Mosin S. Khan

PLoss of heterozygosity (LOH) of deleted in colorectal cancer (DCC) gene and predisposition to colorectal cancer: Significant association in colorectal cancer patients of Kashmir

1 Department of Biochemistry Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Kashmir – 190 011. India. 2 Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Kashmir – 190 011. India. 3 Department of Biotechnology, University of Kashmir, Srinagar Kashmir.190011.India. 4 Department of general surgery, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Kashmir – 190 011. India.

Impact of molecular alterations of BRAF in the pathogenesis of thyroid cancer.

BRAF alterations represent a novel indicator of the progression and aggressiveness of thyroid carcinogenesis. So, the main aim of the study was to elucidate the involvement of BRAF gene mutations and its expression in Kashmiri (North India) patients and investigate their association with clinico-pathological characteristics. Mutational analysis of BRAF gene was performed by polymerase chain reaction followed by DNA sequencing, whereas analysis of BRAF protein expression was done by western blotting. Overall mutations in BRAF was found to be 25% (15 of 60) and all of them were transversions (T>A) affecting codon 600 (valine to glutamine), restricted only to papillary thyroid cancer and well-differentiated grade. Patients with well-differentiated disease and in particular elevated thyroid-stimulating hormone levels were significantly associated with BRAF mutations (P < 0.05). Overall, 90% (54 of 60) of thyroid cancer cases showed increased expression of BRAF and non-smokers being significantly associated with BRAF over-expression. Totally, 86.7% (13 of 15) of BRAF mutation-positive patients were having over-expression of BRAF protein, whereas 91.2% (41 of 45) of patients with wild-type BRAF status were having over-expressed BRAF protein (P > 0.05). We conclude that both mutational events as well as over-expression of BRAF gene is highly implicated in pathogenesis of thyroid cancer and the BRAF protein over-expression is independent of the BRAF mutational status of thyroid cancer patients.

The XRCC1 Arg399Gln Gene Polymorphism and Risk of Colorectal Cancer: a Study in Kashmir

BACKGROUND The DNA repair gene XRCC1 Arg399Gln gene polymorphism has been found to be implicated in the development of various cancers, including colorectal cancer (CRC), in different populations. We aimed to determine any association of this polymorphism with the risk of CRC in Kashmir. MATERIALS AND METHODS A total of 120 confirmed cases of CRC and 146 healthy cancer free controls from the Kashmiri population were included in this study. Genotyping was carried out by the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. RESULTS Genotype frequencies of XRCC1 Arg399Gln observed in controls were 34.2%, 42.5% and 23.3% for GG (Arg/Arg), GA (Arg/Gln), AA( Gln/Gln), respectively, and 28.3%, 66.7% and 5% in cases, with an odds ratio (OR)=5.7 and 95% confidence interval (CI) =2.3-14.1 (p=0.0001). No significant association of Arg399Gln SNP with any clinicopathological parameters of CRC was found. CONCLUSIONS We found the protective role of 399Gln allele against risk to the development of CRC. The XRCC1 heterozygote status appears to be a strong risk factor for CRC development in the Kashmiri population.

Possible Impact of RET Polymorphism and Its Haplotypic Association Modulates the Susceptibility to Thyroid Cancer

Rearranged during Transfection (RET) gene polymorphisms act to influence thyroid cancer in a polygenic and low‐penetrance manner and no study regarding RET alterations in thyroid cancer has undergone from this part of the world (North India). We evaluated RET G691S (rs1799939), L769L (rs1800861), and S904S (rs1800863) polymorphisms to elucidate their possible role as risk factors in papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Polymorphic analysis of RET gene was performed by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism (RFLP). In RET G691S polymorphism, the overall distribution of variant alleles (GA + AA) in cases was 62.9% as against 44.5% in controls (P < 0.05) whereas frequency of RET L769L variant alleles (TG + GG) in cases was 70% versus 88% in controls (P < 0.05). In RET S904S, frequency of variant alleles (CG + GG) in cases was 56% versus 44% in controls (P < 0.05). Interestingly, G691S/L769L variant showed increased risk for the non‐smokers (P < 0.05). RET S904S variant showed association with benign thyroid disease as against those with no history. The over‐representation of homozygotes in G691S and L769L polymorphic variants was not observed, which suggest a “Dominant mode of inheritance.” The S904S polymorphism heterozygote lies almost in the middle of the two homozygotes confirming an “Additive mode of inheritance.” In conclusion, RET gene G691S/S904S polymorphisms were over‐represented and L769L polymorphism was under‐represented in PTC and FTC patients. RET polymorphic variants could act synergistically in the development or progression of PTC and FTC. J. Cell. Biochem. 116: 1712–1718, 2015.

Significant association of TP53 Arg72Pro polymorphism in susceptibility to differentiated thyroid cancer

BACKGROUND Among various polymorphic variants of TP53 gene, codon 72 polymorphism (Arg72Pro) has been found to be associated with cancer susceptibility, but only few studies have investigated their effect on thyroid cancer risk. OBJECTIVE A case control study was conducted to elucidate the possible role of this SNP as risk factor in thyroid cancer development and to examine its correlation with various clinicopathological variables. METHODS In this study, we tested the genotype distribution by PCR-RFLP in 140 thyroid cancer patients and 200 cancer-free controls from Kashmir Valley. RESULTS Genotype frequencies of Arg/Arg (GG), Arg/Pro (GC), and Pro/Pro (CC) genotypes among cases were 0.286, 0.343 and 0.371 while in controls 0.45, 0.37 and 0.18 respectively. Proline allele frequency was significantly higher than arginine frequency in patient group (OR = 2.06, 95% C.I = 1.5-2.8). Significant association was found between variant genotype of codon 72 of TP53 gene and young age group, female gender, urban dwellers, non-smokers and patients with elevated TSH levels (P < 0.05). CONCLUSION It is evident from our study that Arg72Pro SNP of TP53 gene is connected with higher susceptibility to thyroid cancer especially in young age group, female gender, non-smokers and patients with elevated TSH levels, hence, implicated in thyroid carcinogenesis.

Molecular identification of Candida species isolated from cases of neonatal candidemia using polymerase chain reaction-restriction fragment length polymorphism in a tertiary care hospital

Context: Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. Aims: The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1) and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Statistical Analysis Used: Kappa test for agreement. Results: The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. Conclusions: We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

Prognostic Implication of BCR-ABL Fusion Transcript Variants in Chronic Myeloid Leukemia (CML) Treated with Imatinib. A First of Its Kind Study on CML Patients of Kashmir

Background: The prognostic significance of the common BCR-ABL transcripts like e13a2 (b2a2) and e14a2 (b3a2) in Chronic myeloid leukemia (CML) has been reported from patients treated with different tyrosine kinase inhibitors but its impact on clinical response and overall survival remains still unexplored. The aim of this study was to evaluate the prognostic significance of different transcript types in a cohort of CML patients treated with imatinib. Methods: A total 42 confirmed cases of Chronic Myeloid Leukemia (CML) patients were recruited into our cohort study and a multiplex Reverse Transcriptase-Polymerase Chain Reaction technique (RT-PCR) was used to detect 3 main transcript types ‘e1a2’, ‘e13a2’, and ‘e14a2’ found in CML. Results: Only two types of transcripts e13a2 (b2a2) and e14a2 (b3a2) were detected in our CML patients and none had the e1a2 type. All the patients were RT-PCR positive for either e13a2 or e14a2 fusion transcript demonstrating 100% concordance with their Ph+ve cytogenetic status at baseline. TLC count (range of 201-600x103/µl) and platelet count (range of 201-900x103/µl) at baseline were found to be associated more with the e14a2 (b3a2) than the e13a2 (b2a2) transcript type (p-value: 0.001). The two transcripts found did not relate significantly towards sex, age-group or indicated spleen size ranges as well as percentage ranges of blast cells. Conclusion: We conclude that there is no overall prognostic implication of either the e13a2 or the e14a2 transcript type across the spectrum of indicated clinical parameters evaluated. Even the overall survival analysis of the two transcript types revealed no prognostic association whatsoever.

TAZ is an independent prognostic factor in non-small cell lung carcinoma: Elucidation at protein level

BACKGROUND Hippo-LATS pathway is involved in regulating multiple phenotypes. TAZ (transcriptional co-activator with PDZ-binding motif) is a prominent proto-oncogene of this developmental pathway. Elevated TAZ expression has been observed in many cancers including Non-Small Cell lung cancer (NSCLC). OBJECTIVE We elucidated the frequency of protein expression of TAZ gene in NSCLC patients of our population along with its relationship with clinicopathological parameters and determined its prognostic significance concerning survival in patients with resected tumor. METHODS We looked into the protein expression of TAZ gene in sixty nine (n= 69) cases of NSCLC tissue and their corresponding normal lung tissue samples by western blotting. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of TAZ protein expressionon survival. RESULTS Here, we found that TAZ protein expression was elevated in 49.27% (34 of 69) of NSCLC tissues as compared to only 13.04% (09 of 69) in normal lung tissues. TAZ protein expression was significantly associated with smoking, positive lymph node status and vascular invasion (P< 0.05). TAZ protein overexpression increased the hazards ratio by 2.6 (P= 0.005) on univariate analysis. Multivariable analysis confirmed that TAZ protein overexpression along with age and lymph node metastasis increased the hazard of death after adjusting for other clinicopathological factors (P< 0.05). Importantly, TAZ protein overexpression was associated with short overall survival and disease-free survival when compared with normal TAZ expression. CONCLUSION These results indicate that TAZ is an independent prognostic factor and plays an important role in NSCLC progression and may serve as a novel therapeutic target of NSCLC.

Mutational profiling of KRAS and its association with non-small cell lung carcinoma in Indian Kashmiri population

Lung cancer represents commonest cancer worldwide, with a high mortality rate as the disease becomes clinically apparent at advanced stages. The most common molecular alterations observed in NSCLC lie in the mutations of KRAS. These mutations occur in 15–30% of NSCLC and are more frequent in adenocarcinoma. We have screened prospectively all newly diagnosed patients with NSCLC (n=70) for the ability to be analysed for KRAS mutations. Seventy, blood samples of Non-small cell lung cancer were collected from Department of Hematology, Sheri-iKashmir Institute of Medical Sciences (SKIMS). Blood DNA was extracted from these cases. DNA sequencing was performed on all the samples for detection KRAS codon 12, 13 activating mutations. Mutation status was compared with patient clinicopathological characteristics. The prevalence of KRAS mutation rate in NSCLC in the Kashmiri population was 30%. The significant association was seen between KRAS gene mutation and histological types of lung cancer. The higher frequency was seen in adenocarcinoma (ADC) (28.84%) than squamous cell carcinoma (SCC) (6%). The difference was statistically significant (OR=0.81, 95% CI=0.2572.588, p < 0.01). Among the different stages, the higher frequency of KRAS (exon 2) mutation was reported in NSCLC patients in advanced stage (38.09%) than the early stages (17.85%). The difference was statistically significant (OR=0.353, 95% CI= 0.1121.116, p<0.05). A statistically significant difference was reported between smokers and non-smokers with respect to the KRAS (exon 2) mutation (OR= 4.899, 95%CI =1.27318.77, p < 0.01). The significantly higher frequency of this mutation was reported in NSCLC patients (29.16%) with metastasis (OR= 0.941 95% CI= 0.319-2.775, p < 0.03). KRAS (exon 2) mutation is a common molecular alteration in NSCLC and occurs most predominantly on codon 12, 13, characterizing 30% of the total mutations found in Kashmiri population. These mutations are significantly associated with clinicopathological characteristics of patients. Citation: Shah, N.U.D., Ali, M.N., Mudassar, S. and Khan, M.S. Mutational profiling of KRAS and its association with non-small cell lung carcinoma in Indian Kashmiri population [Abstract]. In: Abstracts of the NGBT conference; Oct 02-04, 2017; Bhubaneswar, Odisha, India: Can J biotech, Volume 1, Special Issue, Page 57. https://doi.org/10.24870/cjb.2017-a44

Molecular Alterations and Expression Dynamics in the Etiopathogenesis of Thyroid Cancer

Thyroid carcinoma is the most prevalent endocrine malignancy and accounts for 2% of all human cancers. In the past decade, knowledge of genetic alterations of thyroid cancer (TC) has rapidly expanded, which has provided new insights into thyroid cancer etiology and has offered novel diagnostic tools and prognostic markers that enable improved and personalized management of thyroid cancer patients. Alterations in key signaling effectors seem to be the hallmark of distinct forms of thyroid neoplasia. Mutations or rearrangements in genes that encode Mitogen activated protein kinase (MAPK) pathway effectors seem to be required for transformation. Mutations in BRAF were the most recently identified MAPK effector in thyroid cancer. BRAF V600E is the most common alteration in sporadic papillary carcinoma. Three RAS proto-oncogenes (NRAS, HRAS & KRAS) are implicated in human thyroid tumorigenesis. High incidence of thyroid cancer worldwide indicates the importance of studying genetic alterations that lead to its carcinogenesis. BRAF and RAS alterations represent a novel indicator of the progression and aggressiveness of thyroid carcinogenesis. The GSα-adenylyl cyclase-cyclic AMP (cAMP) cascade is effected in thyroid cancer. Promoter hyperme‐ thylation of multiple genes especially TSHR has been identified to play a role in thyroid cancers, in particular showing a close association with BRAF mutational status. So, the main aim of the study was to elucidate the involvement of BRAF and RAS gene mutations along with BRAF expression and thyroid-stimulating hormone receptor (TSHR) hypermethylation in North Indian patients and investigate their association with clinicopathological characteristics. We screened exon 15 of BRAF gene and exons 1 and 2 of RAS genes (HRAS, KRAS, and NRAS) in 60 consecutive thyroid tissue (tumor and adjacent normal) samples. Overall mutations in BRAF were found to be 25% (15 of 60) affecting codon 600 (valine to glutamine) and restricted only to papillary thyroid cancer and well-differentiated grade.