Molecular binary classification of NSCLC: miR-375 is a potential biomarker to differentiate SQCC from ADCC in Indian NSCLC patients

Background: Current developments in the targeted therapies of non-small-cell lung carcinoma demand accurate classification to dodge the adverse drug response and to yield maximum therapeutic outcome. Accurate classification depends on the classical hematoxylin and eosin staining and immunohistochemistry techniques. In selected critical cases, inter-observer variability, lack of standardization, tumor heterogeneity, and degree of differentiation makes it difficult to classify NSCLC. During the last decade, microRNAs (miRNAs) have been proven to be a promising biomarker in the field of oncology from diagnosis to therapy. The present study developed a binary classification method based on the expression of three well-known miRNAs: miR-205, miR-196b, miR-375, since it is the most demanding criteria to the clinicians trying to provide better therapy to the patients. Methods: Quantitative real-time polymerase chain reaction was performed for 90 NSCLC samples. Receiver-Operator Characteristic Curve and Discriminant Function Analysis was done to classify the NSCLC. A discriminant formula was developed to calculate the Z-score of miR-375 (Z3 = −0.637 + (0.439 x NCt miR-375) + (−0.245 x NCt miR-21). Results: The miR-375 classified NSCLC into SQCC and ADCC with higher accuracy. miR-375 appeared to differentiate SQCC from ADCC accurately in the test and validation set, signifying a sensitivity and specificity of 96.7% and 93.1%, respectively. Discussion: miR-375 is over-expressed in ADCC and suppressed in SQCC. This evidence accentuated the oncogenic and tumor suppressor nature in ADCC and SQCC respectively. Conclusion: miR-375 was proven to be the prominent biomarker of accurate NSCLC classification. The current study developed a molecular binary classification method in adjunctive of IHC which will help the clinicians in better classifying NSCLC and designing therapy (AU)

Molecular analysis of genetic variation in angiotensin I-converting enzyme identifies no association with sporting ability: First report from Indian population.

INTRODUCTION: A polymorphism in the angiotensin-converting enzyme (ACE) gene was the first performance enhancing polymorphisms (PEPs) to be identified and correlated with athletic abilities. This polymorphism (rs. 5186) is the absence (deletion; D allele), rather than the presence (insertion, I allele) of 287bp Alu repeat element in intron 16. However, the association of ACE I/D polymorphism in sports abilities have been contradicted and debated. No study has evaluated the ACE gene polymorphism in Indian athletes so far. Hence, the genotype distribution and allelic frequency of ACE gene in selected Indian athletic and non-athletic population was studied. MATERIALS AND METHODS: A total of 147 athletes and 131 controls were genotyped for the ACE gene polymorphism using PCR. RESULTS: No significant association was observed between the allelic frequencies of ACE gene in controls and athletes on a whole, as well as after sub-categorizing the athletes based on the type of sport they played (P > 0.1). However, a higher representation of I allele was observed in the athletes. CONCLUSION: ACE genotyping studies need to focus on truly elite athletes of a single sporting discipline, to be able to find an association. The ACE I/D polymorphism may not be considered a marker for human performance, but can be further studied in combination with other potent performance enhancing polymorphisms.

Frequency of fokI and taqI polymorphism of vitamin D receptor gene in Indian population and its association with 25-hydroxyvitamin D levels.

BACKGROUND: The VDR protein is at the centre of the vitamin D endocrine system, a complex physiological system with substantial feedback regulatory mechanisms involved in maintaining serum calcium and 1, 25 dihydroxy vitamin D3. Variations in VDR gene are shown to have implications in several diseases and have also been implicated as an important genetic factor affecting bone mass. AIM: To determine the frequency of Fok I and Taq I variants in healthy Indian individuals and its association with 25-OH-Vitamin D levels. SETTINGS AND DESIGN: Blood samples were collected from 143 unrelated normal individuals (Male-84 and Female-59) and their genotypes determined. MATERIALS AND METHODS: After amplification by polymerase chain reaction, each polymorphism was genotyped by restriction fragment length polymorphism. For 100 normal healthy individuals 25-hydroxyvitamin D estimation was done using DiaSorin kit method. STATISTICAL ANALYSIS: Graph pad software was used to calculate the P values from the Chi-square. RESULTS: Out of 143 samples analyzed for FokI and TaqI polymorphisms the following genotypic frequency was obtained FF 59%, Ff 36%, ff 5% and TT 49%, Tt 43%, tt 8% respectively. CONCLUSIONS: Results indicate that the distribution of the polymorphic loci Fok I and Taq I vary considerably not only in different populations, but also within India. Furthermore, when the genotypes were analyzed with respect to 25-OH-Vitamin D levels, a significant association was seen for the Taq 1 SNP but not with the Fok I.

Reference ranges for lymphocyte subsets in adults from western India: Influence of sex, age and method of enumeration.

Background: The enumeration of absolute CD4 counts is of primary importance, since therapeutic protocols for HIV1 patients are based on these. Aims: To establish reference ranges for the CD4 and CD8 T-lymphocytes in the Indian population. Settings and Design: Enumeration of absolute numbers and percentages of lymphocyte subsets was performed in 252 healthy adult Indians. Methods and Materials: The assays for SPT were carried out using the Beckman EPICS XL-MCL flow cytometer and the cytostat tetraCHROME reagent containing CD45/CD8/CD4/CD3 monoclonal antibodies. For comparison with DPT the absolute lymphocyte count was obtained using the Coulter STK-S fully automated hematology analyzer. Statistical Analysis: Regression analysis and Students t test were used for data analysis. Results: Median values were as follows; absolute CD3 counts 1446 cells/mm3 (total), 1361 cells/mm3 (males) and 1511 cells/mm3 (females); absolute CD4 counts are 771 cells/mm3 (total), 705 cells/mm3 (males) and 839 cells/mm3 (females); absolute CD8 counts are 555 cells/mm3 (total), 552 cells/mm3 (males) and 561 cells/mm3 (females). The median CD4/CD8 ratio for the total samples was 1.34, for males 1.22 and for females 1.49. Conclusions: In this study we have established reference ranges for normal Indian adults using the fully automated Single Platform Technology. The lymphocyte subsets values of our population are closer to those of the population from Botswana and China rather than the Western population. The absolute CD3 and CD4 counts and the CD4:CD8 ratio are higher in females than in males. Consistently higher values are obtained by the DPT as compared to the SPT.

Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues are the most invaluable source of diagnostic material for studying pathogenesis of cancer and a variety of other diseases. Unfortunately, DNA extracted from formalin fixed tissues is highly degraded due to cross-linking between nucleic acid strands. Real Time PCR has become the standard for gene copy as well as RNA transcript determination. Thus, optimum standardization of Real Time PCR is crucial for obtaining accurate quantification for both research as well as for clinical diagnosis. However there are various factors which have negative impact . The aim of our study was to establish a simpler method of extraction and Real Time PCR Optimization for FFPE extracted DNA. Five breast cancer tissues that were formalin fixed and paraffin embedded were used for DNA extraction with four different methods. Extracted DNA was amplified with different primer sets that gave amplimers of different size. Optimization of Real Time PCR for EMSY, cyclin D1 and beta-globin genes was carried out on DNA obtained using heat treatment protocol for annealing temperature, primer concentration and template concentration. Highest quantity of DNA was obtained without the use of expensive reagents and in short time frame. PCR positivity was observed in case of shorter amplimer up to 250 bp in length. Amplimers of higher length failed to amplify with paraffin extracted DNA. Optimum annealing temperature for EMSY, Cyclin D1 and beta-globin genes were 60 degrees C, 60 degrees C and 61 degrees C respectively. Good results were seen with a primer concentration of 300 nM and 5 ng of template DNA. This study indicates that DNA obtained from formalin fixed paraffin embedded tissue is highly fragmented and can be used for successful amplification of shorter amplification products up to 250 bp in length. Optimization of real time PCR is important, especially while using SYBR green dye chemistry.

Cytogenetic investigation in chronic myeloid leukemia: study from an Indian population.

Chronic myeloid leukemia (CML) is a malignant neoplasm of hematopoietic cells characterized by abnormal proliferation of myeloid precursors, decreased rates of self destruction and an arrest in cellular differentiation. The bone marrow and peripheral blood accumulates all forms of mature and immature granulocytes, primarily blast cells. It is the most common type of leukemia seen in India, accounting for 30% of all leukemias. Cytogenetic analysis plays a vital and important role in the diagnosis of CML patients. The present study consists of cytogenetic evaluation of 175 CML cases from the Indian population with ages ranging from 6-86 years (mean of 42.8). The study population included 115 males (65.72%) and 60 females (34.28%) with a Male: Female ratio 1.9:1. Out of the 175 cases, 164 (93.7%) were successfully karyotyped while culture failure was observed for 11 (6.3%). Among the 164 reported cases, 53 (32.3%) showed a normal karyotype while within the 111 (67.7%) abnormal cases, 96 cases (86.5%) showed the presence of Philadelphia (Ph') chromosome with standard translocation t(9;22); Ph'+ve along with secondary aberrations was detected in 9 (8.1%) cases. Variants of Ph' chromosome were detected in only one case (0.9%). Ph'-ve CML with other chromosomal aberrations were detected in 5 (4.5%) cases, including +8, del 20q, del 11q and marker chromosome. Furthermore, we believe that availability of more advanced molecular techniques can be used as a supportive tool in CML diagnosis even though it cannot fully replace cytogenetics, which remains the backbone for laboratory investigation of the disease.