|Year : 2015 | Volume
| Issue : 1 | Page : 61-64
No evidence of association of xenotropic murine leukemia virus-related virus with oral cancers: Experience from a tertiary care center in South India
M Sinha1, M Shafiulla1, K Trupti2, NR Namrata3, H Nadimul2, KS Sabitha2, RV Kumar3, RS Jayshree1
1 Department of Microbiology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka, India
2 Department of Oral Surgery, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka, India
3 Department of Pathology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka, India
|Date of Web Publication||3-Feb-2016|
Department of Microbiology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka
Source of Support: Rajiv Gandhi University of Health Sciences,
Bangalore, India., Conflict of Interest: None
Background: Development of oral cancer, a widely prevalent cancer in India, is multifactorial with increased risk in those habituated to smoking, consuming alcohol and chewing paan and tobacco. This does not preclude other etiological factors in the causation of this cancer. Exploratory studies on several oncogenic viruses have found varied associations with oral cancers.Aim: The aim of this study was to explore the association of xenotropic murine leukemia virus-related virus, (XMRV) a retrovirus recently implicated in oncogenesis in humans, with oral cancers.Settings And Design: The presence of XMRV proviral deoxyribonucleic acid (DNA) was evaluated by standard nucleic acid amplification from DNA extracted from representative bits of tumor tissues and adjacent normal tissues from surgically resected specimens sent post-operatively for routine histopathological testing.Materials And Methods: This prospective study comprised 109 patients with a provisional diagnosis of oral cancer who were operated at the Oral Oncology Department of Kidwai Memorial Institute of Oncology, over a period of 10 months.Results: XMRV was not found in any of the tumor tissues (squamous cell carcinomas - 98; verrucous carcinomas - 4) nor in any of the normal tissues. It is thus important that the absence of this oncogenic virus in all the cases makes the association of XMRV with oral cancers very unlikely.Conclusions: There is a need to investigate potentially oncogenic viruses in other solid tumors and in larger sample sizes. Any such association could have implications in detecting, preventing and treating these cancers.
Keywords: Oral squamous cell carcinoma, retrovirus, xenotropic murine leukemia virus-related virus
|How to cite this article:|
Sinha M, Shafiulla M, Trupti K, Namrata N R, Nadimul H, Sabitha K S, Kumar R V, Jayshree R S. No evidence of association of xenotropic murine leukemia virus-related virus with oral cancers: Experience from a tertiary care center in South India. Indian J Cancer 2015;52:61-4
|How to cite this URL:|
Sinha M, Shafiulla M, Trupti K, Namrata N R, Nadimul H, Sabitha K S, Kumar R V, Jayshree R S. No evidence of association of xenotropic murine leukemia virus-related virus with oral cancers: Experience from a tertiary care center in South India. Indian J Cancer [serial online] 2015 [cited 2021 Jan 18];52:61-4. Available from: https://www.indianjcancer.com/text.asp?2015/52/1/61/175595
| » Introduction|| |
Oral cancer is one of the 10 most prevalent cancers in the world with more than 90% of the malignancies being squamous cell carcinomas (SCCs) originating from the oral mucosa. In India, oral cancer is one of the five most common malignancies encountered; the age standardized incidence rates of lip and oral cancers being 3.9/100,000 population (men 5.3; women 2.6).,, In the hospital-based cancer registry (HBCR) of India 2004-2006, oral cancer ranked from first through fifth common cancers in the five regional HBCRs (Mumbai, Bangalore, Chennai, Thiruvananthapuram, Dibrugarh).
Development of oral cancer is multifactorial with no single clearly recognizable cause. The habit of betel quid (”paan”) chewing, tobacco chewing, smoking, alcohol consumption, poor oral hygiene, genetic susceptibility and diet, may all play a role in the development of oral cancer.,, Moreover, the oral epithelium is constantly exposed to a variety of microbial challenges (bacterial, fungal and viral), many of which may gain access by breach of oral epithelium subsequent to the corroding action of betel leaf (paan) and its accompaniments (tobacco, areca nut, calcium hydroxide) resulting in chronic inflammation and eventually oral cancers.
The oral cavity is home to numerous viruses and the role of oncogenic viruses in these cancers cannot be ruled out unless explored. Human papilloma viruses (HPV) in oral cancers have been studied by various researchers; most finding varying degrees of association ,, or refuting this association. Other oncogenic viruses, which have been associated with oral cancers are the Epstein-Barr virus (EBV) and human herpes virus 6 (HHV 6).,, The quest for newer oncogenic viruses and their relationship with various cancers is ongoing.
The association between prostate cancer and antiviral enzyme, RNase L deficiency led to a search for viral sequences in prostate cancers. In 2006, a gammaretrovirus closely related to murine leukemia virus, named xenotropic murine leukemia virus-related virus (XMRV), was found to be associated with 42% cases of prostate cancer (8/19) and subsequently with chronic fatigue syndrome., Whether XMRV is associated or etiologically related to either has been challenged in subsequent studies., However, the apparent un-relatedness of these diseases led us to question the potential association of this virus with other solid tumors. XMRV has been detected in semen samples. This makes sexual transmission of the virus, probably even to unnatural sites like the oral cavity, a possibility. Characteristic of retroviruses, the ability of XMRV to integrate its viral genome into the host deoxyribonucleic acid (DNA) may allow them to maintain a long-term, persistent infection in animal hosts as well as induce tumors by activating proto-oncogenes or disrupting tumor suppressor genes via proviral insertional mutagenesis.
Considering the large number of oral cancers in southern India, there is a need to look at the various possible etiological factors leading to carcinogenesis. Several studies have shown varying association of HPV and oral cancers from this region,, but there may be other factors, including other oncogenic viruses that lead to the development of cancer. Hence, this study was undertaken to investigate the possible association of XMRV with oral cancers.
| » Materials and Methods|| |
This prospective study comprised 109 patients with a provisional diagnosis of oral cancer who were operated at a tertiary care cancer center, over a period of 10 months (June 2010 to March 2011).
The study was approved by the Institutional Ethics Committee and written informed consent was obtained from all subjects. Cancers of buccal mucosa, retromolar area, upper and lower alveolus, floor of mouth, lip and anterior two-third of the tongue, were included in the study.
Representative bits of tumor tissue were collected from surgically resected specimens that were sent for routine histopathological study post-operatively. Due to ethical issues in collecting healthy oral tissue as controls, grossly normal areas of the mucosa at the margins of excision, away from the tumor lesion served as controls. Only those samples where the tumor was well-circumscribed and margins were clear of tumor invasion were included as “normal tissue” in the study. Relevant history such as tobacco or paan chewing, smoking or alcohol consumption was documented.
DNA was extracted from 50 mg of both tumor and normal tissues using published protocol and polymerase chain reaction (PCR) for beta globin gene was done on all extracted DNA to assess DNA adequacy in samples.
A standard semi nested DNA PCR was performed using primers amplifying a conserved region of integrase gene, which was an adaptation of the real time PCR (RT PCR) used by Schlaberg et al. Briefly, the reaction mix for PCR contained 5 μl of PCR buffer (10× Taq buffer containing 1.5 mM MgCl2), 3U of Taq polymerase (Bangalore Genei, Bangalore), 100 pmol of primers (XMRV4552F 5'GAG AGG CAG CCA TGA AGG-3', XMRV4653R1 5'-GAG ATC TGT TTC GGT GTA ATG GAA A-3' and XMRV4673R2 5'-CCC AGT TCC CGT AGT CTT TTG AG-3'), 3 µl of deoxynucleoside triphosphates (2.5 mM, Bangalore Genei) and 5 µl of extracted DNA in a reaction volume of 50 µl. A plasmid with a full-length XMRV (isolate VP62) insert obtained from Addgene Inc., Cambridge, MA, USA (Addgene plasmid 26189) was used as a positive control for each PCR run. Thermocycling conditions were 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s and final extension at 72°C for 5 min. Amplification products of the PCRs were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide and the bands were visualized using a transilluminator at 302 nm.
| » Results|| |
A total of 109 patients, clinically diagnosed as oral cancers were included in the study (33 males; 76 females). Patients' ages ranged from 25 years to 80 years with a median age of 55 years. The histopathological diagnoses of the patients are depicted in [Table 1].
|Table 1: Histopathological diagnoses of the 104 patients with oral cancer|
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Of the 109 patients, only 104 had histologically confirmed carcinomas (100 SCC and 4 verrucous carcinoma). The clinical staging of these 104 oral cancers was T1-5 patients, T2-26 patients, T3-37 patients, T4-36 patients. Node involvement was N0 in 34 and N1 in 70 patients. There were no signs of distal metastasis in any of the patients. By occupation, these 104 patients included agriculturists (n = 18), porters (n = 21), manual laborers (n = 12), teachers (n = 4), businessmen (n = 4), fisherman (n = 1) and housewives (n = 44). Patients' habits of drinking alcohol, chewing of betel leaves (”paan”), tobacco and areca nut and smoking and the surgical management are depicted in [Table 2] and [Table 3]. Five patients did not have any addiction to smoking, chewing or drinking.
|Table 3: Surgical management of the 104 patients with oral malignancy (SCC-98, verrucous carcinoma-4)|
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In two patients with SCC, tumor tissue was not collected since the lesion was very small and sufficient only for histopathological diagnosis and in seven patients with SCC normal tissue was not sampled due to its proximity to visible tumor margin. Hence, DNA was extracted from 102 tumors (SCC - 98; verrucous carcinomas - 4) and 97 normal tissues. DNA from samples histopathologically diagnosed as leukoplakia and hyperplasia were not included in the study.
Beta globin gene was detected in all tumor and normal samples included in the study confirming efficient DNA extraction. XMRV DNA was not detected in any of the samples: Tumor or normal tissue.
| » Discussion|| |
Owing to the high incidence and prevalence of oral cancers in India, there is a need for ongoing studies to look for varied probable etiological agents of this malignancy. That chewing paan and tobacco, smoking and consumption of alcohol are well-known risk factors in the development of oral cancers was corroborated by the present study where the majority of patients were habituated to chewing paan/tobacco (81%), smoking cigarette/beedi (16%) and alcohol consumption (14%). Although these habits are well known risk factors, they do not account for all cases of oral cancer and up to 20% of these cancers are known to develop in persons lacking these habits., In the present study, 4.8% of patients had no habituation to smoking, chewing or drinking. Hence, it is not prudent to oversimplify and restrict tobacco and alcohol as the only risk factors for the development of oral cancer, but to keep exploring other possible etiological factors for this malignancy.
Several oncogenic viruses such as HPV, EBV and HHV 6 have been found to be associated with oral cancers with varied frequencies ,,, and the quest for newer etiological agents is ongoing. Contrary to oropharyngeal cancers, oral cancers and other head and neck SCCs have been found to harbor HPV infrequently. This was also reflected in the multicentric International Agency for Research on Cancer case control study, of which our center was also a part, where HPV DNA was found in 3.9% (30/766) of oral cancer biopsies and 18.3% (26/142) of oropharyngeal cancers.
XMRV has been found to be associated with prostate cancer in recent studies., Using DNA microarray, researchers identified XMRV sequences in complementary DNA samples from around 42% cases of prostate cancer (8/19 cases). They also found XMRV ribonucleic acid (RNA) by RT PCR in 10.5% prostate cancers (9/86 cases) and further molecularly cloned and fully sequenced the full-length viral genome from three XMRV positive prostate cancer tissues (VP35, VP42 and VP62). Furthermore, in XMRV positive tumors the viral nucleic acid was localized within human tumor tissues using fluorescent in-situ hybridization. Other researchers found integrated proviral DNA in 6% and XMRV protein expression in 23% of prostate cancers and also found XMRV to correlate with a higher tumor grade. Evidence of serological reactivity to the virus, the presence of viral proteins in the tumor tissue and provirus found integrated specifically and not randomly with the host genome all point to the fact that this retrovirus is capable of human infection and may have a role in oncogenesis.,,, Researchers have identified unique integration sites in the genome of prostate tumor tissues, validating the identification of XMRV as an authentic human retrovirus and ruling out the possibility that it was a result of laboratory contamination or a PCR artifact. The receptor for XMRV entry is xenotropic and polytropic virus receptor (XPR1), a cell surface protein ubiquitously expressed in many human tissues suggesting that XMRV can potentially infect cells other than prostate.,
Whether this potentially oncogenic virus could be involved with other cancers has not been found and needs further investigation. There is preliminary evidence for XMRV being blood-borne and furthermore being transmitted sexually-viral RNA was detected in prostatic secretions in 13% of men with prostate cancer. Hence, like other sexually transmitted viruses causally associated with oral cancers (e.g., HPV), there could be a possibility of XMRV being associated with oral cancers. However, XMRV DNA was not detected in any of the oral cancers in our study.
Limitations of the present study were that the sample size was reasonable, but not very large. Moreover, standard PCR followed by ultraviolet detection was employed, which is not a very sensitive method for detecting PCR products. RT PCR, which is known to have a higher sensitivity and the ability to quantitate, would have been the ideal detection method, but could not be done due to lack of this system in our present set up.
Literature search on the presence of XPR1 receptor in the oral cavity is scarce, but a very recent study on genetic alterations in oral SCC progression shows deletion of XPR1 gene by array based comparative genomic hybridization in both oral dysplasia and oral SCC in 71% and 86% patients respectively  implying that XMRV is unlikely to infect oral tissues. Furthermore, it is possible that the XMRV tropism is typical to the prostate due to the glucocorticoid response element present in the long terminal repeat of XMRV, which can activate transcription in response to various steroid hormones, maybe androgen, which is essential in the development, growth and maintenance of the prostate.
There is currently, a lot of controversy around XMRV and oncogenesis as evidenced by several studies , and studying its association with cancers from different sites and normal population will shed more light on its occurrence, habitat and oncogenic potential. XMRV may be circulating in the general population at a low rate as evidenced by its presence in 4% of benign controls , while other studies have found XMRV in respiratory secretions. Ours is probably the first study exploring the association of XMRV and oral cancers and indicates that such an association is unlikely.
An interesting finding in our study was female preponderance in oral cancer (70%). The Bombay Population Based Cancer Registry (PBCR), 1986-2000, shows the incidence of oral cancer to be higher in males than in females with a statistically significant decreasing trend in the former, which could be attributed to a decrease in the usage of the pan and tobacco among men. The Bangalore PBCR, 2001-2003, shows annual incidence of oral cancers to be similar in both sexes. Though study design and several other reasons may account for our findings, prevalence of oral cancers in both sexes needs to be corroborated with newer and updated cancer registry data.
To conclude, our study indicated that XMRV is unlikely to be associated with oral cancers. Absence of XMRV from both tumor and normal tissues proves that it is unlikely to be prevalent in the oral cavity, even as an infection. To explore the oncogenic potential of XMRV, the study is intended to be followed on by future studies to look for the presence of this virus in prostate cancers and other solid tumors preferably using RT PCR. The fact that XMRV infects human cells and can integrate as a provirus in the host genome might play an important role in oncogenesis. Hence, despite controversies surrounding XMRV, there is a need to know its association with other solid tumors and in the larger sample sizes. Any such association could have wide implications in detecting, preventing and treating such cancers.
| » Ackmowledgment|| |
This study was funded by a grant from Rajiv Gandhi University of Health Sciences, Bangalore, India. The plasmid with a full-length XMRV (isolate VP62) insert was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: XMRV VP62 cDNA from Drs. Robert H. Silverman and Beihua Dong.
| » References|| |
Hooper SJ, Wilson MJ, Crean SJ. Exploring the link between microorganisms and oral cancer: A systematic review of the literature. Head Neck 2009;31:1228-39.
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893-917.
Sunny L, Yeole BB, Hakama M, Shiri R, Sastry PS, Mathews S, et al
. Oral cancers in Mumbai, India: A fifteen years perspective with respect to incidence trend and cumulative risk. Asian Pac J Cancer Prev 2004;5:294-300.
Chocolatewala NM, Chaturvedi P. Role of human papilloma virus in the oral carcinogenesis: An Indian perspective. J Cancer Res Ther 2009;5:71-7.
Consolidated Report of Hospital Based Cancer Registries: 2004-2006. National Cancer Registry Programme. Bangalore: Indian Council of Medical Research; 2009.
Herrero R, Castellsagué X, Pawlita M, Lissowska J, Kee F, Balaram P, et al
. Human papillomavirus and oral cancer: The international agency for research on cancer multicenter study. J Natl Cancer Inst 2003;95:1772-83.
Al Moustafa AE, Chen D, Ghabreau L, Akil N. Association between human papillomavirus and Epstein-Barr virus infections in human oral carcinogenesis. Med Hypotheses 2009;73:184-6.
Machado J, Reis PP, Zhang T, Simpson C, Xu W, Perez-Ordonez B, et al
. Low prevalence of human papillomavirus in oral cavity carcinomas. Head Neck Oncol 2010;2:6.
D'Costa J, Saranath D, Sanghvi V, Mehta AR. Epstein-Barr virus in tobacco-induced oral cancers and oral lesions in patients from India. J Oral Pathol Med 1998;27:78-82.
Liu JL, Xu ZK, Zhao Y, Jin Y, Dai YM, Yao K. Relationship between human herpesvirus 6 infection and oral squamous cell carcinoma. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2003;17:334-6.
Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, et al
. Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog 2006;2:e25.
Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, et al
. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 2009;326:585-9.
Switzer WM, Jia H, Zheng H, Tang S, Heneine W. No association of xenotropic murine leukemia virus-related viruses with prostate cancer. PLoS One 2011;6:e19065.
Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, et al
. Lack of evidence for xenotropic murine leukemia virus-related virus (XMRV) in German prostate cancer patients. Retrovirology 2009;6:92.
Rusmevichientong A, Chow SA. Biology and pathophysiology of the new human retrovirus XMRV and its association with human disease. Immunol Res 2010;48:27-39.
Adurthi S, Krishna S, Mukherjee G, Bafna UD, Devi U, Jayshree RS. Regulatory T cells in a spectrum of HPV-induced cervical lesions: Cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma. Am J Reprod Immunol 2008;60:55-65.
Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci U S A 2009;106:16351-6.
Wiseman SM, Swede H, Stoler DL, Anderson GR, Rigual NR, Hicks WL Jr, et al
. Squamous cell carcinoma of the head and neck in nonsmokers and nondrinkers: An analysis of clinicopathologic characteristics and treatment outcomes. Ann Surg Oncol 2003;10:551-7.
Arnold RS, Makarova NV, Osunkoya AO, Suppiah S, Scott TA, Johnson NA, et al
. XMRV infection in patients with prostate cancer: Novel serologic assay and correlation with PCR and FISH. Urology 2010;75:755-61.
Kim S, Rusmevichientong A, Dong B, Remenyi R, Silverman RH, Chow SA. Fidelity of target site duplication and sequence preference during integration of xenotropic murine leukemia virus-related virus. PLoS One 2010;5:e10255.
Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, et al
. An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proc Natl Acad Sci U S A 2007;104:1655-60.
Kozak CA. The mouse “xenotropic” gammaretroviruses and their XPR1 receptor. Retrovirology 2010;7:101.
Cha JD, Kim HJ, Cha IH. Genetic alterations in oral squamous cell carcinoma progression detected by combining array-based comparative genomic hybridization and multiplex ligation-dependent probe amplification. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:594-607.
Fischer N, Schulz C, Stieler K, Hohn O, Lange C, Drosten C, et al
. Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract. Emerg Infect Dis 2010;16:1000-2.
[Table 1], [Table 2], [Table 3]