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Squamous cell carcinoma of the uterine cervix (CSCC) is one of the leading causes of death in Indian women. Protein tyrosine phosphatase receptor (PTPR) type J (also known as DEP1) is a recently reported tumour suppressor receptor phosphatase. Critical molecular analysis of PTPRJ/DEP1 (11p11.2) has not performed in CSCC to date. Here, we observed frequent downregulation of cancer samples ( n =31) at the transcriptional level. Immunohistochemistry revealed concordant low expression of PTPRJ protein with a few samples showing intermediate expression. To probe for the cause of such downregulation of the gene in CSCC ( n =155), we analysed the copy number and promoter methylation of PTPRJ. The genetic locus showed deletion (14.8%) and the promoter showed methylation (33.5%) of PTPRJ. To the best of our knowledge, for the first time we explored the molecular status of PTPRJ although we observed no statistically significant association with the prognosis of Indian CSCC patients ( n =76). However, we observed enhanced expression of PTPRJ protein levels that contributes to effective cisplatin chemotherapy in the SiHa cell line. Thus, the present study paves the way for further research into the plausible mechanisms of downregulation of PTPRJ in cervical cancer.

Reports of genetic association of polymorphisms with lung cancer in the Indian subcontinent are often conflicting. To summarise and replicate published evidence for association with lung cancer and its subgroups. We performed a meta-analysis of candidate associations on lung cancer, its histological subtypes and smoking status in the Indian subcontinent following PRISMA guidelines. Multiple testing corrections were done by the Benjamini-Hochberg method through assessment of significance at a false discovery rate of 10%. We genotyped and investigated rs1048943/CYP1A1 in a case-control sample from eastern India, followed by its global meta-analysis using a similar protocol. Meta-analysis of 18 variants of 11 genes reported in 39 studies (7630 cases and 8169 controls) showed significant association of rs1048943/CYP1A1 [2.07(1.49-2.87)] and rs4646903/CYP1A1 [1.48(1.93-1.95)] with overall lung cancer risk at 10% FDR, while nominal association (p < 0.05) was observed for del1/GSTT1, del2/GSTM1, rs1695/GSTP1 and rs17037102/ DKK2. Subtype analysis showed a significant association of del1/GSTT1 with adenocarcinoma, rs4646903/CYP1A1 with squamous carcinoma, and rs1048943/CYP1A1 with both. Association of rs4646903/CYP1A1 in smokers and effect modification by meta-regression analysis was observed. Genotyping of rs1048943/CYP1A1 that presented significant heterogeneity (p < 0.1) revealed an association with adenocarcinoma among eastern Indian smokers, while a global meta-analysis in 10458 cases and 10871 controls showed association with lung cancer and its subgroups. This study identified the susceptibility loci for lung cancer and its covariate-subgroups., (© 2021. The Author(s).)

Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.

Background: CSCC is one of the most common cancer affecting women globally. Though it is caused by the infection of hrHPV but long latency period for malignant outcome in only a subset of hrHPV infected women indicates involvement of additional alterations, primarily CNVs. Here, we showed how CNVs played a crucial role in development of advanced tumors (stage III/IV) in Indian patients., Methods: Initially, high-resolution CGH-SNP microarray analysis pointed out frequent CNVs followed by significantly altered genes. After comparison with TCGA dataset, expressions of the genes were checked in three CSCC datasets to identify key genes followed by Ingenuity® Pathway analysis. Then node effect property analysis was applied on the constructed PPI network to rank the key proteins. Finally, validations in independent samples were performed., Results: For the first time, frequent chromosomal amplifications at 3q13.13-3q29, 1p36.11-1p31.1, 1q21.1-1q44 and 5p15.33-5p12 followed by common deletions at 11q14.1-11q25, 2q34-2q37.3, 4p16.3-4p12 and 13q13.3-13q14.3 were identified in Indian CSCC patients. Integrative analysis found 78 key genes including several novel ones, which were mostly associated with 'Cancer' and may regulate DNA repair and metabolic pathways. Analysis showed PARP1 and ATR were among the top ranking protein interactors., Conclusions: Frequent amplification and over-expression of ATR and PARP1 were further confirmed in cervical lesions, indicating their association with poor prognosis of advanced CSCC patients., General Significance: Our novel approach identified precise CNVs along with several novel genes within these loci and showed that PARP1 and ATR, having biologically significant interactions, may be involved in development of advanced CSCC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Oral cancer is of major public health problem in India. Current investigation was aimed to identify the specific deregulated miRNAs which are responsible for development of resistance phenotype through regulating their resistance related target gene expression in oral squamous cell carcinoma (OSCC). Cisplatin-resistant OSCC cell lines were developed from their parental human OSCC cell lines and subsequently characterised. The resistant cells exhibited enhanced proliferative, clonogenic capacity with significant up-regulation of P-glycoprotein (ABCB1), c-Myc, survivin, β-catenin and a putative cancer-stem-like signature with increased expression of CD44, whereas the loss of E-cadherin signifies induced EMT phenotype. A comparative analysis of miRNA expression profiling in parental and cisplatin-resistant OSCC cell lines for a selected sets (deregulated miRNAs in head and neck cancer) revealed resistance specific signature. Moreover, we observed similar expression pattern for these resistance specific signature miRNAs in neoadjuvant chemotherapy treated and recurrent tumours compared to those with newly diagnosed primary tumours in patients with OSCC. All these results revealed that these miRNAs play an important role in the development of cisplatin-resistance mainly through modulating cancer stem-cell-like and EMT-type properties in OSCC.

Almost all epithelial tumours contain cancer stem-like cells, which possess a unique property of self-renewal and differentiation. In oral cancer, several biomarkers including cell surface molecules have been exploited for the identification of this highly tumorigenic population. Implicit is the role of CD44 in defining CSCs but CD24 is not well-explored. Here we show that CD44(high)CD24(low) cells isolated from the oral cancer cell lines, not only express stem cell related genes but also exhibit Epithelial-to-Mesenchymal transition (EMT) characteristics. This CD44(high)CD24(low) population gives rise to all other cell types upon differentiation. Typical Cancer Stem Cell (CSC) phenotypes like increased colony formation, sphere forming ability, migration and invasion were also confirmed in CD44(high)CD24(low) cells. Drug transporters were found to be over-expressed in CD44(high)CD24(low) sub-population thereby contributing to elevated chemo-resistance. To validate our findings in-vivo, we determined the relative expression of CD44 and CD24 in clinical samples of OSCC patients. CD44 expression was consistently high whereas CD24 showed significantly lower expression in tumour tissues. Further, the gene expression profile of the CSC and non-CSC population unravels the molecular pathways which may contribute to stemness. We conclude that CD44(high)CD24(low) represents cancer stem-like cells in Oral Squamous Cell Carcinoma., (Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.)

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.

Aim: This study examined the prognostic significance of candidate tumor suppressor genes (TSGs) PHD finger protein-2 (PHF2), Fanconi anaemia complementation group C (FANCC) and human homologue of Drosophila patched gene (PTCH1), in head and neck squamous cell carcinoma (HNSCC) treated primarily with surgery, or surgery followed by adjuvant radiotherapy., Patients and Methods: Eighty-four patients with HNSCC were followed-up for recurrence/death for up to five years after diagnosis. Molecular alterations (deletion/methylation) of TSGs and human papilloma virus (HPV) status were determined in previous studies of our group. Statistical analyses of correlation of genetic alterations with treatment response and survival were carried out., Results: Alterations of FANCC and PTCH1 were significantly associated with locoregional recurrence/death. In the surgery with adjuvant radiotherapy-group (n=56), patients showing alterations in FANCC and in PTCH1 were seven- and six-times, respectively, more likely to have locoregional recurrence compared to those with no alterations. In addition, the presence of alterations of both FANCC and PTCH1 had remarkable prognostic significance., Conclusion: FANCC and PTCH1 alterations might be used as molecular markers for prognosis and to develop strategies for effective treatment planning.

Background: Neoadjuvant chemotherapy (NACT) is a treatment modality whereby chemotherapy is used as the initial treatment of HNSCC in patients presenting with advanced cancer that cannot be treated by other means. It leads to shrinkage of tumours to an operable size without significant compromise to essential oro-facial organs of the patients. The molecular mechanisms behind shrinkage due to NACT is not well elucidated., Materials and Methods: Eleven pairs of primary HNSCCs and adjacent normal epithelium, before and after chemotherapy were screened for cell proliferation and apoptosis. This was followed by immunohistochemical analysis of some cell cycle (LIMD1, RBSP3, CDC25A, CCND1, cMYC, RB, pRB), DNA repair (MLH1, p53) and apoptosis (BAX, BCL2) associated proteins in the same set of samples., Results: Significant decrease in proliferation index and increase in apoptotic index was observed in post-therapy tumors compared to pre-therapy. Increase in the RB/ pRB ratio, along with higher expression of RBSP3 and LIMD1 and lower expression of cMYC were observed in post-therapy tumours, while CCND1 and CDC25A remained unchanged. While MLH1 remained unchanged, p53 showed higher expression in post-therapy tumors, indicating inhibition of cell proliferation and induction of apoptosis. Increase in the BAX/BCL2 ratio was observed in post-therapy tumours, indicating up-regulation of apoptosis in response to therapy., Conclusions: Thus, modulation of the G1/S cell cycle regulatory proteins and apoptosis associated proteins might play an important role in tumour shrinkage due to NACT.

Background: Alteration of chromosome 9q22.3 region is an early and frequent event in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to understand the association of candidate tumor suppressor genes PHF2, FANCC, PTCH1, and XPA located in this region in the development of HNSCC., Methods: The alterations (deletion, promoter methylation, mutation, expression) of these genes were analyzed in 65 dysplastic head and neck lesions and 84 primary HNSCC samples. Clinicopathologic correlations were made with alterations of the genes., Results: Overall alterations (deletion, promoter methylation) of FANCC and PTCH1 were high in mild dysplasia and comparable in subsequent stages of tumor progression. However, PHF2 alteration was low in mild dysplasia, but increased in moderate and severe dysplasias. Alterations (deletion, promoter methylation) of FANCC and PTCH1 showed association with each other. Two novel mutations in GLI binding sites of PTCH1 promoter and a novel microsatellite marker hmPTCH1 with four alleles at immediate upstream of the gene were identified. In a case-control study, the (CGG)7 allele of hmPTCH1 was found to be susceptible for HNSCC development. Concordance was seen in the expression (RNA, protein) of these genes with their molecular alterations., Conclusions: Alterations of FANCC and PTCH1 could be used as molecular marker for early diagnosis and prognosis of HNSCC.

Introduction: To understand the role of two interacting proteins LIMD1 and pRB in development of head and neck squamous cell carcinoma (HNSCC), alterations of these genes were analyzed in 25 dysplastic head and neck lesions, 58 primary HNSCC samples and two HNSCC cell lines., Methods: Deletions of LIMD1 and RB1 were analyzed along with mutation and promoter methylation analysis of LIMD1. The genotyping of LIMD1 linked microsatellite marker, hmlimD1, was done to find out any risk allele. The mRNA expression of LIMD1 and RB1 were analyzed by Q-PCR. Immunohistochemical analysis of RB1 was performed. Alterations of these genes were correlated with different clinicopathological parameters., Results: High frequency [94% (78/83)] of LIMD1 alterations was observed in the samples studied. Compare to frequent deletion and methylation, mutation of LIMD1 was increased during tumor progression (P = 0.007). Six novel mutations in exon1 and one novel intron4/exon5 splice-junction mutation were detected in LIMD1 along with a susceptible hmlimD1 (CA)20 allele. Some of these mutations [42% (14/33)] produced non-functional proteins. RB1 deletion was infrequent (27%). Highly reduced mRNA expression of LIMD1 (25.1 +/- 19.04) was seen than RB1 (3.8 +/- 8.09), concordant to their molecular alterations. The pRB expression supported this data. Tumors with LIMD1 alterations in tobacco addicted patients without HPV infection showed poor prognosis. Co-alterations of these genes led the worse patients' outcome., Conclusions: Our study suggests LIMD1 inactivation as primary event than inactivation of RB1 in HNSCC development.

Background: Amplification of the MYC gene is reported to be associated with the development of head and neck squamous cell carcinoma (HNSCC). This study is focused to analyze the correlation between MYC gene amplification and various clinicopathological features and outcome in a cohort of 49 dysplastic and 187 primary head and neck lesions., Methods: MYC gene amplification was assessed by differential polymerase chain reaction using primer sets from the MYC gene as target locus and DRD2 gene as the control locus., Result: The MYC gene amplification was detected in a total of 23.7% (56/236) head and neck lesions comprising 14.2% (7/49) dysplastic lesions and 26% (49/187) HNSCC samples. The clinicopathological association study between MYC gene amplification with the different clinical parameters like sex, tumor stage, tumor differentiation, lymph node status, tobacco habit and HPV 16/18 status determined significant association of MYC amplification with tumor progression (P = 0.009). Kaplan Meier analysis revealed MYC gene has no prognostic significance on survival in HNSCC., Conclusion: In conclusion, our results suggest that MYC gene amplification is associated with tumor progression in HNSCC.

Deletion of chromosomal 3p12.3 was suggested to be associated with dysplastic lesions of head and neck. This region harbors two candidate tumor suppressors ROBO1/DUTT1, ROBO2 and two non-coding RNAs (ncRNAs) located at intron 2 of ROBO1/DUTT1. Aim of this study is to understand the role of these genes in development of head and neck squamous cell carcinoma. A collection of 72 dysplastic lesions and 116 HNSCC samples and two oral cancer cell lines were analyzed for ROBO1/DUTT1 and ROBO2 deletion and promoter methylation. ROBO1/DUTT1, ROBO2 and two ncRNAs mRNA expression were analyzed by Q-PCR. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 was performed. Alterations of these genes were correlated with different clinicopathological parameters. High frequency of molecular alterations (deletion/methylation) was seen in ROBO1/DUTT1 than ROBO2. In mild dysplastic lesions both of these genes showed high molecular alterations and remained more or less constant in subsequent stages. Q-PCR analysis showed reduced expression of these genes and the two ncRNAs. In vitro demethylation experiment by 5-aza-dC showed upregulation of ROBO1/DUTT1 and ROBO2 while the expression of the ncRNAs remained unchanged. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 showed concordance with their mRNA expression and molecular alterations. Poor patients' outcome was predicted in the cases with alteration of ROBO1/DUTT1 along with tobacco addiction and nodal involvement. Our data suggests (a) ROBO1/DUTT1 and the ncRNAs are transcribed from different promoters, and (b) inactivation of ROBO1/DUTT1 could be used as molecular signature for early detection and prognosis of the head and neck cancer.

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.

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 +/- 16.8) > LIMD1 (53.2 +/- 20.1) > CACNA2D2 (23.7 +/- 7.1) > RASSF1A (15.1 +/- 5.6) > CDC25A (5.3 +/- 2.3) > SCOTIN (0.58 +/- 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., ((c) 2008 Wiley-Liss, Inc.)

Aim: To investigate the complex interplay between human papilloma virus (HPV) infection and p53 gene alteration in 92 head and neck squamous cell carcinoma (HNSCC) and 28 leukoplakia samples from eastern India., Methods: DNA isolated from the patient samples was subjected to HPV detection, loss of heterozygosity (LOH) analysis of the chromosome 17p region harbouring p53, genotyping at the p53 codon 72 locus and sequencing of the entire p53 gene to identify somatic mutations. Codon 72 heterozygotes carrying the p53 mutation were further cloned and resequenced to identify the allele harbouring the mutation., Results: HPV positivity in the HNSCC samples was 69%; 21% of the HNSCC were found to harbour p53 mutations in the coding region of the gene. The absence of the p53 mutation in HPV positive tumours was statistically significant compared to the HPV negative tumours (p = 0.01), but the same did not hold true for p53 LOH (p = 1.0). Among the germline p53 codon 72 heterozygotes, the Pro allele was preferentially lost (p = 0.02) while the Arg allele was mutated in the majority of cases. The risk of HPV mediated tumourigenesis increased with the increase in number of Arg alleles at the codon 72 locus., Conclusion: It is proposed that genetic and epigenetic alteration of p53 follow distinct pathways during the development of HNSCC from normal epithelium via dysplasia. The p53 mutation and HPV mediated p53 inactivation possibly constitute two independent pathways of tumourigenesis.

Background: A subset of head and neck squamous cell carcinoma (HNSCC) exhibits a microsatellite instability (MIN) phenotype. The authors correlated alterations in the mismatch-repair genes human mutL homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2) in primary head and neck squamous cell carcinoma (HNSCC) tumors and in samples of leukoplakia with the MIN phenotype., Methods: One hundred twenty-three paired HNSCC normal and tumor tissues and 27 leukoplakia samples were examined for hypermethylation of hMLH1 and hMSH2 promoters. The hypermethylation status of the tissues was confirmed by expression studies. Sixty-three of 123 randomly selected tumors and all 27 leukplakia samples were genotyped with 8 microsatellite markers to determine MIN., Results: Fifty percent of HNSCC tumors and 63% of leukoplakia samples harbored hypermethylation at either or both hMLH1 and hMSH2 promoters. Normal tissues adjacent to methylation-positive tumors also demonstrated hypermethylation of both promoters at a high frequency (25%). A positive correlation between tobacco habit and promoter hypermethylation was observed (P = .001). A correlation was observed between MIN and the frequency of promoter hypermethylation in the leukoplakia samples, but no such trend was observed in the HNSCC tumors. It is noteworthy that patients who had a high frequency of MIN-positive tumors exhibited hypermethylation in both the affected tissues and the adjacent normal tissues (P = .007). Patients with a tobacco habit who had promoter hypermethylation at both the affected tissues and the adjacent normal tissues had tumors that mostly were MIN positive (P = .047)., Conclusions: The current results suggested that tobacco-addicted individuals are more susceptible to promoter hypermethylation of hMLH1 and hMSH2 and that, if such hypermethylation occurs in the normal squamous epithelium of the head and neck region, then those tissues are likely to develop into tumors that involve the MIN pathway.

Defects in the spindle assembly checkpoint are thought to be responsible for an increased rate of aneuploidization during tumorigenesis. Despite a plethora of information on the correlation between BUB-MAD gene expression levels and defects in the spindle checkpoint, very little is known about alteration of another important spindle checkpoint protein, Cdc20, in human cancer and its role in tumor aneuploidy. We observed overexpression of CDC20 in several oral squamous cell carcinoma (OSCC) cell lines and primary head and neck tumors and provide evidence that such overexpression of CDC20 is associated with premature anaphase promotion, resulting in mitotic abnormalities in OSCC cell lines. We also reconstituted the chromosomal instability phenotype in a chromosomally stable OSCC cell line by overexpressing CDC20. Thus, abnormalities in the cellular level of Cdc20 may deregulate the timing of anaphase promoting complex (APC/C) in promoting premature anaphase, which often results in aneuploidy in the tumor cells.

Deletions in chromosome (chr.) 13q occur frequently in head and neck squamous cell carcinoma (HNSCC). Previous studies failed to identify common deleted regions in chr.13q, though several candidate tumour suppressor genes (TSGs) loci, e.g. BRCA2, RB1 and BRCAX have been localized in this chromosome, as well as no prognostic significance of the deletion has been reported. Thus, in the present study, deletion mapping of chr. 13q has been done in 55 primary HNSCC samples of Indian patients using 11 highly polymorphic microsatellite markers of which three were intragenic to BRCA2 gene, one intragenic to RB1 gene and another from BRCAX locus. The deletion in chr.13q was significantly associated with progression of HNSCC. High frequencies (27-39%) of loss of heterozygosity were found in 13q13.1 (BRCA2), 13q14.2 (RB1), 13q21.2-22.1 (BRCAX) and 13q31.1 regions. Deletions in the BRCA2 and RB1 regions were significantly correlated. The four highly deleted regions were associated with clinical stage and histological grades of the tumour as well as poor patient outcome. Deletion in the 13q31.1 region was only found to be associated with HPV infection. High frequencies (11-23%) of microsatellite size alteration (MA) were seen to overlap with the highly deleted regions. Forty per cent of the samples showed rare biallelic alteration whereas loss of normal copy of chromosome 13q was seen in five tumours. Thus, it seems that the putative TSGs located in the BRCAX and 13q31.1 regions as well as the BRCA2 and RB1 genes may have some cumulative effect in progression and poor prognosis of HNSCC. Significant association between deletion in BRCA2 and RB1 gene loci may indicate functional relationship between the genes in this tumour progression.

Intrinsic genomic instability of an incipient tumor cell drives the development of cancer. In colorectal cancer, an inverse relationship between microsatellite instability (MIN) and chromosomal instability (CIN) has been reported. The relationship between MIN and CIN in head and neck squamous cell carcinoma (HNSCC) is uncertain. In the present study, we examined these two types of instabilities in HNSCC using microsatellite markers and arbitrary primed PCR (APPCR) technique. HNSCC tumors showed high frequency of MIN and loss of heterozygosity (LOH). We observed that, in contrast to colorectal tumors, the frequency of LOH increased with the increase in MIN. There was no significant difference between MIN- and MIN+ groups of HNSCC tumors in the extent of overall genomic alterations; rather higher MIN+ tumors exhibited higher incidence of deletion. Thus, in sporadic HNSCC, both MIN and CIN pathways operate simultaneously to drive tumor formation., (Copyright 2006 Wiley-Liss, Inc)

In this study, the alterations (amplification/rearrangement) of 3 D-type cyclins loci were analyzed by Southern blot in 5 dysplastic head and neck lesions and 79 primary head and neck squamous cell carcinoma (HNSCC) of Indian patients to understand the role of the cyclins in development of the disease. No alteration was found in the dysplastic lesions. Overall, 54% of alterations were found in bcl-1/CCND1 locus, whereas amplification was only found in CCND2 and CCND3 loci in 12% and 2% samples, respectively. In bcl-1/CCND1 locus amplification was the major type of alteration; however, rearrangement as well as coalterations had been seen in some samples indicating the common mechanism of activation of this locus in different types of tumors. In bcl-1 region, the breakpoint clustered in the MTC (major translocation cluster) region, whereas in CCND1 the breakpoint located near 3' end of the gene. The coamplification of CCND2 locus with bcl-1, bcl-1/CCND1, and CNND3 loci suggests cumulative effect of these genes in this tumor. The significant association was seen between bcl-1/CCND1 locus alteration with HPV prevalence and poor patient outcome indicating its importance as prognostic marker. This indicates that the genetic instability caused due to HPV infection may induce the alterations in the bcl-1/CCND1 locus, which will provide selective growth advantage to the specific malignant clones resulting poor prognosis of the disease.

The role of 3 p53 polymorphisms (16 bp duplication at intron 3, codon 72 Arg/Pro and intron 6 NciI RFLP at np 13494) as potential markers for indicating cancer risk remains inconclusive. In our case-control study consisting of 197 leukoplakia and 310 oral squamous cell carcinoma (SCC) patients and 348 controls, genotype frequencies at these 3 p53 loci were determined by PCR-RFLP method and analyzed by multiple logistic regression to determine the risks of the diseases. The 2/2 genotype at codon 72 of p53 was at risk for developing leukoplakia (OR = 1.6, 95% CI 1.1-2.3), whereas the combination of 1/2 and 2/2 genotypes at intron 3 and 1/1 and 1/2 genotypes at intron 6 conferred a protective effect against leukoplakia and oral SCC development, respectively (OR = 0.5, 95% CI 0.4-0.8 and OR = 0.6, 95% CI 0.5-0.9, respectively). When subjects were stratified according to specific tobacco habit, the risk/protection estimates improved significantly in some cases. Specifically, the exclusive smokers with p53 codon 72 2/2 genotype showed a higher risk of developing leukoplakia (OR = 2.7, 95% CI 1.2-6.3). Furthermore, a particular p53 haplotype 1-2-2 was at risk for both tobacco-associated leukoplakia and oral SCC (OR = 1.5, 95% CI 1.1-1.9 and OR = 1.3, 95% CI 1.1-1.7, respectively). Our results show that both specific p53 genotype and haplotype can indicate risk of tobacco-associated leukoplakia, but risk of development of tobacco-associated oral SCC can be predicted by specific p53 haplotype only., (Copyright 2005 Wiley-Liss, Inc)

Amplification of the MYC gene is reported to be associated with the development of head and neck squamous cell carcinoma (HNSCC). However, there are no data concerning whether the amplification is confined to the MYC gene itself or spans distant 5' and/or 3' regions of this gene in HNSCC as seen in different lymphomas, colon carcinoma, and uterine cervical carcinoma (CaCx). In this study, we analyzed the alterations (amplification/rearrangement) in the 580 Kb surrounding of the MYC gene to understand the status of this locus in primary HNSCC of Indian patients. The MYC alterations were analyzed by Southern blot using the pal-1/MYC/MLVI4 probes. The alterations in the MYC locus (adjacent region of the c-myc gene) were then correlated with the various clinicopathological parameters. The overall amplification involving the MYC locus was seen in 46% of the samples. The MYC gene, pal-1 region, and MLVI4 region were amplified in about 38%, 24%, and 20% of the samples, respectively. Some samples showed co-amplification encompassing pal-1-MYC-MLVI4 or pal-1-MYC or MYC-MLVI4 regions. No significant association was observed between the amplification in the MYC locus and the different clinicopathological parameters except for tumor differentiation. Thus, it seems that, similar to other tumors, the MYC gene may be activated by amplification in its surrounding 5' and/or 3' region in HNSCC.

Background: The predictive value of codon 72 arginine homozygosity at the p53 gene for human papilloma virus associated cervical cancer risk remains inconclusive. It has also been proposed that the inheritance of specific germline haplotypes based on three biallelic polymorphisms of p53 (intron 3 16 bp duplication, codon 72 Bst UI (Arg/Pro), and intron 6 Nci I restriction fragment length polymorphism at nucleotide 13494) is a better predictor of various cancer risks., Aims: To determine the genotype and haplotype frequency of these three p53 polymorphisms in 61 patients with cervical squamous cell carcinoma and 94 ethnically matched controls from the eastern region of India and estimate the risk, if any, of specific genotypes and haplotypes., Methods: Samples were genotyped by polymerase chain reaction followed by variant specific restriction enzyme digestion. Haplotypes were estimated by the maximum likelihood method using the expectation maximisation algorithm., Results: Genotype distributions of the three polymorphisms in patients and controls showed a good fit to the Hardy-Weinberg equilibrium. The p53 codon 72 arginine homozygous genotype was significantly over represented in patients compared with controls. Those with the homozygous arginine genotype exhibited a 2.59 fold higher risk of developing squamous cell carcinoma of the uterine cervix. A significant risk was also seen with a combination of two haplotypes, 1-2-1 and 1-2-2., Conclusion: p53 codon 72 arginine homozygotes appear to be at greater risk of developing squamous cell carcinoma of the uterine cervix. The high risk haplotypes 1-2-1 and 1-2-2 also contain the arginine allele, further strengthening this conclusion.

In this study we performed detailed deletion mapping of two broad regions in the short arm (p) of chromosome 3 (i.e., 3p21.2 approximately p22 and 3p12 approximately p13), which were shown to have a high rate of deletions in head and neck lesions in our previous study. Using 18 highly polymorphic microsatellite markers, the deletion mapping was done in 35 dysplastic lesions and 46 primary head and neck squamous cell carcinoma (HNSCC) samples from Indian patients. Within the 21.6-megabase (Mb) region of 3p21.1 approximately p21.33, we have identified four areas (D1, 3p21.33; D2, 3p21.32; D3, 3p21.31; D4, 3p21.1) that showed a high frequency (46%-69%) of deletions in our samples. In the 3p12 approximately p13 region, we narrowed down the deletion within the 0.7-Mb region (D5, 3p12.1). Among these five regions (D1-D5), deletion in D3 is suggested to be necessary for the development of early dysplastic lesions, whereas the deletion in D2 may be necessary for dysplastic lesions and tumor progression. On the other hand, the deletion in D5 is significantly associated with progression of the lesions from mild/moderate to severe dysplasia. The deletions in D1 and D4, however, are required for tumor progression. As in our previous study, microsatellite size alterations (MA) were observed to be high in and around the highly deleted regions and gradually increased during the progression of the tumor. Loss of normal copy/interstitial alterations of chromosome 3 in the late stages of the tumor as well as rare biallelic alterations around the highly deleted regions also were seen in our samples. Human papilloma virus infection has been found to be associated with the deletion in the D5 region and MA in the D1 region, whereas nodal involvement of the tumor correlated only with the MA in D1 and D5. Thus, this study indicates that multiple tumor suppressor genes whose differential deletions are associated with the development of HNSCC may be present in 3p.

Background: Genetic instability of chromosome 11 is a frequent event in many solid tumours, including head and neck squamous cell carcinoma (HNSCC)., Aims: To perform allelic imbalance analysis of cytogenetically mapped altered regions of human chromosome 11 in patients with HNSCC from eastern India., Methods: Genomic alterations were investigated using highly polymorphic microsatellite markers in both HNSCC and leukoplakia tissues., Results: Microsatellite markers D11S1758 from 11p13-15 and D11S925 from 11q23.3-24 had the highest frequency (38% and 32%, respectively) of loss of heterozygosity among all the markers analysed. Allelic loss at the marker D11S925 was seen in both leukoplakia and in all stages of HNSCC tumour tissues suggesting that it is an early event in HNSCC tumorigenesis. Microsatellite size alteration was also found to be high (> 20%) in several markers. In leukoplakia samples microsatellite instability was seen at a higher frequency than loss of the allele, indicating such alterations might initiate the process of tumorigenesis in HNSCC., Conclusions: The high rate of chromosomal alterations at 11q21-24 in HNSCC suggests the presence of a putative tumour suppressor gene in this region.

Genomic instability in simple repeated sequences has been observed in several human cancers. We have analyzed 50 squamous cell carcinomas of the head and neck (SCCHN) and 5 pre-malignant severe dysplastic tissues from Indian patient populations for microsatellite instability in 18 different loci spread over eight different chromosomes. Among the tumors analyzed, 45% exhibited instability at two or more loci, and 15% exhibited instability at 40% of the markers tested. Similar analysis of SCCHN tumors from other populations (British, American and French) showed much less frequency of instability. SCCHN tumors in the present study did not show any instability in the mononucleotide repeat sequences. There is also a clear distinction in the nature of the instability in these tumors in comparison with colorectal tumors. These results suggest that the underlying mechanism generating this type of instability is different from those reported for colorectal tumors.