A allele of -5144A>T polymorphism and T allele of C4138T polymorphism which were known to be influencing ATM signaling capacity are significantly associated with enhanced risk for CML independently and also in combination (evident from the haplotype and diplotype analyses). Significant elevation in the frequencies of both the risk alleles among high risk groups under European Treatment and Outcome Study (EUTOS) score suggests the possible role of these polymorphisms in predicting the prognosis of CML patients. CONCLUSIONS This study provides the first evidence of association of functional ATM gene polymorphisms with the increased risk of CML development as well as progression.

LCN2 Promoter Methylation Status as Novel Predictive Marker for Microvessel Density and Aggressive Tumor Phenotype in Breast Cancer Patients.

LCN2 (Lipocalin 2) is a 25 KD secreted acute phase protein, reported to be a novel regulator of angiogenesis in breast cancer. Up regulation of LCN2 had been observed in multiple cancers including breast cancer, pancreatic cancer and ovarian cancer. However, the role of LCN2 promoter methylation in the formation of microvessels is poorly understood. The aim of this study was to analyze the association of LCN 2 promoter methylation with microvessel formation and tumor cell proliferation in breast cancer patients. The LCN2 promoter methylation status was studied in 64 breast cancer tumors by methylation specific PCR (MSP). Evaluation of microvessel density (MVD) and Ki67 cell proliferation index was achieved by immunohistochemical staining using CD34 and MIB-1 antibodies, respectively. LCN2 promoter unmethylation status was observed in 43 (67.2%) of breast cancer patients whereas LCN2 methylation status was seen in 21 (32.8%). Further, LCN2 promoter unmethylation status was associated with aggressive tumor phenotype and elevated mean MVD in breast cancer patients.

Role of the MDM2 Promoter Polymorphism (-309T>G) in Acute Myeloid Leukemia Development

BACKGROUND The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator of Tp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpression thereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate the possible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. MATERIALS AND METHODS A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for the MDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCR method. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantified MDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypes and the MDM2 expression were correlated with disease free survival (DFS) rates among patients who have achieved complete remission (CR) after first induction chemotherapy. RESULTS MDM2-309T>G polymorphism was significantly associated with AML development (p<0.0001). The presence of either GG genotype or G allele at MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p=0.003) increased AML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates (16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2 expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+ve cases (p=0.0036). CONCLUSIONS The MDM2-309T>G polymorphism might be involved in AML development and also serve as a good prognostic indicator.

Abstract 4625: Influence of intrinsic apoptotic pathway gene polymorphisms on the development and progression of acute myeloid leukemia: Case-control study

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Apoptosis plays a pivotal role in maintaining cellular homeostasis mainly in rapidly proliferating tissue like hematopoietic tissue. Defective apoptosis is one of the hallmarks of cancer as it plays a crucial role in malignant transformation through disruption of homeostasis mechanism resulting in acquired chemo-resistance. Aim: The functional single nucleotide polymorphisms (SNPs) in apoptotic genes can influence the gene expression leading to altered apoptosis that may promote malignancy. Moreover, these SNPs might also influence the treatment outcome, since most of the chemotherapeutic drugs being used are aimed to induce apoptosis of malignant cells. The present study mainly aims to evaluate the role of SNPs in BCL2 (-938C>A (rs2279115); BAX (-248G>A (rs4645878), CASP9 [-1263A>G (rs4645978); -712C>T (rs4645981); -293del (rs4645982); Ex5+32G>A (rs1052576)] with development of acute myeloid leukemia (AML) and treatment outcome in terms of disease free survival (DFS) among patients who have achieved complete remission(CR) rates after first induction chemotherapy. Methods: The study includes 225 AML cases and 307 age-gender matched controls for case-control comparison. The study was approved by ethical committee of Osmania University. Before collecting 5ml of blood sample, each participant had provided written informed consent. Baseline line clinical characteristics and follow-up data were collected from the tumor registries with the help of oncologist. Genomic DNA was extracted and SNPs were genotyped using PCR, RFLP, Tetra primer techniques. The genotype data was subjected to various statistical analyses to correlate with disease occurrence and treatment outcome in terms of CR and DFS rates using SPSS Software. We have also performed non parametric analysis to know gene-gene interaction through Multi Dimensionality Reduction (MDR). Results: The single SNP analysis had revealed that BCL2 (rs2279115), BAX (rs4645878), CASP9 (rs4645978) and CASP9 (rs4645982) were significantly associated with the origin of AML. Survival analysis had shown that minor alleles of both CASP9 (rs4645978 and rs4645982) SNPs were associated with reduced DFS rates (log rank p = 0.02). Further, cox regression analysis had also confirmed these observations where CASP9 rs4645978 (HR = 1.91; 95%CI = 1.19-3.07; p = 0.008) and rs4645982 (HR = 1.88; 95%CI = 1.06-3.35; p = 0.03) had elevated risk for relapse and shorter DFS rates. The MDR analysis had revealed that CASP9 (rs4645981) is the highest predicting variable. The best interaction model was found to be CASP9 (-1263A>G (rs4645978); -712C>T (rs4645981); and BCL2 (rs2279115) with CVC = 8/10 and TAB = 0.604. Conclusion: Our results suggest that CASP9 (rs4645978 and rs4645982) SNPs might influence the AML development and DFS rates after first induction chemotherapy. Citation Format: Anuradha Cingeetham, Sugunakar Vuree, Nageswara Rao Dunna, Manjula Gorre, Raghunadharao Digumarti, Sudha Sinha, Vishnupriya Satti. Influence of intrinsic apoptotic pathway gene polymorphisms on the development and progression of acute myeloid leukemia: Case-control study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4625. doi:10.1158/1538-7445.AM2015-4625

Abstract 3858: Genetic polymorphisms of DNA repair genes in the origin and progression of chronic myeloid leukemia

Background: The origin of Chronic Myeloid Leukaemia (CML) involves the formation of Double Strand Breaks (DSBs) which are initially sensed by the Ataxia Telangiectasia Mutated (ATM) signal kinase. Additionally, Bcr-Abl transformed CML cells produce high levels of Reactive Oxygen Species (ROS) that induce more DSBs. In such kind of cells, proper functioning of ATM and NHEJ repair mechanisms are required for ensuring the accurate repair of DSBs. Aim: The present study is planned to understand the role of DNA repair, specifically NHEJ repair in the occurrence of CML and its progression. Methodology: We have analysed seven genetic polymorphisms in ATM, XRCC5, XRCC6 and XRCC7 genes in 476 Ph+ve CML cases and 449 age and sex matched controls without family history of cancers. Direct Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) analyses were carried out using the DNA isolated from blood samples through non-enzymatic/salting out method. Statistical analyses were performed through SPSS (IBM SPSS Statistics 20) software. Results: The T allele of ATM C4138T polymorphism and A allele of ATM -5144A>T polymorphism were found to be significantly associated with enhanced risk of CML in our study. The CC genotype of XRCC6 -61C>G polymorphism was found to have independent effect with CML risk as confirmed by the LD and MDR analyses. Lower repeat (0R) of XRCC5 VNTR and TT genotype of XRCC7 6721G>T polymorphisms had shown borderline association with CML risk. Even though the XRCC5 2408G>A and XRCC6 -1310C>G polymorphisms did not show significant association with CML independently, the AA and GG genotypes of these polymorphisms have been found to confer enhanced risk for CML in combination with other SNPs of the respective genes in haplotype analysis. Interestingly, combined analysis demonstrated cumulative effect of risk genotypes in CML development as well as progression where the fold change increased with the number of adverse genotypes. Conclusion: The risk associated genotypes were known to influence the expression and functioning of genes in NHEJ pathway. Therefore, results of the present study suggested that defective DNA damage sensing and deregulated repair due to variations in ATM and NHEJ genes might influence CML development as well as progression. Key words: CML, ATM, NHEJ, DNA repair, polymorphisms Citation Format: Manjula Gorre, Prajitha Edathara Mohandas, Sailaja Kagita, Anuradha Cingeetham, Sugunakar Vuree, Sandhya Annamaneni, Raghunadharao Digumarti, Vishnupriya Satti. Genetic polymorphisms of DNA repair genes in the origin and progression of chronic myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3858. doi:10.1158/1538-7445.AM2015-3858

Incidence of internal tandem duplications and D835 mutations of FLT3 gene in chronic myeloid leukemia patients from Southern India

Abstract Objective To screen two important FLT3 mutations (internal tandem duplication (ITD) and D835 point mutations) in chronic myeloid leukemia (CML) patients from Southern India and report their incidence. Methods Screened 350 CML patients and 350 controls for the two FLT3/mutations through polymerase chain reaction and restriction fragment length polymorphism methods. Results ITDs were detected in 12 of the 350 CML patients (3.4%) and D835 mutations in only four cases (1.14%), relatively low in frequency as compared to those reported earlier from non-Indian populations. None of the cases showed simultaneous occurence of both ITD and D835 mutations. Discussion These FLT3 mutations seem to be very rare in CML, and it is possible that these could be found only in a subset of patients who are in the progressive stage and/or with varied drug response. Prospective studies are needed to confirm the role of FLT3 mutations in CML pathogenesis, which may help devising therapeutic interventions.