Detection of Human Papillomavirus in Squamous Cell Carcinomas of the Lung by Polymerase Chain Reaction

Teresa Bohlmeyer, Tuan N. Le, A. Laurie Shroyer, Neil Markham, and Kenneth R. Shroyer

Department of Pathology and Division of Internal Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and Department of Cardiac Research, Veteran's Affairs Medical Center, Denver, Colorado

	Abstract

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Existing evidence supports the hypothesis that human papillomavirus (HPV) may play an etiologic role in the malignant transformation of squamous epithelial cells. Although HPV DNA has been identified in a high proportion of squamous cell carcinomas (SCC) of the cervix, anorectum, skin, and upper airways, few studies have tested for HPV in SCC of the lung. To confirm the presence of HPV in lung SCC, we tested for HPV DNA extracted from formalin-fixed tissues of 34 patients by polymerase chain reaction (PCR). DNA amplification was performed using HPV L1 consensus sequence primers (MY11 and MY09; Perkin-Elmer Cetus, Norwalk, CT) which recognize a broad spectrum of HPV types including 6, 11, 16, 18, 31, and 33, among many other known types, as well as at least 20 other unidentified types. PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization with [³²P]-labeled generic HPV probes. HPV DNA positive cases were subsequently analyzed by slot-blot hybridization of the PCR products with specific probes for HPV types 6, 11, 16, 18, and 33. HPV type 18 was detected in two cases, including one case from a 44-year-old female and one from a 64-year-old male, with the remaining 32 cases negative. In situ hybridization for HPV DNA failed to detect HPV types 6/11, 16/18, or 31/33/35 in any of the cases. We conclude that a small proportion of cases of primary pulmonary SCC test positive for HPV type 18 but that the great majority of cases are not associated with HPV.

	Introduction

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Human papillomaviruses (HPVs) are cutanotropic infectious agents (1) associated not only with cervical dysplasias that can progress to malignancies (2), but also with malignant changes in the skin and numerous other sites including esophagus, nasal sinuses, and bladder (8). In the upper respiratory tract, HPV 6 and 11 have been associated with juvenile laryngeal papillomatosis, a benign process which can undergo malignant changes in patients exposed to radiation therapy (17). HPV types 6, 16, and 18 have been detected in laryngeal carcinoma as well (21). In the lower respiratory tract, HPV as a causative agent in squamous cell carcinoma (SCC) of the lung has been investigated because of the morphologic evidence of condylomatous-like lesions of bronchial mucosa adjacent to invasive bronchial SCC in about 25% of cases (24) and because of the potential for HPV infection of traumatized bronchial mucosa in cigarette smokers (28).

Southern blot hybridization is considered by many the "gold standard" for detection of HPV in host tissue due to its high sensitivity and ability to suggest the physical state of DNA in the cell (29). Stremlau and colleagues (30) reported over 10 years ago that Southern blot hybridization using probe cocktails for HPVs 1, 2, 4, 8, 9, 10, 11, 13, 16, and 18 was negative in nine of nine cases of pulmonary SCC. By contrast, HPV type 16 was detected in one of five cases of anaplastic carcinoma of the lung. Southern blot hybridization has not been reported in additional studies on the association of HPV with pulmonary SCC, perhaps due to the difficulty in applying the method in archival tissue analysis where DNA is highly fragmented and modified in the tissue fixation process (7).

More recently, in situ hybridization has been applied to HPV DNA detection due to the advantages of cellular localization, utility in routinely fixed/archival tissues, and morphologic correlation with HPV types. Syrjanen and coworkers (31) reported in 1987 that HPV DNA could be detected by in situ hybridization in five of 99 cases of pulmonary SCC. This study utilized a generic HPV probe cocktail which was capable of recognizing HPV types 6, 11, 16, 18, and 30 but did not attempt to identify specific HPV types in positive cases. Since the time of the initial report by Syrjanen and associates (31), additional studies have confirmed the presence of HPV DNA by in situ hybridization in 9 to 30% of cases of sporadic pulmonary SCC (32- 39). Among these studies, "high risk" cervical-type HPVs including HPV types 16, 18, and 31/33/35 have been found in a high proportion of the positive cases.

Most recent studies on the association of HPV with pulmonary SCC have relied on the application of polymerase chain reaction (PCR)-based methods, due to the high level of sensitivity provided by this method for the detection of HPV in archival fixed tissues. PCR for HPV, using consensus sequence primers capable of detecting a broad range of HPV types, has generally shown a relatively low prevalence of HPV in cases of sporadically occurring pulmonary SCC. Szabo and coworkers (40), using consensus primers designed to detect HPVs 6, 11, 16, 18, 31, 33, 52b, and 58, failed to detect HPV DNA in any of 40 cases of pulmonary SCC. Two more-recent studies, however, have reported the detection of HPV in a small proportion of pulmonary cases, including six of 66 cases tested by Al-Ghamdi and colleagues (32) and two of 43 cases reported by Fong and coworkers (34).

In the current paper, we confirm that HPV DNA can be detected in only a relatively small proportion of cases of pulmonary SCC.

	Materials and Methods

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Thirty-four archival, formalin-fixed, paraffin-embedded tissue sections and biopsies from 34 nonimmunocompromised patients with SCC of the lung at the University of Colorado Health Sciences Center and the Denver VA Medical Center were tested for HPV DNA by PCR (Figure 1). Sections of pulmonary metastases of an HPV type 16 positive cervical SCC were included as a positive control. Duplicates of 9-µm-thick tissues were placed into individual 1.5-ml capped sterile centrifuge tubes and digested with proteinase K as previously described (7, 41). Ten µl aliquots of the proteinase K digested samples were amplified in an automated thermocycler (Perkin-Elmer, Norwalk, CT) programmed for 40 cycles (7). HPV L1 consensus sequence primers (MY11 and MY09; Perkin-Elmer-Cetus, Norwalk, CT) were used. Recombinant plasmids containing HPV 6, 11, 16, and 18 DNA were used as positive controls. -globin primers (PC04 and GH20; Perkin-Elmer-Cetus) were used as amplification controls. Reagent controls lacking target DNA were included as negative controls to monitor for sample contamination and primer annealing. The absence of product in the negative control reactions indicated that there was no contamination by sample DNA, that products in the experimental reaction were template-specific, and that they were not caused by primer dimers. After DNA amplification, the PCR products were analyzed by agarose gel electrophoresis, ethidium bromide staining, and ultraviolet (UV) light visualization. The PCR products were hybridized, using the Southern blot method, with a generic [³²P]-labeled HPV probe mix which recognizes a broad spectrum of HPV types including 6, 11, 16, 18, 31, and 33, among many other known types, and at least 20 other unidentified types (42). The hybridizable band containing the HPV DNA were detected by autoradiography (Figure 2). HPV-positive cases were confirmed by repeating the entire assay starting from the original tissue block in each case. PCR products of cases which tested positive for HPV DNA by Southern blot hybridization were subsequently analyzed by slot-blot hybridization with type-specific probes for HPV types 6, 11, 16, 18, and 33 (42).

View larger version (151K): [in this window] [in a new window]   Figure 1.   Well-differentiated SCC of the lung. Occasional cells show binucleation with perinuclear cytoplasmic clearing, suggestive of HPV effect. (H&E stain).

View larger version (42K): [in this window] [in a new window]   Figure 2.   I. PCR amplification with HPV L1 consensus sequence primers, pulmonary SCC, representative cases (lanes A-G). HPV L1 DNA was amplified over 40 cycles. Amplified products were analyzed by agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light. An HPV L1 positive case (lane G) shows a band at about 450 bp (arrowhead). HPV 6, 11, 16, 18 plasmid DNA positive control are shown in the right lanes. A negative (aqueous) control is in lane H. -globin products, used as an amplification control, show a band at about 268 bp (double arrowheads). II. Southern blot hybridization of the electrophoresed products with a generic [32P]-labeled HPV probe mix. Autoradiographic exposure reveals a band at about 450 bp for HPV L1 in lane G (arrowhead). HPV 6, 11, 16, 18 plasmid DNA controls show a strong positive signal.

HPV DNA in situ hybridization was performed on all cases that tested positive for HPV DNA by PCR. The hybridization followed the manufacturer's protocol and used three different biotin-labeled DNA probe cocktails that recognize HPV types 6/11, 16/18, and 31/33/35 (ViraType In Situ; Life Technologies, Silver Spring, MD). Each hybridization assay included the manufacturer's positive and negative DNA controls. The assay involves incubation of deparaffinized tissue sections in digestion reagent at 37°C for 15 min, denaturation of target DNA at 100°C for 5 min, and hybridization with biotin-labeled probe reagents at 37°C for 2 h. After hybridization, sections were reacted with an alkaline phosphatase-antibiotic antibody conjugate and developed using nitroblue tetrazolium and 5'-bromo-4-chloro-3-indoyl phosphate.

	Results

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The patients ranged from 40 to 79 years of age (mean 60.76 years, population standard deviation 9.39 years), were males in 29 of 34 cases, and all had a history of cigarette smoking.

Histologic examination of the tumors revealed focal areas with features suggestive of HPV effect in 11 of the 34 cases, including 1 of 2 well-differentiated SCC, 7 of 15 moderately differentiated SCC, and 3 of 17 poorly differentiated SCC (Table 1). These changes consisted of tumor cells with perinuclear cytoplasmic clearing (koilocytosis) and occasional cells with binucleation (Figure 1). The changes, however, were less pronounced than those typically seen in condylomatous lesions and thus were considered suspicious for, but not diagnostic of, HPV effect.

View this table: [in this window] [in a new window]   TABLE 1 Summary of test cases (pulmonary SCC)

HPV DNA was detected by PCR in two cases (Table , Figure 2). In these two cases, a strong signal for HPV was found by Southern blot hybridization of the PCR products with a generic HPV probe and by slot-blot hybridization of the PCR products with a probe specific for HPV type 18. The remaining 32 cases of pulmonary SCC tested negative for HPV by PCR. In situ hybridization showed no evidence of staining for HPV DNA in any of the cases. Of the two cases that tested positive for HPV type 18, one (case 2) was from a 44-year-old female and the other (case 20) was from a 64-year-old male. Both HPV positive cases showed no evidence of koilocytosis binucleation and both were moderately differentiated (grade 2) carcinomas.

	Discussion

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HPV DNA is found in a high proportion of SCC of the female genital tract but its association with carcinomas of other tissues is generally less well defined. Pulmonary SCCs clearly have a multifactorial pathogenesis, with cigarette smoking associated with a high proportion of cases. It is clear, however, that no single risk factor predicts the subsequent development of pulmonary SCC.

The potential association of HPV with pulmonary SCC was suggested, in part, by the fact that HPV has been found in carcinomas of the upper respiratory tract, including SCC of the larynx (16, 21). In general, it appears that HPV-induced lesions have a propensity to occur at sites of squamo-columnar mucosal junctions, such as the squamo-columnar junctions of the cervix, the anorectal junction, and the junction of squamous mucosa with respiratory mucosa in the larynx (3, 8). Cigarette smoking induces multifocal squamous metaplasia throughout the bronchial tree and a high proportion of bronchogenic SCCs appear to arise from metaplastic squamous bronchial epithelium (33). Thus in the cigarette smoker, in contrast to the nonsmoker, the lower respiratory tract will often have innumerable squamo-columnar junctions which could serve as a "fertile soil" for infection by HPV.

In the current study, we tested the hypothesis that HPV DNA is associated with at least some cases of pulmonary SCC. If this hypothesis is correct, then HPV infection could be an important cofactor in the malignant transformation of bronchial mucosa, perhaps interacting with other agents, such as cigarette smoke, in the pathogenesis of pulmonary SCC.

In our study, PCR for HPV DNA was positive in two of 34 cases of pulmonary SCC. Slot-blot hybridization of the PCR products identified HPV type 18 in these two cases. In situ hybridization for HPV DNA was negative in all cases. The failure to detect HPV DNA by in situ hybridization in cases that tested positive by PCR could reflect the relatively greater sensitivity of the latter method and may indicate that the viral copy number per cell is relatively low compared with other HPV-associated tumors such as cervical or anorectal SCC (43, 44).

Our results are similar to those of several previous PCR-based studies, which have shown a relatively low prevalence of "high risk" HPVs in pulmonary SCC (32, 34, 35) but differ from the study of Szabo and colleagues, who found no evidence of HPV among 40 cases tested using E6 and E7 consensus primers (40). Similar to the results of our study, Kinoshita and associates (35) found HPV type 18 DNA in 3 of 36 cases, including 1 of 10 SCCs and 2 of 22 adenocarcinomas of the lung. The finding of HPV type 18 in lung carcinoma was somewhat unexpected since HPV type 18 is relatively uncommon in SCCs of other tissues.

Our studies showed that histologic evidence suggestive of HPV (koilocytosis) did not predict the detection of HPV by PCR. The lack of correlation between HPV detection and koilocytosis suggests that koilocytotic change in pulmonary SCC is not definitive evidence of HPV effect. Consistent with this idea, histologic studies have shown condylomatous-like lesions adjacent to invasive pulmonary SCC in about 25% of cases (24) but PCR-based studies have generally shown a lower prevalence of detectable HPV DNA.

In summary, we found evidence of HPV DNA in a relatively small proportion of cases of pulmonary SCCs. Thus HPV infection appears unlikely to play an etiologic role in the great majority of cases of sporadically occurring pulmonary SCC.

	Footnotes

Address correspondence to: Kenneth R. Shroyer, M.D., Ph.D., University of Colorado Health Sciences Center, Dept. of Pathology, B216, 4200 E. Ninth Ave., Denver, CO 80262-0216. E-mail: Ken.Shroyer{at}UCHSC.edu

(Received in original form May 27, 1997 and in revised form August 12, 1997).

Acknowledgments: A. L. S. was funded and sponsored, in part, by the Department of Veterans Affairs, Office of Quality Management, and Program for Cooperative Studies in Health Services Research.

Abbreviations HPV, human papillomavirus; PCR, polymerase chain reaction; SCC, squamous cell carcinoma.

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