Effect of p53 codon 72 polymorphism on the survival outcome in advanced stage cervical cancer patients in India.

BACKGROUND & OBJECTIVES: The Arg>Pro polymorphism in codon 72 of p53 gene is known to affect the susceptibility of cervical cancer differently in different population worldwide although information regarding its role in determining survival status and disease outcome in patients is lacking. The present study was conducted to determine the genotype frequency and prognostic role of p53 codon 72 Arg>Pro polymorphism in patients with advanced stage cervical cancer in India. METHODS: The p53 codon 72 polymorphism was determined in tumour biopsies (n = 107) and matched blood samples (n = 19) in cervical cancer patients using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Effect of p53 genotype on the overall survival (OS) and recurrence-free survival (RFS) was analyzed. Individual Arg or Pro alleles were studied for their significance on survival as Pro carriers (Pro/Pro + Arg/Pro) versus Arg/Arg individuals or Arg carriers (Arg/Arg + Arg/Pro) versus Pro/Pro individuals. RESULTS: The frequencies for Arg/Arg, Arg/Pro and Pro/Pro genotypes were 27.2, 49.5 and 23.3 per cent, respectively. There was no significant difference in the genotypes with respect to patients' OS or RFS. INTERPRETATION & CONCLUSIONS: The findings of our study indicated that p53 codon 72 polymorphism might not be an independent marker in predicting clinical outcome in advanced stage cervical cancer patients. Further studies need to be done in larger samples to confirm these findings.

Human papillomavirus (HPV) genome status & cervical cancer outcome--A retrospective study.

BACKGROUND & OBJECTIVES: Persistent infections with high-risk (HR) human papillomaviruses such as HPV 16, 18, 31, 33 and 45 have been identified as the major aetiological factor for cervical cancer. The clinical outcome of the disease is often determined by viral factors such as viral load, physical status and oncogene expression. The aim of the present study was to evaluate the impact of such factors on clinical outcome in HPV16 positive, locally advanced cervical cancer cases. METHODS: One hundred and thirty two pretreatment cervical tumour biopsies were selected from patients undergoing radiotherapy alone (n=63) or concomitant chemo-radiation (n=69). All the samples were positive for HPV 16. Quantitative real time-PCR was carried out to determine viral load and oncogene expression. Physical status of the virus was determined for all the samples by the ratio of E2 copies /E7 copies ; while in 73 cases, the status was reanalyzed by more sensitive APOT (amplification of papillomavirus oncogene transcripts) assay. Univariate analysis of recurrence free survival was carried out using Kaplan-Meier method and for multivariate analysis the Cox proportional hazard model was used. RESULTS: The median viral load was 19.4 (IQR, 1.9- 69.3), with viral integration observed in 86 per cent cases by combination of the two methodologies. Both univariate and multivariate analyses identified viral physical status as a good predictor of clinical outcome following radiation treatment, with episomal form being associated with increased recurrence free survival. INTERPRETATION & CONCLUSIONS: The present study results showed that viral physical status might act as an important prognostic factor in cervical cancer.

Evidence for the presence of high risk human papillomavirus in retinoblastoma tissue from nonfamilial retinoblastoma in developing countries.

BACKGROUND: The prevalence of human papillomavirus (HPV) infection in India is high. HPV infection is known to cause cervical cancer and has also been implicated in the pathogenesis of retinoblastoma (RB), a common intraocular malignant tumor of childhood which can be familial or sporadic. Despite the high incidence of RB in India, its familial form is rare. Hence this study was undertaken to investigate whether high-risk HPV types 16 and 18 are involved in the development of RB. METHODS: Formalin fixed paraffin embedded RB tissues (n = 76) including prospective cases with corresponding maternal cervical smears (n = 10) were analyzed for the presence of HPV DNA sequences. Expression of the cell cycle regulatory proteins viz; p105, p107, p30, p16, E2F-1, E2F-4, and MiB-1 was studied by immunohistochemistry (IHC) (n = 34). RESULTS: A total of 53 out of 76 (69.7%) cases were positive for HPV, of these 3 cases were positive for HPV-16, 23 for HPV-18, and 27 for both HPV-16 and -18. Of the prospective cases (n = 10) studied, five cases along with the corresponding maternal cervical cytology smear had identical HPV status. HPV-16 positive tumors were classified as well differentiated (P = 0.013). Nuclear expression of pRB2/p130 showed significant association with HPV-16 infection (P = 0.04) or dual infection of HPV-16/-18 (P = 0.02). CONCLUSIONS: Our study lends support to the hypothesis that infection of HPV-16/-18 may play an important role in the development of nonfamilial form of RB in children in India.

Establishment & characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract & healthy individuals.

BACKGROUND & OBJECTIVES: A major drawback for genetic studies as well as long-term genotype-phenotype correlation studies in cancer is lack of representative human cell lines providing a continuous source of basic biomolecules and a system to carry out various experimental investigations. This can be overcome to some extent by establishing lymphoblastoid cell lines (LCLs) by infecting peripheral blood lymphocytes with Epstein Barr virus (EBV) which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines. The present study involves preparation and characterization of LCLs generated from patients with multiple primary neoplasms (MPN) of upper aero-digestive tract (UADT). METHODS: Thirty seven LCLs were established from UADT MPN patients and healthy age, sex and habit matched controls using EBV crude stock. Characterization was done with respect to expression of CD-19 (Pan B-cell marker), CD3 (T cell specific marker), CD56 (NK-cell specific marker), cell morphology, ploidy analysis, genotype and gene expression comparison with the parent lymphocytes. RESULTS: LCLs showed rosette morphology with doubling time of approximately 24 h. Ploidy analysis showed diploid DNA content which was maintained for at least 30 population doublings. When compared with parent lymphocytes there appeared no change at genetic and gene expression level. INTERPRETATION & CONCLUSIONS: Our results show that lymphoblastoid cell lines are a good surrogate of isolated lymphocytes bearing their close resemblance at genetic and phenotypic level to parent lymphocytes and are a valuable resource for understanding genotype-phenotype interactions.

HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse model.

Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model.

Proton sponge trick for pH-sensitive disassembly of polyethylenimine-based siRNA delivery systems.

Small interfering RNAs offer novel opportunities to inhibit gene expression in a highly selective and efficient manner but depend on cytosolic translocation with synthetic delivery systems. The polyethylenimine (PEI) is widely used for plasmid DNA transfection. However, the water-soluble PEI does not form siRNA polyplexes stable enough in extracellular media for effective delivery. We previously showed that rendering PEI insoluble in physiological media, without modifying drastically its overall cationic charge density, by simple conjugation with natural hydrophobic alpha-amino acids, can lead to effective siRNA delivery in mammalian cells. In here, we comprehensively investigated the mechanism behind the excellent efficacy of the leading PEIY vector. Our data revealed that the underlining proton sponge property is key to the effectiveness of the tyrosine-polyethylenimine conjugate as it may allow both endosomal rupture and siRNA liberation via an optimal pH-sensitive dissolution of the PEIY self-aggregates. Altogether, these results should facilitate the development of novel and more sophisticated siRNA delivery systems.

Effect of suicide gene therapy in combination with immunotherapy on antitumour immune response & tumour regression in a xenograft mouse model for head & neck squamous cell carcinoma.

BACKGROUND & OBJECTIVES: Prodrug activation strategy as well as immunotherapy have been widely used for cancer gene therapy. In the present study, using a head and neck squamous cell carcinoma (HNSCC) xenograft nude mouse model, we have investigated whether the two therapies in combination could improve tumour cell kill. We also investigated induction of immune effector cells viz., NK (DX5+) and DC (CD11c+) in vivo, post-combination gene therapy. METHODS: A retroviral vector producing cell line (PLTK47.1 VPC) carrying Herpes simplex virus thymidine kinase gene (HSVtk) was used for intratumoural injection into NT8e xenograft tumours followed by the prodrug ganciclovir (GCV). IL-2 plasmid DNA was injected intramuscularly. Immune cells were analyzed by flow-cytometry. Non parametric ANOVA was performed with Kruskal Wallis test. RESULTS: IL-2 could induce proliferation of both NK cells (DX5+) and dendritic cells (CD11c+) in vivo. Apoptosis was higher in combination therapy group as compared to HSVtk/GCV alone or IL-2 alone and was mediated through caspase-3 dependent pathway. Significant reduction in tumour volume was seen in all 3 treatment arms as compared to controls. INTERPRETATION & CONCLUSIONS: Combination of suicide gene therapy and immunotherapy leads to successful tumour regression in a HNSCC xenograft mouse model. Immunotherapy could help in a systemic long lived anti-tumour immune response which would prove powerful for the treatment of metastatic cancers, and also for minimal residual disease. The results of this study may form the basis for Phase 1 clinical trials.

Expansion and characterization of cells from surgically removed intervertebral disc fragments in xenogen-free medium.

Low back pain due to degeneration of intervertebral disc (IVD) is a major health problem resulting in significant disability as well as adding to the economic burden. Discectomy is a very common procedure done worldwide to relieve this pain. At present all the surgically removed disc tissue is mostly discarded. However, there are reports that state that progenitor cells in the IVD can be grown ex vivo and have the potential to be used for IVD repair and regeneration. We report here that viable cells can be harvested from surgically removed, herniated disc tissue and can be potentially used in cell based therapy. Further, we have successfully replaced xenogenic supplements such as foetal bovine serum with either autologous serum or human platelet lysate for culturing IVD cells from patient's surgically removed disc tissue, without loss of any cell characteristics, including cell surface markers, growth factor secretion in the conditioned medium and osteogenic and chondrogenic differentiation potential in vitro. The present work will not only contribute to overcoming some of the major barriers in carrying out human clinical trials, but also provide a cheap, alternate source of proteins and growth factors for growing IVD cells ex vivo for therapy.

Cholic acid-modified polyethylenimine: in vitro and in vivo studies.

Low-molecular-weight polyethylenimine has lower cytotoxicity than high molecular weight polyethylenimine, but it is not an efficient transfection agent because of limitations of DNA delivery into the cytoplasm. Therefore, in the present study, the hydrophobic modification of low-molecular-weight polyethylenimine (PEI 2 kDa [PEI2]) by cholic acid (ChA) was performed to form PEI2-ChA, and in vitro and in vivo studies were performed. Results indicate that the nanoplexes of PEI2-ChA with gWIZ-GFP have greater transfection efficiency (27%) in NT8e cell lines as evaluated by flow cytometry and also observed by fluorescence imaging. The present study concluded that the transferrin-containing nanoplexes of PEI2-ChA conjugates with plasmid p53 warrant clinical trials in humans after exhaustive animal studies for use as a novel gene delivery system.

Metastasis of squamous cell carcinoma of the oral tongue is associated with down-regulation of epidermal fatty acid binding protein (E-FABP).

Squamous cell carcinoma (SCC) of the tongue is one of the most common cancers encountered in India, due to the prevalent habits of tobacco chewing and smoking. Up to 40% of the early stage tumours (clinical N0 M0) presenting at the Tata Memorial Hospital have occult cervical lymph node metastasis. Therefore, features in the primary tumour that would predict metastasis would be very useful for designing therapeutic approaches. Hence, we aimed at detecting genotypic markers of the metastatic sub-clones within the heterogeneous primary tumour population. We studied the differential expression of mRNAs between the primary tumour samples of SCC of the oral tongue and their metastasis by differential display analysis and identified a gene, FABP5, coding for Epidermal fatty acid binding protein (E-FABP-GenBank Accession ). Its expression was up to 4-fold higher in the primary tumours (67%) as compared to the corresponding metastatic lymph nodes by northern blot analysis.

Somatic Variations in Cervical Cancers in Indian Patients.

There are very few reports that describe the mutational landscape of cervical cancer, one of the leading cancers in Indian women. The aim of the present study was to investigate the somatic mutations that occur in cervical cancer. Whole exome sequencing of 10 treatment naïve tumour biopsies with matched blood samples, from a cohort of Indian patients with locally advanced disease, was performed. The data revealed missense mutations across 1282 genes, out of 1831 genes harbouring somatic mutations. These missense mutations (nonsynonymous + stop-gained) when compared with pre-existing mutations in the COSMIC database showed that 272 mutations in 250 genes were already reported although from cancers other than cervical cancer. More than 1000 novel somatic variations were obtained in matched tumour samples. Pathways / genes that are frequently mutated in various other cancers were found to be mutated in cervical cancers. A significant enrichment of somatic mutations in the MAPK pathway was observed, some of which could be potentially targetable. This is the first report of whole exome sequencing of well annotated cervical cancer samples from Indian women and helps identify trends in mutation profiles that are found in an Indian cohort of cervical cancer.

Expression of enhancing factor/phospholipase A2 in skin results in abnormal epidermis and increased sensitivity to chemical carcinogenesis.

Enhancing factor (EF), a growth factor modulator, is the mouse homologue of human secretory group II phospholipase A(2). EF exhibits growth-promoting activity in vitro, in the presence of epidermal growth factor, and also brings about phenotypic transformation of normal cells. In order to ascertain the role of EF in vivo, a human keratin-14 promoter was used to drive the expression of EF ectopically to squamous epithelial cells. The founder mouse and its progeny showed abnormal whiskers and a scaly, beaded tail. In these mice, keratinization pattern of the epidermis was disturbed and parakeratosis and acanthosis were noted. The transgenic mice, TgK14-EF, expressed EF in the suprabasal layers of tail epidermis as well as in the epithelial cells of hair follicle and sebaceous glands of skin. Expression of EF along with hyperplasia was also observed in other squamous epithelia such as buccal mucosa, tongue and oesophagus. TgK14-EF mice homozygous for the transgene showed delayed and scanty hair growth although the mice were healthy and fertile. The hemizygous TgK14-EF mice were sensitive to a two-stage chemical carcinogenesis and developed a higher number of papillomas than their normal littermates over the course of the experiment. The conversion rate of papilloma to carcinoma was two fold higher in the transgenic mice.

Females with paired occurrence of cancers in the UADT and genital region have a higher frequency of either Glutathione S-transferase M1/T1 null genotype.

Upper Aero digestive Tract (UADT) is the commonest site for the development of second cancer in females after primary cervical cancer. Glutathione S-transferase (GSTM1 and / or T1) null genotype modulates the risk of developing UADT cancer (primary as well as second cancer). The aim of this study was to evaluate the difference in GST null genotype frequencies in females with paired cancers in the UADT and genital region as compared to females with paired cancers in the UADT and non-genital region. Forty-nine females with a cancer in the UADT and another cancer (at all sites-genital and non-genital) were identified from a database of patients with multiple primary neoplasms and were analyzed for the GSTM1 and T1 genotype in addition to known factors such as age, tobacco habits, alcohol habits and family history of cancer. Frequencies of GSTM1 null, GSTT1 null, and either GSTM1/T1 null were higher in females with paired occurrence of cancer in the UADT and genital site (54%, 33% and 75% respectively) in comparison to females with paired occurrence of cancer in the UADT and non-genital sites (22%, 6% and 24% respectively). The significantly higher inherited frequency of either GSTM1/T1 null genotype in females with a paired occurrence of cancers in UADT and genital region (p = 0.01), suggests that these females are more susceptible to damage by carcinogens as compared to females who have UADT cancers in association with cancers at non-genital sites.

Genotype, phenotype and cancer: role of low penetrance genes and environment in tumour susceptibility.

Role of heredity and lifestyle in sporadic cancers is well documented. Here we focus on the influence of low penetrance genes and habits, with emphasis on tobacco habit in causing head and neck cancers. Role of such gene-environment interaction can be well studied in individuals with multiple primary cancers. Thus such a biological model may elucidate that cancer causation is not solely due to genetic determinism but also significantly relies on lifestyle of the individual.

Glutathione S-transferase M1 or T1 null genotype as a risk factor for developing multiple primary neoplasms in the upper aero-digestive tract, in Indian males using tobacco.

In this study conducted amongst Indian male tobacco users with upper aero-digestive tract (UADT) squamous carcinoma, 30 patients with multiple primary neoplasms (MPN) were compared with 28 age and sex matched patients with a single primary neoplasm (SPN) for various environmental factors (form of tobacco use, alcohol, radiotherapy for index cancer) and genetic parameters (family history of UADT cancers and GSTT1/GSTM1 genotype). The GSTM1/T1 null genotype, seen in 60% patients with MPN versus 33% patients with SPN (P=0.03) had an odds ratio of 3.7 [CI=1.14-11.99; P=0.03] for developing MPN. Tobacco use in the form of smoking with or without chewing, as opposed to only chewing, and regular alcohol intake were the two other factors with almost three fold increased risk for the development of MPN, although, the effect was not statistically significant. All three patients with a family history of UADT cancer developed MPN, suggesting an inherited predisposition.

Comparison of the activities of wild type and mutant enhancing factor/mouse secretory phospholipase A2 proteins.

Enhancing factor (EF) protein, an isoform of secretory phospholipase A2 (PLA2), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA2 was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressed in vitro. Comparison of enhancing activity as well as phospholipase A2 activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities

Apoptosis induction and anti-cancer activity of LeciPlex formulations.

PURPOSE: Cationic agents have been reported to possess anti-neoplastic properties against various cancer cell types. However, their complexes with lipids appear to interact differently with different cancer cells. The purpose of this study was to (i) design and generate novel cationic lecithin nanoparticles, (ii) assess and understand the mechanism underlying their putative cytotoxicity and (iii) test their effect on cell cycle progression in various cancer-derived cell lines. In addition, we aimed to evaluate the in vivo potential of these newly developed nanoparticles in oral anti-cancer delivery. METHODS: Cationic lecithin nanoparticles were generated using a single step nanoprecipitation method and they were characterized for particle size, zeta potential, stability and in vitro release. Their cytotoxic potential was assessed using a sulforhodamine B assay, and their effect on cell cycle progression was evaluated using flow cytometry. The nanoparticle systems were also tested in vivo for their anti-tumorigenic potential. RESULTS: In contrast to cationic agents alone, the newly developed nanoformulations showed a specific toxicity against cancer cells. The mechanism of toxic cell death included apoptosis, S and G2/M cell cycle phase arrest, depending on the type of cationic agent and the cancer-derived cell line used. Both blank and drug-loaded systems exhibited significant anti-cancer activity, suggesting a synergistic anti-tumorigenic effect of the drug and its delivery system. CONCLUSIONS: Both in vitro and in vivo data indicate that cationic agents themselves exhibit broad anti-neoplastic activities. Complex formation of the cationic agents with phospholipids was found to provide specificity to the anti-cancer activity. These formulations thus possess potential for the design of effective anti-cancer delivery systems.

Genetic susceptibility to multiple primary neoplasms in the upper aero-digestive tract: genotype score and phenotype correlation.

Early detection and treatment of head and neck cancer has led to increased patient survival. However such patients are at a high risk for multiple primary neoplasm(s) (MPN). In order to study the genetic susceptibility to MPN, 22 candidate SNPs were genotyped based on which a distinctive Genotype Score was created using Additive, Dominant and Recessive models. Using lymphoblastoid cell lines (LCLs) generated from these individuals, the Genotype Score was correlated with carcinogen sensitivity in vitro. LCLs from MPN patients exhibited significantly higher Genotype Score and showed resistance to genotoxic agents compared to matched controls. This report demonstrates quantitative assessment of cumulative effect of gene polymorphisms and its correlation with carcinogen sensitivity for predicting susceptibility to MPN.

Oligobenzylethylenimine enriches linear polyethylenimine with a pH-sensitive membrane-disruptive property and leads to enhanced gene delivery activity.

We report here the synthesis of a diblock linear polymer of oligo(benzylethylenimine)-b-polyethylenimine (OBzEI-PEI) and investigate its gene delivery properties. The linear copolymer OBzEI-PEI was prepared in a straightforward manner by acidic hydrolysis of a diblock polyoxazoline, which had been made by sequential polymerization of 4-benzyl-2-ethyl-2-oxazoline followed by 2-ethyl-2-oxazoline. pH titration and DNA complexation profiles of the new polymer are similar to regular linear PEIs, but with higher gene transfection efficiencies in various cell lines despite a decreased cellular uptake of plasmid DNA. Further experiments suggest that the OBzEI tail complements the intrinsic proton-sponge endosomolytic activities of PEI with an acid pH-sensitive membrane-perturbing activity.