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.

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.

Peripheral blood lymphocyte subpopulations in Indian women with cervical intraepithelial neoplasia and invasive cancer--an immunocytochemical study using monoclonal antibodies.

The peripheral blood lymphocyte subpopulations in Indian women with various grades of cervical intraepithelial neoplasia [33], invasive cancer [22] and matched controls [30] were studied using specific monoclonal antibodies and biotin avidin immunofluorescence technique. Our results showed a significant fall in peripheral T (CD2) B(CD22) cells in patients with CIN III and invasive cervical cancer lesions compared to controls (P less than 0.001). Similarly the quantum of T helper cells (CD4) decreased according to the severity of cervical lesions (P less than 0.01 - P less than 0.001), whereas the T suppressor cells (CD8) depicted an increasing trend in patients with higher grade of cervical lesions (CIN III and invasive cancer, P less than 0.001) as compared to controls. Further, the CD4/CD8 ratio showed a significant downfall with the severity of cervical lesions (P less than 0.01 - P less than 0.001), indicating a perturbance in the homeostasis of host cellular immunity.

Double-minute chromosomes in the leukocytes of a patient with a previous history of cervical carcinoma.

Double-minute chromosomes (DMs) were observed in repeated samples in the leukocytes of a patient with a previous history of cervical carcinoma. The most interesting cytogenetic finding was the coexistence of DMs and a dicentric chromosome along with chromosome- and chromatid-type breaks and gaps. This observation suggests that DMs might originate through the breakage of existing chromosomes. The presence of DMs in leukocytes may also indicate the possibility that certain common agents cause DMs in tumor cells as well as in normal cells.

Spontaneous chromosomal aberrations in patients with precancerous and cancerous lesions of the cervix uteri.

Chromosomal aberrations were studied in metaphases from peripheral blood cultures of 52 women with cancer of the cervix uteri, 89 cases of various grades of cervical precancerous lesions, and 47 age-matched normal (control) women. The frequency of metaphases with chromosome and chromatid aberrations was 17.24% in cancer patients, 10.41% in those with precancerous lesions, and 6.39% in control women. There was a significant (p less than 0.001) increase in the frequency of chromosome aberrations in patients with cervical precancerous and cancerous lesions, compared with controls. After the exclusion of the treated cases, cancer patients also revealed a highly significant (p less than 0.001) increase in the frequency of chromosome aberrations, compared with controls. The results of the present study indicate the existence of chromosomal instability in the majority of cervical cancer patients and in some cases of precancerous lesions. The increased frequency of spontaneous chromosome aberrations in patients with precancerous lesions may be of importance for the understanding of their biological behavior.

Nucleolar organizer regions in patients with precancerous and cancerous lesions of the uterine cervix.

Nucleolar organizer regions (Ag-NORs) were studied in patients with cervical precancerous and cancerous lesions along with controls. The data revealed a statistically significant decrease of Ag-NORs in cancer patients, as well as in women with severe precancerous lesions as compared with controls. A similar decrease in the amount of Ag-staining was also observed in both cancer and severe dysplasia cases. The study suggests a possible relationship of Ag-NOR activity to malignancy.

Baseline frequency of sister-chromatid exchanges (SCE) in newborn lymphocytes and its relationship to in vivo aging in humans.

Heparinised cord blood from newborns and peripheral venous blood from three other age groups of individuals (1-75 years) have been cultured in vitro to obtain baseline frequencies of SCE and to see if the frequency of baseline SCE in vitro varies as a function of aging in vivo. The results demonstrate an age-dependent variation in the frequency of SCEs. Although the SCE frequency was lowest (5.10/cell) in 1-5-year-old infants, a significantly higher (P less than 0.001) frequency (8.97/cell) was observed in the cord blood of newborns. In old age, the level of SCE also increased. The plausible reason(s) for such observations is discussed.

Cytological monitoring of femal genital tract in women using Cu-IUD.

PIP: Cytological monitoring being conducted along with clinical trials of the CuT-200 and the Cu 7-200 has, to date, shown no significant progressive cytomorphological abnormalities. 1 of the 2135 women followed for 24 months developed an initially severe dysplasia which progressed into carcinoma in situ, but this could not be related to the IUD. Of the 1797 women who had previously used contraception, 60 revealed dysplastic changes. Vaginal discharge was the most common symptom; 52% of all dysplastic cases revealed healthy cervix on pelvic examination. At 24 months, 40.9% had inflammatory smears, compared with 39.8% preinsertion. A long-term study is recommended. In the meantime careful follow-up of all women wearing an IUD is advised.^ieng

Copper containing intrauterine devices and cervical carcinogenesis--48 months follow up.

PIP: An analysis of data on 815 acceptors of Cu-IUD who had 48 months of followup was conducted in order to study the role of Cu-IUD in cervical carcinogenesis. The acceptors were registered during the period from 1971 to 1972; this cohort would complete 48 months of use by the end of December 1976, the cut off date adopted for the study. Smears from ectocervix, endocervix and endometrial aspiarate were obtained from each women. The analysis was based on exocervical smears collected from women using Cu-IUD. Cytomorphological evaluation of the smears was based on the criteria established by the World Health Organization. Before IUD acceptance, 30 women had dysplastic lesion. An equal number developed dysplasia during the followup period. The rate of initial dysplastic lesion among acceptors was 3.7%. At the end of the 48 month period, all the cases of dysplasia observed either initially or during followup periods regressed to normalcy. The developed dysplasia cases were slightly younger than the initial cases. The mean age of developed dysplasia cases was 29.3 years as compared to 30.2 years for initial cases. The initial dysplasia cases were more with 1-2 parity; the newly developed dysplasia cases were in 3-4 parity group. As yet there is no conclusive evidence regarding the carcinogenic properties of copper containing IUDs.^ieng

Microphotometric nuclear DNA analysis in cervical dysplasia of the uterine cervix: its relation to the progression to malignancy and regression to normalcy.

The present study was attempted to reveal the predictive value of DNA ploidy pattern of uterine cervical dysplasia cases in relation to cervical carcinogenesis. Microphotometric nuclear DNA analysis using Feulgen stain was carried out in 310 cervical smears of 80 dysplasia cases consisting of 53 cases which progressed to malignancy and 27 cases which regressed to inflammation or normalcy during their follow-up periods. Aneuploid DNA pattern was observed in initial as well as follow-up smears in 69.8% of cases of the progressive group, and in 7.4% of cases of the regressive group. This difference is statistically highly significant (chi 2 27.88, p less than 0.001). In the progressive group, an aneuploid DNA value was observed in 40.0% of mild dysplasia, 71.9% of moderate dysplasia and 90.9% of severe dysplasia. In the regressive group, DNA aneuploidy was observed in only 9.1% of moderate dysplasia. This difference is statistically significant. These findings indicate that an aneuploid DNA value is a risk indicator for malignant potential of dysplasia cases.

A simulation approach for estimating the loss of womanyears due to cervical cancer and probability of developing cervical cancer.

Using a simulation approach, the number of womanyears that can be saved by preventing cancer of the uterine cervix was estimated. Probabilities of developing cervical cancer in the lifetime were also estimated. The number of years saved varied from 42 in the women aged between 20 and 24 years to 2 years in women aged 70 years and above. Probabilities of developing cervical cancer in the lifetime varied from 5.2% in women aged between 20 and 24 years to 0.6% in women aged 70 years and above. The results indicate that a noticeable incidence starts only after 35 years of age and about 90% of the total years can be saved by preventing the disease in women aged between 35 and 64 years.

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.

Risk for development of cancer in three urban areas of India.

The probability of developing cancer by using life table approach has been computed for the population of three metropolitan cities of India based on data of population based cancer registries located at Bangalore, Bombay and Madras. It was observed that the risk for development of malignancy of all sites from 20 to 64 years ranged from 4.73% to 5.28% in males, whereas it was 6.76% to 9.18% in females. The increased risk in females was mainly due to the high risk of development of cancer of the uterine cervix and breast. The available morbidity indices such as cumulative incidence rate and cumulative risk do not account for the mortality experiences of population. The present exercise will be useful in evaluating the changes in the disease spectrum as a result of change in the mortality experiences and population structure.

Efficacy of a hospital based cytology screening program.

From 1976 to 1986, a total of 117,471 women attending gynecologic outpatient departments of six hospitals in Delhi, India, were screened cytologically. The cytodiagnosis revealed 30,399 (25.9%) normal finding, 84,889 (72.3%) inflammatory changes, 1910 (1.6%) dysplasia of various grades and 213 (0.2%) malignant lesions. Of the 213 cases detected as malignant, clinical suspicion of cervical cancer was not present in 125 women (58.7%). Histologically malignancy was confirmed in 192 women (90.1%) of the 213 cytologically diagnosed malignant cases. The diagnosis revealed 94 (49.0%) as carcinoma in situ and the rest of the cases were invasive lesions. This was in contrast to only 5.2% (10/194) of cases with carcinoma in situ seen at the cancer clinic during 1983-1986 in one of the major collaborating hospitals of Delhi. The analysis of data according to age revealed that median age at detection of mild/moderate, severe dysplasia, carcinoma in situ (CIS) and invasive cancers was 34.0, 37.9, 38.6, and 47.8 years, respectively, indicating a latency period of one and a half decade from the onset of precursor lesions to invasive disease. Mass population screening in our country is not feasible in the near future and this may be true also for other developing countries. In its absence cytological screening of patients attending hospitals and maternity homes can give a large yield of early cervical cancers, which are curable.

Immunocytological demonstration of HSV-II antigen on exfoliated cells from precancerous and cancerous lesions of the uterine cervix.

Cancer of the uterine cervix is the most common malignancy among Indian women. Evidence has shown that cervical cancer is preceded by dysplastic, or precancerous, lesions. HSV-II has been associated with this process of cervical carcinogenesis. We collected 167 cervical smears that had been diagnosed as normal, dysplastic, or carcinoma-in-situ and found a significantly high positivity of HSV-II antigen in women with cervical dysplasia (38.8%) and carcinoma-in-situ (33.3%) versus women with normal cytology (17.02%). Latent HSV-II infection of cervical epithelium may be considered of prognostic value to identify a population at high risk for cervical cancer.

Supportive role of image analysis and DNA ploidy pattern in the diagnosis of thyroid tumors.

To evaluate the supportive role of image cytometry and DNA ploidy pattern in the diagnosis of thyroid tumors, a preliminary study was performed on fine needle aspirates of 30 cases. Of these, 10 cases each were of colloid goiter, follicular neoplasm, and papillary carcinoma. The nuclear area and DNA value of 50 cells in each case were measured. The mean nuclear area in colloid goiter (69.50 + 12.62 sq.microns) was significantly lower than the mean nuclear area in a follicular neoplasm (88.71 + 15.51 sq.microns) (P less than 0.05). Similar differences between the mean nuclear area in colloid goiter and papillary carcinoma (124.0 + 12.27 sq microns) was also highly significant (P less than 0.001). The results obtained by image cytometry were compared with estimated DNA ploidy pattern of follicular cells from the same cases. All colloid goiter had mean nuclear area below 100 sq.microns with diploid DNA value. However, papillary carcinoma showed aneuploid DNA pattern in eight cases (80.0%), but mean nuclear area was above 100 sq.microns. A diagnostically useful finding obtained in two of the 10 cases of follicular neoplasm was the association of aneuploid DNA pattern with mean nuclear area of the follicular cells above 100 sq.microns indicating a high probability of carcinoma and thus demanding an urgent open biopsy. These cases were readily distinguished from other cases of the same category showing diploid DNA pattern and mean nuclear area of follicular cells below 100 sq.microns.

Cytodiagnosis of nipple discharge: a study of 602 samples from 484 cases.

To find out the gross and microscopic differentiating features between nipple discharges (ND) due to various breast lesions, smears of 602 ND samples from 484 cases were reviewed by one of the investigators (D.K.D.). The reviewed cytodiagnoses were as follows: benign nipple discharge (59.1%), inflammatory ND (6.5%), ?papillary lesions (2.5%), papillary lesions (20.6%), papillary lesions with atypia (3.8%), duct cells with atypia (0.2%), suspicious for malignancy (0.5%), malignant ND (1.2%), and inadequate (5.6%). Following review, samples with epithelial abnormalities (?papillary lesion, papillary lesion with and without atypia, duct cells with atypia, suspicious for malignancy, and malignancy) increased from 16.6% to 30.4% of adequate samples (P < 0.0001). 37.9% unilateral ND samples showed epithelial abnormalities, as opposed to 18.9% of bilateral ND samples (P < 0.0001). Bloodstained ND showed epithelial abnormalities in 41.5% samples, as compared to 22.1% of ND with other specified gross characteristics (P < 0.0001). The samples with epithelial abnormalities differed significantly from benign and inflammatory ND in respect of frequency of benign duct cells, duct cells with atypia, papillary clusters with or without atypia, malignant cells, columnar cells, red blood cells, inflammatory cells, and background lipid vacuoles (P < 0.01 to < 0.0001). The ND samples with suspicious and malignant cytology, besides the presence of malignant cells (P < 0.0001), differed significantly from rest of the lesions in respect of foam cells (P < 0.0001), red blood cells (P < 0.01), and inflammatory cells (P < 0.05). When compared with histopathological diagnosis in 20 cases, the benign or malignant nature of the lesion was correctly identified in ND in 80% cases. The ND cytologies in 7 histologically proved malignant cases were malignancy (3 cases), suspicious for malignancy (1 case), papillary lesion with atypia (1 case), papillary lesion (1 case), and benign ND (1 case).

False negative cytologic diagnosis of breast carcinoma.

OBJECTIVE: To study the reasons for interpretive errors in false negative diagnosis of breast carcinoma on fine needle aspiration cytology material. STUDY DESIGN: We reviewed only those histologically proved malignant cases where the cytologic material was abnormal and to some extent misinterpreted. RESULTS: There were four lobular carcinomas and one each case of in situ, infiltrating duct, medullary and tubular carcinoma. Smears of lobular carcinomas were hypocellular overall, and the cells showed minimal nuclear pleomorphism. In situ, medullary and tubular carcinoma were associated with fibrocystic changes. The presence of bipolar cells and stromal fragments was misleading in cases of infiltrating duct carcinoma. CONCLUSION: The presence of associated fibrocystic disease may be a misleading factor since it may mask a malignancy. Hypocellularity and relatively nuclear monomorphism were the most common reasons for failure to diagnose malignant breast lesions. Careful attention should be paid to extreme nuclear monomorphism and absence of naked bipolar cells. A cytologically atypical or suspicious diagnosis together with radiologic suspicion should suggest a diagnosis of malignancy.

Fine needle aspiration cytology in the diagnosis of sinuses and ulcers of the body surface (skin and tongue).

Both fine needle aspiration (FNA) and conventional scraping were used to obtain cytodiagnostic samples from ulcers and sinuses of the skin (18 cases) and tongue (3 cases). These included 14 cases of tuberculosis, 5 squamous-cell carcinomas and 2 cases of nonspecific infection. The FNA smears contained characteristic tuberculous granulomas or epithelioid cells in 10 of the 14 tuberculosis cases and ample diagnostic material in 4 of the 5 carcinoma cases; in contrast the scraping smears predominantly contained necrotic material on which a diagnosis was not possible. Of the 19 cases of tuberculosis and malignancy, 12 were diagnosed only by FNA, 6 were diagnosed by both methods and the FNA sample was negative while the scrape was positive in 1 case. Hence, the diagnosis was made in 18 of 19 by FNA cytology, but in only 7 of 19 cases by scraping cytology, strongly indicating the diagnostic superiority of the former in diagnosing many lesions of the body surface.