ANTICANCER RESEARCH 27: 2697-2704 (2007)

Short Review Human Papillomavirus Infections in Lung . Detection of E6 and E7 Transcripts and Review of the Literature

LAURA GIULIANI1, CARTESIO FAVALLI1, KARI SYRJANEN2 and MARCO CIOTTI1

1Laboratory of Clinical Microbiology and Virology, University Hospital Tor Vergata, Rome, Italy; 2Department of and Radiotherapy, Turku University Hospital, Turku, Finland

Abstract. Lung cancer is the leading cause of cancer related Lung cancer is the leading cause of cancer-related death death in Western countries. Several factors have been implicated in Western countries (8). Cigarette smoking, radiation, in its aetiology: cigarette smoking, environmental pollution, environmental pollution and exposure to asbestos are asbestos and genetic factors. The possible involvement of human considered the main risk factors for lung cancer. However, papillomavirus (HPV) in bronchial squamous cell lesions was they cannot explain all lung cancer cases and there is a first suggested in 1979 by Syrjänen. Since then, several studies possibility that additional risk factors can play a role in have confirmed the presence of HPV DNA in about 20% of bronchial as suggested in the early 1980’s by lung cancer cases examined, with HPV16 and 18 as the two Syrjanen who described in bronchial epithelial most frequently found oncogenic viral types. More recently, these changes resembling those typical of HPV-induced exophytic data have been supported by the detection of E6 and E7 and flat condylomas of the genital tract (9- 11). These transcripts in HPV-positive lung cancer cases, reinforcing the morphological studies were later confirmed by reports on hypothesis that oncogenic HPVs could act as cofactors in detection of HPV-DNA in bronchial carcinomas (11-15). bronchial carcinogenesis. This published literature is briefly In contrast other authors did not find HPV DNA in reviewed and new data of the authors on detection of E6 and lung tumors and failed to confirm such association (16- E7 transcripts in lung cancer samples are presented. 19). One of the reasons for these negative findings could be the different sensitivity of the techniques used or High risk human papillomaviruses (HPVs) are the prime inappropriate choice of primers. In fact, during the etiological agents for of the uterine . They integration of the virus into the host cell genome, part of are actually found in almost 100% of cases the L1 gene may be lost as well as the E2 gene and the (1, 2). It has been estimated that 15-20% of all human consensus primers MY09/MY11, frequently used to screen malignancies could be related to oncogenic HPVs (2-4). In these biopsies could then be inadequate for amplification. recent years, data have accumulated implicating the How does the virus get into this location? It has been involvement of HPV in squamous cell tumors at other speculated that HPV reaches the lungs via the bloodstream mucosal sites, such as the oral cavity, oesophagus, sino-nasal coming from distant infected sites such as the genital tract. tract, larynx and bronchi (2). The oncogenic power of HPV HPV DNA was detected in the blood of women with is due to its ability to interfere with the cell-cycle and cervical infection (20). In the light of the data reported tumour suppressive functions of the cell through its above, demonstration of E6 and E7 and their oncoproteins E6 and E7, which inactivate p53 and Rb transcripts is crucial in order to elucidate the role of HPV in proteins, respectively (3-7). lung carcinogenesis.

Lung Cancer

Correspondence to: Dr. Marco Ciotti, Laboratory of Clinical Lung cancer is one of the leading -related Microbiology and Virology, University of Tor Vergata, Viale deaths worldwide. Cigarette smoking is responsible for Oxford, 81, 00133, Rome, Italy. Tel: +39 06 20902087, Fax: +39 06 20902078, e-mail: [email protected] about 90% of lung cancer cases. However, less than 20% of smokers develop lung cancer (21). Therefore, other Key Words: HPV infections, HPV-DNA, E6 and E7 transcripts, etiological agents, including genetic factors, functional lung cancer, short review. inactivation of tumor suppressor genes such as p53, Rb, p16,

0250-7005/2007 $2.00+.40 2697 ANTICANCER RESEARCH 27: 2697-2704 (2007) and infection with oncogenic types of HPV have been We recently studied the expression of E6 and E7 implicated in lung carcinogenesis (16, 22). Some reports oncogenes in six fresh lung tumour biopsies positive for have stressed an increased risk of developing lung cancer in HPV16 (80). To our knowledge, only one previous study women with anogenital malignancies (23, 24). An increased using in situ hybridization has addressed this issue in lung risk has been also observed in meat handlers (25-28). All cancer (34), despite the fact that transcriptional activity this epidemiological evidence supports a possible role for of E6 and E7 has been widely analysed in cervical HPV in lung carcinogenesis. carcinomas and in HPV transformed cell lines (82-87). In these studies, the transcription process of HPV appears Evidence for HPV Involvement to be quite complex. A common promoter (P97 in HPV16) controls the transcription of both E6 and E7 The accumulating data implicating a relationship between oncogenes using an alternative splicing mechanism. A HPV infection and lung carcinogenesis is derived from the common splice donor site at nt 226 and two different following approaches. splice acceptor sites at nts 409 and 526 give rise to three different transcripts. An unspliced E6/E7 transcript Morphological evidence. In 1979, Syrjanen reported encodes the E6 protein. The E7 protein in turn is morphological changes in squamous cell carcinomas of the encoded by the splicing products E6*I (acceptor site at lung closely resembling those seen in genital HPV lesions nucleotide 409) and E6*II (acceptor site at nucleotide (10, 11). These observations led to the hypothesis that HPV 526) (83). A novel promoter with a major start site could have a role also in bronchial carcinogenesis. around 670 nt (P670) in the E7 ORF was identified in an HPV16 transformed cell line derived from a vulvar Immunohistochemistry. Immunohistochemistry (IHC) has intraepithelial (84). been used only sporadically to detect HPV antigens in lung In our study, the transcription pattern in lung tumors was cancer because it was soon replaced by the hybridisation deduced from three different PCR protocols carried out in assays in HPV detection. Hence, only two cases of HPV16-positive patients and in control CaSki cells (Figure squamous cell (SCPs) were analyzed by IHC as 1). Amplification with primers encompassing the E7 ORF reported by Trillo et al. (29). promptly showed the presence of E7 specific transcripts in five out of six patients as well as in CaSki cells (Figure 1, Detection of HPV DNA. Several different techniques have experiment A). been used to detect HPV-DNA in lung cancer cases: dot A more complex pattern was obtained when the E6 blot, Southern blot, ISH and PCR. These studies are transcription was investigated. The nested RT-PCR with summarised in Table I, (12-14, 29-81). The detection rate of primers encompassing the E6 ORF gave negative results in HPV DNA vary from 0% to 100% probably due either to four of these patients, while the remaining two showed a the different sensitivity of the techniques used and to 365-bp transcript (corresponding to the unspliced form of primers choice. Of all bronchial carcinomas analysed to E6) and a 183-bp transcript corresponding to the E6*I date, about 20% contain HPV DNA. HPV16 and 18 are the isoform, respectively (Figure 1, experiment B). The third most frequently found, while HPV6 and 11 are less RT-PCR protocol carried out with primers encompassing frequent. Multiple infections are sporadically described. the E6/E7 region showed a 166-bp transcript in all patients Among the high-risk group HPV31, 33 and 35 have also except two (Figure 1, experiment C). This band was also been found in scattered lesions. seen in CaSki cells together with two minor additional upper bands. Interestingly, the unspliced form (365-bp) of Detection of oncogenic transcripts. The possible role of E6 transcript was identified in only one tumor. It is oncogenic HPV types in lung carcinogenesis is suggested by important to emphasize that the synthesis of E6 protein is the presence of HPV DNA in bronchial carcinomas (Table mainly derived from a bicistronic mRNA, containing full- I). However, the presence of HPV DNA alone is not length E6 and E7 sequences (85). sufficient to support an active role of the virus in lung Taken together, our small study showed that all tumors carcinogenesis. Synthesis of E6 and E7 oncoproteins is a except one contained E6 and E7 RNA transcripts. The condition sine qua non for cell transformation and absence of viral transcripts in one of these patients could be development of cancer. Indeed, the early oncoprotein E6 due to bystander cells or to an inactive status of the virus. binds and degrades the p53 protein, while the E7 protein The latter seems most likely, since HPV DNA was not binds and inactivates the protein Rb, resulting in abnormal found in the surrounding normal tissue. The finding of cell proliferation and tumour growth (5-7). Thus, oncogenic transcripts in this series of non-small cell lung demonstration of HPV transcripts is necessary to provide cancer further support the role for oncogenic HPVs in further confirmatory evidence. bronchial carcinogenesis.

2698 Giuliani et al: Oncogenic HPVs in Lung Cancer (Short Review)

Table I. Detection of HPV-DNA in lung tumours¨.

Type of Technique No. of HPV type detected % HPV+ First author lesion used cases 6 6/11 11 16 16/18 18 Other

SQC SB 9 0 Stremlau (12) ASC SB 2 0 LCC SB 7 0 ASC SB 5 1 20 SQC SB 1 1 100 Byrne (13) SQC ISH 99 5 5 Syrjanen (14) SCP EM 1 100 Trillo (29) SQC ISH 131 2 7 7 Syrjanen (30) SCP ISH 1 1 100 Kerley (33) SCP ISH 15 1 7 Carey (35) SQM ISH 10 1 10 Bejui-Thivol (36) SQC ISH 33 1 1 1 3 18 SQC ISH 1 1 100 Bejui-Thivol (37) SQC FISH 5 2 40 Kulski (38) SCP ISH 6 6 100 Popper (39) SQC ISH 5 5 100 SQC ISH 1 1 100 Di Lorenzo (40) SQM ISH 17 1 1 12 Yousem (41) SQC ISH 20 1 2 15 ASC ISH 12 0 ASSC ISH 16 0 SQC ISH 4 0 LCC ISH 6 1 17 SCP ISH 2 2 100 SQC PCR, SB 29 3 10 Ogura (42) SCP ISH 1 1 100 Katial (43) SQC PCR, ISH 49 1 5 1 14.2 Liu (44) SCP PCR 20 3 6 2 55 Popper (31) SQC PCR 11 1 9 SQC PCR 85 0 Shamanin (32) LSSC PCR 7 0 Szabo (45) SQC PCR 40 0 SQC PCR, ISH 49 4 2 2 16.3 Xing (46) SQC PCR 66 3 1 2 9 Al-Ghamdi (47) SQC DB, SB, PCR 10 1 10 Kinoshita (34) AdC DB, SB, PCR 26 2 9 SQC DB, PCR 50 10 1 5 32 Li (48) ASSC ISH, PCR 22 2 3 1 1 10 36 Nuorva (49) AdC DB, PCR 16 2 12 SQC DB, PCR 2 1 50 SQC PCR 8 1 12.5 Sagawa (50) SQC PCR 18 1 1 11 Thomas (51) SQC PCR 9 9 100 Da (52) SQC PCR 16 8 50 AdC PCR 12 5 42 AdC PCR 4 1 1 50 Thomas (53) LCC PCR 2 0 SQC PCR 7 2 28 SQC PCR, ISH 73 11 26 4 56 Hirayasu (54) AdC PCR 41 2 6 20 Noutsou (55) SQC PCR 41 2 2 10 LCC PCR 10 3 2 20 SQC PCR 7 0 SQC ISH, PCR 28 5 4 6 4 1 67 Soini (56) AdC ISH, PCR 12 1 2 2 3 5 75 SQC ISH, PCR 3 0

Table I. continued

2699 ANTICANCER RESEARCH 27: 2697-2704 (2007)

Table I. continued

Type of Technique No. of HPV type detected % HPV+ First author lesion used cases 6 6/11 11 16 16/18 18 Other

SQC PCR ND ND ND ND 79 Nakazato (57) SQC ISH, PCR 32 0 Welt (19) SQC ISH, PCR 6 0 SQC ISH, PCR 34 2 6 Bohlmeyer (58) SCP ISH, PCR 14 5 36 Flieder (59) SQC PCR, SB 52 6 11 69 Papadopoulou (60) ASC ISH, PCR 23 18 78 Tsuhako (61) ASC PCR 35 0 Wistuba (62) SQC PCR, ISH 31 0 Gorgoulis (18) AdC PCR, ISH 32 0 LCC PCR, ISH 5 0 SQC PCR 1 1 100 Henning (63) SQC ISH, PCR 75 12 1†† 18 (5)* 1 49 Henning (64) AdC PCR, EM 285 1 0.4 Hiroshima (65) SCP PCR 1 1 100 Kawaguchi (66) SQC HCII 185 5¨ 2.7 Clavel (67) SCP ISH, PCR 1 1 1 100 Harada (68) SQC ISH, PCR 44 ND 57 Iwamasa (69) SQC ISH, PCR 157 9 16 22 25 5 49 Miyagi (70) SQC ISH, PCR 141 77 55 Cheng (71) SQC ISH 26 1 2 11.5 Kaya (72) SQC ISH 22 1 4.5 Miasko (73) AdC ISH 13 1 7.6 LCC ISH 5 1 20 SQC ISH, PCR 59 29 49 Miyagi (74) AdC ISH, PCR 62 12 19.4 SQC PCR 24 1 4 Papadakis (75) LCC PCR 1 1 100 NSCLC PCR 40 2 5Zafer (76) SQC PCR 40 2 5 Jain (77) NSCLC ISH 122 0 Brouchet (78) SQC Line blot 126 1 0.7 Coissard (79) AdC Line blot 80 LCC Line blot 11 3 3.7 Line blot 1 SQC PCR 16 1 1 1 18 Ciotti (80) AdC PCR 15 5 33.3 SQC PCR, SB 14 4 29 Castillo (81) AdC PCR, SB 13 2 1 23 SC PCR, SB 9 3 33

††HPV11/16; *HPV6/16; ¨Oncogenic types in HCII assay. SQC, ; AdC, ; ASC, adenosquamous carcinoma; ASSC, alveolar carcinoma: SC, small cell; SB, Southern blot hybridisation; DB, dot blot hybridisation; ISH, in situ hybridisation; LCC, large cell carcinoma; PCR, polymerase chain reaction; SCP, squamous cell ; SQM, squamous cell metaplasia (modified with permission 16).

Conclusion The role of HPV as co- was first hypothesized in 1979 by Syrjanen, who has described a proportion of lung Lung cancer is one of the leading causes of cancer related tumors morphological changes closely resembling those death worldwide and cigarette smoking is the main risk seen in genital HPV lesions. This observation prompted factor. However, only 20% of smokers develop lung research groups around the world to look for the presence cancer, leaving plenty of space for additional risk factors of the HPV genome in lung cancer. The detection rate to be involved. varied from 0% to 100%, depending on the technique and

2700 Giuliani et al: Oncogenic HPVs in Lung Cancer (Short Review)

Figure 1. The presence of the oncogenic transcripts (E6 and E7) was evaluated by nested RT-PCR. The experiments A and B aimed at identifying the presence of the E7 and E6 transcripts, respectively. The arrows indicate the borders of the E6 and E7 genes, respectively. Donor and acceptor sites involved in the splicing process are indicated by dashed lines. With experiment C, we investigated the presence of the E6/E7 transcript with primers encompassing the E6/E7 sequence. primer set used. Among the lung cancer cases examined to 3 zur Hausen H: Cervical carcinoma and human papillomavirus: date, HPV DNA has been found in 22%, mainly in on the road to preventing a major human cancer. J Natl Cancer squamous cell carcinomas. As in cervical lesions, the Inst 93: 252-253, 2001. 4 zur Hausen H: Papillomaviruses and cancer: from basic studies oncogenic types most frequently found are HPV16 and 18. to clinical application. Nature Rev Cancer 2: 342-350, 2002. Establishment of immortalized lung cell lines from HPV- 5 Werness BA, Levine AJ and Howley PM: Association of human positive cases would be a valuable model to study the papillomavirus types 16 and 18 E6 proteins with p53. Science transcription process of the virus and expand our 248: 76-79, 1990. understanding of the carcinogenic process. Strictly speaking, 6 Kessis TD, Slebos RJ, Nelson WG, Nelson WG, Kastan MB, the detection of HPV DNA alone is not sufficient to Plunkett BS, Han SM, Lorincz AT, Hedrick L and Cho KR: confirm an active role of the virus in this process. Human papillomavirus 16 E6 expression disrupt the p53- mediated cellular response to DNA damage. Proc Natl Acad Sci 90: 3988-3992, 1993. References 7 Dyson N, Howley PM, Munger K and Harlow E: The human papilloma virus-16 E7 oncoprotein is able to bind to the 1 IARC: On the Evaluation of Carcinogenic Risks to Humans. retinoblastoma gene product. Science 243: 934-937, 1989. IARC Monograph. Lyon; 1995, pp. 1-409. 8 Ferlay J, Bray F, Pisani P and Parkin DM: GLOBOCAN 2000: 2 Syrjänen K and Syrjänen S: Papillomavirus Infections in Human Cancer Incidence, Mortality and Prevalence Worldwide, Pathology. New York. J. Wiley and Sons, 2000. Version 1.0. In: IARC Cancer Base 5. Lyon, IARC Press, 2001.

2701 ANTICANCER RESEARCH 27: 2697-2704 (2007)

9 Syrjänen K: Papillomavirus infections and cancer. In: 27 Benton EC: in butchers a cause for concern? Lancet 343: Papillomaviruses and Human Disease. Syrjänen K, Gissmann 11-14, 1994. L, Koss LG (eds.). Heidelberg, Springer-Verlag, pp. 468-503, 28 Durusoy R, Boffetta P, Mannetje A, Zaidze D, Szeszenia- 1987. dabrowska N, Rudnai P, Lissowska J, Fabianova E, Cassidy A, 10 Syrjanen KJ: Condylomatous changes in neoplastic bronchial Mates D, Bencko V, Salajka F, Janout V, Fevotte J, Fletcher T . Report of a case. Respiration 38: 299-304, 1979. and Brennan P: Lung cancer risk and occupational exposure to 11 Syrjanen KJ: Epithelial lesions suggestive of a condylomatous meat and live animals. Int J Cancer 118: 2543-2547, 2006. origin found closely associated with invasive bronchial 29 Trillo A and Guha A: Solitary condylomatous papillomas of the squamous cell carcinomas. Respiration 40: 150 160, 1980. bronchus. Arch Pathol Lab Med 112: 731-733, 1988. 12 Stremlau A, Gissmann L, Ikenberg H, Stark M, Bannasch P and 30 Syrjanen K, Syrjanen S, Kellokoski J, Karja J and Mantyjarvi R: zur Hausen H: Human papillomavirus type 16 related DNA in Human papillomavirus (HPV) type 6 and 16 DNA sequences an anaplastic carcinoma of the lung. Cancer 55: 1737-1740, in bronchial squamous cell carcinomas demonstrated by in situ 1985. DNA hybridisation. Lung 167: 33-42, 1989. 13 Byrne JC, Tsao MS, Fraser RS and Howley PM: Human 31 Popper HH., el-Shabrawi Y, Wockel W, Hofler G, Kenner L, papillomavirus-11 DNA in a patient with chronic laryngo- Juttner-Smolle FM and Pongratz MG: Progostic importance of tracheobronchial papillomatosis and metastatic squamous-cell human papilloma virus typing in of the carcinoma of the lung. N Engl J Med 317: 873-878, 1987. bronchus: comparison of in situ hybridisation and the 14 Syrjänen KJ and Syrjänen SM: Human papillomavirus DNA in polymerase chain reaction. Human Pathol 25: 1191-1197, 1994. bronchial squamous cell carcinomas. Lancet 1: 168-169, 1987. 32 Shamanin V, Delius H and de-Villiers EM: Development of a 15 Syrjänen K: HPV infections in the respiratory tract. In: broad spectrum PCR assay for papillomaviruses and its Papillomavirus Infections in Human Pathology. Syrjänen K and application in screening lung cancer biopsies. J Gen Virol 75: Syrjänen SJ (eds.). New York, Wiley and Sons, pp. 355-378, 1149-1156, 1994. 2000. 33 Kerley SW, Buchon-Zalles C, Moran J and Fishback JL: 16 Syrjänen KJ, Chang F and Syrjänen SM: HPV infections in Chronic cavitary respiratory papillomatosis. Arch Pathol Lab etiology of benign and malignant sinonasal, bronchial and Med 113: 1166-1169, 1989. oesophageal squamous cell lesions. In: 4th International 34 Kinoshita I, Dosaka-Akita H, Shindoh M, Fujino M, Akie K, Kato Multidisciplinary Congress EUROGIN 2000. Monduzzi (ed.). M, Fujinaga K and Kawakami Y: Human papillomavirus type 18 Bologna, pp. 169-179, 2000. DNA and E6-E7 mRNA are detected in squamous cell carcinoma 17 Syrjanen K: HPV infections and lung cancer. J Clin Pathol 55: and adenocarcinoma of the lung. Br J Cancer 71: 344-349, 1995. 885-891, 2002. 35 Carey FA, Salter DM, Kerr KM and Lamb D: An Investigation 18 Gorgoulis VG, Zacharatos P, Kotsinas A, Kyroudi A, into the role of human papillomavirus in endobronchial Rassidakis AN, Ikonomopoulos JA, Barbatis C, Herrington CS papillary squamous tumours. Resp Med 84: 445-447, 1990. and Kittas C: Human papillomavirus (HPV) is possibly involved 36 Bejui-Thivolet F, Liagre N, Chignol MC, Chardonnet Y and in laryngeal but not in lung carcinogenesis. Human Pathol 30: Patricot LM: Detection of human papillomavirus DNA in 274-283, 1999. squamous bronchial metaplasia and squamous cell carcinomas 19 Welt A, Hummel M, Niedobitek G and Stein H: Human of the lung by in situ hybridization using biotinylated probes in papillomavirus infection is not associated with bronchial paraffin-embedded specimens. Huma Pathol 21: 111-116, 1990. carcinoma: evaluation by in situ hybridization and the 37 Bejui-Thivolet F, Chardonnet Y and Patricot LM: Human polymerase chain reaction. J Pathol 181: 276-280, 1997. papillomavirus type 11 DNA in papillary squamous cell lung 20 Chiou HL, Wu MF, Liaw YC, Cheng YW, Wong RH, Chen CY carcinoma. Wirchows Arch Pathol Anat Histopathol 417: 457- and Lee H: The presence of human papillomavirus type 16/18 461, 1990. DNA in blood circulation may act as a risk marker of lung 38 Kulski JK, Demeter T, Mutavdzic S, Sterret GF, Mitchell KM cancer in Taiwan. Cancer 97: 1558-1563, 2003. and Pixley EC: Survey of histologic specimens of human cancer 21 Wood ME, Kelly K, Mullineaux LG and Bunn PA: The for human papillomavirus types 6/11/16/18 by filter in situ inherited nature of lung cancer: a pilot study. Lung Cancer 30: hybridization. Am J Clin Pathol 94: 566-570, 1990. 135-144, 2000. 39 Popper HH, Winsberger G, Juttner-Smolle FM, Pongratz MG 22 Matakidou A, Eisen T and Houlston RS: TP53 polymorphisms and Sommersgutter M: The predictive value of human and lung cancer risk: a systematic review and meta-analysis. papilloma virus (HPV) typing in the prognosis of bronchial Mutagenesis 18: 377-385, 2003. squamous cell papillomas. Histopathology 21: 323-330, 1992. 23 Frisch M and Melbye M: Risk of lung cancer in pre- and post- 40 Di Lorenzo TP, Tamsen A, Abramson AL and Steinberg BM: menopausal women with anogenital malignancies. Int J Cancer Human papillomavirus type 6a DNA in the lung carcinoma of a 62: 508-511, 1995. patient with recurrent is characterized 24 Rabkin CS, Biggar RJ, Melbye M and Curtis RE: Second by a partial duplication. J Gen Virol 73: 423-427, 1992. primary following anal and cervical carcinoma: evidence 41 Yousem SA, Ohori NP and Sonmez-Alpan E: Occurrence of of shared etiologic factors. Am J Epidemiol 136: 54-58, 1992. human papillomavirus DNA in primary lung . Cancer 25 Coggon D, Pannett B, Pippard EC and Winter PD: Lung cancer 69: 693-697, 1992. in meat industry. Br J Ind Med 46: 188-191, 1989. 42 Ogura H, Watanabe S, Fukushima K, Masuda Y, Fujiwara T 26 Kristensen TS and Lynge E: Lung cancer among butchers and and Yabe Y: Human papillomavirus DNA in squamous cell slaughterhouse workers. Scand J Work Environ Health 19: 137- carcinomas of the respiratory and upper digestive tracts. Jpn J 147, 1993. Clin Oncol 23: 221-225, 1993.

2702 Giuliani et al: Oncogenic HPVs in Lung Cancer (Short Review)

43 Katial RK, Ranlett R and Whitlock WL: Human papilloma 58 Bohlmeyer T, Le TN, Shroyer AL, Markham N and Shroyer virus associated with solitary squamous papilloma complicated KR: Detection of human papillomavirus in squamous cell by bronchiectasis and bronchial stenosis. Chest 106: 1887-1889, carcinomas of the lung by polymerase chain reaction. Am J 1994. Respir Cell Mol Biol 18: 265-269, 1998. 44 Liu HR, Xing LQ and Si JY : A study of human papillary virus 59 Flieder DB, Koss MN, Nicholson A, Sesterhenn IA, Petras RE infection by in situ hybridization and histopatology in squamous and Travis WD: Solitary pulmonary papillomas in adults: a cell carcinoma of the lung. Chung Hua Ping Li Hsueh Tsa Chih clinicopathologic and in situ hybridization study of 14 cases 23: 299-301, 1994. combined with 27 cases in the literature. Am J Surg Pathol 22: 45 Szabo I, Sepp R, Nakamoto K, Maeda M, Sakamoto H and 1328-1342, 1998. Uda H: Human papillomavirus not found in squamous and 60 Papadopoulou K, Labropoulou V, Davaris P, Mavromara P and large cell lung carcinomas by polymerase chain reaction. Cancer Tsimara-Papastamatiou H: Detection of human papillo- 73: 2740-2744, 1994. maviruses in squamous cell carcinomas of the lung. Virchows 46 Xing LQ, Liu HR and Si JY: Analysis of the characteristics of Arch 433: 49-54, 1998. human papilloma virus infection in 85 neoplasms of the 61 Tsuhako K, Nakazato I, Hirayasu T, Sunakawa H and Iwamasa respiratory system in adult patients. Zhonghua Zhong Liu Za T: Human papillomavirus DNA in adenosquamous carcinomas Zhi 16: 424-427, 1999. of the lung. J Clin Pathol 51: 741-749, 1998. 47 al-Ghamdi AA, Sanders CM, Keefe M, Coggon D and 62 Wistuba II, Behrens C, Milchgrub S, Virmani AK, Jagirdar J, Maitland NJ: Human papillomavirus DNA and TP53 Thomas B, Ioachim HL, Litzky LA, Brambilla EM, Minna JD mutations in lung cancers from butchers. Br J Cancer 72: 293- and Gazdar AF: Comparison of molecular changes in lung 297, 1995. cancers in HIV-positive and HIV-indeterminate subjects. 48 Li Q, Hu K, Pan X, Yang J and Hu S: Detection of human JAMA 279: 1554-1559, 1998. papillomavirus types 16, 18 DNA related sequences in 63 Henning EM, Di Lonardo A, Venuti A, Holm R, Marcante bronchogenic carcinoma by polymerase chain reaction. Chin ML and Nesland JM: HPV 16 in multiple neoplastic lesions Med J 108: 610-614, 1995. in women with CIN III. J Exp Clin Cancer Res 18: 369-377, 49 Nuorva K, Soini Y, Kamel D, Pollanen R, Bloigu R, 1999. Vahakangas K and Paakko P: P53 protein accumulation and the 64 Henning EM, Suo Z, Karlsen F, Holm R, Thoresen S and presence of human papillomavirus DNA in bronchial-alveolar Nesland JM: HPV positive bronchopulmonary carcinomas in carcinoma correlate with poor prognosis. Int J Cancer 64: 424- women with previous high-grade cervical intraepithelial 429, 1995. neoplasia (CIN III). Acta Oncol 38: 639-647, 1999. 50 Sagawa M, Saito Y, Endo C, Sato M, Usuda K, Kanma K, 65 Hiroshima K, Toyozaki T, Iyoda A, Ohwada H, Kado S, Takahashi S, Chin E, Sakurada A and Aikowa K: Detection of Shirasawa H and Fujisawa T: Ultrastructural study of human papillomavirus type 16, 18 and 33 DNA in stage I intranuclear inclusion bodies of pulmonary adenocarcinoma. (pT1N0M0) squamous cell carcinoma of the lung by polymerase Ultrastruct Pathol 23: 383-389, 1999. chain reaction. Kyobu Geka 48: 360-362, 1995. 66 Kawaguchi T, Matumura A, Iuchi K, Yamamoto S, Inoue Y, 51 Thomas P, De-Lamballerie X, Garbe L, Douagui H and Sunami T, Naka N, Okishio K, ueno K, Atagi S, Ogawara M, Kleisbauer JP: Detection of human papillomavirus DNA in Hosoe S and Kawahara M: Solitary squamous papilloma of the primary lung carcinoma by nested polymerase chain reaction. bronchus associated with human papilloma virus type 11. Intern Cell Mol Biol 41: 1093-1097, 1995. Med 38: 817-819, 1999. 52 Da J, Chen L and Hu Y: Human papillomavirus infection and 67 Clavel CE, Nawrocki B, Bosseaux B, Poitevin G, Putand IC, p53 mutation in primary lung cancer. Chung Hua Chung Liu Mangeonjean CC, Monteau M and Birembaut PL: Detection of Tsa Chih 18: 27-29, 1996. human papillomavirus DNA in bronchopulmonary carcinomas 53 Thomas P, De-Lamballerie X, Garbe L, Castelnau O and by hybrid capture II: a study of 185 tumours. Cancer 88: 1347- Kleisbauer JP: Detection of human papillomavirus by 1352, 2000. polymerase chain reaction in primary lung carcinoma. Bull 68 Harada H, Miura K, Tsutsui Y, Mineta H, Urano M, Abe M, Cancer 83: 842-846, 1996. Kurada M and Kasahara M: Solitary squamous cell papilloma 54 Hirayasu T, Iwamasa T, Kamada Y, Kovanagi Y, Usuda H and of the lung in a 40-years old woman with recurrent laryngeal Genka K: Human papillomavirus DNA in squamous cell papillomatosis. Pathol Int 50: 431-439, 2000. carcinoma of the lung. J Clin Pathol 49: 810-817, 1996. 69 Iwamasa T, Miaygi J, Tsuhako K, Kinjo T, Kamada Y, Hirayasu 55 Noutsou A, Koffa M, Ergazaki M, Siafakas NM and Spandidos T and Genka K: Prognostic implication of human DA: Detection of human papillomavirus (HPV) and K-ras papillomavirus infection in squamous cell carcinoma of the mutations in human lung carcinomas. Int J Oncol 8: 1089-1093, lung. Pathol Res Prat 196: 209-218, 2000. 1996. 70 Miyagi J, Tsuhako K, Kinjo T, Iwamasa T and Hirayasu T: 56 Soini Y, Nuorva K, Kamel D, Pollanen R, Vahakangas K, Recent striking changes in histological differentiation and rate Lehto VP and Paakko P: Presence of human papillomavirus of human papillomavirus infection in squamous cell carcinoma DNA and abnormal p53 protein accumulation in lung of the lung in Okinawa, a subtropical island in southern Japan. carcinoma. Thorax 51: 887-893, 1996. J Clin Pathol 53: 676-684, 2000. 57 Nakazato I, Hirayasu T, Kamada Y, Tsuhako K and Iwamasa 71 Cheng YW, Chiou HL, Sheu GT, Hsieh LL, Chen JT, Chen T: Carcinoma of the lung in Okinawa, Japan: with special CY, Su JM and Lee H: The association of human reference to squamous cell carcinoma and squamous papillomavirus 16/18 infection with lung cancer among non metaplasia. Pathol Int 47: 659-672, 1997. smoking Taiwanese women. Cancer Res 61: 2799-2803, 2001.

2703 ANTICANCER RESEARCH 27: 2697-2704 (2007)

72 Kaya H, Kotiloglu E, Inanli S, Ekicioglu G, Bozkurt SU, 82 Smotkin D and Wettstein FO: Transcription of human Tutkun A and Kullu S: Prevalence of human papillomavirus papillomavirus type 16 early genes in a cervical cancer and a (HPV) DNA in larynx and lung carcinomas. Pathologica 93: cancer-derived cell line and identification of the E7 protein. 531-534, 2001. Proc Natl Acad Sci USA 83: 4680-4684, 1986. 73 Miasko A, Niklinska W, Niklinski J, , Chyczewska E, Naumnik 83 Smotkin D, Prokoph H and Wettestein FO: Oncogenic and W and Chyczewski L: Dectection of human papillomavirus in nononcogenic human genital papillomaviruses generate the E7 non-small cell lung cancer by polymerase chain reaction. Folia mRNA by different mechanisms. J Virol 63: 1441-1447, 1989. Histochem Cytobiol 39: 127-128, 2001. 84 Cornelissen MT, Smits HL, Briet MA, van-den TJ, Struyk AP, 74 Miyagi J, Kinjo T, Tsuhako K, , Higa M, Iwamasa T, Kamada Y van-der NJ and ter Schegget J: Uniformity of the splicing and Hirayasu T: Extremely high Langerhans cell infiltration pattern of the E6/E7 transcripts in human papillomavirus type contributes to the favourable prognosis of HPV-infected 16-transformed human fibroblasts, human cervical premalignant squamous cell carcinoma and adenocarcinoma of the lung. lesions and carcinomas. J Gen Virol 71: 1243-1246, 1990. Histopathology 38: 355-367, 2001. 85 Sotlar K, Stubner A, Diemer D, Menton S, Menton M, Dietz 75 Papadakis ED, Soulitzis N and Spandidos DA: Association K, Wallwiener D, Kandolf R and Bultmann B: Detection of of p53 codon 72 polymorphism with advanced lung cancer: high-risk human papillomavirus E6 and E7 transcripts the Arg allele is preferentially retained in tumours arising in in cervical scrapes by nested RT-Polymerase chain reaction. J Arg/Pro germline heterozygotees. Br J Cancer 87: 1013-1018, Med Virol 74: 107-116, 2004. 2002. 86 Stacey SN, Jordan D, Snijders PJF, Mackett M, Walboomers 76 Zafer E, Ergun MA, Alvear G, Sahin FI, Yavuzer S and JMM and Arrand JR: Translation of the human papillomavirus Ekmekci A: Detection and typing of human papillomavirus in type 16 E7 oncoprotein from bicistronic mRNA is independent non-small cell lung cancer. Respiration 71: 88-90, 2004. of splicing events within the E6 open reading frame. J Virol 69: 77 Jain N, Singh V, Hedau S, Kumar S, Daga MK, Dewan R, 7023-7031, 1995. Murthy NS, Husain SA and Das BC: Infection of human 87 Grassmann K, Rapp B,Maschek H, Petry KU and Iftner T: papillomavirus type 18 and p53 codon 72 polymorphism in lung Identification of a differentiation-inducible promoter in the E7 cancer patients from India. Chest 128: 3999-4007, 2005. open reading frame of human papillomavirus type 16 (HPV16) 78 Brouchet L, Valmary S, Dahan M, Didier A, Galateau-Salle F, in raft cultures of a new cell line containing high copy numbers Brousset P and Degano B: Detection of oncogenic virus of episomal HPV16 DNA. J Virol 70: 2339-2349, 1996. genomes and gene products in lung carcinoma. Br J Cancer 92: 743-746, 2005. 79 Coissard CJ, Besson G, Polette MC, Monteau M, Birimbaut PL and Clavel CE: Prevalence of human papillomaviruses in lung carcinomas: a study of 218 cases. Mod Pathol 18: 1606-1609. 80 Ciotti M, Giuliani L, Ambrogi V, Ronci C, Benedetto A, Mineo TC, Syrjänen K and Favalli C: Detection and expression of human papillomavirus oncogenes in non-small cell lung cancer. Oncol Rep 16: 183-189, 2006. 81 Castillo A, Aguayo F, Koriyama C, Shuyama K, Akiba S, Herrera-Goepfert R, Carrascal E, Klinge G, Sanchez J and Received February 21, 2007 Eizuru Y: Human papillomavirus in lung carcinomas among Revised April 3, 2007 three Latin American countries. Oncol Rep 15: 883-888, 2006. Accepted April 23, 2007

2704