Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 8, Num. 2, 2002, pp. 69-72

Indian Journal of Human Genetics, Vol. 8, No. 2, Jul-Dec, 2002 pp. 69-72

The Micronucleus Test in Urothelial Cells of Cervix Cancer Patients

Gursatej Gandhi, Pankaj Sharma

Department Of Human Genetics,Guru Nanak Dev University, Amritsar 143 005, India
Address for correspondence: Dr. Gursatej Gandhi, Department Of Human Genetics,Guru Nanak Dev University, Amritsar 143 005, India, E-jrgandhi@sancharnet.in

Code Number: hg02014

Cervix cancer continues to be a common malignancy in Indian women in the absence of routine cervix examination. Most cases come to light as referrals of advanced gynecologic complaints. It was proposed to look for micronuclei in bladder cells (as a non-invasive method) of such women subsequently diagnosed with cervix cancer. Urine samples of just diagnosed cancer patients (n=25; 21-80 yrs) and of controls (with other gynecological problems; n=25; 21-70 yrs) were processed for the micronucleus test using standard protocols.

Significantly elevated frequency of cells with micronuclei was observed in 72% of the patients as compared to that among controls (16.7%). Percent frequencies of micronucleated cells were highest in patients in stage III; in older patients; in those with younger ages-at-marriage; who had highest number of pregnancies; and were of low socio-economic status. The test in urothelial cells indicates damage in a tissue, which is not the site where cervix cancer develops. As it utilizes a non-invasive procedure of sampling, if validated it may find use in mass screening of cervix cancer.

Key words: micronuclei, uterine cancer, bladder cells.

Introduction

Carcinoma of the cervix has been reported to be the commonest malignancy in women in india comprising around 24% of all cancers in females^1. Almost 20 per 100,000 indian women have cervix cancer and it has been estimated² that one in 63 is likely to suffer from it in her life- time since the recognized risk factors for it^3,4 like illiteracy, low socio-economic status, early menarche, early marriage, multiparity, first child birth at an early age, poor genital hygiene and genital infections are widely prevalent in this population.

However, the early detection of this cancer has reduced morbidity and mortality from it among the screened persons.⁵ In fact in developed countries, 80% of cases are curable because of early detection, however in developing countries, 80% of cases are incurable at the time of detection, if they are detected at all.⁶ The National Cancer Control Programme⁷ has the goal of screening for cervix and oral cancers. Unfortunately it has not been strictly implemented.

The clinical detection and management of cervical cancer can be improved with emerging knowledge base of its etiology, risk factors and epidemiology. Hence, the present study was an attempt to study the population of cervix cancer patients in the local hospitals for the etiology of the disease and the micronucleus (mn) assay was utilised to score for any cytogenetic damage in urothelial cells of patients subsequently diagnosed with cervix cancer. Micronuclei are important cytogenetic end-points for scoring aneugenic and clastogenic damage in various cell types.^8-10 The use of exfoliated cells in MN test has found applications in recognizing population groups at an elevated risk for cancer, to estimate synergistic and additive effects of carcinogen exposure and to pinpoint the site in an organ from which most carcinomas will develop.¹¹

Materials and methods

A survey of the patients attending the local hospitals for gynecological complaints (post-coital bleeding, inter-menstrual bleeding, leuchorrhea and prolongation of menstrual period) was made. Of these, 25 tested positive with Pap smear and hence comprised the patient group. Those testing negative (age-and socio-economic status matched) formed the control sample as a routine cervix examination is hardly ever undertaken by healthy women here.

Individual records were maintained for age (or age-at-detection, both being the same), age-at-marriage, age-at-first-pregnancy, reasons for consultation, economic status, dietary history, smoking and alcohol drinking patterns, any disease incidence, any environmental or medical exposure, etc. Each individual was explained the purpose of this study. Those who signed the consent form voluntarily were requested to provide mid-stream urine samples (~5 ml) before commencing with treatment. The samples collected in aseptic vials were transported in an ice-box to the laboratory and were processed within 3-6 hours of collection.

The protocol used by Chakrabarti and Dutta¹² with some alterations for the MN test in the urothelial cell population was followed. The urine sediment, after centrifuging at 1200 rpm for 30', was washed three times in phosphate buffered saline by centrifuging each time at 1200 rpm for 10 minutes. Smear preparations from the pellet (2-3slides/individual) were made and allowed to air dry. Fixation of cells was then carried out in absolute methanol for 20' followed by staining in May Grunwald's stain (0.25%; Hi-media, India) for 5'. Following a rinse in distilled water, counter-staining with Giemsa (1%; Hi-media, India) for 3 minutes was carried out. The preparations were mounted in d.p.x. (Hi-media, India) ,coded and scored blind thereafter. Depending on the cell population available, about 200-500 cells for each individual were scored under the low power (40x) of a binocular microscope, while the presence of micronucleated cells was confirmed under oil immersion (100x). The Student's t-test was used for the analysis of the data.

Results and discussion

The results of the individual interviews pertaining to the life-style and personal information revealed that the cancer patients (all females) were in the age range of 28-75 yrs and they had mostly married young (15 yrs). The number of abortions (n=21) and pregnancies was also higher in the cancer patients. Except for one subject who was a daily smoker of 2-3 cigarettes, no other patient or control individual had this habit. The pedigree records of the patients did not reveal any family history of cervix cancer.

Micronucleated cells were observed in 17 (72%) patients while in the control individuals, these were observed in only four of the 24 samples (one sample failed to yield cells). The frequency of mnd cells in patients was significantly higher as compared to that in controls (t_cal =6.244 t _tab 5% =2.014, 1% = 2.690, df = 47). The number of micronucleated cells varied from 1 to 3 per individual with the cells scored varying as per the amount of urine sample. The highest frequency of MND cells ( 0.928) was observed in a patient in stage iiib . She had also had an early marriage (15 years) though her age at detection was 60 years. She belonged to a low socio-economic status family and her reproductive history included four live-births and one spontaneous abortion.

The frequency of MND cells in lymphocytes (0.18), in buccal (2.40) smears,⁹ and in urothelial (0.73±0.22) cells¹⁰ of control groups has been reported to be generally low in literature. Rather, the North Indian (mostly male population) lacked micronuclei in their buccal epithelial cells^13,14 which the authors suggested could be due to good nutritive habits.

The percent MN frequencies in stage iii cancer patients in comparison to that in stage i was statistically significant (t_cal = 5.580, t_tab 5%=2.131 1%= 2.947, df = 15) as was also the case when MN data in stages ii and iii were compared (t_cal = 4.93, t_tab 5%=2.131, 1%= 2.947, df = 15). However, non-significance was seen when damage in stage I was compared to that in stage ii. Nonetheless, these data in comparison with that in the control were observed to be significant both at 5% and 1% levels.

The elevated MN frequencies in the present study finds a parallel with cytogenetic damage reported in literature for cervix cancer patients. Chromosomal breaks, sister chromatid exchanges and C-band heteromorphisms of chromosome 1 in patients with progressive cancerous and pre-cancerous lesions besides other chromosomal rearrangements involving chromosomes 1,4,5,11,14,15,17 have been reported in cervical carcinoma cell lines and in the cells of cervix cancer patients.^15,16 Modal chromosome counts of 47 and 48 in pre-invasive and invasive cervix cancer patients¹⁷ and trisomy 8 among cases of invasive squamous cell carcinoma¹⁸ were also observed. In cervical epithelial cells and leucocytes of patients with pre-cancerous and cancerous lesions of cervix, significant step-wise increase in the mean basal dna damage using Single Cell Gel Electrophoresis (scge) assay has been reported¹⁹ with the comet tail lengths greater in cervix epithelial cells. A slight increase in uterine smears of patients with micronuclei in moderate and severe dysplasia cases compared to that in the inflammatory and mild dysplasia ones had also been earlier recorded.¹²

Younger women indulging in sexual activity have been reported to be at a higher risk for cervix cancer.^20-23 The frequency of MND cells was observed to be maximum in patients belonging to 11-15 yrs' marriage age-group in the present study. Statistically significant results were obtained when the MN data in different age-groups was compared with the data in the respective parallel control and total control groups. When damage in 11-15 years' age-group of cancer patients was compared to that in 16-20 yrs' age group (t_cal=4.69, t_tab 5%=2.080, 1%= 2.831, df = 21) and to that in the patients marrying at 26 years (t_cal=5.68, t_tab 5%=12.706, 1%= 63.657, df= 1), significant and non significant results were obtained, respectively. A similar trend was observed for the 16-20 years' age group on comparison with 26-30 years' age group(t_cal=2.93, t_tab 5%=2.093, 1%= 2.861, df =19). In cervix smears of these patients, maximum MN frequency in the 11-15 years' age-group was also observed.²⁴

Invasive carcinoma was observed to be commoner in women older than 50 years with a prevalence rate of 0.47 per 1000 though mild, moderate and severe dysplasias and carcinoma in situ were reported in women aged between 25-39 years ²⁵. Other studies have reported the mean age range of cervix cancer patients as 49.08-50.30 years.^26,27 The frequency of mnd cells observed in 51-60 yrs' age-group was two folds higher to that in the 21-30 yrs'group. In other intermittent age-groups, though no trend for step-wise increase in MN frequency was noticed, yet significant damage with respect to the values in control was there. Generally, significant results were also observed on comparing the frequencies of MND cells within the study groups and their respective parallel counter parts.

In the cancer patients the number of pregnancies per individual were more as compared to those in controls. Multiparity is a recognized risk factor in cervix cancer ^22,23,28. Student's `t' test for the data in the 1-3 and 4-6 pregnancies' groups to their parallel control groups, highly significant differences for MN induction were obtained (t_cal=3.870, t_tab 5%=2.110, 1%= 2.898, df =17; t_cal=4.540, t_tab 5%=2.086, 1%= 2.845, df = 20, respectively). A similar significance has been detected for the total number of pregnancies and the mnd cells in the patients' and total control groups. However except for comparison of damage in the 1-3 pregnancy group to that in 4-6 group in cancer patients, all other categories showed significant results.

Low socio-economic status (ses) has been correlated with poor genital hygiene²⁷ and hence is a significant risk for inducing cervix cancer.^28,29 The samples for the present study were collected from government and charitable hospitals and comprised those in low and medium ses. On analysis of the MN data for the low ses cancer patients with the values in the parallel control and the total control groups, highly significant differences were obtained (t_cal=4.07, t_tab 5%=2.052, 1%= 2.771, df =27; t_cal=5.67, t_tab 5%=2.021, 1%= 2.704, df = 41, respectively). Statistically significance of results for middle ses cancer group in comparison with its parallel and total control groups was also observed (t_cal=5.41, t_tab 5%=2.101, 1%= 2.88, df =18 ; t_cal=5.84, t_tab 5%=2.050, 1%= 2.763, df = 28, respectively). Non-significant values were however obtained when both cancer groups were compared to each other (t_cal=0.729, t_tab 5%=2.069, 1%= 2.807, df =23).

Hence, cytogenetic damage in bladder exfoliated cells of cervix cancer patients by MN test has been observed. Though the collection of urine samples is much easier than collection of blood samples or cervix scrapings, yet the number of exfoliated cells obtainable were rather less. For this, a larger urine sample size is required. This is often not feasible as it gets to be contaminated with blood in patients with advanced stages of cervix cancer. However, the use of the MN test in non-invasive tissues of cancer patients is still attractive. The test (after validation) can find applications in pilot screening programmes. As india is a developing country, so tests like mnt which are not too expensive and have easy sampling methodologies can be suggested as a part of routine gynecological examination. The economics of the assay for early detection alongwith appropriate treatment measures can eventually assist in bringing down the morbidity and mortality associated with this cancer if our policy makers take a note of it.

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Editorial Comment

This interesting paper is published here to stimulate more work in this area, particularly to see positive predictive value and negative predictive value of this test in a large number of cases and to investigate the molecular pathology and cytogenetic changes if such findings, correlating with the malignant changes, before it is recommeneded as "test".

Copyright 2002 - the Indian Society of Human Genetics