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CONTRIBUTION TO HUMAN PAPILLOMAVIRUS RESEARCH AND CERVICAL CANCER RESEARCH

Immunobiology of Cervical Cancer

 

A multiparameter analysis of immune function was done on patients with squamous cell carcinoma of the uterine cervix to look for any variable(s) that could be correlated with the clinical stage of the disease. Five immunological variables, viz., CD4+ lymphocytes, CD4/CD8 ratio, natural killer cells, concanavalin A-induced suppressor index, and circulating immune complexes, were found to consistently vary with tumor load. When these variables were subjected to a multiple regression and multivariate analysis, an equation for a diagnostic index curve was derived. Application of this equation led to an immunological staging system that could be used as an excellent prognostic indicator. The immunological staging system showed that patients classified into a particular clinical (FIGO) stage behaved in a heterogeneous way immunologically and that patients developing recurrent disease could easily be identified from those remaining disease free, even before treatment. Subsequent follow-up of these patients further confirmed this observation, with the recurrent disease group easily identifiable. These results point out the immense potential of such a staging system and the importance of immunological evaluation in the preliminary management of patients with malignant cervical neoplasia.

 

Relevant Publications

 

1. 1. Pillai MR, Balaram P, Abraham T, Nair MK. Lymphocyte populations in premalignant and malignant lesions of the oral cavity. Neoplasma 34: 469-479,1987.
2. 2. Pillai MR, Balaram P, Padmanabhan TK, Nair MK. Monoclonal antibody defined phenotypes of peripheral blood lymphocytes in cancer of the uterine cervix. American Journal of Reproductive Immunology and Microbiology 14: 141-143,1987.
3. 3. Balaram P, Pillai MR, Padmanabhan TK, Abraham T, Hareendran NK, Nair MK. Immune function in malignant cervical neoplasia: a multiparameter analysis. GynecologicalOncology31:409-423,1988.
4. 4. Pillai MR, Balaram P, Abraham T, Padmanabhan TK, Nair MK. Natural cytotoxicity and serum blocking in malignant cervical neoplasia. American Journal of Reproductive Immunology and Microbiology 16:158-162, 1988.
5. 5. Pillai MR, Balaram P, Padmanabhan TK, Nair MK. Immunological profiles of T lymphocytes in malignant cervical neoplasia. Journal of Experimental and Clinical Cancer Research 7:251-257,1988.
6. 6. Pillai MR, Balaram P, Padmanabhan TK, Abraham T, Nair MK. Interleukin 2 and alpha interferon induced in vitro modulation of spontaneous cell mediated cytotoxicity in patients with cancer of the uterine cervix undergoing radiotherapy. Acta Oncologica 28: 39-44,1988.
7. 7. Pillai MR, Balaram P, Hareendran NK, Bindu S, Abraham T, Padmanabhan TK, Nair MK. Immune reactive proteins as prognostic and clinical markers in malignant cervical neoplasia. Journal of Cancer Research and Clinical Oncology 115: 583-591, 1989.
8. 8. Pillai MR, Balaram P, Bindu S, Hareendran NK, Padmanabhan TK, Nair MK. Interleukin 2 production in lymphocyte cultures: a rapid test for cancer associated immunodeficiency in malignant cervical neoplasia. Cancer Letters 47: 205-210,1989.
9. 9. Pillai MR, Balaram P, Bindu S, Hareendran NK, Padmanabhan TK, Nair MK. Radiation associated eosinophilia and monocytosis in carcinoma of the uterine cervix: a simple reliable prognostic and clinical indicator. Neoplasma 37: 91-96,1990.
10. 10. Balaram P, Pillai MR, Padmanabhan TK. Stage related augmentation of depressed immunity by interferon alpha and interleukin 2 in cancer of the uterine cervix. Biomedicine 10: 3-8,1990.
11. 11. Pillai MR, Balaram P, Hareendran NK, Bindu S, Padmanabhan TK, Nair MK. Clinical and prognostic significance of Concanavalin A induced suppressor cell activity in malignant cervical neoplasia. British Journal of Obstetrics and Gynaecology 97: 357-361,1990.

 

Histogenesis of cervical cancer

 

Cervical cancer is the fifth most common neoplasm worldwide and is second only to breast cancer as a cause of cancer death in women. It is a major public health problem in India, since not only is the incidence high, but at least 70% of cases present with advanced stages of the disease. The entire concept of morphological tumor markers is based on the fact that tumor cells express a specific, though sometimes limited program of differentiation which normally forms at least part of the differentiation program of the putative cell of origin. Since most tumors of uterine cervix are squamous origin, epithelial tumor markers should contribute to understanding the biologic behavior of cervical lesions and may also help in assessing the clinical behavior of suchtumors.

 

Biological markers of malignant transformation in uterine cervix were elucidated in the present study. Cytoskeletal proteins, Involucrin, Oncoproteins ras and c-myc, Basement Membrane (BM) associated proteins, laminin, collagen-IV and fibronectins (FN) and cell adhesion receptors ( Integrins a2b1- a collagen IV receptor, a3b1 a laminin receptor and a5b1-a FN receptor) have been analyzed and compared during cervical cancer progression. Intense cytoplasmic immunoreactivity was seen for CK types 13 and 16 1, 10 and 11 in the spinal cell layers with the basal cells showing no positivity in normal cervical epithelium with CK 19 and 14 expressing only in the basal cells. Changes from the normal expression pattern were seen in HGSILs and invasive SCCs. Involucrin a cytoplasmic protein synthesized during squamous maturation is known to be a marker of terminal differentiation. In normal cervical epithelium and LGSILs Involucrin showed intense and homogenous cytoplasmic reactivity in the spinal cell layers with the basal cells negative. In HGSILs and WDSCC, a patchy local immunoreactivity and loss of expression in PDSCCs was evident. Involucrin may thus be a sensitive marker in identifying the differentiation status of the lesions while the absence of Involucrin in PDSCC may be helpful in differential diagnosis. Many of the molecular alterations involve growth promoting cancer genes or oncogenes. This study addresses the presence and localization of ras and c-myc proteins by IHC in cervical carcinogenesis. Only N-ras showed increasing immunoreactivity with increasing histologic abnormality. The % of nuclei positive for c-myc also showed a gradual increase from LGSIL through HGSIL and Invasivecancer.

 

The BM is an important component of cellular structure which plays a key role in epithelial carcinogenesis during invasion and metastasis. Transition from SIL to early stromal invasion and to a micro invasive carcinoma could therefore involve breakdown of the BM. This study aimed to demonstrate any possible alterations in the deposition of BM proteins laminin, collagen–IV and FN as well as the extent of BM continuity during cervical cancer progression. IHC analysis showed a regular, thick and continuous BM in normal cervical epithelium and LGSILs. Interruptions and discontinuity was evident in HGSILs. In SCCs the distribution of Laminin collagen IV and FN was related to the degree of cellular differentiation. Carcinomas are characterized by invasion of malignant cells into the underlyingconnective tissue and by an ability to metastasize to different sites. This process involves an alteration of cell matrix and cell interactions which may be associated with changes in the expression of function of cell molecules such as Integrins. In apparently normal cervical epithelium integrin a2 and a3 showed strong pericellular immunoreactivity along the basal cell layer, but few samples showed positivity along the whole cervical epithelium. In HGSILs integrins showed diffuse positivity and in Invasive SCCs the immunoreactivity of integrins a2 and a3 showed a gradual decrease with PDSCC showing negativereaction.

 

The information provided about CKs and Involucrin analysis is in principle independent of morphological features, while it is a fact that majority of tumors can be diagnosed using conventional morphological techniques, these markers provide valuable data in equivocal cases. The study on ras and c-myc in cervical carcinogenesis has shown that the two oncoproteins can be used as markers to study the biological aggressiveness of cervical cancer. The role of BM tumor interactions has to be considered as part of a complex multistep process that leads to invasion and metastasis necessarily involving other factors such as tumor growth fraction cell motility and angiogenesis. As BM proteins tend to exhibit peculiar alterations in different grades of cervical lesions they can serve as important biological markers in cervical cancer progression. Integrins, the cell adhesion molecules play a crucial role for cell-cell and cell-matrix interactions. From this study, it may be suggested a2 and a3 integrins may be helpful in predicting the invasive potential of the tumor and loss of integrin reactivity may aid in identification of highly aggressive tumors having a metastatic potential. Therapeutic gains in oncology are likely to stem from improved understanding of tumor biology. Targeted intervention during the invasive and metastatic process may arrest the progress of tumorcells.

 

Relevant Publications

 

1. 1. Nair AS, Nair MB, Jayaprakash PG, Rajalekshmi TN, Nair MK, Pillai MR. Involucrin and tumor progression in the uterine cervix. Pathobiology 64: 333-338, 1997
2. 2. Nair AS, Nair MB, Jayaprakash PG, Rajalekshmi TN, Nair MK, Pillai MR. Increased expression of cytokeratins 14, 18 and 19 correlates with tumor progression in the uterine cervix. Pathobiology 65: 100-107, 1997.
3. 3. Nair AS, Nair MB, Jayaprakash PG, Rajalekshmi TN, Nair MK, Pillai MR. The basement membrane and tumor progression in the uterine cervix. General and Diagnostic Pathology 142:297-303,1997.
4. 4. Nair AS, Nair MB, Jayaprakash PG, Rajalekshmi TN, Nair MK, Pillai MR. Cytokeratin and the evaluation of tumor differentiation in squamous lesions of the uterine cervix. General and Diagnostic Pathology 143: 15-22,1997.
5. 5. Lakshmi S, Nair MB, Jayaprakash PG, Rajalekshmi TN, Nair MK, Pillai MR. The c-erbB2 oncoprotein and epidermal growth factor receptor in cervical lesions. Pathobiology 65: 163-168,1997.
6. 6. Nair AS, Nair BM, Jayaprakash PG, Rajalekshmy TN, Nair MK, Pillai MR. ras and c-myc Oncoproteins during tumor progression in the uterine cervix. Tumori 5: 583-588,1998.
7. 7. Hariharan R, Babu JM, Rema P, Pillai MR. Mutational analysis of Smad7 in human cervical cancer. Oncology Reports 21 (4): 1001-1004, 2009.
8. 8. Klug SJ, Ressing M, Koenig J, Abba MC, Agorastos T, Brenna SM, Ciotti M, Das BR, Del Mistro A, Dybikowska A, Giuiliano AR, Gudleviciene Z, Gyllensten U, Haws AL, Helland A, Herrington CS, Hilesheim A, Humbey O, Jee SH, Kim JW, Madeleine MM, Menczer J, Ngan HY, Nishikawa A, Niwa Y, Pegoraro R, Pillai MR, Ranzani G, Rezza G, Rosenthal AN, Roychoudhury S, Saranath D, Schmitt VM, Sengupta S, Settheetham-Ishida W, Shirasawa H, Snijders PJ, Stoler MH, Suarez-Rincon AE, Szarka K, Tachezy R, Ueda M, van der Zee AG, von Knebel Doeberitz M, Wu MT, Yamashita T, Zehbe I, Blettner M. TP53 codon 72 polymorphism and cervical cancer: a pooled analysis of individual data from 49 studies. Lancet Oncology 10 (8): 772-784,2009.

 

Human papillomaviruses and development of epithelial cancer

 

High-risk human papillomaviruses (HPVs) have been shown to be involved in the pathogenesis of many squamous carcinomas, particularly those of the uterine cervix. A number of random studies have also reported association of high-risk HPV subtypes with cancers of the oral cavity, larynx, hypopharynx, and esophagus. The roles of other molecular factors involved during HPV infection in these tumors still remain unclear. Recent findings from our laboratories have suggested possible mechanisms associated with HPV-mediated carcinogenesis. Both p53 mutation-dependent and mutation-independent pathways may be associated with HPV-mediated carcinogenesis, the former mainly in upper aerodigestive tract tumors (UADT) and the latter in cervical tumors. In cervical tumors, inactivation of the p53 tumor suppressor protein by the E6 gene product of high-risk HPVs and mutation of the p53 gene in UADT is associated with alterations in the apoptotic regulatory bcl-2 and bax genes, leading to downregulation of programmed cell death (PCD) and increased cell proliferation. HPV infection is also associated with increased tissue angiogenesis and activation of telomerase. Altered kinetics of telomere fragments is evident in HPV-infected tissue. We therefore believe that the combined manifestations of all these factors may contribute to development of a "condemned mucosa syndrome" facilitating development UADT and cervical cancers. A distinct step in the pathogenesis of both types of tumors may only be in the mode of p53 inactivation, whereas all other events appear to be strongly correlated to the presence of HPV. The development and validation of such a molecular model has significant clinical priority. It can be used to identify target populations or individuals for intervention, to monitor effects of intervention, and to determine which individuals or groups are at increased risk of developingcancer.

 

Relevant Publications

 

1. 1. Ho L, Chan SY, Burk R, Das BC, Fujinaga K, Icenogle JP, Kahn T, Kiviat N, Lancaster W, Mavromara P, Rosembaum SM. Norrilid B, Pillai MR, Stoerker J, Syrjaenen K, Syrjaenen S, Tay SK, Villa L, Wheeler CM, Williamson AL, Bernard HU. The genetic drift of Human papillomavirus Type 16 is a means of reconstructing prehistoric viral spread and movement of ancient human populations. Journal of Virology 67: 6413-6423,1993
2. 2. Lakshmi S, Nair MB, Rajalakshmi TN, Jayaprakash PG, Nair MK, Pillai MR. P53 protein and tumorigenesis in the uterine cervix. General and Diagnostic Pathology 142: 281-287,1997.
3. 3. Lakshmi S, Pillai MR, Rajalekshmy TN, Gopalakrishnan K, Nandini VR, Nair AS, Nair MK. Human papillomavirus infection and cervical pre-cancer: implications for management and control. Journal of Clinical & Experimental Cancer Research 68: 45-52,1995
4. 4. Pillai MR, Halabi S, McKalip A, Jayaprakash PG, Rajalekshmi TN, Nair MK, Herman B. The presence of human papillomavirus 16/18 E6, p53 and bcl-2 protein expression in cervicovaginal smears from patients with invasive cancer. Cancer Epidemiology, Biomarkers and Prevention 1996,5:329-335.
5. 4. Pillai MR, Lakshmi S, Sreekala S, Devi TG, Jayaprakash PG, Rajalakshmi TN, Devi CGC, Nair MK, Nair MN. High risk human papillomavirus infection and E6 protein expression in lesions of the uterine cervix. Pathobiology 66: 240-246,1998.
6. 6. Nair P, Gangadevi T, Jayaprakash PG, Nair MB, Nair MK, Pillai MR. Increased angiogenesis in the uterine cervix associated with human papillomavirus infection. Pathology Research & Practise 195: 163-169, 1999. Nair P, Jayaprakash PG, Nair MK, Pillai MR. Telomerase, p53 and human papillomavirus infection in the uterine cervix. Acta Oncologica 39 (1): 65-70,2000.
7. 7. Pillai MR, Nair MK. Development of a condemned mucosa syndrome and pathogenesis of human papillomavirus associated upper aerodigestive tract and uterine cervical tumors. Experimental and Molecular Pathology 69233-241, 2000.
8. 8. Pillai MR, Chacko P, Kesari AL, Jayaprakash PG, Jayaram HN, Antony AC. Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer. Journal of Clinical Pathology 56:569-574,2003.
9. 9. Pillai MR, Sreevidya S, Pollock B, Jayaprakash PG, Herman B. Polymorphism at codon 72 of p53, human papillomavirus and cervical cancer in South India. Journal of Cancer Research and Clinical Oncology 128: 627-631,2002.
10. 10. Pillai MR, Sreevidya S, Pollock B, Jayaprakash PG, Herman B. Human papillomavirus type 16 E6 and E7 variations in Indian cervical cancer. Gynecologic Oncology 87:268-273,2002.:233-241,2000.
11. 11. Pillai MR, Janki MBV, Jissa VT, Lakshmi S, Chiplunkar SV, Patkar M, Tongaonkar H, Reddy BKM, Chakka KN, Siddiqui M, Roychoudury S, Abraham P, Peedicayil A, Gnanamony M, Subhashini J, Ram TS, Dey B, Singh N, Singh A, Jain SK, Jayshree RS. Region-wise distribution of high-risk human papillomavirus types in squamous cell carcinomas of the cervix in India. International Journal of Gynecological Cancer 20 (6): 1046-1051,2010.
12. 12. Reshmi G, Surya R, Jissa VT, Babu PS, Preethi NR, Santhi WS, Jayaprakash PG, Pillai MR. C-T variant in a miRNA target site of BCL2 is associated with increased risk of human papillomavirus related cervical cancer - an in-silico approach. Genomics 98 (3): 189-193,2011.
13. 13. G and Pillai MR. Interplay between HPV Oncoproteins and MicroRNAs in Cervical Cancer. Human Papillomavirus and Related Diseases - From Bench to Bedside - Research aspects, Davy Vanden Broeck (Ed.), ISBN: 978-953-307-855-7, InTech,2012.
14. 14. Joshi, J.M. Babu, D. Jayalakshmi, V. Kulkarni, U. Divate, R. Muwonge, T.Gheit, M. Tommasino, R. Sankaranarayanan, Pillai MR. Human papillomavirus infection among human immunodeficiency virus-infected women in Maharashtra, India. Vaccine 32 (2014) 1079–1085.
15. 15. GheitT,AnantharamanD,HolzingerD,AlemanyL,TousS,LucasE,PrabhuPR,PawlitaM,RidderR, Rehm S, Bogers J, Maffini F, Chiocca S, Lloveras B, Kumar RV, Somanathan T, de Sanjosé S, Castellsagué X, Arbyn M, Brennan P, Sankaranarayanan R, Pillai MR, Gangane N, Tommasino M. Role of mucosal high risk human papillomavirus types in head and neck cancers in central India.; HPV-AHEAD study group. International Journal of Cancer. 2017 Jul 1; 141(1): 143-151. doi: 10.1002/ijc.30712. Epub 2017 Apr19.

 

 

The Human Papillomavirus (HPV) Vaccine and prevention of Cervical Cancer

 

An increase in worldwide HPV vaccination could be facilitated if fewer than three doses of vaccine are as effective as three doses. We originally aimed to compare the immunogenicity and frequency of persistent infection and cervical precancerous lesions caused by vaccine-targeted HPV after vaccination with two doses of quadrivalent vaccine on days 1 and 180 or later, with three doses on days 1, 60, and 180 or later, in a cluster-randomised trial. Suspension of the recruitment and vaccination due to events unrelated to our study meant that some enrolled girls could not be vaccinated and some vaccinated girls received fewer than the planned number of vaccinations by default. As a result, we re-analysed our data as an observational cohort study. Our study was designed to be done in nine locations (188 clusters) in India. Participants were unmarried girls aged 10-18 years vaccinated in four cohorts: girls who received three doses of vaccine on days 1, 60, and 180 or later, two doses on days 1 and 180 or later, two doses on days 1 and 60 by default, and one dose by default. The primary outcomes were immunogenicity in terms of L1 genotype-specific binding antibody titres, neutralising antibody titres, and antibody avidity after vaccination for the vaccine-targeted HPV types 16, 18, 6, and 11 and incident and persistent infections with these HPVs. Analysis was per actual number of vaccine doses received. This study is registered with ISRCTN, number ISRCTN98283094; and with ClinicalTrials.gov, number NCT00923702. Vaccination of eligible girls was initiated on Sept 1, 2009, and continued until April  8, 2010. Of 21 258 eligible girls identified at 188 clusters, 17 729 girls were recruited from 178 clusters before suspension. 4348 (25%) girls received three doses, 4979 (28%) received two doses on days 1 and 180 or later, 3452 (19%) received two doses at days 1 and 60, and 4950 (28%) received one dose. Immune response in the two-dose HPV vaccine group was non-inferior to the three-dose group (median fluorescence intensity ratio for HPV 16 1·12 [95% CI 1·02-1·23] and for HPV 18 1·04 [0·92-1·19]) at 7 months, but was inferior in the two-dose default (0·33 [0·29- 0·38] for HPV 16 and 0·51 [0·43-0·59] for HPV 18) and one-dose default (0·09 [0·08-0·11] for HPV 16 and 0·12 [0·10-0·14] for HPV 18) groups at 18 months. The geometric mean avidity indices after fewer than three doses by design or default were non-inferior to those after three doses of vaccine. Fewer than three doses by design and default induced detectable concentrations of neutralising antibodies to all four vaccine-targeted HPV types, but at much lower concentration after one dose. Cervical samples from 2649 participants were tested and the frequency of incident HPV 16, 18, 6, and 11 infections was similar irrespective of the number of vaccine doses received. The testing of at least two samples from 838 participants showed that there was no persistent HPV 16 or 18 infections in any study group at a median follow-up of 4·7 years (IQR 4·2-5·1). Despite the limitations imposed by the suspension of the HPV vaccination, our findings lend support to the WHO recommendation of two doses, at least 6 months apart, for routine vaccination of young girls. The short-term protection afforded by one dose of HPV vaccine against persistent infection with HPV 16, 18, 6, and 11 is similar to that afforded by two or three doses of vaccine and merits further assessment.

 

Relevant Publications

 

1. 1. Hariharan I, Pillai MR. Genotypes of the human papillomavirus: relevance to Indian field trials of the vaccine. Indian Journal of Medical Research 130 (3): 247-260,2009.
2. 2. Rengaswamy Sankaranarayanan, Priya R. Prabhu, Michael Pawlita, Tarik Gheit, Neerja Bhatla, Richard Muwonge, Bhagwan M. Nene, Pulikottil O. Esmy, Smita Joshi,UshaRaniReddyPoli,Parimal Jivarajani, Yogesh Verma, Eric Zomawia, Maqsood Siddiqi, Surendra S. Shastri, Kasturi Jayant, Sylla G Malvi, Eric Lucas, Angelika Michel, Julia Butt, Janki Mohan Babu, Subha Sankaran, Kannan TR, Rintu Varghese, Uma Divate, Shila Thomas, Geeta Joshi, Martina Willhauck-Fleckenstein, Tim Waterboer, Martin Müller, Peter Sehr, Sanjay Hingmire, Alka Kriplani, Gauravi Mishra, Sharmila Pimple, Radhika Jadhav, Catherine Sauvaget, Massimo Tommasino, and Pillai MR for the Indian HPV vaccine study group. Immunogenicity and HPV infection following one, two and three doses of quadrivalent vaccine: Early results from a multi-center cohort study in India. Lancet Oncology Published Online December 1, 2015; http://dx.doi.org/10.1016/S1470-2045(15)00414-3
3. 3. Neerja Bhatla, Bhagwan M. Nene, Smita Joshi, Pulikottil O. Esmy, Usha Rani Reddy Poli, Geeta Joshi, Yogesh Verma, Eric Zomawia, Sharmila Pimple, Priya R. Prabhu, Partha Basu, Richard Muwonge, Sanjay Hingmire, Catherine Sauvaget,Eric Lucas, Michael Pawlita, Tarik Gheit, Kasturi Jayant, Sylla G Malvi, Maqsood Siddiqi, Angelika Michel, Julia Butt, Subha Sankaran, Kannan TR, Rintu Varghese, Uma Divate, Martina Willhauck-Fleckenstein, Tim Waterboer, Martin Müller, Peter Sehr, Alka Kriplani, Gauravi Mishra, Radhika Jadhav, Ranjit Thorat, Massimo Tommasino, Pillai MR, and Rengaswamy Sankaranarayanan*, for the Indian HPV vaccine study group. Are two doses of human papillomavirus vaccine sufficient for older girls aged 15-18 years? Results from a cohort study in India. Papillomavirus Research, 2018, 5:163-171
4. 4. Sankaranarayanan R, S Joshi, P. Basu, PO Esmy, P. Prabhu, Muwonge R, Bhatla N,BM Nene, UR Reddy Poli, Joshi G, Verma Y, Zomawia E, Pimple S, Shaw J, Hingmire S, Lucas E, Sauvaget C,Pawlita M, Gheit T, Jayant K, Malvi SG, Siddiqi M, Michel A, Butt J, Sankaran S, Kannan TR, Varghese R, Divate U, Fleckenstein MW, Waterboer T, Müller M, Sehr P, Kriplani K, Mishra G, Jadhav R, Thorat R, Patel R, Chiwate A, Tommasino M, Pillai MR for the Indian HPV vaccine study group. Can a single dose of human papillomavirus (HPV) vaccine prevent cervical cancer? Early findings from an Indian study. Vaccine (2018), https:// doi.org/ 10.1016/ j.vaccine. 2018.02.087

 

CONTRIBUTION TO CANCER STEM CELL RESEARCH

Drug-induced senescence generates chemo-resistant stem-like cells with low reactive oxygen species.

 

A major initial focus of the lab was to understand whether neo adjuvant chemo modulates tumor recurrence mediated through tumor stem cell modulation. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells with stem cell properties after chemotherapy are poorly defined. Our study provided evidence for the rare escape of tumor cells from drug-induced cell death, after an intermediate stay in a non-cycling senescent stage followed by unstable multiplication characterized by spontaneous cell death. However, some cells appear to escape and generate stable colonies with an aggressive tumor stem cell-like phenotype. These cells displayed higher CD133 and Oct-4 expression. Notably, the drug-selected cells that contained low levels of reactive oxygen species (ROS) also showed an increase in antioxidant enzymes. Consistent with this in vitro experimental data, we observed lower levels of ROS in breast tumors obtained after neoadjuvant chemotherapy compared with samples that did not receive preoperative chemotherapy. These latter tissues also expressed enhanced levels of ROS defenses with enhanced expression of superoxide dismutase. Higher levels of Oct-4 and CD133 were also observed in tumors obtained after neoadjuvant chemotherapy. Further studies provided evidence for the stabilization of Nrf2 due to reduced 26 S proteasome activity and increased p21 association as the driving signaling event that contributes to the transition from a high ROS quiescent state to a low ROS proliferating stage in drug-induced tumor stem cell enrichment. [Journal of Biological Chemistry, 2011 Oct 28; 286(43):37813-29]

 

Multiple drug resistant, tumorigenic stem-like cells in oral cancer.

 

We developed an in vitro cell line model to exemplify tumor stem cell concept in oral cancer. We were able to identify CD147 expressing fractions in SCC172 OSCC cell line with differing Hoechst dye efflux activity and DNA content. In vivo tumorigenic assay revealed three fractions enriched with stem-like cells capable of undergoing mesenchymal transition and a non-tumorigenic fraction. The regeneration potential and transition of one fraction to other imitated the phenotypic switch and functional disparities evidenced during oral tumor progression. Knowledge of these additional stem-like subsets will improve understanding of stem cell based oral epithelial tumor progression from normal to malignant lesions [Cancer Letters, 2013; 338 (2): 300 –

316, 2013].

 

Identification of heat shock protein 90 inhibitors to sensitize drug resistant side population tumor cells using a cell based assay platform.

 

Current cancer therapeutics is identified based on initial tumor regression screens that mostly kill differentiated tumor cells, sparing the rare cancer stem cells (CSCs). Being rare and difficult to characterize, it remains a challenge to identify compounds active against them. Side population (SP) cells identified in multiple cancer cell line panels expressing mitochondrial Cytochrome C-EGFP were evaluated for identifying possible drug candidates utilizing high-throughput imaging. We identified heat shock protein 90 inhibitors as potential agents to sensitize SP cells to anticancer drugs. Hsp90 inhibitors induced down regulation of Akt leading to proteasomal degradation of survivin and consequent mitochondrial apoptosis. A successful screening platform for identifying compounds targeting drug resistant side population cells was developed. [Cancer Letters 2012 Apr 1;317(1):78-88]

 

Persisisting cancer cell population after drug treatment: visualizing its fate and re emergence

 

Our previous studies suggest that molecular events related to emergence of drug resistant cells after chemotherapy is associated with reactivation of antioxidant defence signaling which help in the transition of these cells from high ROS to low ROS state. But the key molecular signature events that contribute to the immediate cell survival signaling after alethal dose of drug or high stress are still poorly understood. Since the immediately surviving cells contribute for later emergence of drug tolerant cells, we focused our studies to understand the signaling involved in the immediate survival of cells. In depth analysis of cells surviving immediately after higher dose of anticancer drugs, reveals an unusual secondary acquisition of cells with increased autophagy and mitophagy, coupled with constitutive activation of the redox masters. The study suggests that quick shift to low proteasome activity associated with induction of autophagy regulators such as LC-3 and the adaptor protein p62 prepares a fraction of cells to enter into chronic autophagy followed by Parkin dependant mitophagy. Chronic mitophagy appears to promote long-term survival of drug escaped cells in low nutrient condition and intracellular protein stress. This chronic mitophagy-mediated mitochondrial quality measures eventually lead to the generation of low ROS stem cell enriched fraction. Using stable cancer cells expressing a sensitive redox sensor allowed us to detect and quantify spontaneous emergence of cells with low intracellular ROS cells in drug-exposed cells. The study reveals unique cascades of signaling and key cell state transitions involved in the evolution of drug resistance. In order to identify key targets in each phase and its dynamics, Immediate Drug escaped cells, emerging drug tolerant colonies and Drug tolerant expanded colonies were utilized for transcriptomics analysis. Our study found an oscillation of many proteins, noticeably stemcell markers, during the acquisition of drug resistance by cancer cells. An abrupt difference in gene expression pattern was observed in the immediate drug tolerant cells with significant up-regulation seen in genes involved in cell cycle regulation and stem cell markers. Interestingly, genes belonging to the Forkhead family of transcription   factors   (shown   to   play   important   roles   in   the   expression     of genes involved in cell growth, proliferation, differentiation) displayed a considerable down regulation in immediate drug tolerant cells. A proteomics and transcriptomics analysis of cells at different phases was employed to study the dynamics of “Persisters” cells and key cell transitions. Pathways including protein folding, proteasome activity, oxidation-reduction and negative regulation of apoptosis were significantly altered during the process of drug -induced chemo resistance. (BioMed Research International 2013:517237. doi: 10.1155/ 2013/ 517237, 2013).

 

CONTRIBUTION TO PHOTODYNAMIC THERAPY AND DEVELOPMENT OF NEAR INFRA RED (NIR) DYES

Photodynamic therapy (PDT) is one of the emerging anti-cancer treatment modality that functions via photosensitizer mediated oxidative cytotoxicity. We first designed and developed LED array using 630 nM LEDs and Sodium vapor lamp with 610 BP filter and heat cut off system for In vitro - photodynamic experiments. Our findings based on In vitro cytotoxic analysis (drug sreening) indicate that of the three squaraines, DRAG 105 showed significant phototoxicity in NIR region. Of the porphyrins, SJR 103, BMR 102 & NCP S0₃ showed significant phototoxicity and these compunds showed negligible dark cytotoxicity. DRAG 105 & SJR 103 within an hour of addition to the cells showed good cellular intake and was found to be localized in the nucleus. Hence we conducted detailed studies on selected drug candidates including DRAG 105 from Squarines, SJR 103 from hydroxyl porphyrin group and NCP SO3 from N confused porphyrin group.

 

Squaraines are a class of dyes possessing all beneficial characteristics of a photosensitizer and considered to be a potent candidate for clinical PDT. In this study we used iodo derivative of squaraine called Diiodo-squaraine (Bis (3,5-diiodo- 2,4,6-trihydroxyphenyl) squaraine) which is well known for its tumor specificity but least studied for its molecular basis of action. In this study we validated various molecular events initiated by oxidative stress in squaraine based PDT. To delineate various molecular pathways, we performed proteomic profiling followed by analysis using DAVID Bioinformatics Resources. Protein profiling with LC-MS/MS followed by bioinformatics analysis in breast adenocarcinoma MDA MB 231 cells showed activation of response to unfolded protein (RUP), cell redox homeostasis (CRH), actin cytoskeletal organization (ACO) and programmed cell death (PCD) pathways. Further confirmation and role of key molecules involved was studied using Western blotting and Real time PCR and established relation oxidative stress an unfolded protein response (UPR) and endoplasmic reticulum(ER) stress. Confocal microscopy and Flow-cytometric analysis for oxidative stress, Mitochondrial Membrane Potential (MMP) and actin cytoskeletal changes demonstrate regulation of Reactive Oxygen Species (ROS) mediated cell death pathways. Our study reveals how oxidative stress activates both protective and death inducing pathways in squaraine based PDT.

 

We also synthesized a novel water-soluble porphyrin THPP and its metalated derivative Zn-THPP having excellent triplet excited state quantum yields and singlet oxygen generation efficiency. When compared to U.S. Food and Drug Administration approved and clinically used sensitizer Photofrin, THPP showed ca. 2−3-fold higher in vitro photodynamic activity in different cell lines under identical conditions. The mechanism of the biological activity of these porphyrin systems has been evaluated through a variety of techniques: 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay, comet assay, poly(ADP-ribose)- polymerase (PARP) cleavage, CM-H2DCFDA assay, DNA fragmentation, flow cytometric analysis, fluorescence, and confocal microscopy, which confirm the apoptotic cell death through predominantly reactive oxygen species (ROS). Moreover, THPP showed rapid cellular uptake and are localized in the nucleus of the cells as compared to Hoechst dye and Photofrin, thereby demonstrating its use as an efficient sensitizer in photodynamic therapy and live cell NIR nucleus imaging applications.

 

Relevant Publications

 

1. 1. Thomas AP, Saneesh Babu PS, Nair AS, Ramakrishnan S, Ramaiah D, Chandrashekar TK, Srinivasan A, Pillai MR. Meso-Tetrakis (p-sulfonatophenyl) N-Confused Porphyrin Tetrasodium Salt: A Potential Sensitizer for Photodynamic Therapy. Journal of Medicinal Chemistry. 55 (11) : 5110-20,2012.
2. 2. Suneesh C. Karunakaran,P. S. Saneesh Babu,Bollapalli Madhuri,Betsy Marydasan, Albish K. Paul, Nair AS,K. Sridhar Rao,A. Srinivasan,Tavarekere K. Chandrashekar,Ch. Mohan Rao, Pillai MR and Danaboyina Ramaiah. Water Soluble Neutral Porphyrin Derivative: An Efficient Sensitizer for Photodynamic Therapy and NIR Nuclear Imaging Applications. ACS Chemical Biology 8: 127 – 132, 2013.

 

CONTRIBUTION TO VIRAL DISEASE BIOLOGY

 

H1NI VIRUS STUDIES

 

Mankind is constantly under the threat of emerging new viruses that causes epidemics and pandemics. Influenza virus, commonly known as the Flu virus, infects predominantly humans, birds and swine. The first influenza pandemic of the 21^st century was reported in Mexico in 2009. A novel H1N1 influenza virus, generated from the re-assortment of two preexisting swine influenza viruses, caused this. In India, the infection was first reported at Hyderabad on 13 May 2009 and the first case in Kerala was reported in June2009.

 

Surveillance for outbreaks of influenza viruses is non-existentin Kerala. Following special emergency requests from Government of India and State Government of Kerala, Rajiv Gandhi Centre for Biotechnology (RGCB), started providing diagnostic support for human febrile cases as per the guidelines prescribed by the United States Centre for Disease Control and Prevention (CDC). The Kerala State Health Department collected human nasal swab samples from various districts across Kerala and sent them to RGCB for diagnosis. In August of 2009, RGCB reportedthe first laboratory confirmed case of H1N1 from Kerala. Since then, for the past 8 years, RGCB has provided H1N1 diagnosis services to the State of Kerala. In 2009-10 alone, RGCB collected and analyzed over 4000 nasal-wash samples from patients suspected of Influenza virus infection. Out of this, 40.66% were positive for influenza A virus, of which 74.25 % and 25.75 % were positive for pandemic H1N1 2009 and Seasonal Influenza A.

 

This large sample base led us on to begin the H1N1 research program. Uponanalyzing the clinical and demographic data we observed that severe disease and mortality in the influenza A (H1N1) pdm2009 infection predominantly affected relatively healthy adolescents and adults between the age group of 10 and 50 years. There wasn’t any gender-related susceptibility for influenza infection; both men and women were equally afflicted. Most of the fatal cases reported had at least one of the coexisting conditions like pregnancy, diabetes or were age (>70years). A month-wise analysis of influenza virus-positive cases revealed that influenza  (H1N1) pdm 2009 infections, since August 2009 in Kerala, occurred with two successive peaks. The first was during the monsoon season (September- November 2009) and another during the summer months (May – July 2010).

 

To determine whether the influenza viruses that circulated in Kerala had changed over time we sequenced the two surface glycoproteins of the virus, hemagglutinin (HA) and neuraminidase (NA) which plays a significant role in causing antigenic variation of influenza viruses and helped us mainly to determine if key mutations affecting receptor binding and drug binding region were present in the influenza viruses circulating in Kerala. Phylogeny analysis using Bayesian method combined with divergence time estimation indicated that the Kerala H1N1 HAs and NAs are derived from the 2009 pdm H1N1 lineage, with most recent common ancestor seen in the year 2012. We observed two mutations T200A and D225N that were known to impact HA receptor binding specificities and virulence of the virus. Additionally, we also noted a key change in the Sa antigenic site, K166Q receptor; the functional consequence of this mutation is presently being analyzed. Our analysis revealed that there are no drug-resistant viruses circulating in Kerala and points to the continued effectiveness of oseltamivir for influenza treatment. The sequencing data provided new perspectives on the evolution of H1N1 virus in Kerala. Still some questions remain unanswered - are there other virulence markers (e.g. E627K in  the PB2 gene)? To address this question we did a whole-genome sequencing of pandemic (2009) and post-pandemic (2014, 2015, 2017) viruses. Majority of amino acid substitutions observed during the 2009 pandemic period—namely, those for PB2-I354L, PB1-I397M, I435T, PA-P224S, NS-I123V, and NP-V100I—persisted in the isolates from 2014 to 2017, with the accumulation of several new mutations in PB2-R54K, M66I, D195N, R293K, V344M, R368R, W537L, V731I, S453T, R299K, T303S; PB1-V113I, G154D; PA-I621V, G684R, R266H, S522G, R559K, L649I, V100I, N321K, I330V, K361R, R362K; Ns-G51S, and NP-A22T. With the basic genetic characteristics we have identified key mutations and would further try to understand the effect of these mutations on viral infection and replication and its impact on the host using experiments in cell lines as well as animal models.

 

In addition to the genetic analysis of the virus, we also studied the baseline overall antibody reactivity of the Keralite population to the 2009 pandemic virus. In collaboration with the Trivandrum Medical College Community Health department we collected 1250 human serum samples between the age group of 10 years to >70 years after the post pandemic period and tested against A/California/04/2009 virus. Upon analysis we observed that the reactivity of humans aged 50-90 was very low when compared to the younger age group.

 

The consequence of antigenic drift observed in our previous study presents serious public health concerns. Along with the usual symptoms, influenza viruses can lead to complications especially in the very young, old and immune-compromised individuals. Even though vaccination is still considered to be the most feasibleand cost-efficient way to protect against influenza, it has been observed to induce varying levels of protection for recipients, especially within older immunosenescent adults. Accurate markers or models that explain the development ofprotective immunity to influenza, or that predict vaccine failure in our population is incredibly useful in developing appropriate vaccines and immunization schedules relevant forthe Indian population. RGCB developed a research project in collaboration with Mayo Clinic, USA to create innovative immune profile signatures that explain and predict variations in immune responses to influenza A/H1N1 vaccines mainly among the elderly. In Kerala, the percentage of senior citizens who lost their lives due to H1N1 infection during the 2009 pandemic outbreak was reported to be very high. Hence focusing on the elderly allows us to have the twin advantages of examining a segment of the population most affected by influenza as well as providing a greater diversity in immune response study that is relevant globally. For the study, we recruited 150 healthy volunteers between the age group of 50-74years andyounger subjects as controls ranging in age from 19-35 years. All Subjects who took part in the study signed the written consent form, which was reviewed by the on field doctor. The sampling and influenza vaccine administration was carried out at a private hospital in Trivandrum. The first sampling day was taken as the 0th day which included a pre-bleed (baseline) followed by intramuscular vaccination with the 2014-2015 trivalent, inactivated seasonal influenza vaccine, Vaxigrip (Sanofi Pasteur). Subsequent bleeds for sample collection were made on the 3rd, 28th and the 75th day post vaccine administration. Immune responses of post bleed samples were compared with the baseline data. The greatest achievement with regard to this study is the possibility to create awareness on vaccination among the Kerala population especially among the aged individuals, a category that is highly neglected in terms of influenza vaccination. Since these projects are futuristic in their approach, we also anticipate our findings to have serious implications especially in influencing health policy makers so that we can realize themoto “bench to bed-side”. The project also represents a mutually beneficial research enterprise that will set the stage for larger and more varied collaborations in the future.

 

Relevant Publications:

 

1. 1. V C Dhanya, P J Sara, D Sanjai, F Amar, P M Deepa, G R Santosh, V T Jissa and Pillai MR. Demographic and Clinical Characteristics of Pandemic Influenza A (H1N1) 2009 Outbreak in Kerala, Southern India. British Microbiology Research Journal. 2014 Oct; 4(10):1142-1153
2. Jones S, Prasad R, Nair AS, Dharmaseelan S, Usha R, Nair RR, Pillai MR. Whole-genome sequences of influenza A (H1N1) pdm 09 virus isolates from Kerala, India. Genome Announc. 2017 Jul 13;5(28).

 

RESEARCH ON MEASLES VACCINE RESPONSE (COMMENCED IN JULY 2017 AND WORK IN PROGRESS)

 

Measles is the most transmissible human virus known, with more than 20 million measles infections occurring worldwide each year. Measles vaccination was introduced in India in the 1980s and a two-dose vaccination schedule started in 2011; resulting in dramatic decreases in the morbidity and mortality of measles in India. In 2010 India accounted for nearly half of the global measles mortality. Even in locations, such as Kerala, where vaccination rates are among the highest in  India, there continue to be outbreaks of disease. Previously vaccinated children comprise a significant portion (20- 30%, and in one case, >95%) of the measles cases during these outbreaks, indicating that single dose vaccine failure is a significant issue. If recent outbreaks among highly vaccinated populations in North America and Europe are any indication, even with the two-dose schedule, vaccine failure will still account for a small but sizeable percentage (1%-24%) of measles cases. Environmental factors such as malnutrition, vitamin deficiencies, overcrowding and poor sanitation may exacerbate the problem as they are risk factors for severe disease and are likely to negatively impact vaccine response.

 

Thus measles and measles vaccine failure remain major public health problems worldwide and are of particular importance in India. Measles vaccine (MV) sero- conversion rates in India are routinely around 90%, but vary and can be as low as 72%. Unfortunately, efficacy rates in the low 90s are insufficient given the high transmissibility of measles and the need for 96-98% herd immunity. The reason for these lower sero-conversion rates is not known; in fact, this lack of understanding is a critical barrier to progress in measles eradication in India.

 

The proposed international collaboration in infectious disease research (ICIDR) involving the Mayo Clinic, Emory University, and the Rajiv Gandhi Centre for Biotechnology (RGCB) seeks to address this gap in scientific knowledge regarding measles immunity and vaccine failure. The objectives of the proposal are to:

 

1. 1. Understand how southern Indian children respond to the measlesvaccine.
2. 2. Verify the effect of maternal antibody on thatresponse.
3. 3. Examine the effect of vitamin A deficiency on measles vaccine response in Indian children.
4. 4. Assess the contribution of genetic variation within vitamin A receptor pathways on measles vaccine responses and disease susceptibility.
5. 5. Conduct a detailed analysis of the effect of vitamin A on the transcriptome, antibody repertoire, and phenotype of measles-specific B cells among vaccinated children.
6. 6. Sequence measles strains that cause outbreaks in southern India and assess the extent to which measles vaccine induced immunity cross-reacts with circulating strains.

 

Successful completion of these objectives will improve our understanding of: how measles immunity is generated and the effect of nutritional and genetic (host andpathogen) factors on that immunity. Our objectives allow us to identify key differences between vaccinated children who subsequently become infected (vaccine failure) and vaccinated children who are immune (vaccine success). In turn this data may inform the: improvement of vaccination schedules, identification of biomarkers of vaccine efficacy, development of new vaccines, and the evaluation of therapeutic and/or supportive care during measles infection.