Amlan Ghosh

Frequent alterations of the candidate genes hMLH1, ITGA9 and RBSP3 in early dysplastic lesions of head and neck: Clinical and prognostic significance

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin α 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)

Alterations of 3p21.31 Tumor Suppressor Genes in Head and Neck Squamous Cell Carcinoma: Correlation With Progression and Prognosis

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.

SH3GL2 and CDKN2A/2B loci are independently altered in early dysplastic lesions of head and neck: correlation with HPV infection and tobacco habit

To understand the association of candidate tumour suppressor genes SH3GL2, p16INK4a, p14ARF, and p15INK4b 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, p16INK4a, and p14ARF showed a higher frequency of overall alterations (60–70%) than in p15INK4b (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 p16INK4a/p14ARF 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 p15INK4b have a significant poor patient outcome. The alterations of p16INK4a, p14ARF, and p15INK4b 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 p16INK4a/p14ARF/p15INK4b alterations with significant poor patient outcome in comparison to group II. Our findings suggest that deregulation of SH3GL2‐associated signalling and p16INK4a/p14ARF/p15INK4b‐mediated G1–S/G2–M checkpoints of cell cycle are independent pathways for the development of early dysplastic lesions of the head and neck. Copyright

Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes

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.

Association of FANCC and PTCH1 with the Development of Early Dysplastic Lesions of the Head and Neck

BackgroundAlteration 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.MethodsThe 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.ResultsOverall 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.ConclusionsAlterations of FANCC and PTCH1 could be used as molecular marker for early diagnosis and prognosis of HNSCC.

Alterations of ROBO1/DUTT1 and ROBO2 loci in early dysplastic lesions of head and neck: clinical and prognostic implications

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.

Reduced Expression of Limd1 in Ulcerative Oral Epithelium Associated with Tobacco and Areca Nut

PURPOSE The aim of this study was to cast light on initiating molecular events associated with the development of premalignant oral lesions induced by tobacco and/or areca nut. METHOD Immunohistochemical analyses of cell cycle regulatory proteins (LIMD1, RBSP3, p16, RB, phosphorylated RB, p53), EGFR and SH3GL2 (EGFR associated protein) were performed with inflammatory/ ulcerative epithelium and adjacent hyperplastic/mild dysplastic lesions. RESULTS No change in expression of the proteins was seen in inflammatory epithelium. Reduced nuclear expression of LIMD1 was evident in ulcerative epithelium. In hyperplastic lesions, reduced expression of RBSP3, p16, SH3GL2 and overexpression of p-RB and EGFR were apparent. Reduced nuclear expression of p53 was observed in mild dysplastic lesions. CONCLUSION Our data suggest that inactivation of LIMD1 in ulcerative epithelium might predispose the tissues to alterations of other cell cycle regulatory and EGFR signaling proteins needed for the development of premalignant oral lesions.

Sensitivity to House Dust Mites Allergens with Atopic Asthma and Its Relationship with CD14 C(-159T) Polymorphism in Patients of West Bengal, India

Abstract India is the home to around 15–20 million asthmatics, and asthma prevalence is increasing in Indian metropolitan area, including Kolkata, West Bengal. Complex interactions of genetic and environmental factors are involved in asthma. Genome-wide search for susceptible loci regulating IgE response (atopy) have identified a candidate gene CD14 which is most important in the context of allergic responses of respiratory system. This study was aimed to investigate the role of house dust and house dust mites in development of bronchial asthma and to explore the possible association of candidate gene CD14 with disease manifestation among Kolkata patient population. Skinprick test was done among 950 asthmatic patients against 8 aeroallergens, including house dust and house dust mites and total serum IgE and allergen-specific IgE were measured. Polymerase chain reaction-restriction fragment length polymorphism was done in patients and nonasthmatic control (n = 255 in each) to characterize a functional polymorphism, C(-159)T, of CD14, a positional candidate gene for allergy. We identified house dust as the most common aeroallergen sensitizer among atopic patients in Kolkata followed by Dermatophagoides pteronyssinus and Dermatophagoides farinae Hughes (Acari: Pyroglyphidae) mites. Patients sera contain significantly higher IgE level than that of control. Allergen-specific IgE antibody test revealed that 76.36% patients had specific IgE antibody against D. pteronyssinus mite. There was a significant difference in the distribution of alleles and genotypes for CD14 polymorphism with an increase in disease severity. So, in Kolkata, house dust mite is a common aeroallergen and D. pteronyssinus is predominant among mites. The present study revealed that bronchial asthma has a genetic background.

Role of offending out-door aero-allergen and CD14 C(-159)T polymorphism in development and severity of asthma in a Kolkata patient population.

Objective Present study involved identification of offending out-door aero-allergens and associated genetic pathway in nasso-bronchial asthma among Kolkata population. Methods Skin-prick test was done among 950 asthmatic patients against 11 common aero-allergens and total serum IgE concentration was measured. PCR-RFLP was done in patients and non-asthmatic control (n=220 in each) to characterize functional polymorphism, C(-159)T, of CD14, a positional candidate gene for allergy. Association of genetic polymorphisms was made with clinico-pathological conditions. Results We identified Cocos nucifera as the most common aero-allergen sensitizer among atopic patients in Kolkata. Patients with childhood-onset asthma were significantly more sensitive towards aero-allergens and had significantly higher serum IgE level than those of adult-onset (p< 0.0001). No significant difference was found in distribution of SNP genotypes of CD14 among case and control (p=0.178). However among patients, frequency of C allele is significantly higher in childhood-onset group than that of adult-onset and concordantly in former CC genotype was associated with significant higher level of serum IgE than CT and TT. Conclusion In Kolkata, pollen is a common out-door aero-allergen and Cocos nucifera is predominant among pollens. Childhood-onset and adult-onset of asthma showed significant difference in allergen sensitivity as well as genetic background with respect to CD14 polymorphism.

Diurnal Activity Pattern of Golden Jackal ( Canis aureus Linn.) in an Urban Landscape of Kolkata, India

The Golden jackal is known to have a wide distribution range and has been put under “Least Concern” category in the IUCN (ver 3.1) Red Data Book. However, the species once quite common along with the Bengal Fox in the eighteenth and nineteenth century of Kolkata has faced serious challenge due to land use and land cover changes in their urban habitats. As such, the Golden jackal has become “locally endangered” in the city of Kolkata, India. Kolkata, one of the largest cities in India, with population density of 14,112,536 is one such example where the land use change is profound. The Tollygunge Club, Kolkata is a social club with restricted entry at the heart of the city of Kolkata and harbours a healthy breeding population of the Golden jackal (Canis aureus Linn.). The present study focuses on the adaptability of the jackals to human proximity and its capacity to switch to natural food (fruits and leaves of available plants in the club, molluscs etc.) present in the club when the club refuse is not available. Their diurnal activity has been studied. These animals show diurnal activity in the open terrains of the club as they are least threatened by the humans. Only few intraspecific and interspecific (with the stray dogs in the club premises) conflicts have been noticed. In is interesting to note that at the heart of a city, in an urban area, such social clubs with restricted entry can provide suitable habitat to sustain a healthy breeding populations of the animals which are showing constant decline in their population due to change in land use pattern.