A multivariate analysis of prognostic indicators in complete hydatidiform moles (CHM).

OBJECTIVE: To analyse prognostic factors in complete hydatidiform moles using multiple logistic regression analysis. METHODS: Evaluation of host and tumour related parameters including (a) gestational age, patient age, parity, molar phenotype, grade of proliferation of the tumour and cytological atypia, (b) expression of beta-HCG, EGF, EGFR, TGF-alpha, TGF-beta, IL1-alpha, IL1-beta by immunohistochemistry, (c) serial monitoring of serum beta-HCG levels by ELISA, and (d) lectin binding using jack fruit lectin histochemistry as indices for persisting trophoblastic disease (PTD). RESULTS: Serum beta-HCG levels at 4 weeks, cellular atypia, lectin binding, expression of TGF-alpha and IL1-beta showed highly significant correlation with persistence of the tumour (P<0.001). The sensitivity and specificity at 4 weeks in combination with cytological atypia to identify spontaneously regressing lesions was 100% and those requiring chemotherapeutic intervention was 80%. CONCLUSION: The concentration of serum beta-HCG 4 weeks post evacuation(<300 mIU/ml) combined with cytological abnormalities could identify nearly 100% of the spontaneously regressing lesions (low risk) and 80% of those needing chemotherapeutic intervention (high risk), thereby suggesting that patients who have a serum beta-HCG at 4 weeks of evacuation <300 mIU/ml with no cytological atypia of the trophoblasts need only be followed up at long intervals, while those having a serum beta-HCG at 4 weeks of evacuation >300 mIU/ml accompanied with cytological atypia of the trophoblasts should be closely followed up.

Immunolocalization and quantitation of transforming growth factor alpha in hydatidiform mole.

Complete hydatidiform mole (CHM), a condition related to abnormal gestation, occurs predominantly in the young reproductive age group and has a high prevalence rate in the Trivandrum region, occurring in 1.2% of deliveries. Transforming growth factor alpha (TGF-alpha) is an important growth regulatory molecule, the location and function of which at the human fetomaternal interface in CHM remains to be determined. The present study examined the presence of TGF-alpha in the normal and complete molar placenta and decidua throughout gestation. A total of 149 complete molar placental tissue samples and 96 normal placental tissue samples were evaluated for TGF-alpha expression by immunohistochemistry and 50 each of CHM and normal placental tissue for TGF-alpha concentration by radioimmunoassay. The peptide was localized immunocytochemically with a monoclonal anti-TGF-alpha antibody on paraffin-embedded tissue using the avidin-biotin complex peroxidase technique with aminoethyl carbazole as the chromogen. In molar placenta, villous trophoblast cells (syncytiotrophoblasts and cytotrophoblasts) showed intense cytoplasmic staining at all gestational ages compared to normal placenta of the same gestational age. The tissue concentration of TGF-alpha was highly overexpressed in the molar placenta (10-1000 fold) compared to that in the normal placenta. The results indicate that TGF-alpha is present in trophoblasts throughout human gestation and may provide additional growth advantage to maintenance of the hyperproliferative condition in trophoblastic tumors.

ras and c-myc oncoproteins during tumor progression in the uterine cervix.

AIMS AND BACKGROUND: Altered oncogenic activity is a feature associated with many malignant and premalignant conditions. Among the many oncogenes, ras and myc are commonly altered in many tumors. This study aims to evaluate the expression of ras and c-myc oncoproteins in a total of 204 cervical tissue samples, including premalignant and malignant lesions as well as apparently normal cervical tissue. METHODS AND STUDY DESIGN: Mouse monoclonal antibodies against the three mammalian ras gene products (c-H-ras, c-K-ras, c-N-ras) and the c-myc protein were used to evaluate oncoprotein expression by immunocytochemistry. RESULTS: None of the samples analyzed displayed immunoreactivity for H-ras and K-ras. Normal cervical epithelium showed minimal immunoreactivity for N-ras with about 33% of the samples expressing the protein. More conspicuous expression in normal tissue was displayed by c-myc, with about 90% of the samples expressing the protein (mean value of cells positive=34%). The immunoreactivity for N-ras increased with increasing histological abnormality from low-grade squamous intraepithelial lesions (SIL) to invasive carcinoma. Increased immunoreactivity for N-ras was evident in the basaloid cells of malignant lesions, with the maximum value of 66% found in poorly differentiated squamous cell carcinoma (PDSCC). The percentage of nuclei positive for c-myc also showed a gradual increase from low-grade SIL onwards, the highest positivity being found in PDSCC, where the mean value was 85%. Statistical analysis revealed a good correlation between the expression of N-ras (r=0.8922, P=0.001) and c-myc (r=0.8856, P=0.001) and various histological stages of tumor progression in the cervical epithelium. CONCLUSIONS: These results therefore suggest that c-myc and N-ras oncoproteins are important during tumor progression in the uterine cervix.

Lack of association of gestational trophoblastic diseases (GTD) with syphilis and AIDS.

The association between human immunodeficiency virus (HIV) infection and syphilis infection as an etiological factor in Gestational Trophoblastic Disease (GTD) was investigated by means of micro-enzyme linked immunosorbent assay (Micro-ELISA) and Treposcreen-Rapid Plasma Reagin Card Test in 138 sera from patients with Gestational Trophoblastic Disease. We have found only one sample to be positive for HIV infection and one for VDRL. These findings suggest a lack of an etiologic role for the HIV and Syphilis infection in GTD.

Proliferating cell nuclear antigen (PCNA) expression in gestational trophoblastic diseases (GTD).

Gestational Trophoblastic Disease is an abnormal condition of the placenta, the incidence of which is very high in the state of Kerala, India. The proliferative rate of molar placentas in comparison with the normal placentas of comparable gestational age group was done in order to find out its role in the prognosis of this tumor by assessing the expression of PCNA in trophoblasts. PCNA expression was evaluated in 149 trophoblastic tumors and 96 normal placental tissue. The percentage of positive cells was significantly increased in molar placentas of the 1st trimester in comparison to the normal placentas. Correlation of the staining score to the regression pattern of the tumor showed a significant increase in the chemotherapy group when compared to the spontaneously regressing group. But no correlation was found between the percentage of PCNA positive cells with histological grade of the tumor proliferation.

Cytokeratins and the evaluation of tumor differentiation in squamous lesions of the uterine cervix.

The differential expression of cytokeratins in epithelial or squamous cells has been demonstrated to be altered during the process of carcinogenesis. This altered expression of cytokeratins (CKs) may be closely related with epithelial differentiation and may remain stable in malignant tumors. In the present study an analysis using two monoclonal antibodies, CK 8.12 antibody specific for CK type 13 and 16 and CK 8.60 antibody specific for CK type 1, 10 and 11 was done in different grades of lesions in the uterine cervix. Changes from the normal expression pattern were seen in high grade Squamous intraepithelial lesions (SIL) (CIN-2/3) and invasive squamous cell carcinoma (SCC). No conspicuous difference in the staining expression between normal/benign cervical tissue and low grade SIL (CIN-I) was evident. Statistical analysis also revealed a significant correlation between the expression of these CK types to the differentiation status of the cervical lesions analyzed. Alterations in the expression of these CKs can be correlated to the differentiation pathway which may be deregulated during cervical carcinogenesis. The findings of the present study suggest that the expression of CK types 13 and 16 and 1, 10 and 11 using CK 8.12 and CK 8.60 antibodies respectively may serve as markers of differentiation in cervical squamous neoplasms.

p53 protein and tumorigenesis in the uterine cervix.

The relationship between molecular abnormalities of p53 tumor suppressor gene product and cancer has been well documented. That correlation may exist between immunocytochemically detectable amount of p53 protein and neoplasia is evidenced by several studies. Detection of p53 protein by immunocytochemistry varies depending on the methods and antibodies used. It has been suggested that the quantitative aspect of p53 protein expression and the proportion of cells expressing p53 may be of clinical importance in human malignancies. In the present study, we have examined the expression of p53 protein in various grades of lesions of the uterine cervix. Statistical analysis showed a good correlation between expression of p53 protein and histologic grade of lesions. Increased expression of p53 in dysplastic and malignant lesions compared to non dysplastic lesions suggests that p53 protein accumulation may be an early event in carcinogenesis.

The basement membrane and tumor progression in the uterine cervix.

Immunocytochemical localization of the basement membrane (BM) proteins laminin, type-IV collagen and fibronectin were analyzed in normal cervical epithelium, low grade squamous intraepithelial lesions (SILs), high grade SILs and invasive squamous cell carcinoma (SCC) of the uterine cervix. A regular, thick and continuous BM was present in normal cervical epithelium and low grade SIL. Interruptions and discontinuity of the BM were more evident in high grade SILs. There was a good correlation between increasing severity of the lesion and increasing number of breaks. In SCC, the distribution of laminin, collagen IV and fibronectin was related to the degree of cellular differentiation, with decreased immunoreactivity being evident in moderately and poorly differentiated tumors. As the invasive potential of the tumor increased, the fragmentation and loss of BM was more evident. Fibronectin showed only moderate to mild immunoreactivity in normal cervical epithelium and low grade SILs. However, the intensity of expression increased in high grade SILs especially in the peritumoral stroma. It may therefore be concluded from these results that snythesis and reabsorption of BM proteins may be related to shifts in cellular metabolism during tumorigenesis.

Expression of epidermal growth factor in gestational trophoblastic disease (GTD).

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Increased expression of cytokeratins 14, 18 and 19 correlates with tumor progression in the uterine cervix.

The expression of cytokeratins (CKs) in normal cervical epithelium, low grade squamous intraepithelial lesions (SIL), high grade SILs and squamous cell carcinoma (SCC) were analyzed using four different monoclonal antikeratin antibodies. In normal cervical epithelium, CK 18 showed strong immunoreactivity in basal and parabasal layers. CK 19 and 14 were expressed only in the basal layer while CK 13 was found selectively n the spinal cells. As the lesions progressed from low grade SIL to high grade SIL, immunoreactivity of CK 18, 19 and 14 in the basal cell compartment increased while the expression of CK 13 decreased. In SCC, as well-differentiated tumors showed decreased immunoreactivity for CK 18, 19 and 14 with CK 13 showing a strong and focal (localized) immunoreactivity. Undifferentiated carcinomas totally lacked CK 13 reactivity. Our findings therefore suggest that expression of CK 18, 19 and 14 may be directly related to tumor grade and CK 13 may be a marker of differentiation in cervical lesions.

c-erbB-2 oncoprotein and epidermal growth factor receptor in cervical lesions.

Epidermal growth factor receptor (EGF-R) and the oncogene product of c-erbB-2 have been shown to be expressed in human tumors and in some cases relate to clinical outcome. This study investigates the correlation between the presence of these receptor proteins and various histological grades of cervical tissues. Immunocytochemical analysis of benign, premalignant and malignant cervical tissues showed a highly significant correlation between the expression of the two proteins and the histological grade of the lesions. Moreover, the overall frequency of coexpression of these proteins was similar. This study suggests that these proteins may be involved in cellular proliferation and have a significant role in the progression of cervical cancer.

Expression of epidermal growth factor receptor in gestational trophoblastic diseases.

Gestational trophoblastic disease is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of this disease is vague and methods to determine the malignant potential are limited. Placentae of normal (50) and molar pregnancies (95), including 32 showing persistent disease, were used for the study. Epidermal growth factor (EGFR), expression was evaluated by immunohistochemistry using monoclonal antibody, and quantified using the 125I-EGF-binding assay. EGFR was expressed more strongly in molar placentae than in normal placentae of similar gestational age, and was strong in the molar placentae of all gestational ages, whereas expression was mainly restricted to the first and second trimesters in normal placentae. Moles with early presenting symptoms (before 16 weeks) were at a higher risk of developing persistent disease. To conclude, the immunohistochemical study shows high expression of EGFR in early normal placentae and in moles, linking its role to the proliferative and differentiating activity of trophoblasts. Tumours with a histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGFR. The present observations also suggest the possibility of mutated EGF receptors being present in persistent trophoblastic disease.

Involucrin and tumor progression in the uterine cervix.

The expression of involucrin, a cytoplasmic protein synthesized during squamous maturation, was assessed by immunocytochemistry in different grades of cervical lesions. In normal/benign cervical epithelium and low-grade squamous intraepithelial lesions [SILS or cervical intraepithelial neoplasia (CIN)-1] involucrin showed intense and homogenous cytoplasmic expression in the spinal layers of 75 and 57% of samples, respectively. The basal cell layers showed no expression of involucrin. In high-grade SILs (CIN-2/3) 40% of the samples showed diffuse and focal cytoplasmic expression of involucrin in the differentiated basaloid cells. In the squamous cell carcinomas (SCCs) analyzed, well-differentiated tumors showed intense focal expression in 61% of the cases, moderately differentiated SCCs showed intense expression in 33% of the cases, while poorly differentiated SCCs (PDSCC) showed only a mild focal expression in 7% of cases. With increasing severity of the lesions, patchy expression of involucrin with a mixture of reactive and nonreactive cells predominated. Patterns of immunocytochemical staining for involucrin in cervical lesions of different grades, from low-grade to high-grade SILs, and invasive carcinoma may be of critical importance, if loss of involucrin expression is used as a criterion for neoplastic transformation in cervical epithelium. Our findings suggest that involucrin may be a sensitive marker in identifying the differentiation status of the lesion while the absence of involucrin in PDSCC may be helpful in differential diagnosis.

Argyrophilic nucleolar organizer regions (AgNORs) in inflammatory pre-malignant and malignant lesions of the uterine cervix.

Nuclear organizer regions (NORs) code for ribosomal RNA and are associated with non-histone nucleoproteins, which can be identified by silver staining (AgNORs). AgNORs have been correlated to proliferative activity of tumors and hence may be prognosis-related. The present study evaluates AgNOR counts in inflammatory lesions of the uterine cervix, cervical intra-epithelial neoplasia, and invasive cervical squamous carcinoma. Significant variation in AgNOR counts were observed between the three study groups, with invasive carcinoma showing maximum counts. Further, a highly significant positive correlation was observed between AgNOR counts and tumor progression. These results therefore suggest that AgNOR counts may be of significance in the evaluation of cervical carcinogenesis and could elaborate histopathological diagnosis of cervical lesions.