Prabha Balaram

Pathogenesis of oral submucous fibrosis. Relationship to risk factors associated with oral cancer

Oral submucous fibrosis (OSF) is a chronic disease of the oral mucosa characterized by inflammation and a progressive fibrosis of the lamina propria and deeper connective tissues. It is a condition predominantly seen among people of Indian origin, and an epidemiologic survey done a decade ago showed no less than 250,000 cases in India, a figure that must have increased sharply. OSF is a condition with a high risk of malignant transformation; to date, no conclusive etiologic agent has been identified, although plenty of data have been generated on various aspects of the disease. These include genetic, carcinogenic, immunologic, viral, nutritional, and autoimmune possibilities, all of which also have been implicated in the development of oral cancer. This article reviews existing evidence on the pathogenesis of OSF and its relation to oral cancer and suggests a possible multifactorial model to explain the disease process.

Mutations and polymorphisms in the p53, p21 and p16 genes in oral carcinomas of Indian betel quid chewers

India has one of the worlds highest incidences of oral cancer. It is believed that the widespread habit of betel quid chewing is an important risk factor as it exposes the oral mucosa to known carcinogens. It also induces physical abrasions, which may create mitogenic environments during wound healing as gateways for infections. A recent study from our laboratories identified human papillomavirus (HPV) DNA, mostly of the high‐risk types HPV‐16 and HPV‐18, in 67 of 91 oral cancer lesions from a cohort of Indian patients consisting mostly of betel quid users. This suggested a viral etiology of some lesions but tumorigenesis in the absence of viruses in other lesions. Here, we examined whether the p53 gene, whose function is abrogated by the product of the HPV gene E6, would be mutated in those oral cancers that were free of HPV DNA, and we found point mutations at known hot spots for mutational alteration of p53 in 4 of 23 lesions. We also considered the possibility that p21, a target of regulation by the p53 protein, may be mutationally altered in tumors with a functional p53 gene. While we did not identify mutations in the p21 gene, 6 of 11 lesions contained a polymorphism that may be associated with cancer. Interestingly, 3 of 23 lesions had mutations in the p16 gene, a third regulator of the cell cycle which is frequently mutated in melanoma but rarely in other cancers, with 1 lesion even having a mutation in the p53 as well as in the p16 gene. Our data point to p53 and p16 as gene targets of oral carcinogenesis, with chemicals in the betel quid possibly functioning in these tumors as carcinogens.

Cadherins as Predictive Markers of Nodal Metastasis in Breast Cancer

Adhesion molecules, particularly cadherins play a pivotal role in cancer invasion and metastasis. Because the therapeutic management of tumors with and without nodal metastasis differs considerably, our idea was to identify tumors with metastatic potential. We studied the expression of E-cadherin and P-cadherin immunohistochemically in 51 cases of breast cancer that included 29 node-negative and 22 node-positive cases. Expression of the cadherins was mainly membranous, with cytoplasmic staining in a few lesions. Both E-cadherin and P-cadherin showed significant down-regulation of their expression in node-positive tumors in comparison to node-negative tumors. Logistic regression analysis revealed that the positive expression of E-cadherin and P-cadherin showed low odds ratios of 0.1 and 0.2, respectively, and were statistically significant. On multivariate analysis, both the cadherins were found to be of independent prognostic value. This suggests that cadherin expression could be a marker of nodal metastasis. An observation of interest was that the expression of E-cadherin and P-cadherin were highly correlated (correlation coefficient = 0.5873), which requires further evaluation for confirmation of a common regulatory pathway that could be activated in the early onset of nodal metastasis.

Down regulation of endothelial adhesion molecules in node positive breast cancer: Possible failure of host defence mechanism

Endothelial cell adhesion molecules (CAMs) are important in tumorigenesis and host defense mechanism. Their status in breast cancer with regard to nodal invasion is not yet known. Hence we looked at the expression of three important CAMs: VCAM, ICAM and E-selectin. A downregulation of all these CAMs was noted in node positive breast cancer in comparison to node negative cases. This suggests shedding of these molecules in cases with nodal metastasis which might help the tumor cells to escape the host defense mechanism. On multivariate analysis, VCAM alone emerged as an independent predictor of nodal metastasis.

Development of an immunological staging system to prognosticate disease course in malignant cervical neoplasia

A multiparameter analysis of immune function was done on patients with squamous cell carcinoma of the uterine cervix to look for any variable(s) that could be correlated with the clinical stage of the disease. Five immunological variables, viz., CD4+ lymphocytes, CD4/CD8 ratio, natural killer cells, concanavalin A-induced suppressor index, and circulating immune complexes, were found to consistently vary with tumor load. When these variables were subjected to a multiple regression and multivariate analysis, an equation for a diagnostic index curve was derived. Application of this equation led to an immunological staging system which could be used as an excellent prognostic indicator. The immunological staging system showed that patients classified into a particular clinical (FIGO) stage behaved in a heterogeneous way immunologically and that patients developing recurrent disease could easily be identified from those remaining disease free, even before treatment. Subsequent follow-up of these patients further confirmed this observation, with the recurrent disease group easily identifiable. These results point out the immense potential of such a staging system and the importance of immunological evaluation in the preliminary management of patients with malignant cervical neoplasia.

Quantitation of Fc receptor-bearing T-lymphocytes (TG and TM) in oral cancer

The lymphocytes from the peripheral blood of 30 untreated patients with oral cancer and 15 apparently healthy normal controls were studied for quantitation of T‐cell subsets. When compared to age‐matched controls, the patients as a group showed a significant increase in the proportion and number of TG cells (IgG Fc receptor‐bearing T‐cells) and a significant reduction in the TM cells (IgM Fc‐bearing T‐cells). The levels of total lymphocytes, T‐cells, and B‐cells remained in the control range. The discrepancy in the TG subset was evident in the early stages of the disease, while in TM cells, it was evident only in the advanced stages.

Interferon activation of latent natural killer cells and alteration in kinetics of target cell lysis: Clinical implications for oral precancerous lesions

Reduced natural killer cell activity was observed in patients with oral leukoplakia and submucous fibrosis compared with normal control subjects. However, the number of target binding lymphocytes was found to be normal in these precancers. Treatment of effector cells with interferon-alpha resulted in highly elevated active killer cell activity, although no change was observed in target binding lymphocyte counts. This finding could imply that precytotoxic cells that are activated by interferon exist in peripheral blood or that direct recruitment of a new cell population takes place. In addition, altered target lysis kinetics was observed, with interferon-activated killer cells demonstrating a tremendous lytic activation that is completed so quickly that a statistical kinetic analysis could not be accurately done. Because natural killer cell activity is an important effector system in immunosurveillance against tumors, its modulation with interferon may be an exciting clinical possibility in the control of malignant transformation or oral precancers.

Interleukin 2 and Alpha Interferon Induced in Vitro Modulation of Spontaneous Cell Mediated Cytotoxicity in Patients with Cancer of the Uterine Cervix Undergoing Radiotherapy

In vitro modulation of spontaneous cell mediated cytotoxicity by interferon and interleukin 2 was carried out using peripheral blood lymphocytes from patients with cancer of the uterine cervix before and at different intervals after commencement of radiation treatment. A total of 150 patients with various stages of the disease were included and cytotoxicity was measured using the single cell cytotoxic assay. These results indicate a beneficial effect in vitro of interleukin 2 and interferon in augmenting spontaneous cell mediated cytotoxicity, a possibly vital antitumour immune mechanism in patients with relatively early cervix cancer. Natural killer cell, lymphokine activated killer cell and interferon activated killer cell activity was depressed immediately following radiotherapy. The activity of these cell types later on increased above pretreatment levels in patients with stages I, IIA and IIB. A similar rebound above pretreatment levels was not observed in patients with stages III and IV.

Alterations in Expression of Terminal Differentiation Markers of Keratinocytes during Oral Carcinogenesis

Expression of cytoskeletal proteins has been shown to be dependent on the differentiation status of the tissue. In the present study, expression of two cytoskeletal proteins normally present in terminally differentiated keratinocytes, namely cytokeratin types 10/11 and involucrin, were studied in different stages of tumour progression in the oral mucosa. Results showed that cytokeratins 10/11 and involucrin strongly correlated with the differentiation status of cells. High expression was observed in non-dysplastic hyperplastic epithelium as compared to normal, dysplastic and neoplastic epithelium. In addition, various grades of dysplasia showed an inverse correlation with expression of these proteins. Statistical analysis of the results also showed a negative correlation between the differentiation of upper spinal cells and the stage of tumour progression. It therefore appears that the proteins studied may be useful as markers for epithelial carcinogenesis.

Lymphocyte subset distribution after radiation therapy for cancer of the uterine cervix. Possible prognostic implications and correlation with disease course

An analysis of lymphocyte subpopulations was done in patients with cancer of the uterine cervix before and at different intervals after the commencement of radiation therapy. A common feature was a duration of T‐cell and B‐cell lymphopenia after therapy. The findings relating to the T‐cell subsets were interesting. Although the CD4/CD8 ratio remained unchanged in Stages I/IIA for 24 months after treatment, patients with Stages IIB and III showed a lowering of the ratio immediately after treatment. Distinctive patterns of lymphocyte subset distribution were seen in a comparison between patients who were disease‐free and those with recurrent disease. The CD4+ cell counts and CD4/CD8 ratio differed between the two groups, with consistent lowered values during the follow‐up associated with recurrent disease. This study demonstrates the effects of radiation therapy in altering lymphocyte subset distribution, resulting in characteristic patterns which could be used as clinical and prognostic indicators.