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      Study of association and molecular analysis of human papillomavirus in breast cancer of Indian patients: Clinical and prognostic implication

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          Abstract

          Objectives

          Human papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile.

          Methods

          HPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done.

          Results

          High prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20–45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants.

          Conclusion

          Thus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.

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          Most cited references46

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          In vivo half-life of a protein is a function of its amino-terminal residue.

          When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.
            • Record: found
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            Human papillomavirus type 16 sequence variation in cervical cancers: a worldwide perspective.

            We examined intratype human papillomavirus type 16 (HPV-16) sequence variation in tumor samples that were collected and analyzed in an international study of invasive cervical cancer. The collection included tumors from 22 countries in five continents. Using our recently developed E6 and L1 PCR-based hybridization systems to distinguish HPV-16 variant lineages, we analyzed material from tumors previously found to contain HPV-16 DNA. Of 408 specimens analyzed in the E6 hybridization assay, 376 (92.2%) belonged to previously reported HPV-16 variant lineages. The remaining 32 specimens (7.8%) harbored HPV-16 variants with novel hybridization patterns, novel nucleotide changes, or both. Nucleotide sequences (1,203 bp) were determined for the E6, the MY09/11 region of L1, and the long control region of each novel variant and representative specimens from each hybridization pattern observed. Based on E6 hybridization patterns, most of the variants from European and North American samples were phylogenetically classified as European prototype (E) while samples from Africa contained primarily African 1 (Af1) or African 2 (Af2) variants. The majority of Asian (As) variants were observed in Southeast Asia, and almost all Asian American (AA) variants were from Central and South America or Spain. A single North American 1 (NA1) variant was detected in a tumor from Argentina. Nucleotide changes previously shown to covary between the MY09/11 region of L1 and the E6 coding region were examined in a subset of 249 specimens. We observed 22 combined E6-L1 hybridization patterns, of which 11 (in 21 samples) were novel. No unanticipated nucleotide covariation was observed between the E class and the AA-Af1-Af2-NA1 classes, suggesting the absence or rarity of genomic recombination between HPV-16 lineages. This extensive description of HPV-16 variants forms a basis for further examining the relationship between intratype variation and basic functional differences in biological activities. HPV-16 variants may prove important for the determination of the risk of cervical neoplasia and for the design of HPV-16 vaccine strategies.
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              Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer.

              Integration of human papillomavirus type 16 (HPV-16) into the host DNA has been proposed as a potential marker of cervical neoplastic progression. In this study, a quantitative real-time PCR (qRT-PCR) was used to examine the physical status of HPV-16 in 126 cervical carcinoma in situ and 92 invasive cervical cancers. Based on criteria applied to results from this qRT-PCR assay, HPV-16 was characterized in carcinoma in situ cases as episomal (61.9%), mixed (i.e., episomal and integrated; 29.4%), and integrated (8.7%) forms. In invasive cervical cancer samples, HPV-16 was similarly characterized as episomal (39.1%), mixed (45.7%), and integrated (15.2%) forms. The difference in the frequency of integrated or episomal status estimated for carcinoma in situ and invasive cervical cancer cases was statistically significant (P = 0.003). Extensive mapping analysis of HPV-16 E1 and E2 genes in 37 selected tumors demonstrated deletions in both E1 and E2 genes with the maximum number of losses (78.4%) observed within the HPV-16 E2 hinge region. Specifically, deletions within the E2 hinge region were detected most often between nucleotides (nt) 3243 and 3539. The capacity to detect low-frequency HPV-16 integration events was highly limited due to the common presence and abundance of HPV episomal forms. HPV-16 E2 expressed from intact episomes may act in trans to regulate integrated genome expression of E6 and E7.

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                28 February 2017
                2017
                : 12
                : 2
                : e0172760
                Affiliations
                [1 ]Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
                [2 ]Department of Pathology, North Bengal Medical College and hospital, Sushruta Nagar, Darjeeling, West Bengal, India
                [3 ]Department of pathology, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
                [4 ]Department of Surgical Oncology, Chittaranjan National Cancer Institute37, Kolkata, West Bengal, India
                [5 ]Department of Epidemiology & Biostatistics, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
                [6 ]Saroj Gupta Cancer Centre and Research Institute, Thakurpukur, Kolkata, India
                University of Navarra, SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: SI CKP.

                • Data curation: SI A. Roy GKM.

                • Formal analysis: SI SM.

                • Funding acquisition: HD CKP.

                • Investigation: SI HD RB NM.

                • Methodology: SI CKP.

                • Project administration: CKP SR.

                • Resources: NA GKM CKP JB.

                • Supervision: CKP.

                • Validation: A. Roychowdhury SI HD.

                • Visualization: SI A. Roychowdhury.

                • Writing – original draft: SI.

                • Writing – review & editing: A. Roychowdhury CKP SR.

                Author information
                http://orcid.org/0000-0002-0854-6978
                Article
                PONE-D-17-00022
                10.1371/journal.pone.0172760
                5330495
                28245287
                502173fd-91f3-4167-98f3-f7f6d98ad3b1
                © 2017 Islam et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 January 2017
                : 9 February 2017
                Page count
                Figures: 7, Tables: 1, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001501, University Grants Commission;
                Award ID: F.2-3/2000 (SA-I) (Sr. No. 2061030813, Ref. No.: 20-06 / 2010
                Award Recipient :
                Funded by: Council of Scientific and Industrial Research, Government of India
                Award ID: 60(0111)/14/EMR-II of dt 03/11/2014
                Award Recipient :
                This work was supported by University Grant Commission Fellowship grant F.2-3/2000 (SA-I) (Sr. No. 2061030813, Ref. No.: 20-06 / 2010) to Mrs. H. Dasgupta and grants from Council of Scientific and Industrial Research, Government of India [no. 60(0111)/14/EMR-II of dt 03/11/2014] to Dr. C. K. Panda. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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