Meera Mathur

pRb and p16 protein alterations in human oral tumorigenesis

Cyclin dependent kinase inhibitor 2/multiple tumour suppressor gene 1 (CDKN2/MTS1) and retinoblastoma (Rb) tumour suppressor genes play important roles in the regulation of the cell cycle. The protein products of these genes p16INK4 (p16) and pRb, respectively, like p53 protein inhibit progression from G1 to S phase. p16 exerts its function through inhibition of CDK4-mediated phosphorylation of pRb. The pRb/p16 pathway is a critical target for molecular aberration at the G1-S checkpoint in a wide range of primary human tumours. The expression of p16 and pRb proteins was analyzed by immunohistochemistry in 35 cases of oral squamous cell carcinomas (SCCs), 22 cases of premalignant oral lesions and 30 normal oral tissues. Lack of pRb expression was observed in 23/35 (66%) oral SCCs and 14/22 (64%) premalignant lesions. Lack of p16 expression was observed in 22/35 (63%) oral SCCs and 13/22 (59%) premalignant lesions. Weak p16 and pRb immunoreactivities were observed in normal oral mucosal epithelium. The status of p16 and pRb was correlated with clinicopathological characteristics of the patients. Alteration in p16 expression showed significant correlation with tumour staging and progression (P = 0.024). Alteration in pRb/p16 expression correlated with heavy consumption of betel and tobacco. Our results suggest that alterations in the p16/pRb pathway are early events in oral tumorigenesis and may be involved in the development of betel- and tobacco-related oral malignancies.

Stromelysin 3, Ets-1, and Vascular Endothelial Growth Factor Expression in Oral Precancerous and Cancerous Lesions: Correlation with Microvessel Density, Progression, and Prognosis

Purpose: Identification of molecular changes characteristic of development and progression of oral cancer are of paramount importance for effective intervention. Stromelysin 3 (MMP11) is a unique matrix metalloproteinase shown to have dual function during cancer progression. The transcription factor Ets-1 and vascular endothelial growth factor (VEGF) are important proangiogenic factors in cancer. This study was designed to test the hypothesis that concomitant expression of stromelysin 3, Ets-1, and/or VEGF affects the development, progression, and prognosis of oral cancer. Patients and Methods: Immunohistochemical analysis of stromelysin 3, Ets-1, VEGF, and platelet/endothelial cell adhesion molecule 1 (a marker for intratumoral microvessel density) was carried out in serial paraffin embedded tissue sections of 220 oral squamous cell carcinomas (OSCC), 90 precancerous lesions (59 hyperplasias and 31 dysplasias), and 81 matched histologically normal oral tissues. Results: Ets-1, VEGF, and stromelysin 3 expression independently correlated with increased intratumoral microvessel density in precancerous lesions (P = 0.05, 0.001, and 0.026, respectively) as well as in SCCs (P = 0.005, 0.01, and 0.031, respectively). Logistic regression analysis revealed that concomitant expression of stromelysin 3 and Ets-1 (stromelysin 3+/ Ets-1+ phenotype; odds ratio, 3.7; P = 0.001) was the most significant predictor for transition to precancerous stage, whereas dual expression of stromelysin 3 and VEGF (stromelysin 3+/ VEGF+ phenotype; odds ratio, 2.07; P = 0.004) was the most important predictor for progression from precancerous stage to frank malignancy. Intriguingly, Ets-1 expression was significantly associated with VEGF expression and stromelysin 3 expression in precancerous tissues as well as OSCCs. Follow-up data for 144 patients for a maximum period of 115 months showed that VEGF [hazards ratio (HR), 4.532; P = 0.004] and Ets-1 (HR = 2.182; P = 0.049) expression significantly correlated with reduced disease-free survival in univariate analysis. In bivariate analysis, patients harboring Ets-1+/VEGF+ phenotype had the worst survival (median disease-free survival, 50 months; HR, 2.943; P = 0.003). Multivariate analysis using Coxs proportional hazards model showed that increased VEGF expression was the most significant adverse prognosticator in OSCC patients (HR, 4.470; P = 0.004). Conclusions: In conclusion, this study provides the first evidence of concomitant expression of stromelysin 3, VEGF, and Ets-1 in clinical specimens in different stages of development of oral cancer. In early stages, concomitant expression of stromelysin 3 and Ets-1 favors the development of a precancerous state, whereas dual expression of stromelysin 3 and VEGF is associated with progression from precancerous to cancerous state. VEGF expression is an adverse prognosticator for disease-free survival.

Alterations of rb pathway components are frequent events in patients with oral epithelial dysplasia and predict clinical outcome in patients with squamous cell carcinoma.

Objective: This study was designed to test the hypothesis that alterations in expression of G1/S modulators cyclin D1, p16 and pRb occur in patients with oral epithelial dysplasia, considered to be at increased risk for malignant transformation. In addition, the analysis of expression of all three markers in the same set of oral cancer patients would provide a unique opportunity to determine whether these alterations have cooperative or synergistic effects on oral cancer development and prognosis. Patients and Methods: A prospective study was undertaken to carry out immunohistochemical analysis of cyclin D1, p16 and pRb proteins in serial paraffin-embedded tissue sections of 220 oral squamous cell carcinomas (OSCCs), 90 potentially malignant lesions (52 oral hyperplastic lesions, 38 dysplasias) and 81 matched histologically normal oral tissues and correlated them with clinicopathological parameters. Ninety-eight OSCC patients were followed up for a maximum period of 94 months with overall median survival of 21 months. Results: Seventy-five of 90 (83%) potentially malignant lesions and 198 of 220 (90%) OSCCs showed altered expression of at least one of the proteins in the pRb pathway, while 10 of 90 (11%) patients with potentially malignant lesions and 40 (18%) of 220 OSCC patients showed all three alterations. Loss of p16 was the earliest event in oral tumorigenesis. In a multivariate model, loss of pRb was associated with transition from hyperplasia to dysplasia (OR = 3.727, p = 0.005). The transition of potentially malignant lesions to malignant stage was associated with pRb–/cyclin D1+ phenotype (OR = 2.294, p = 0.001) and p53+ phenotype (OR = 2.230, p = 0.002). Loss of pRb and accumulation of p53 (pRb–/p53+) phenotype was associated with histologic progression of the tumors and acquisition of invasive potential. Multivariate analysis using Cox’s proportional hazards model revealed that pRb–/p53+ phenotype was the most significant adverse prognosticator for disease-free survival (hazards ratio, (HR) = 2.642, p = 0.004). Conclusions: Deregulation of the p16/pRb/cyclin D1 pathway is an early event in acquisition of dysplasia, but deregulation of both pRb and p53 pathways is associated with malignant transformation and adverse prognosis in oral tumorigenesis.

Hepatocellular carcinoma in a hepatitis B ‘x’ transgenic mouse model: A sequential pathological evaluation

Background: The introduction of transgenic technology has made it possible to study the steps of carcinogenesis and directly establish the link between viral subgenomic fragments and specific types of cancer. Research directed at hepatitis B virus (HBV)‐related carcinogenesis has benefited from this technology. We present a detailed pathological evaluation of the sequential steps of hepatocarcinogenesis in a hepatitis B ‘x’ (HBx) transgenic mouse model. In this model, the transgene incorporates the region encoding amino acids 58–154 of the HBV X protein and the murine c‐myc gene. This model demonstrated changes in the liver from birth with foci of multicentric dysplasia evolving into nodules and overt hepatocellular carcinoma between 20 and 28 weeks.

Novel polymorphism in p21 waf1/cip1 cyclin dependent kinase inhibitor gene: association with human esophageal cancer

p21waf1/cip1, an important regulator of the cell cycle, binds to PCNA and acts as a mediator of the growth suppressing and apoptosis promoting functions of p53. We report a hitherto unobserved polymorphism in the carboxy terminal domain (codon 149) of p21waf1/cip1 gene, the domain encoding the PCNA binding motif. The codon 149 polymorphism (GAT→GGT) was observed in 42 of 50 (84%) esophageal squamous cell carcinomas (ESCCs) and eight of 50 (16%) normal individuals. The resultant amino acid substitution from aspartate to glycine may have vital implication in PCNA mediated cell cycle regulation by p21waf1/cip1. The second polymorphism at codon 31, involving a C→A transversion at nucleotide 168 (AGC→AGA) changing the amino acid from serine to arginine, was observed in 2/50 (4%) ESCCs at a relatively lower frequency in the Indian population than that reported in the West. No significant association was observed between p21waf1/cip1 polymorphism at codon 149 and p21waf1/cip1 protein expression in ESCC in this cohort of patients. Interestingly, the frequency of p21waf1/cip1 variants (codon 149) in ESCCs (18 of 19 cases) with wild-type p53 was significantly higher than in tumors with p53 mutations, suggesting that this polymorphism affects the p53 pathway and may play an important role in esophageal tumorigenesis. Analysis of p21waf1/cip1 expression in relation to p53 gene and protein status revealed its induction by p53-dependent as well as independent pathways in esophageal tumorigenesis.

Circulating p53 antibodies, p53 gene mutational profile and product accumulation in esophageal squamous-cell carcinoma in India†

Esophageal cancer (EC) in the Indian population presents in advanced stages with poor prognosis and warrants the identification of a non‐invasive marker for early detection and better prognostic assessment. We have previously reported high prevalence of p53 protein accumulation in esophageal squamous‐cell carcinomas (ESCCs). The present study was designed to determine (i) if esophageal cancer patients elicit a humoral immune response to intra‐tumoral p53 protein accumulation and (ii) their relationship with p53 gene mutations. The goal was to compare the cellular events, p53 protein accumulation and gene mutations with the presence of serum anti‐p53 antibodies (p53‐Abs) and to assess the utility of serological p53‐Ab analysis as a surrogate marker for p53 alterations in esophageal cancer. A high prevalence of circulating p53‐Abs was observed in 36 of 60 (60%) ESCC patients. In a subset of 44 ESCCs, exons 5–9 of the p53 gene were examined for mutations by PCR and direct sequencing of PCR products. Mutational data have been correlated with p53‐Abs and p53 protein accumulation in ESCCs. Circulating p53‐Abs in ESCC patients were significantly associated with intra‐tumoral p53 protein accumulation (p=0.0005). A strong correlation observed between humoral immune response against p53 protein, missense gene mutations and protein accumulation warrants the application of serological p53‐Abs as a non‐invasive surrogate marker in screening high‐risk populations for early detection of malignancy. Int. J. Cancer 85:791–795, 2000.

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; Odds 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 [Hazards 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.

Stromelysin-2 overexpression in human esophageal squamous cell carcinoma: potential clinical implications.

The degradation of the extracellular matrix (ECM) is a major step in the process of tumor invasion and metastasis, mediated by matrix metalloproteinases (MMPs). Knowledge of alterations in the expression of stromelysin-2 (ST-2) or MMP-10 in human esophageal squamous cell carcinoma (ESCC) is meager. Immunohistochemical analysis of ST-2 expression was carried out in surgically resected ESCCs (50 cases) and paired distal histologically normal esophageal tissues (50 cases), correlated with clinicopathological parameters. Overexpression of ST-2 protein, in tumor cell cytoplasm and stromal elements, was observed in 37 of the 50 (74%) ESCCs localized in tumor cell cytoplasm and stromal elements. Low levels of ST-2 were observed in 8 of the 50 (16%) matched histologically normal esophageal tissues. Significant associations were observed between ST-2 overexpression and tumor size (r = 0.02, P = 0.04), local invasiveness of the tumor (r = -0.30, P = 0.002) and distant organ metastasis (r = -0.227, P = 0.02), suggestive of its involvement in development and progression of ESCCs. The data underscore the significance of ST-2 expression in context to the aggressive tumor characteristics observed in ESCCs in the Indian population, wherein extremely poor prognosis has been ascribed to extensive local invasion and metastasis.

Loss of heterozygosity of a locus on 17p13.3, independent of p53, is associated with higher grades of astrocytic tumours

Amongst the human astrocytic tumours, the commonest of primary brain tumours, the clinical outcome of astrocytoma (AS) is significantly better than anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM). Often, low grade tumours can progress to or recur with a more malignant phenotype. Recent loss of heterozygosity (LOH) reports suspect the involvement of a tumour suppressor gene, different from p53, in the 17p13.3 region of the human chromosome. However, the effect of LOH of 17p13.3 region on tumour histology at presentation and prognosis is as yet undefined. As a first step to define the role of this putative oncogene in astrocytic tumour progression, we correlated the LOH of a locus, D17S379, in 17p13.3 region and the p53 locus in 17p13.1 region with the histopathology of astrocytic tumours by PCR based microsatellite and restriction fragment length polymorphism of DNA extracted from microdissected paraffin sections of 45 astrocytic tumours of different histopathological grades. LOH of D17S379 was significantly associated (P=0.02) with AA and GBM (high grade malignancy), while no such preferential association was found with LOH of p53. There were no mutations in the exons 5 to 9 of p53 gene in the five tumours with LOH of D17S379 but not of p53 region. In a case of AA with a heterogenous microscopic appearance, heterozygosity of D17S379 was lost only in the area with a more malignant histology while both areas had no LOH or mutation of p53. A locus at the 17p13.3 region, independent of the p53 locus, is involved in a large subset of astrocytic tumours during transformation into a more malignant phenotype, and thus may be a link in the chain of genetic events occurring in astrocytic tumour progression.

COMBINED EVALUATION OF EXPRESSION OF TELOMERASE, SURVIVIN, AND ANTI-APOPTOTIC BCL-2 FAMILY MEMBERS IN RELATION TO LOSS OF DIFFERENTIATION AND APOPTOSIS IN HUMAN HEAD AND NECK CANCERS

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers, and it accounts for 5% of all adult cancers worldwide. Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumor progression. Little is known about the distribution of inhibitors of apoptosis in HNSCC or how they correlate with other biomarkers of malignancy, such as telomerase, an enzyme that plays a critical role in cellular immortalization. The objective of this study was to assess the protein expression of anti‐apoptotic members of Bcl‐2 family and survivin and correlate them with telomerase activity.