Phylogenetic analysis of Human papillomavirus 16 variants isolated from Indian Breast cancer patients showed difference in genetic diversity with that of cervical cancer isolates.

The genetic variations of HPV16 in Breast Cancer (BC) are not well studied unlike HPV16 in Cervical Cancer (CACX). In this study, the genetic variations of HPV16 in BC were compared with HPV16 in CACX. In sequencing analysis of LCR, E6 and E7 regions of HPV16 in BC and CACX the A lineage was seen to be 64.2% and 66.6% respectively. The other lineages showed differential frequency in BC and CACX. The mutation frequency index of the regions in BC and CACX was in the following order: LCR>E6>E7. However, the inter-patient genetic diversity in LCR and E6/E7 regions was high in BC than CACX. The LCR region showed more variations than the E6/E7 region in BC. Apart from some common variations, some unique tissue specific variants in LCR and E6/E7 region were seen in BC and in CACX. Besides the selection of some common variants in both BC and CACX, some unique variants in BC (D98Y; 395 G>T) and CACX (R48W; 245 G>T) were observed. The 7521 G>A variant of LCR showed association with Luminal B subtype of BC and progression of CACX. Whereas, 145 G>T (Q14H) and 335 C>T (H78Y) variants of E6 showed association with either early invasiveness of BC and/or poor outcome of the patients. Thus, this study indicates that there may be a difference in the genetic variation of HPV16 in BC and in CACX.

Inactivation of SLIT2-ROBO1/2 pathway in premalignant lesions of uterine cervix: clinical and prognostic significances.

The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion:ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation:SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥ 5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (<19 years) had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease.

Prevalence of human papillomavirus in women without cervical cancer: a population-based study in Eastern India.

Despite the high incidence of cervical cancer, population-based data on prevalence of human papillomavirus (HPV) are limited in India. This study aimed to evaluate the prevalence of any HPV type and type-specific prevalence of HPV 16/18 in women without cervical cancer. HPV viral load was measured and correlated with cytologic abnormalities of the cervix. A total of 2501 women between 25 and 65 years of age and without cervical cancer were screened by pap smear cytology. HPV DNA was detected from cervical scrapes by nested polymerase chain reaction. Detection of HPV 16/18 was carried out by polymerase chain reaction using type-specific primers and was confirmed by Southern hybridization. Viral load was determined by absolute real-time polymerase chain reaction. Population prevalence of any HPV was found to be 9.9%. The risk of HPV infection was higher in women aged 25 to 34 years (odds ratio, 1.11), in married women below 20 years of age (odds ratio, 1.80), and in women with parity ≥4 (odds ratio, 1.04). Prevalence of HPV 18 (1.4%) was greater than that of HPV 16 (0.6%) in the overall screened population. High-grade squamous intraepithelial lesion cytology was more frequent in women infected with HPV 16 than in those infected with HPV 18 and other types. A gradual increase in HPV copy numbers was associated with progressive cytologic severity. In this study, HPV prevalence is comparable to HPV prevalence reported by other studies among Indian and Asian women. Although the prevalence of HPV 18 was more than that of HPV 16, type 16 infection was associated with higher oncogenicity.

Alterations of RASSF1A in premalignant cervical lesions: clinical and prognostic significance.

This study aimed to understand the importance of RASSF1A and CACNA2D2, located in chromosomal 3p21.31 region, in the development of uterine cervical carcinoma (CACX). To this end, firstly the expression (RNA) profiles of RASSF1A and CACNA2D2 were screened in primary cervical carcinoma (CACX) samples which indicated highly reduced expression for both genes. Thereafter alterations (deletion/methylation) of these genes were analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, deletion was observed only for RASSF1A (26%), whereas methylation was in the following order: RASSF1A (35%) > CACNA2D2 (9%). However, in CACX their deletion frequencies were the same (50%) and methylation frequencies were comparable RASSF1A (33%), CACNA2D2 (27%). The reduced expression and molecular alterations of these genes were concordant. Overall alterations of RASSF1A showed association with CIN lesions and CACNA2D2 with disease progression from CIN → stage I/II. Interestingly, alterations of these genes showed significant association in CACX suggesting possible functional synergism during tumor progression. Alterations of RASSF1A and CACNA2D2 predicted poor prognosis for the patients. Moreover, RASSF1A alterations along with multiparity (≥5 yr) and early sexual debut (<19 yr) were determinants of worse prognosis. Our data suggests the association of RASSF1A and CACNA2D2 in cervical carcinogenesis and its importance in early diagnosis and prognosis of the tumor.

Genetic and epigenetic changes of HPV16 in cervical cancer differentially regulate E6/E7 expression and associate with disease progression.

OBJECTIVE: The study was aimed at understanding the complex interactions of genetic and epigenetic events in expression of HPV16 E6/E7 and progression of cervical carcinoma. For this, expression of E6/E7 was done in 36 samples, along with the physical status, methylation and LCR sequence variations. Later, the genetic and epigenetic studies were extended to 239 samples to find out the association of these factors with progression of cervical cancer. METHODS: E6/E7 expression was quantified by real-time PCR. Physical status of HPV16 was determined by mutiplex-PCR of whole E2 ORF using overlapping primers and E6 ORF and validated by real-time PCR. Methylation status of P97 promoter/enhancer was analyzed by methylation sensitive restriction analysis (MSRA). Viral lineage and variations in LCR was ascertained by sequencing LCR/E6/E7 ORFs. RESULTS: Samples with episomal unmethylated virus showed comparatively high expression of E6/E7 than episomal methylated, integrated unmethylated and integrated methylated forms of HPV16. Variations in the LCR, particularly in the binding sites of negatively regulating transcription factors, also contribute to high expression of E6/E7. The integrated form significantly increases with decrease of episomal form during tumor progression. Methylation of the promoter/enhancer gradually decreased with tumor progression and is inversely correlated to integration. Two novel variants were observed in E6 gene in European- and North-American-1-lineages. Log-rank test revealed better prognosis of the patients with episomal methylated HPV16 compared to the other forms. CONCLUSION: Our results show higher expression of E6/E7 in samples with episomal unmethylated virus having sequence variations in LCR.

Alterations of ATM and CADM1 in chromosomal 11q22.3-23.2 region are associated with the development of invasive cervical carcinoma.

To understand the importance of chr11q22.3-23.2 region in the development of cervical cancer, we have studied the genetic and epigenetic alterations of the candidate genes ATM, PPP2R1B, SDHD and CADM1 in cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CACX) samples. Our study revealed low expression and high alterations (methylation/deletion) (55-59%) of ATM and CADM1 genes along with poor patient outcome. The alterations of ATM and CADM1 are associated with the progression of tumor from CIN to Stage I/II, thus implying their role in early invasiveness. The two genes, PPP2R1B and SDHD, lying in between ATM and CADM1, have low frequency of alterations, and majority of the alterations are in CACX samples, indicating that their alterations might be associated with disease progression. Expressions (mRNA/protein) of the genes showed concordance with their molecular alterations. Significant co-alteration of ATM and CADM1 points to their synergic action for the development of CACX. Mutation is, however, a rare phenomenon for inactivation of ATM. Association between the alteration of ATM and CHEK1 and poor survival of the patients having co-alterations of ATM and CHEK1 points to the DNA damage response pathway disruption in development of CACX. Thus, our data suggest that inactivation of ATM-CHEK1-associated DNA damage response pathway and CADM1-associated signaling network might have an important role in the development of CACX.

Inactivation of CHEK1 and EI24 is associated with the development of invasive cervical carcinoma: clinical and prognostic implications.

To understand the importance of frequent deletion of chromosomal 11q23.3-24.3 region in cervical carcinogenesis, alterations (deletion/methylation/mutation/expression) of the candidate genes LOH11CR2A, EI24 and CHEK1 located in the region were analyzed in 29 cervical intraepithelial neoplasia (CIN), 112 cervical carcinoma (CACX) samples and two CACX cell lines. The deletion frequency of these genes was low in CIN than in CACX [CIN: CHEK1: 28%, EI24: 21%, LOH11CR2A: 15% and CACX: CHEK1: 51%, EI24: 41%, LOH11CR2A: 36%]. Similar trend was seen in promoter methylation of these genes [CIN: CHEK1: 10%, EI24: 3%, LOH11CR2A: 3% and CACX: CHEK1: 55%, EI24: 31%, LOH11CR2A: 14%]. Mutations of the genes are a rare event. Overall alterations (deletion and methylation) of CHEK1 and EI24 were associated with progression of CACX. Quantitative mRNA expression analysis showed reduced expression of the three genes in concordance to their molecular alterations. A shorter isoform of CHEK1 lacking exon 8, hence impaired in substrate binding capacity, was found in two samples. Immunohistochemical analysis showed nuclear expression of Chek1, p-Chek1 and Ei24 in tumor tissues, whereas the cell lines exhibited both nuclear and cytoplasmic expression of Chek1 and Ei24, as is also evident from Western blot analysis suggesting differential localization of the proteins. Alterations of CHEK1 and EI24 coupled with tumor stage and early sexual debut (≤ 19 years) predicted worst prognosis. Thus, our data suggest that inactivation of EI24 and CHEK1 through two independent mechanisms contributes to the development of CACX.

Amplification of CyclinL1 in uterine cervical carcinoma has prognostic implications.

The chromosomal 3q25.31 region was consistently amplified in primary cancer of cervix (CACX). CyclinL1 is a candidate gene of this region and already have been implicated as an oncogene in head and neck cancers. In this study, we aimed to investigate the involvement of CyclinL1 in cervical carcinogenesis and for this purpose its copy number variation (CNV) was studied in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN lesions CyclinL1 was not amplified; however, the amplification frequency was 16% (9/56) in stage I/II tumors which remained comparable during subsequent stages of tumorigenesis. This implied association of CyclinL1 amplification with development of early invasiveness. Quantitation of mRNA expression revealed 2.6 ± 1.53-fold overexpression of this gene in primary CACX. The amplification/copy number gain of CyclinL1 and its mRNA profile were concordant, in tumors. Immunohistochemical (IHC) analysis in primary CACX, cell lines: SiHa and HeLa revealed intense nuclear expression of cyclinL1, which was further confirmed by Western blot in the cell lines. However 47% (7/15) CACX samples expressed high/intermediate level of cyclin L1. Kaplan-Meier survival analysis indicated CyclinL1 amplification as a determinant of poor patient outcome. Tumor radio-resistance developed as a consequence of CyclinL1 amplification. Cox multivariate analysis revealed that multiparous (≥5) CACX patients with amplified CyclinL1 locus along with advanced tumor stage (III/IV) had worst prognosis. Our data suggest importance of CyclinL1 in cervical carcinogenesis with its associated pathways viz: pre-mRNA splicing, cell-cycle regulation (G0/G1 and G2/M) being potential targets of therapeutic interventions in CACX.

RBSP3 is frequently altered in premalignant cervical lesions: clinical and prognostic significance.

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4-9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN --> Stage I/II and for ITGA9 from CIN --> Stage I/II and also from Stage I/II --> Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (>or=5)/early sexual debut (

Variant haplotypes at XRCC1 and risk of oral leukoplakia in HPV non-infected samples.

BACKGROUND: One of the mechanisms in human papillomavirus (HPV)-related carcinogenesis is inhibition of DNA repair by HPV oncoprotein. In this study, we investigated whether polymorphisms at XRCC1, one of the DNA repair loci, could modulate the risk of tobacco-related leukoplakia and cancer in HPV-infected individuals. METHODS: Tissue DNA from 83 oral cancer, 91 leukoplakia and 100 healthy controls were screened for HPV 16/18 infection and polymorphisms at XRCC1 by PCR-RFLP to estimate the risk of diseases independently and jointly. RESULTS: Human papillomavirus infection was significantly associated with increased risk of leukoplakia and cancer (OR = 2.8, 95% CI = 1.2-6.5 and OR = 5.5, 95% CI = 1.6-19, respectively). Independently, genotypes at three polymorphic sites on XRCC1 did not modulate the risk of diseases but pooled variant haplotypes increased the risk of leukoplakia in overall and HPV non-infected (OR = 1.8, 95% CI = 1.2-2.8; OR = 2.2, 95% CI = 1.2-4.0, respectively) samples but not that of cancer. CONCLUSION: The association between variant haplotypes at XRCC1 and risk of leukoplakia is pronounced in non-infected individuals since HPV oncoprotein could inhibit directly the DNA repair activity of XRCC1. But more samples of leukoplakia and cancer are essential to validate these results.

Influence of regular black tea consumption on tobacco associated DNA damage and HPV prevalence in human oral mucosa.

Black tea is more widely consumed than green tea worldwide, particularly in India. Therefore, it is necessary to focus attention on black tea with respect to its health promoting and anti-cancer actions. In order to establish the concept that black tea is a potential candidate for cancer prevention, it is important to provide epidemiological evidence derived from investigations of human populations. In view of this, the objective of the present study was to determine the correlation between nature of black tea consumption and DNA damage in normal subjects with or without tobacco habit and oral cancer patients, taking the latter as positive controls. Much experimental evidence points to associations between tobacco habit and HPV 16 and HPV 18 (Human Papilloma virus) infection. But no studies have taken into account the possible confounding effect of black tea consumption on DNA damage along with HPV infection. A pilot study was therefore undertaken. Comet assay was used to evaluate the DNA damage among normal subjects including tobacco users (n = 86), non-tobacco users (n = 45) and Oral cancer patients (n = 37). Percentage of damaged cells was scored in the buccal squamous cells of all subjects mentioned above. HPV analysis was performed on 79 samples (including 37 oral cancer patients). The evaluation of various confounding factors like age, tenure of tobacco habit and tea habit showed significant associations with DNA damage. The observations strongly indicate that regular intake of black tea at least above four cups can reduce tobacco associated DNA damage among normal tobacco users. HPV prevalence was not seen to be associated with age, tenure of tobacco habit or the tea drinking habit.

Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene.

The aim of this study was to locate the candidate tumor suppressor genes (TSGs) loci in the chromosomal 4p15-16, 4q22-23 and 4q34-35 regions associated with the development of uterine cervical carcinoma (CA-CX). Deletion mapping of the regions by microsatellite markers identified six discrete areas with high frequency of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2: 35-38%), 4p15.2 (D3: 37-40%), 4q22.2 (D4: 34%), 4q34.2-34.3 (D5: 37-59%) and 4q35.1 (D6: 40-50%). Significant correlation was noted among the deleted regions D1, D2 and D3. The deletions in D1, D2, D5 and D6 regions are suggested to be associated with the cervical intraepithelial neoplasia (CIN), and deletions in the D2, D3, D5 and D6 regions seems to be associated with progression of CA-CX. The deletions in the D2 and D6 regions showed significant prognostic implications (P = 0.001; 0.02). The expression of the candidate TSG SLIT2 mapped to D2 region gradually reduced from normal cervix uteri -->CIN --> CA-CX. SLIT2 promoter hypermethylation was seen in 28% CIN samples and significantly increased with tumor progression (P = 0.04). Significant correlation was seen between SLIT2 deletion and its promoter methylation (P = 0.001), indicating that both these phenomena could occur simultaneously to inactivate this gene. Immunohistochemical analysis showed reduced expression of SLIT2 in cervical lesions and CA-CX cell lines. Although no mutation was detected in the SLIT2 promoter region (-432 to + 55 bp), CC and AA haplotypes were seen in -227 and -195 positions, respectively. Thus, it indicates that inactivation of SLIT2-ROBO1 signaling pathway may have an important role in CA-CX development.