The aim of our study was to analyze the alterations of some candidate tumor suppressor genes (TSGs) viz. LIMD1, LTF, CDC25A, SCOTIN, RASSF1A and CACNA2D2 located in the chromosomal region 3p21.31 associated with the development of early dysplastic lesions of head and neck. In analysis of 72 dysplastic lesions and 116 squamous cell carcinoma of head and neck, both deletion and promoter methylation have been seen in these genes except for CDC25A and SCOTIN where no methylation has been detected. The alteration of LIMD1 was highest (50%) in the mild dysplastic lesions and did not change significantly during progression of tumor indicating its association with this stage of the disease. It was evident that alterations of LTF, CDC25A and CACNA2D2 were associated with development of moderate dysplastic lesions, while alterations in RASSF1A and CACNA2D2 were needed for progression. Novel somatic mutations were seen in exon 1 of LIMD1 (7%), intron 3/exon4 splice junction of LTF (2%) and exon 7 of cdc25A (10%). Quantitative RT-PCR analysis revealed mean reduced expression of the genes in the following order: LTF (67.6 6 16.8) > LIMD1 (53.2 6 20.1) > CACNA2D2 (23.7 6 7.1) > RASSF1A (15.1 6 5.6) > CDC25A (5.3 6 2.3) > SCOTIN (0.58 6 0.54). Immunohistochemical analysis of CDC25A showed its localization both in cytoplasm and nucleus in primary lesions and oral cancer cell lines. In absence of HPV infection, LTF and RASSF1A alterations jointly have adverse impact on survival of tobacco addicted patients. Thus, our data suggested that multiple candidate TSGs in the chromosomal 3p21.31 region were differentially associated with the early dysplastic lesions of head and neck. ' 2008 Wiley-Liss, Inc.Key words: dysplastic lesions; expression; head and neck squamous cell carcinoma; mutation; 3p21.31; survival study; tumor suppressor genes; methylation Head and neck squamous cell carcinoma of (HNSCC) is the sixth most common cancer worldwide and it accounts for 30-40% of all cancer types in Indian subcontinent.1 HNSCC is a heterogeneous epithelial malignant disease arising from mucosa of the upper aerodigestive tract (oral cavity, larynx, oropharynx and hypopharynx) with complex molecular abnormalities. Although the major and common environmental carcinogenic risk factors of tobacco, alcohol, betel quid and HPV infection have long been recognized, details of the genetic and epigenetic events leading to the development and spread of HNSCC remain largely unknown. Array-based gene expression profiling focused the transition of normal mucosa to premalignant lesion as the most important event with majority of transcriptional alterations rather than transition from premalignant lesion to invasive carcinoma during the progression of HNSCC.2 In microcell hybrid system, it has been shown that the introduction of chromosome 3 can suppress the tumorigenicity of oral cancer cell lines, suggesting the presence of at least 1 tumor suppressor gene (TSG) in this chromosome associated with the development of HNSCC.3 According to a recent tumor progr...

The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC.

To understand the association of candidate tumour suppressor genes SH3GL2, p16(INK4a), p14(ARF), and p15(INK4b) in the pathogenesis of head and neck squamous cell carcinoma (HNSCC), we studied the deletion, mutation, and methylation of these genes in 61 dysplastic lesions and 94 HNSCC samples. In mild dysplasia, SH3GL2, p16(INK4a), and p14(ARF) showed a higher frequency of overall alterations (60-70%) than in p15(INK4b) (40%). However, in subsequent stages of tumour progression, the alteration frequency of these genes did not change significantly. One novel mutation in common exon 2 of p16(INK4a)/p14(ARF) and three in exon 9 of SH3GL2 were seen. Concordance was seen in the expression of these genes with their molecular alterations. Deletions of INK4A-ARF and p15(INK4b) have a significant poor patient outcome. The alterations of p16(INK4a), p14(ARF), and p15(INK4b) were positively correlated with tobacco and inversely with HPV, while SH3GL2 alterations were independent of these factors. Based on aetiological factors, four tumour subtypes were recognized: HPV(-)tobacco(-) (I), HPV(+)tobacco(-) (II), HPV(-)tobacco(+) (III), and HPV(+)tobacco(+) (IV). Groups III and IV showed a high frequency of p16(INK4a)/p14(ARF)/p15(INK4b) alterations with significant poor patient outcome in comparison to group II. Our findings suggest that deregulation of SH3GL2-associated signalling and p16(INK4a)/p14(ARF)/p15(INK4b)-mediated G1-S/G2-M checkpoints of cell cycle are independent pathways for the development of early dysplastic lesions of the head and neck.

Previously, we reported that B-cell chronic lymphocytic leukemia (CLL) patients contained elevated levels of microvesicles (MVs). However, given the quiescent nature of CLL B-cells and the relative indolence of the disease, the dynamics of MV generation and their unique phenotypes are not clearly defined. In this study, we find that CLL B-cells generate MVs spontaneously and can be further induced by B-cell receptor-ligation. Most interestingly, CLL B-cells predominantly generate CD52+ MVs, but not CD19+ MVs in vitro, suggesting preferential usage of CD52 into leukemic-MVs and that the CLL plasma MV phenotypes corroborate well with the in vitro findings. Importantly, we detected increased accumulation of CD52+ MVs in previously untreated CLL patients with progressive disease. Finally, sequential studies on MVs in pre- and post-therapy CLL patients demonstrate that while the plasma CD52+ MV levels drop significantly after therapy in most and remain at low levels in some patients, a trend of increased accumulation of CD52+ MVs was detected in majority of post-therapy CLL patients (25 of 33). In total this study emphasizes that dynamic accumulation of CD52+ MVs in plasma can be used to study CLL progression and may be a useful biomarker for patients as they progress and require therapy.

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin a RLC (ITGA9), RB1 serine phosphates from human chromosome 3 (RBSP3) at chromosomal 3p22.3 region and development of head and neck squamous cell carcinoma (HNSCC), alterations (deletion ⁄ promoter methylation ⁄ expression) of these genes were analyzed in 65 dysplastic lesions and 84 HNSCC samples. Clinicopathological correlations were made with alterations of the genes. In HNSCC, deletion frequencies of hMLH1, ITGA9, and RBSP3 were comparatively higher than APRG1. Overall alterations (deletion ⁄ methylation) of hMLH1, ITGA9, and RBSP3 were high (45-55%) in mild dysplasia and comparable in subsequent stages of tumor progression. Quantitative RT-PCR analysis showed reduced expression of these genes in tumors concordant to their molecular alterations. An in vitro demethylation experiment by 5-aza-2¢-deoxycytidine confirmed the promoter hypermethylation of RBSP3 in Hep2 and UPCI:SCC084 cell lines. Functionally less-active RBSP3A isoform was predominant in tumor tissues contrary to the adjacent normal tissue of tumors where more active RBSP3B isoform was prevalent. In immunohistochemical analysis, intense nuclear staining of hMLH1 and pRB (phosphorylated RB, the substrate of RBSP3) proteins were seen in the basal layer of normal epithelium. In tumors, concordance was seen between (i) low ⁄ intermediate level of hMLH1 expression and its molecular alterations; and (ii) intense nuclear staining of pRB and RBSP3 alterations. Poor patient outcome was seen with hMLH1 and RBSP3 alterations. Moreover, in absence of human papilloma virus (HPV) infection, tobacco-addicted patients with hMLH1, RBSP3 alterations, and nodal invasions showed poor prognosis. Thus our data suggests that dysregulation of hMLH1, ITGA9, and RBSP3 associated multiple cellular pathways are needed for the development of early dysplastic lesions of the head and neck. (Cancer Sci 2010; 101: 1511-1520 H ead and neck squamous cell carcinoma (HNSCC) accounts 30-40% of all cancer types in the Indian subcontinent and is associated with prolonged tobacco habit, alcohol abuse, and human papilloma virus (HPV) infection, particularly types 16 and 18.(1) Despite improvements in treatment modalities, HNSCC is associated with high rates of recurrence and mortality and 5-year survival rates remain around 50%.(2) If identified early, the prognosis of HNSCC is excellent, so the implication of being able to classify genetic alterations during tumor progression is significant.(3) In a microcell hybrid system, there was evidence of suppression of tumorigenicity of oral cancer cell lines following introduction of chromosome (chr.) 3p, suggesting the presence of at least one tumor suppressor gene (TSG) in the chromosomal arm.(4) In a preliminary tumor progression model of HNSCC, allelic loss in chr. 3p was suggested to be associated with the transition of hyperplasia to dysplasia.Our previous study on HNSCC of Ind...