Thangarajan Rajkumar

Cervical carcinoma and reproductive factors: Collaborative reanalysis of individual data on 16,563 women with cervical carcinoma and 33,542 women without cervical carcinoma from 25 epidemiological studies.

The International Collaboration of Epidemiological Studies of Cervical Cancer has combined individual data on 11,161 women with invasive carcinoma, 5,402 women with cervical intraepithelial neoplasia (CIN)3/carcinoma in situ and 33,542 women without cervical carcinoma from 25 epidemiological studies. Relative risks (RRs) and 95% confidence intervals (CIs) of cervical carcinoma in relation to number of full‐term pregnancies, and age at first full‐term pregnancy, were calculated conditioning by study, age, lifetime number of sexual partners and age at first sexual intercourse. Number of full‐term pregnancies was associated with a risk of invasive cervical carcinoma. After controlling for age at first full‐term pregnancy, the RR for invasive cervical carcinoma among parous women was 1.76 (95% CI: 1.53–2.02) for ≥≥7 full‐term pregnancies compared with 1–2. For CIN3/carcinoma in situ, no significant trend was found with increasing number of births after controlling for age at first full‐term pregnancy among parous women. Early age at first full‐term pregnancy was also associated with risk of both invasive cervical carcinoma and CIN3/carcinoma in situ. After controlling for number of full‐term pregnancies, the RR for first full‐term pregnancy at age <17 years compared with ≥≥25 years was 1.77 (95% CI: 1.42–2.23) for invasive cervical carcinoma, and 1.78 (95% CI: 1.26–2.51) for CIN3/carcinoma in situ. Results were similar in analyses restricted to high‐risk human papilloma virus (HPV)‐positive cases and controls. No relationship was found between cervical HPV positivity and number of full‐term pregnancies, or age at first full‐term pregnancy among controls. Differences in reproductive habits may have contributed to differences in cervical cancer incidence between developed and developing countries.

HUMAN PAPILLOMAVIRUS AND RISK FACTORS FOR CERVICAL CANCER IN CHENNAI, INDIA: A CASE-CONTROL STUDY

To evaluate the role of human papillomavirus (HPV) and other risk factors in the aetiology of invasive cervical carcinoma (ICC), we conducted a hospital‐based case‐control study in Chennai, Southern India. A total of 205 ICC cases (including 12 adenocarcinomas) and 213 frequency age‐matched control women were included. HPV DNA in cervical cells was evaluated by means of a polymerase chain‐reaction assay. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were computed by means of unconditional multiple logistic regression models. HPV infection was detected in all but one ICC cases and in 27.7% of control women (OR = 498). Twenty‐three different HPV types were found. HPV 16 was the most common type in either cases or controls, followed by HPV 18 and 33. The association of ICC with HPV 18 and HPV 16‐associated types was somewhat stronger than the one with HPV 16. Multiple HPV infections did not show a higher OR for ICC than single infections. Other than HPV infection, high parity (OR for >4 vs. ≤2 births = 7.3), a womans report of her husbands extramarital sexual relationships (OR = 10.0) and early menopause (OR for <45 vs. ≥45 years = 4.2) were significantly associated with ICC, also after restricting the analysis to HPV‐positive cases and controls. Poor hygienic conditions were associated with an increased risk of HPV infection among control women but not with ICC risk among HPV‐positive women. A vaccine against HPV 16 and 18 may be effective in more than three‐quarters of ICC in the study area.

Identification and validation of genes involved in cervical tumourigenesis

BackgroundCervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer.Materials and methodsA total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay.ResultsOur study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells.ConclusionsSeveral novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a therapeutic target in cervical cancer.

Identification and validation of genes involved in gastric tumorigenesis

BackgroundGastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets.Materials and methodsWe used 24 gastric cancers, 20 Paired normal (PN) and 5 apparently normal gastric tissues obtained from patients with non-gastric cancers (Apparently normal - AN) for the microarray study followed by validation of the significant genes (n = 63) by relative quantitation using Taqman Low Density Array Real Time PCR. We then used a custom made Quantibody protein array to validate the expression of 15 proteins in gastric tissues (4 AN, 9 PN and 9 gastric cancers). The same array format was used to study the plasma levels of these proteins in 58 patients with gastric cancers and 18 from patients with normal/non-malignant gastric conditions.ResultsSeventeen genes (ASPN, CCL15/MIP-1δ, MMP3, SPON2, PRSS2, CCL3, TMEPAI/PMEPAI, SIX3, MFNG, SOSTDC1, SGNE1, SST, IGHA1, AKR1B10, FCGBP, ATP4B, NCAPH2) were shown to be differentially expressed between the tumours and the paired normal, for the first time. EpCAM (p = 0.0001), IL8 (p = 0.0003), CCL4/MIP-1β (p = 0.0026), CCL20/MIP-3α (p = 0.039) and TIMP1 (p = 0.0017) tissue protein levels were significantly different (Mann Whitney U test) between tumours versus AN & PN. In addition, median plasma levels of IL8, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, PDGFR-B and TIMP1 proteins were significantly different between the non-malignant group and the gastric cancer group. The post-surgical levels of EpCAM, IGFBP3, IL8, CXCL10/IP10, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, SPP1/OPN and PDGFR-B showed a uniform drop in all the samples studied.ConclusionsOur study has identified several genes differentially expressed in gastric cancers, some for the first time. Some of these have been confirmed at the protein level, as well. Some of these proteins will need to be evaluated further for their potential as diagnostic biomarkers in gastric cancers and some could be useful as follow-up markers in gastric cancer.

Antibodies against high-risk human papillomavirus proteins as markers for invasive cervical cancer

Different human papillomavirus (HPV) genes are expressed during the various phases of the HPV life cycle and may elicit immune responses in the process towards malignancy. To evaluate their association with cervical cancer, antibodies against proteins from HPV16 (L1, E1, E2, E4, E6 and E7) and HPV18/31/33/35/45/52/58 (L1, E6 and E7) were measured in serum of 307 invasive cervical cancer cases and 327 controls from Algeria and India. Antibody response was evaluated using a glutathione S‐transferase‐based multiplex serology assay and HPV DNA detected from exfoliated cervical cells using a GP5+/6+‐mediated PCR assay. Among HPV16 DNA‐positive cases, seroprevalence of HPV16 antibodies ranged from 16% for HPV16 E1 to 50% for HPV16 E6 and all were significantly higher than controls. Seroprevalence of E6, E7 and L1 antibodies for HPV18 and for at least one of HPV31/33/35/45/52/58 were also higher in cases positive for DNA of the corresponding type (50% and 30% for E6 of HPV18 and HPV31/33/35/45/52/58 combined, respectively). E6 and E7 antibodies were rarely found in controls, but cross‐reactivity was evident among cancer cases positive for DNA of closely phylogenetically‐related HPV types. E6 or E7 antibodies against any of the eight HPV types were detected in 66.1% of all cervical cancer cases, as compared to 10.1% of controls. E6, and to a lesser extent E7, antibodies appear to be specific markers of HPV‐related malignancy. However, even among cases positive for the same type of HPV DNA, approximately one‐third of cervical cancer cases show no detectable immune response to either E6 or E7.

Role of glutathione-s-transferase and CYP1A1*2A polymorphisms in the therapy outcome of south Indian acute lymphoblastic leukemia patients.

Background: Polymorphisms in the drug-metabolizing enzymes are found to be associated with the inter-individual variation in response to a particular drug. Glutathione S-transferases (GSTs) are involved in the metabolism of several anticancer drugs, including alkylating agents, anthracyclines, and cyclophosphamides. Aim: The present study is aimed to examine the association of GST and CYP1A1*2A polymorphisms in the susceptibility to acute lymphoblastic leukemia (ALL) and the prognostic significance. Materials and Methods: A total of 92 immunophenotyped patients and 150 cord blood controls were genotyped by PCR for GSTM1 and GSTT1, RQ-PCR allelic discrimination assay for GSTP1 and PCR-RFLP for CYP1A1*2A polymorphism. Results: We have previously reported the significant association of GSTM1 (null) and combined GSTP1 {(Ile/Val)/ (Val/Val)} /GSTM1 (null) genotype with the susceptibility to ALL. No significant association was observed with GSTT1 (P=0.75) and CYP1A1*2A (P=0.61 for +/- and P=0.86 for -/- respectively) in the susceptibility to ALL. Survival analysis was performed in 50 of the 92 patients who were followed for three years. Kaplan-Meier survival analysis for three years showed significant lower event-free survival in patients harboring GSTP1 (Ile/Val) and GSTP1 (Val/Val) (P=0.038 and 0.0001, respectively) genotype. Cox regression analysis revealed GSTP1 as an independent prognostic marker with 6-fold higher risk with Val/Val genotype (P=0.003). Conclusions: Our results show that GSTP1 (Ile/Val) polymorphism has a role in the susceptibility to ALL and also influence treatment outcome.

A 7 gene expression score predicts for radiation response in cancer cervix

BackgroundCervical cancer is the most common cancer among Indian women. The current recommendations are to treat the stage IIB, IIIA, IIIB and IVA with radical radiotherapy and weekly cisplatin based chemotherapy. However, Radiotherapy alone can help cure more than 60% of stage IIB and up to 40% of stage IIIB patients.MethodsArchival RNA samples from 15 patients who had achieved complete remission and stayed disease free for more than 36 months (No Evidence of Disease or NED group) and 10 patients who had failed radical radiotherapy (Failed group) were included in the study. The RNA were amplified, labelled and hybridized to Stanford microarray chips and analyzed using BRB Array Tools software and Significance Analysis of Microarray (SAM) analysis. 20 genes were selected for further validation using Relative Quantitation (RQ) Taqman assay in a Taqman Low-Density Array (TLDA) format. The RQ value was calculated, using each of the NED sample once as a calibrator. A scoring system was developed based on the RQ value for the genes.ResultsUsing a seven gene based scoring system, it was possible to distinguish between the tumours which were likely to respond to the radiotherapy and those likely to fail. The mean score ± 2 SE (standard error of mean) was used and at a cut-off score of greater than 5.60, the sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) were 0.64, 1.0, 1.0, 0.67, respectively, for the low risk group.ConclusionWe have identified a 7 gene signature which could help identify patients with cervical cancer who can be treated with radiotherapy alone. However, this needs to be validated in a larger patient population.

Perspectives of long non-coding RNAs in cancer

A recent advance in transcriptomics has spawned the ‘Decade of non-coding RNAs’ by potentiating the growing numbers of long non-coding RNA in cancer. LncRNA involvement in cancer denotes its significance beyond our perception as they participate in tumor suppression and promoting oncogenesis, which raises them as a mighty class of effectors or regulators. Aberrantly expressed lncRNAs interact with major protein and coding partners, which ultimately deregulate normal cellular processes and drive the cell towards malignant state. Identification of theses interactions are utmost important as lncRNAs can be ideal targets for therapy. Dysregulation of lncRNAs by genomic alterations like single nucleotide variations and gene fusions are also potential modulators of their secondary structure. In this review, we discuss the various molecular interactions of lncRNAs with major bio-molecules and genetic variations in lncRNA genes and their importance in cancer. This systematic review outlines the vivid role of lncRNAs in cancer context and opens up future conceptual applications.

Role of Circulating Cell-Free DNA in Cancers

Liquid biopsy is a term used to describe non-invasive tests, which provide information about disease conditions through analysis of circulating cell-free DNA and circulating tumor cells from peripheral blood samples. In patients with cancer, the concentration of cell-free DNA increases, and structural, sequence, and epigenetic changes to DNA can be observed through the disease process and during therapy. Furthermore, cell-free DNA released by the tumor contains the same variants as those in the tumor cells. Therefore, cell-free DNA allows non-invasive assessment of cancer in real time. This review summarizes the origin of cell-free DNA, recent advancements in the detection of cell-free DNA, a possible role in metastasis, and its importance as a non-invasive diagnostic assay for cancer.

Genetic variants in nicotine addiction and alcohol metabolism genes, oral cancer risk and the propensity to smoke and drink alcohol: a replication study in India.

Background Genetic variants in nicotinic acetylcholine receptor and alcohol metabolism genes have been associated with propensity to smoke tobacco and drink alcohol, respectively, and also implicated in genetic susceptibility to head and neck cancer. In addition to smoking and alcohol, tobacco chewing is an important oral cancer risk factor in India. It is not known if these genetic variants influence propensity or oral cancer susceptibility in the context of this distinct etiology. Methods We examined 639 oral and pharyngeal cancer cases and 791 controls from two case-control studies conducted in India. We investigated six variants known to influence nicotine addiction or alcohol metabolism, including rs16969968 (CHRNA5), rs578776 (CHRNA3), rs1229984 (ADH1B), rs698 (ADH1C), rs1573496 (ADH7), and rs4767364 (ALDH2). Results The CHRN variants were associated with the number of chewing events per day, including in those who chewed tobacco but never smoked (P =  0.003, P =  0.01 for rs16969968 and rs578776 respectively). Presence of the variant allele contributed to approximately 13% difference in chewing frequency compared to non-carriers. While no association was observed between rs16969968 and oral cancer risk (OR =  1.01, 95% CI =  0.83– 1.22), rs578776 was modestly associated with a 16% decreased risk of oral cancer (OR =  0.84, 95% CI =  0.72– 0.98). There was little evidence for association between polymorphisms in genes encoding alcohol metabolism and oral cancer in this population. Conclusion The association between rs16969968 and number of chewing events implies that the effect on smoking propensity conferred by this gene variant extends to the use of smokeless tobacco.