Prediction of recurrence-free survival using a protein expression-based risk classifier for head and neck cancer.

Loco-regional recurrence in 50% of oral squamous cell carcinoma (OSCC) patients poses major challenge for oncologists. Lack of biomarkers that can predict disease aggressiveness and recurrence risk makes the scenario more dismal. On the basis of our earlier global proteomic analyses we identified five differentially expressed proteins in OSCC. This study aimed to develop protein biomarkers-based prognostic risk prediction model for OSCC. Sub-cellular expression of five proteins, S100A7, heterogeneous nuclear ribonucleoproteinK (hnRNPK), prothymosin α (PTMA), 14-3-3ζ and 14-3-3σ was analyzed by immunohistochemistry in test set (282 Indian OSCCs and 209 normal tissues), correlated with clinic-pathological parameters and clinical outcome over 12 years to develop a risk model for prediction of recurrence-free survival. This risk classifier was externally validated in 135 Canadian OSCC and 96 normal tissues. Biomarker signature score based on PTMA, S100A7 and hnRNPK was associated with recurrence free survival of OSCC patients (hazard ratio=1.11; 95% confidence interval 1.08, 1.13, P<0.001, optimism-corrected c-statistic=0.69) independent of clinical parameters. Biomarker signature score stratified OSCC patients into high- and low-risk groups with significant difference for disease recurrence. The high-risk group had median survival 14 months, and 3-year survival rate of 30%, whereas low-risk group survival probability did not reach 50%, and had 3-year survival rate of 71%. As a powerful predictor of 3-year recurrence-free survival in OSCC patients, the newly developed biomarkers panel risk classifier will facilitate patient counseling for personalized treatment.

Soluble VEGFR-1 in pathophysiology of pregnancies complicated by hypertensive disorders: the Indian scenario.

An imbalance between angiogenic and anti-angiogenic factors has been hypothesized to have a major role in hypertensive disorders during pregnancy, which account for significant morbidity and mortality for the mother and neonate in India. There is a paucity of information on soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, anti-angiogenic factor) concentrations in different subgroups of pregnancy-induced hypertensive (PIH) disorders particularly in gestational hypertension (GH) and eclampsia during pregnancy. This cross-sectional study was conducted in order to test the above hypothesis and to get more insight into the role of sVEGFR-1 in these disorders. The concentrations of sVEGFR-1 in serum were measured from women with 22-39 weeks of gestation in the control (n=180) and gestationally matched hypertensive (n=360) pregnant mothers by ELISA. These sVEGFR-1 concentrations were found to be significantly elevated in the study groups as the severity of disease increases from GH to eclampsia (24 076 pg ml(-1); 42000 pg ml(-1), P=0.0001) as compared with controls (3360 pg ml(-1)). According to Receiver operating characteristic curve analysis, at ≤ 34 weeks, the concentration cutoff, sensitivity, specificity for sVEGFR-1 in differentiating GH, pre-eclampsia and eclampsia were ≥ 7619.2 pg ml(-1), 75%, 75%; ≥ 16726.6 pg ml(-1), 89.1%, 89.1%; ≥ 26222.8 pg ml(-1), 91.6%, 91.6%, respectively. The gradual upregulation of sVEGFR-1 concentrations in these groups may be due to its intimate involvement in the etiopathogenesis of severity of various hypertensive disorders by antagonizing effects of VEGF during the placental development. These observations indicate the clinical utility of sVEGFR-1 in differentiating PIH disorders and also will be helpful for the evaluation of increased monitoring system for successful pregnancy.

Clinical significance of 14-3-3 zeta in human esophageal cancer.

We recently found 14-3-3 zeta to be overexpressed in esophageal squamous cell carcinomas (ESCCs) by differential display. In the present study we determined the clinical significance of 14-3-3 zeta in esophageal tumorigenesis. Immunohistochemical analysis was carried out in 61 ESCCs, 33 dysplasia samples, 14 hyperplasia samples and 7 matched histologically normal esophageal tissues and correlated with clinicopathological parameters. Cytoplasmic expression of 14-3-3 zeta protein was observed in 95% of ESCCs; 63% of tumors also showed nuclear localization. All hyperplastic and dysplastic tissues distant from ESCCs as well as dysplastic endoscopic biopsies showed cytoplasmic immunopositivity for 14-3-3 zeta, while nuclear localization was observed in 58% of dysplasia and 36% of hyperplasia samples. Matched distant histologically normal epithelia either showed basal cytoplasmic expression of 14-3-3 zeta or no detectable nuclear expression of the protein. Interestingly, immunopositivity observed in normal esophageal tissues and early hyperplasia was confined to cytoplasm only, though significant nuclear expression was detected in dysplasia and ESCC. Immunoblotting and RT-PCR analyses further confirmed 14-3-3 zeta expression in dysplasia and ESCC. To our knowledge, this is the first report demonstrating overexpression of 14-3-3 zeta in esophageal hyperplasia, dysplasia and squamous cell carcinoma, suggesting that alteration in its expression occurs in early stages and is associated with esophageal tumorigenesis.

Clinical, pathological, cellular and molecular lesions caused by oral smokeless tobacco--a review.

While carcinogenicity of smokeless tobacco (ST) to humans is well established the oral lesions that precede development of cancer are less well characterized. The clinical appearances of ST-associated lesions are variable. Epidemiological studies show a strong significant association of risk with chronic daily use but population differences are noted because of various commercial products in use. Morphological features observed are some what different to oral lesions caused by smoking and oral dysplasia in ST-associated lesions is less common. Effects of ST on oral keratinocytes observed in vitro include alterations in cell proliferation, apoptosis and activation of inflammatory markers. Genetic aberrations caused by ST include activation of ras, uncommon in smokers but mutational hot spots in p53 encountered are similar to those in smokers.

Evaluation of endometrial changes and p53 expression in tamoxifen treated women: comparison of various methods.

OBJECTIVE: To compare transvaginal sonography (TVS), sonohysterography (SHG), hysteroscopy and endometrial aspiration (EA) and p53 expression in assessing endometrial abnormalities in women on tamoxifen. METHODS: In a cross sectional study of 50 pre- and post-menopausal women receiving tamoxifen for > 2 years, all participants underwent TVS and EA. Those with endometrial thickness > 4 mm on TVS underwent hysteroscopy and SHG. Serum p53 antibody and p53 immunohistochemistry were tested in all women. RESULTS: The sensitivity and specificity when compared with histopathology as the reference standard were as follows: TVS 100% and 33.3%, SHG 85.7% and 50%, hysteroscopy 92.8% and 80.8%, serum p53 50% and 83.3%, and p53 immunohistochemistry 57.1% and 61.1%. Prevalence of endometrial abnormalities was not significantly different in asymptomatic and symptomatic women. CONCLUSION: Tamoxifen-users require routine testing for endometrial evaluation. TVS followed by hysteroscopy and biopsy is an effective option. p53 expression correlates with histological abnormalities.

Alterations in expression of retinoid receptor beta and p53 in oral submucous fibrosis.

OBJECTIVE: Knowledge of the molecular pathogenesis of oral submucous fibrosis (OSF), a potentially malignant condition with high risk of transition to oral cancer, is meagre. Alterations in the expression of retinoic acid receptor beta (RARbeta) and tumor suppressor gene, p53 are early events in oral tumorigenesis. The aim of this study was to investigate the alterations in the expression of RARbeta and p53 in OSF lesions and determine their association with disease pathogenesis. METHODS: The expression of RARbeta and p53 proteins was analyzed by immunohistochemistry in 50 cases of OSF and 30 histologically normal oral tissues. RESULTS: No detectable RARbeta expression was observed in 35 of 50 (70%) OSF cases. p53 protein accumulation was observed in 24 of 50 (48%) OSF cases analyzed. Thirty-six percent OSF lesions showed loss of RARbeta and p53 overexpression. Interestingly, 41 of 50 (82%) of OSF lesions showed altered expression of at least one of these two proteins. CONCLUSION: Altered expression of either RARbeta or p53 in majority of OSF lesions suggests their association with disease pathogenesis and warrants follow-up to determine whether OSF lesions harboring concomitant alterations in RARbeta and p53 are at a high risk of transition to malignancy.

Increased expression of MMP-2 and MMP-9 in esophageal squamous cell carcinoma.

PURPOSE: Matrix metalloproteinases (MMPs) are known to play an important role in extracellular matrix remodeling during the process of tumor invasion and metastasis. However, little is known about their role in preinvasive lesions and early esophageal carcinomas. METHOD: Immunohistochemical analysis of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinoma (ESCC) (58 cases) and paired distal normal esophageal tissues (44 cases) and correlated with clinicopathological parameters. RESULT: Overexpression of MMP-2 and MMP-9 proteins was observed in 39 (67%) and 32 (55%) of the 58 ESCCs, respectively localized in tumor cell cytoplasm and stromal elements. Histological evaluation of hematoxylin- and eosin-stained 44 matched distal normal esophageal tissue sections revealed that 26 comprised of normal epithelium, while 15 tissues showed evidence of dysplasia and three tissues showed hyperplasia. Interestingly, 12 (80%) and 13 (87%) of these 15 dysplasias showed immunostaining for MMP-2 and MMP-9 proteins, respectively. Low levels of MMP-2 and MMP-9 were observed in 10 (38%) and 6 (23%) of 26 matched histologically normal esophageal tissues, respectively. Higher MMP-2 immunopositivity was observed in well and moderately differentiated SCCs in comparison with poorly differentiated tumors. The expression of MMP-2 was significantly reduced with the progressive de-differentiation of esophageal SCCs ( P =0.03). Overexpression of MMP-2 and MMP-9 in dysplasia as well as SCC suggests that these alterations occur in early stages of esophageal tumorigenesis. CONCLUSION: Increased levels of MMP-2 and MMP-9 proteins in ESCCs as compared to normal esophageal tissues suggest their association with esophageal tumorigenesis. Increased levels of these MMPs are observed in majority of dysplasias analyzed herein, indicating that these alterations may be early events in esophageal tumorigenesis. In-depth studies are warranted to determine their role in development and progression of esophageal cancer.

Retinoids as chemopreventive agents.

Retinoids are promising agents for cancer chemoprevention. The myriad effects of retinoids on biological processes including development, differentiation, homeostasis, carcinogenesis and apoptosis are mediated through their molecular targets, the retinoid and rexinoid receptors. Tissue specific expression patterns, ligand specificities, receptor numbers, their distinct functions and functional redundancy make retinoid signaling highly complex. The cross-talks of these receptors with cell surface receptors signaling pathways, as well as their interactions with multiple co-activators and co-repressors further add to the complexity of the pleiotropic effects of retinoids. Elucidation of retinoid signaling pathways and indepth understanding of the mechanisms that underlie the anti-proliferative and apoptotic action of retinoids has paved the way for designing synthetic retinoids for effective chemoprevention and therapy of cancer. Development of receptor selective synthetic retinoids is a major focus of molecular retinoid development. Other new avenues encompass identification of novel retinoid regulated genes, orphan-receptor ligands/functions, novel retinoid mechanisms involving receptor-independent apoptosis inducing activity and synergistic combinations with other agents for cancer prevention and therapy. This review focuses on recent advances in the understanding of molecular mechanisms underlying the action of retinoids and retinoid molecular targeting studies designed primarily to develop retinoids with reduced toxicity, while maintaining or enhancing activity in context of chemoprevention. The clinical efficacy of retinoid based chemoprevention trials is discussed.

Prognostic factors in betel and tobacco related oral cancer.

Paucity of well-defined prognostic molecular markers severely hampers prediction of the clinical course of squamous cell carcinoma (SCC) of oral cavity. The aim of the study was to evaluate the prognostic significance of impairments in the expression of proteins involved in cell cycle regulation and locoregional spread in oral SCC of habitual betel and tobacco chewers. A prospective study was performed in 105 betel and tobacco consumers with oral SCCs during the period 1988-1999, to evaluate the prognostic relevance of impairments in the expression of proteins involved in cell cycle regulation and locoregional spread. Alterations in the expression of p53, pRb, p16, MDM2, p21, and Ets-1 proteins were determined by immunohistochemical analysis in formalin fixed, paraffin embedded tissue sections from oral SCCs. Analysis of multiple molecular biological factors showed overexpression of p53 in 69/105 (66%) cases, MDM2 in 72/105 (69%) cases, p21 in 57/105 (54%) cases and Ets-1 in 64/105 (61%) cases. Loss of pRb was observed in 58/105 (55%) cases and p16 loss was observed in 72/105 (69%) cases. Interestingly, multivariate analysis revealed loss of pRb as the most significant predictor of advanced tumour stage [P=0.001; Odd's Ratio (OR)=3.5] and overexpression of Ets-1 protein was an independent risk factor for lymph node metastasis (OR: 10.1; P<10(-6)). Multiple logistic regression models showed that pRb loss [Hazard's Ratio (HR): 3.93] and p53 overexpression (HR: 2.97) may serve as adverse prognosticators for disease free survival of the patients. The data demonstrate multiple impairments in p53/MDM2/p21/Ets-1 and p16/pRb pathways in betel and tobacco related oral tumourigenesis.

Coexpression of Ets-1 and p53 in oral carcinomas is associated with P-glycoprotein expression and poor prognosis.

PURPOSE: To determine the association between the expression of P-gp with Ets-1 and p53 proteins in oral squamous cell carcinomas (SCCs). MATERIALS AND METHODS: Immunohistochemical analysis of Ets-1, P-glycoprotein (P-gp), and p53 proteins was carried out in 40 formalin-fixed, paraffin-embedded tissue sections from oral SCCs using specific antibodies for these proteins. RESULTS: Expression of Ets-1 protein was observed in 27/40 (68%) cases, P-gp was overexpressed in 27/40 (68%) cases, and p53 accumulation was observed in 26/40 (65%) cases. Twenty-two of 27 (82%) SCCs showed concomitant overexpression of Ets-1 and P-gp underpinning an association between the expression of these two proteins ( P=0.007). Twenty-one of 27 (78%) Ets-1 overexpressing oral SCCs showed accumulation of p53 protein ( P=0.015). Nineteen of the 27 (70%) P-gp expressing tumours showed p53 accumulation. Concomitant Ets-1 and P-gp overexpression was significantly associated with poor prognosis ( P=0.002). In multivariate analysis using Cox's proportional hazards model, P-glycoprotein emerged as the most significant adverse predictor of disease-free survival (HR=6.2, P=0.003). The hallmark of the study was the significant association between the expression of Ets-1, P-gp, and p53 proteins in oral SCCs and their association with poor prognosis. Oral cancer patients showing concomitant expression of Ets-1, P-gp, and p53 proteins had shorter disease-free survival (median time of no recurrence=18 months) and worst prognosis ( P=0.001) as compared to the cases overexpressing any of these proteins. CONCLUSION: Concomitant expression of Ets-1, P-gp, and p53 proteins adversely affects the clinical outcome in oral SCCs.

Alterations in p53 and pRb pathways and their prognostic significance in oesophageal cancer.

The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis. To evaluate which of these cell cycle regulatory proteins are related to patients' prognosis, a comprehensive analysis of alterations in these components was carried out in 100 ESCCs (oesophageal squamous cell carcinoma) using immunohistochemistry and correlated with clinicopathological parameters by univariate analysis. Overexpression of p53, MDM2 and cyclin D1 proteins was observed in 73, 42 and 67% of the cases, respectively, while loss of expression of p21, p16 and pRb was observed in 36, 45 and 75% of the cases, respectively. Multiple logistic regression analysis revealed that loss of p16 immunoreactivity was a significant risk factor for tumour stage (pT) (Odds Ratio (OR)=3.3), whereas the loss of pRb was a significant risk factor for nodal metastasis (pN) (OR=8.8). MDM2 overexpression emerged as the most significant risk factor for distant organ metastasis (pM) (OR=4.6). Of the ESCC patients who underwent oesophagectomy, 50 cases were followed-up for a maximum period of 44 months and median of 16 months. Survival analysis revealed that Cyclin D1 overexpression is an adverse prognosticator for disease-free survival, as well as overall survival, and tumour stage (pT) is an adverse prognosticator for disease-free survival. In conclusion, these data support a model of oesophageal cancer pathogenesis in which both the pRb and p53 pathways are inactivated and suggests an in-depth evaluation of the clinical utility of these putative markers is warranted.

Prognostic impact of Ets-1 overexpression in betel and tobacco related oral cancer.

In oral squamous cell carcinoma (SCC), the presence of lymph node metastasis is one of the most important prognostic factors, correlating locoregional spread, recurrence, distant metastasis and survival. However, the biological factors implicated and the mechanisms underlying these events are not completely elucidated. We reported Ets-1 expression in oral SCC and proposed that it may serve as a plausible marker of invasive potential and lymph node metastasis. Herein, we investigate the prognostic significance of Ets-1 expression in oral cancer. One hundred oral SCCs assessed for Ets-1 expression showed significant association with tumor stage (P = .027), lymph node involvement (P < 10(-6)) and distant organ metastasis (P = .007). The transcription factor Ets-1 regulates the expression of several genes involved in extracellular matrix remodeling that may account for its association with lymph node and distant organ metastasis. Kaplan-Meier survival analysis entailed significant association of Ets-1 expression in oral SCCs with reduced disease free survival (P = .0041), suggesting its utility as a prognosticator for oral cancer. In conclusion, these findings underscore the role of Ets-1 in oral tumor invasion and metastasis and may thus account for its association with diminished disease free survival.

Alterations in MDM2 expression in esophageal squamous cell carcinoma: relationship with p53 status.

In view of the significance of MDM2 as a regulator as well as critical target of wild type p53, this study was undertaken to determine the alteration in MDM2 expression in esophageal squamons cell carcinoma (ESCC) and its relationship to clinicopathological parameters as well as p53gene and protein status. Immunohistochemical analysis of MDM2 and p53 proteins on paraffin embedded sections from 64 surgically resected ESCCs and matched histologically normal tissues showed overexpression of MDM2 protein in 23/64 (36%) ESCCs, while the histopathologically normal esophageal tissues did not show detectable level of MDM2 immunoreactivity. Interestingly, MDM2 /p53 + phenotype was observed in 37/64 (58%) cases. None of the cases with p53 mis-sense mutations (12/30, 40%) showed detectable level of MDM2 protein. Missense p53 mutations were significantly associated with discordant p53 + /MDM2 immunophenotype (p= 0.004). The most intriguing feature of the study was accumulation of MDM2 in the absence of detectable p53 in 11% of and overexpression of MDM2 and p53 in 25% of ESCCs, suggesting a p53-independent role for MDM2 in a subset of tumors. These results underscore the involvement of MDM2 in p53-dependent and -independent pathways in the pathogenesis of esophageal cancer in the Indian population.

Esophageal squamous cell carcinomas with DNA replication errors (RER+) are associated with p16/pRb loss and wild-type p53.

PURPOSE: Microsatellite instability (MSI) as a determinant of propensity to esophageal squamous cell carcinoma (ESCC) at seven microsatellite markers at 2p (2p15-16), 3p (3p13, 3p14.1-3, 3p25, and 3p26) and 16q (16q12.1-3) was investigated to analyze their putative role as indicators of predisposition to esophageal malignancies. METHODS: Seven microsatellite loci were amplified by polymerase chain reaction, from surgically resected tumor tissues from 30 ESCC patients from Indian population, to assess the loss of heterozygosity (LOH) and replication error repeats (RER) and to correlate these alterations with aberrations in major cell cycle regulatory proteins and histopathological parameters. RESULTS: LOH and RER analyses at these loci demonstrated moderate microsatellite alterations, suggesting the involvement of MSI in esophageal tumorigenesis in a subset of the Indian population. MSI, defined as RER in at least two or more of the loci studied, was observed in ten of 30 (33%) patients. Twenty-two of 30 patients (73%) showed LOH at one or more loci, while 17 of the 30 patients (60%) showed RER in at least one of the loci studied. RER-positive patients showed a trend towards better prognosis when compared to RER-negative patients. MSI demonstrated a significant association with concomitant loss of p16 and pRb (p16-/pRb- phenotype) (P=0.046). Interestingly, we observed an inverse correlation between MSI and p53 mutations (P=0.03) suggesting that MSI may provide a p53-independent pathway for esophageal tumorigenesis in RER+ patients. MSI showed a trend towards longer survival and absence of distant organ metastasis (P=0.06). CONCLUSIONS: The present study demonstrates the probable role of MSI in esophageal squamous cell carcinoma in the Indian population. Instability associated with the repetitive sequences--the revealing marks of loss of DNA replication fidelity may serve as an indicator of predisposition to esophageal cancer.

Correlation between p53 gene mutations and circulating antibodies in betel- and tobacco-consuming North Indian population.

Alterations in p53 tumour suppressor gene and its expression may be implicated in the pathogenesis of betel- and tobacco-related oral cancer. There is wide regional variation in betel- and tobacco-consuming habits in different parts of the Indian subcontinent. The purpose of this study was to determine the correlations between p53 gene mutations, protein accumulation and serum antibodies in oral precancer and cancer. We analysed 30 potentially malignant oral lesions (leukoplakia) and 30 oral squamous cell carcinomas (SCCs) from northern India because the betel quid-consuming habits are different from those prevalent in other regions of India. p53 mutations were analysed by polymerase chain reaction amplification of genomic DNA and direct sequencing, p53 protein accumulation by immunohistochemical analysis and circulating p53 antibodies by ELISA. p53 gene mutations, analysed within exons 5-9, were observed in five out of 30 (17%) potentially malignant oral lesions and seven out of 30 (23%) oral SCCs. All the mutations were base substitution mutations. Three missense and two nonsense mutations were observed in potentially malignant oral lesions, while six missense and one nonsense mutations were identified in oral SCCs. The probable hot spots for the mutations were identified at codons 126, 136 and 174, which have not been observed thus far. A good correlation was observed between p53 missense mutation, p53 antibodies and p53 protein accumulation in matched potentially malignant and malignant oral lesions. All the potentially malignant and cancerous lesions harbouring missense mutations showed accumulation of p53 protein and the majority of these patients showed circulating p53 antibodies suggesting that serological detection of p53 antibodies may serve as a surrogate marker for p53 alterations in oral lesions.

Co-expression of colligin and collagen in oral submucous fibrosis: plausible role in pathogenesis.

The high incidence of oral submucous fibrosis (OSF), a potentially malignant condition of the oral cavity, in the Indian subcontinent is causally associated with commonly prevailing habit of chewing areca nut and tobacco. Knowledge of molecular alterations in OSF is meagre. OSF is characterised by progressive accumulation of collagen fibres in lamina propria and oral submucosa. Colligin/HSP47 is a 47KDa stress protein which acts as a chaperone for collagen. We hypothesized that since colligin plays a vital role in folding and assembling collagen it may be involved in the pathogenesis of OSF. The present study was undertaken in tobacco and areca nut chewing Indian OSF patients to investigate the correlation, if any, between the expression of colligin and collagen type I proteins in OSF lesions. Immunohistochemical analysis showed overexpression of colligin and collagen type I proteins in 16/23 (70%) and 15/23 (65%) of OSF cases, respectively. The hallmark of the study was the significant association between the increased expression of type I collagen and its chaperone, colligin, in OSF lesions (P=0.0494). The data suggest that the increased levels of colligin in OSF may contribute to the deposition of collagen and consequent increased fibrosis in the oral submucosa in OSF lesions.

Alterations in cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population.

PURPOSE: The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alterations in expression of tumour suppressor gene products, p16 and pRb in esophageal cancer. Knowledge of alterations in cyclin D1, a vital component of this pathway in esophageal carcinomas from the Indian subcontinent, where the etiology and pathogenesis may be confounded by various unique dietary and environmental factors, is presently scanty. In order to bridge the gap between the accentuating incidence of esophageal cancer and aberrations in the components of this vital pathway, we analysed cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population. METHOD: Immunohistochemical analysis of cyclin D1 expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinomas (ESCC) (70 patients) and matched with histopathologically normal esophageal tissues from a distant site. The findings were correlated with clinicopathological parameters. RESULTS: Overexpression of cyclin D1 was observed in the tumour nuclei in 41 out of 70 (59%) patients. We found concomitant alterations in 16 and cyclin D1 (p16-/CycD1+ phenotype) in 16 of the 70 patients (23%), while alterations of pRb and cyclin D1 (pRb-/CycD1+) were observed in 36 of the 70 (51%) patients of ESCCs. Cyclin D1 overexpression was significantly associated with the loss of p16 immunoreactivity (P = 0.005). The pRb- and p16-/pRb-/Cyc D+ phenotypes showed significant association with differentiation of the tumour (P = 0.005, 0.05, respectively). Kaplan-Meier analysis for disease recurrence showed increased disease recurrence in cyclin D1 overexpressed patients. Median time to disease recurrence in the cyclin D1+ group was 15 months as against 18 months observed in the cyclin D1- patients (P = 0.067; log-rank test). CONCLUSION: Alterations in at least one of the components of the p16/cyclin D1/pRb pathway in majority of the 70 patients analysed herein, and concomitant alterations in all the three proteins in 19 patients (35%) underscore the critical role of this pathway in esophageal tumourigenesis. The results of the present study taken together with our previous findings on p16 and pRb alterations in ESCCs suggest that these alterations are not mutually exclusive and may cooperatively provide greater tumour growth advantage. The prognostic significance of alterations in the expression of these components cyclin D1, p16, and pRb remains to be established in a larger cohort.

Expression of RARalpha and RARbeta in human oral potentially malignant and neoplastic lesions.

Retinoids reverse potentially malignant lesions and inhibit the development of second primary cancers in patients with head-and-neck cancer. Many of the effects of retinoids result from modulation of gene expression by 2 distinct classes of nuclear receptor, RARs and RXRs; alterations in their expression can lead to tumorigenesis. To determine whether aberrations in expression of the receptors are related to the development of betel- and tobacco-related oral cancer, we used specific monoclonal antibodies against RARalpha and RARbeta to detect expression of these proteins in 30 histopathologically normal tissues, 45 potentially malignant lesions (leukoplakia) with histological evidence of either hyperplasia (31 cases) or dysplasia (14 cases) and 64 oral squamous-cell carcinomas (SCCs) by immunohistochemistry. Of the 30 normal oral tissues analysed, 8 cases showed detectable levels of RARalpha protein, while 10 cases did not show detectable RARbeta immunoreactivity. Immunostaining for RARalpha protein was observed in 12/31 (39%) hyperplastic lesions, 6/14 (43%) dysplastic lesions and 43/64 (67%) oral SCCs. Expression of RARalpha in oral SCC was significantly associated with the histological differentiation status of tumours (p = 0.016). In contrast, lack of detectable immunoreactivity was observed in 19/31 (61%) hyperplastic lesions, 8/14 (57%) dysplastic lesions and 21/64 (33%) oral SCCs. The hallmark of the study was the significant increase in RARalpha immunopositivity in oral SCCs compared to normal tissue (p = 0.0005) and hyperplastic lesions (p = 0.016). One intriguing feature was the significant decrease in RARbeta immunopositivity in hyperplastic lesions compared with normal oral mucosa (p = 0.05) as well as in oral SCCs compared with normal tissues (p = 0.0008).

Frequent alterations in the expression of tumor suppressor genes p16INK4A and pRb in esophageal squamous cell carcinoma in the Indian population.

PURPOSE: Alterations in the cell cycle regulatory p16INK4a/Cyclin D1/pRb pathway play a pivotal role in tumorigenesis. Knowledge of alterations in the tumor suppressor protein pRb and its negative regulator, p16CDKN2/MTS1/INK4a in esophageal squamous cell carcinoma (ESCC) from the Indian subcontinent is meager. To gain insight into the mechanisms underlying tumorigenesis and to search for diagnostic molecular markers for ESCC, we analyzed the expression of p16INK4a and pRb in ESCCs in the Indian population. METHODS: Immunohistochemical analysis of pRb and p16INK4a proteins was carried out in paraffin-embedded sections from 61 surgically resected ESCCs and matched normal tissues, and the results correlated with clinicopathological parameters using chi square and Fisher's exact tests. Dual immunohistochemical analysis has been carried out to demonstrate the concomitant loss of expression of p16INK4a and pRb. RESULTS: Fifty-nine of 61 (97%) cases showed aberration(s) in either or both of these proteins confirming their critical role in esophageal tumorigenesis. Loss of pRb was observed in 51 of the 61 (84%) and loss of p16INK4a was observed in 35 of 61 (57%) cases. Loss of pRb showed significant association with dedifferentiation of the tumor (P = 0.004). p16-/pRb-, and p16+/pRb- phenotypes were significantly associated with nodal metastasis (P = 0.017 and 0.027, respectively), while p16-/pRb+ phenotype was associated with dedifferentiation of the tumor (P = 0.012). CONCLUSION: pRb/p16INK4a pathway plays a critical role in esophageal tumorigenesis in the Indian population. The dual hits (concomitant loss) of pRb and p16INK4a expression suggest that these two components are not mutually exclusive, and can both be altered in a significant proportion of primary ESCCs serving as putative diagnostic markers for esophageal cancer. However, the impact of dual hit on tumor behavior and disease prognosis remains to be determined.

Induction of MDM2-P2 transcripts correlates with stabilized wild-type p53 in betel- and tobacco-related human oral cancer.

MDM2, a critical element of cellular homeostasis mechanisms, is involved in complex interactions with important cell-cycle and stress-response regulators including p53. The mdm2-P2 promoter is a transcriptional target of p53. The aim of this study was to determine the association between mdm2-P2 transcripts and the status of the p53 gene in betel- and tobacco-related oral squamous cell carcinomas (SCCs) to understand the mechanism of deregulation of MDM2 and p53 expression and their prognostic implications in oral tumorigenesis. Elevated levels of MDM2 proteins were observed in 11 of 25 (44%) oral hyperplastic lesions, nine of 15 (60%) dysplastic lesions, and 71 of 100 (71%) SCCs. The intriguing feature of the study was the identification and different subcellular localization of three isoforms of MDM2 (ie, 90 kd, 76 kd, and 57 kd) in oral SCCs and their correlation with p53 overexpression in each tumor. The hallmark of the study was the detection of mdm2-P2 transcripts in 12 of 20 oral SCCs overexpressing both MDM2 and p53 proteins while harboring wild-type p53 alleles. Furthermore, mdm2 amplification was an infrequent event in betel- and tobacco-associated oral tumorigenesis. The differential compartmentalization of the three isoforms of MDM2 suggests that each has a distinct function, potentially in the regulation of p53 and other gene products implicated in oral tumorigenesis. In conclusion, we report herein the first evidence suggesting that enhanced translation of mdm2-P2 transcripts (S-mdm2) may represent an important mechanism of overexpression and consequent stabilization and functional inactivation of wild-type p53 serving as an adverse prognosticator in betel- and tobacco-related oral cancer. The clinical significance of the functional inactivation of wild-type p53 by MDM2 is underscored by the significantly shorter median disease-free survival time (16 months) observed in p53/MDM2-positive cases as compared to those which did not show co-expression of these proteins (median time, 26 months; P = 0.02).