Allelic variations in CYP2D6 gene and susceptibility to cervical cancer.

Epidemiological studies have identified a number of risk factors that contribute to the development of cervical cancer precursors and cervical cancer. These include infection with certain oncogenic types of human papillomaviruses (HPVs) and other socio-economic factors. Tobacco smoking is an independent risk-factor for cervical neoplasia. It has been found that polymorphism at loci that encode carcinogen-metabolizing enzyme such as cytochrome P450 2D6 (CYP2D6) catalyzing the detoxification of carcinogens may determine susceptibility to cervical cancer. Therefore, it is likely that an understanding of these allelic differences is important for determining an individual's risk of cancer and susceptibility to potentially toxic agents. The aim of the present study was to elucidate the role of CYP2D6 polymorphism and susceptibility to squamous cell carcinoma of the uterine cervix in Indian population. Therefore, the genotype frequencies at this locus in females suffering with low-grade CIN, high-grade CIN and squamous cell carcinoma were compared. The control group consisted of 77 females with normal cervical cytology and the cases comprised of 61 mild/moderate dysplasia, 48 severe dysplasia and 45 cases of squamous cell carcinoma of uterine cervix. The individuals were divided into poor metabolizers (PM) and extensive metabolizers (EM) on the basis of their ability to metabolize certain drugs and carcinogens. Comparison of the frequency distribution for the combination of CYP2D6 EM genotype and smoking between mild/moderate and severe dysplasia was statistically significant (p=0.047) suggesting that women with cervical intraepithelial neoplasia I/II (CIN I/ CIN II) and CYP2D6 EM genotype who smoke appears to have more chances for the lesions to progress to CIN III. Whereas, frequency distribution for the same combination between severe dysplasia and squamous cell carcinoma failed to attain any statistical significance suggesting that CIN III with CYP2D6 EM genotype has less chance to progress to cervical cancer. Increased frequency of CYP2D6 EM and tobacco smoking show strong association with CIN III, indicating that not all lesions with the histopathological high grade CIN are premalignant. Conversely some squamous cell carcinomas may not be preceded by CIN.

Polymorphisms at GSTM1 and GSTT1 gene loci and susceptibility to cervical cancer in Indian population.

Multiple allelism at loci encoding detoxifying enzymes is associated with cancer risk. Glutathione S-transferase (GSTs) catalyzes the conjugation of glutathione to numerous potentially genotoxic compounds. This study evaluates the influence of genetic polymorphisms of GST M1 and GST T1 on susceptibility to cervical cancer. A multiplex polymerase chain reaction method was used to detect the presence or absence of the GSTM1 and GSTT1 genes in genomic DNA isolated from cases with cervical cancer (n=142) and normal controls (n=96). The results showed that the frequency of homozygous GSTM1 null genotype was higher in cervical cancer cases (57.0%) as compared to controls (34.4%) and the differences were significant (p<0.05), OR=2.5, 95% CI: 1.4--4.5. The frequency of homozygous GSTT1 null genotype in cancer cases was 19.7% in comparison to 12.5% in controls, however, the difference was not statistically significant (OR=1.7, 95% CI: 0.8-3.8). Significant difference was found between the cases and controls in the distribution of the null genotype of GST M1 in individuals aged above 45 years (p=0.04), but this difference was not significant in individuals aged below 45 years (p=0.06). No significant differences were found in cervical cancer cases and controls when data were analyzed according to age group for GSTT1 null genotype. Further, the combined analysis of both GSTM1 null and GSTT1 null genotypes did not appear to influence the susceptibility to cervical cancer, suggesting that polymorphisms of other detoxifying enzymes may play a significant role in cervical carcinogenesis.

Atypical squamous cells of undetermined significance: is it worthwhile to qualify them further?

BACKGROUND: In Bethesda System of reporting cervico-vaginal smears, the equivocal epithelial cell abnormalities have been categorised as "atypical squamous and glandular cells of undetermined significance" (ASCUS and AGUS). These abnormalities may harbour minor lesions attributed to mere inflammatory changes to potentially serious high grade lesions. It is recommended to further qualify these lesions according to whether they favor a reactive or a neoplastic outcome. AIMS: We undertook the present study to assess the significance of ASCUS qualifiers. MATERIAL AND METHODS: A total of 12071 women were screened for early detection of cancer cervix. The women with ASCUS diagnosis were further qualified as ASCUS favor reactive and ASCUS favor SIL (Squamous intraepithelial lesion) according the Bethesda 1991 system of reporting. ASCUS-reactive cases were followed up by a repeat smear examination and persistent ASCUS cases were taken up for colposcopy and biopsy if indicated. All ASCUS-SIL cases were taken up for immediate colposcopy. RESULTS: The pick up rate of high grade lesions was not significantly different (P=0.47; Chi square test) on the follow up of ASCUS-reactive (2/222) and ASCUS-SIL (2/130) groups. The low grade lesions were picked up in 10/222 ASCUS-reactive and 58/130 in ASCUS-SIL, which was statistically significant (P < 0.001). CONCLUSIONS: The results of our study indicate that it is not worthwhile to qualify these lesions as majority of ASCUS-SIL also revealed only low grade epithelial which have a benign biological behaviour.

A survey on risk factors associated with cervical cancer.

Carcinoma of the cervix is one of the leading malignancies in the developing countries including India. In view of this health care program might have to be redefined. Most of the data are available from the developed countries, which rely mainly on cytology screening on regular basis. This however may not be feasible in developing countries because of various constraints. Thus alternative approaches are needed based on risk reduction modalities. This article while dealing with the control approaches based on secondary prevention, reviews several risk factors associated with cervical cancer. The various articles were approached through Medline search including cross-references. The important life styles associated with cervical cancer and which are amenable to primary prevention strategies through health education, behavioral interventions, legislative approaches and modifying the health care seeking behavior were identified through the review process. These factors mainly pertain to early sexual debut, multiple sexual partners, menstrual hygiene and unprotected sex. Role of male partners has also been delineated in the process of cervical carcinogenesis. These factors are essentially conducive to the transmission of an etiological agent; the high-risk types human papillomaviruses, the more proximal cause in the web of causation. Barrier method of contraception and prophylactic vaccine in future could help to check the transmission of the virus. Role of smoking and oral contraceptives has also been discussed. Till the facilities for mass scale screening are developed in developing countries the primary prevention approaches could certainly help to check the incidence of the disease.

Genetic deletion and human papillomavirus infection in cervical cancer: loss of heterozygosity sites at 3p and 5p are important genetic events.

"High-risk" human papillomavirus (HPV) types 16/18 are recognized as being associated with cervical cancer. We have already reported frequent loss of heterozygosity (LOH) at 3p, 3q, 4q, 5p and 5q in primary cervical cancer, the frequency at these sites ranging from 31.0 to 56.3%. The present analysis deals with these frequent-deletion sites in relation to HPV infection. HPV frequency, as determined by PCR and by Southern-blot, was 87.0%. Out of 45 HPV-positive tumours, 26 (57.8%) were "high-risk" HPV-16/18-positive, with HPV type 16 predominant (24 of 26). Among these 24, 14 (58.3%) tumours had HPV-16 DNA integrated with the host genome. LOH on 3p revealed significant association with HPV-16/18 infection; 64.0% of LOH at 3p was in HPV16/18-positive tumours, vs. 23.3% in the tumours that did not reveal the presence of HPV16/18. On the other hand, 78.6% of LOH at 5p, which was the most frequent (56.3%), was in tumours without HPV16/18 infection, vs. 43.7% in tumours that had HPV 16/18 infection, suggesting independence of HPV 16/18 infection. At other sites, LOH did not differ, irrespective of the presence or absence of "high-risk" HPV infection.

Frequent amplification of C-erbB2 (HER-2/Neu) oncogene in cervical carcinoma as detected by non-fluorescence in situ hybridization technique on paraffin sections.

Sixty primary untreated squamous cell carcinomas were studied for C-erbB2 gene amplification by non-fluorescence in situ hybridization technique. Amplified tumour cells showed intranuclear dark brown, often paired signals under light microscopy. Twenty-two out of 60 tumours (36.6%) showed signs of amplification of different degree (+ to +). Considerable heterogeneity of C-erbB2 amplification per cell was seen within each amplified tumour. The proportion of amplified cells ranged between 10 and 90% in these tumours. The arrangement of C-erbB2 in clusters in most amplified tumours suggests that C-erbB2 amplification occurs intrachromosomally. No significant difference in the frequency of amplification was observed according to clinical stage or the histological patterns of the tumours. The results of the present study showed frequent amplification of C-erbB2 in cervical carcinoma on paraffin sections. This technique was found to be more sensitive than the Southern blot technique which could detect amplification in 14% of the tumours of the same cohort.

Genetic alterations at 5p15: a potential marker for progression of precancerous lesions of the uterine cervix.

BACKGROUND: Development of uterine cervical cancer is preceded by preneoplastic proliferative changes in the cervical epithelium called "intra-epithelial neoplasia" or "dysplasia." The genetic basis of the origin and progression of such preneoplastic lesions is not known. By analysis of carcinomas for loss of constitutional heterozygosity (LOH), we have previously shown a high frequency of allelic loss in the short arm of chromosome 5 (5p), suggesting loss of a candidate tumor suppressor gene located in 5p and associated with the development of this tumor. PURPOSE: To further understand the role of genetic alterations that affect 5p in cervical carcinogenesis, we evaluated the status of microsatellite polymorphisms at five loci mapped to 5p14-ter in precancerous and cancerous lesions. METHODS: Biopsy specimens from two groups of patients were analyzed for genetic alterations affecting 5p. One group comprised 14 cases of precancerous lesions (i.e., dysplasias) and five cases of carcinoma in situ (CIS); the second group comprised 46 previously untreated patients with invasive carcinoma. Tumor and normal DNAs were analyzed by polymerase chain reaction for genetic losses and instability at five polymorphic microsatellite loci (D5S392, D5S406, D5S208, D5S117, and D5S432) mapped to 5p. RESULTS: LOH was observed in 25 (55.6%) of 45 informative invasive carcinomas, one (20%) of five cases of CIS, and three (21%) of 14 precancerous lesions. Among the loci tested, D5S406 (5p15.1-15.2) exhibited LOH in 12 (48%) of 25 invasive carcinomas, one (33%) of three cases of CIS, and three (60%) of five precancerous lesions, suggesting this to be the site in 5p of the novel candidate tumor suppressor gene. In addition, replication error-type alterations were noted in the 5p14-ter region in six (13%) of 46 invasive carcinomas, two (40%) of five cases of CIS, and three (21%) of 14 precancerous lesions. Instability affected D5S406 in eight (66.7%) of 12 instances that showed microsatellite instability. CONCLUSION: These observations suggest that allelic loss and microsatellite instability in the region of D5S406 may play a role early in the development of cervical carcinoma and identify the site of a candidate tumor suppressor gene. These genetic markers (allelic loss and microsatellite instability) may also define CIS and precancerous lesions at high risk for progression to invasive cancer. IMPLICATIONS: The future molecular cloning of the candidate tumor suppressor gene at 5p15.1-15.2 may provide new insights into the genetic mechanisms of cervical carcinogenesis. Analysis and clinical follow-up of a large cohort of prospectively ascertained cases of precancerous lesions would help to validate the usefulness of these markers.

Allelotype analysis of cervical carcinoma.

To identify the genetic events which may play a role in the development of cervical carcinoma, we performed a detailed allelotype analysis utilizing DNA from 53 primary tumors and corresponding normal cells and 57 polymorphic probes mapped to each of the chromosomal arms, excluding the short arms of the acrocentric chromosomes. Loss of heterozygosity (LOH) of > 25% was observed at sites on 11 chromosomal arms, which included 1q (26%), 3p (35%), 3q (31%), 4q (46%), 5p (53%), 5q (38%), 6p (28%), 10q (28%), 11p (42%), 18p (38%), and Xq (26%). The most frequent LOH was noted on 4q (ADH3) and 5p (D5S19), suggesting that loss of candidate tumor suppressor genes on these chromosomal arms may play a role in the development of cervical carcinoma. The two sites of deletions identified on 5p and Xq represent novel candidate tumor suppressor gene sites which have so far not been reported in any other tumor type. Human papilloma virus status did not correlate with any of the sites which showed frequent LOH. TP53 mutation analysis by single-strand conformation polymorphism analysis was performed in 17 tumors that either showed 17p deletions (TP53, D17S5, or D17S28) or were human papilloma virus negative. One of the 7 human papilloma virus-negative tumors, which also showed LOH at the D17S28 locus, had a mutation in exon 5. This study represents the first comprehensive genetic analysis of this cancer and identifies several novel features of significance to genetic etiology of cervical carcinoma.

i(5p) and del(6q) are nonrandom abnormalities in carcinoma cervix uteri. Cytogenetics of two newly developed cell lines.

The karyotypes of two recently developed cell lines derived from squamous cell carcinomas of uterine cervix have been analyzed and are described here. Several clonal rearrangements were identified in both cell lines. Abnormalities common to these two cell lines were i(5)(p10) and del(6)(q15q26). The i(5p) was confirmed by use of whole chromosome 5 painting probe in fluorescence in situ hybridization. Based on these findings and review of published data on the cytogenetics of carcinoma of uterine cervix these two lesions are considered to be nonrandom abnormalities associated with this tumor.

ERBB2 (HER2/neu) oncogene is frequently amplified in squamous cell carcinoma of the uterine cervix.

We evaluated a panel of 22 protooncogenes for amplification in 50 primary, untreated squamous cell carcinomas of the uterine cervix. The tumors studied belonged to clinical stages II and III; histologically, the majority of them were moderately to well differentiated. Amplification represented by 5 or more copies was observed for the genes MYCL1, SEA, CCND1, BCL1, and GLI in one case each (2%); HRAS in 2 cases (4%); and ERBB2 in 7 cases (14%). Amplification of ERBB2 ranged from 5 to 68 copies. In addition, 2 tumors with ERBB2 amplification showed additional restriction fragments suggesting possible mutation or rearrangement of the gene. The high incidence of ERBB2 amplification in cervical cancer suggests that this gene may play an important role in tumorigenesis.

Cervix tumour cell line established from tumour after long term passages as heterotransplant in nude mice.

One human cancer of the uterine cervix xenograft was established in tissue culture only after repeated passages in nude mice suggesting that with the repeated passages in nude mice, tumour cells acquire some properties which allow them to grow in vitro. Attempts to establish cell line tumours from earlier passages were not successful. The established cell line is tumourigenic. On inoculation of cultured cells in nude mice tumour take was found to be 100%. Karyotypic analysis revealed human origin.

Risk factors related to biological behaviour of precancerous lesions of the uterine cervix.

In a study of factors related to cervical carcinogenesis, a cohort of 1,107 cervical dysplasia along with 1,077 controls matched for age and parity were followed up prospectively. During the follow up 75 dysplasia cases progressed to carcinoma in situ. The overall rate of progression of dysplasia to malignancy was observed to be 15.7% at the end of 108 months of follow-up. The analysis of progression rates in relation to various factors revealed significantly higher progression rates for initially higher grade of dysplastic lesions, and early age at consummation of marriage (ACM). The other factors, such as religion, literacy status of the patient, number of pregnancies, presence of cervical erosion, history of fetal loss and positivity to HSV-II antibodies, did not reveal statistical significance. The case-control comparison for detection of HPV 16/18 by in situ hybridisation revealed the presence of HPV 16/18 sequences in 67.3% of the dysplasia subjects progressed to carcinoma in situ while 27.3% of precancerous cases regressed to normalcy. The difference was found to be statistically significant (P less than 0.001).

Chromosomal phenotypes in patients with precancerous lesions of the uterine cervix progressed to cancer during follow-up.

A cytogenetical analysis of chromosomal aberrations, sister chromatid exchanges (SCEs), C-band heteromorphisms and nucleolar organizer regions was done in lymphocytes of 30 patients with precancerous lesions of the uterine cervix progressed to carcinoma and of 30 patients with nonprogressed lesions in a period of 60 months follow-up, in order to identify if any differences existed between low-risk and high-risk dysplasias. Increased frequency of chromosomal breaks, SCEs and C-band heteromorphisms of chromosome 1 and decreased activity of ribosomal genes was observed in patients with dysplasias progressed to carcinoma compared to that of nonprogressed precancerous lesions. The present study thus suggests an intimate relationship between the constitutional chromosomal phenotypes and development of cancer, and these may serve as useful biological markers in defining dysplasias at high risk to progress to cancer.

Heteromorphisms of C-bands in patients with precancerous and cancerous lesions of the cervix uteri.

C-band heteromorphisms of chromosomes 1, 9 and 16 were studied in 62 patients with cervical cancer, 100 women with various grades of precancerous lesions and 47 normal women as controls. The data showed an increased frequency of heteromorphisms of chromosome 1 in patients with cancer (48.39%) and severe dysplasias (40%) as compared to controls (29.8%) and lower grades of dysplastic lesions, i.e. mild and moderate (28.8%). The increase in the incidence of chromosome 1 heteromorphisms in cancer was found to be statistically significant (p less than 0.05) compared to controls. The present study indicates that C-band heteromorphisms may play some role in the development of malignancy of the uterine cervix.

Mitomycin C induced chromosomal aberrations and sister chromatid exchanges (SCEs) in lymphocytes of patients with precancerous and cancerous lesions of the uterine cervix.

Baseline and mitomycin C (MMC)-induced chromosomal aberrations and sister chromatid exchanges (SCEs) in blood lymphocytes of patients with cervical precancerous and cancerous lesions and normal women were studied. The baseline frequency of chromosomal aberrations and SCEs revealed a significant increase in higher grades of precancerous (moderate and severe dysplasias) and cancerous lesions compared to those of controls. The MMC-induced chromosomal aberrations and SCEs did not show any differential response in the different groups studied. The results thus indicate that chromosomal instability as observed in precancerous and cancerous lesions is not associated with their sensitivity to mitomycin C.

The number of silver-staining NORs (rDNA) in lymphocytes of newborns and its relationship to human development.

The number of silver-staining NORs (rDNA)/cell and their pattern of distribution were studied in phytohaemagglutinin-stimulated blood lymphocyte chromosomes of different age group individuals starting from newborns to old age (0-75 years) in order to investigate if the number of Ag-NORs or rDNA genes varies during development in humans. The results indicate presence of a relatively high modal number of NORs in newborns and infants (9.00 and 8.00/cell, respectively) and a significantly reduced number in old individuals (6.00/cell) as compared to that of normal adults (7.00/cell). These data are complimentary as well as comparable to the previous findings of Denton et al. (Mech. Ageing Dev., 15 (1981) (1-7). It is suggested that at young age due to an obvious enhanced growth and differentiation more gene sites may be transcriptionally active showing higher number of silver-stained NORs but as the development proceeds and the age advances many of these may be gradually repressed or inactivated.

Sister chromatid exchanges in patients with precancerous and cancerous lesions of cervix uteri.

The frequency of sister chromatid exchanges (SCEs) was studied in leucocytes from 46 patients with cervical carcinoma, 89 precancerous lesions, and 43 age-matched control women. The frequency of SCEs was found to be 10.15 +/- 2.49 in cancer, 8.83 +/- 2.15 in precancerous lesions, and 7.55 +/- 2.24 in controls. The analyses of SCE data revealed a highly significant (P less than 0.001) increase in precancerous and cancerous lesions compared to controls. The intra-chromosomal distribution of SCEs revealed a random increase in various chromosomal groups in patients with cancer and dysplasia compared to controls. The mean SCE level among various groups of precancerous lesions according to severity of pathological condition did not show significant differences. However, 70.8% of dysplasia cases revealed SCE levels higher than the average in controls. The increased frequencies of SCEs in the majority of cancer patients and a few precancerous lesions indicate that individuals with high SCE levels may be at a high risk of developing cancer. Thus the usefulness of SCE levels as a preclinical marker to identify the high risk group of dysplasias needs to be ascertained by follow-up studies; these are in progress.