Presence of Human Papillomavirus Type 16 Related Sequences in Verrucous Carcinoma of the Larynx'

[CANCER RESEARCH 46, 2185-2188, April 19861 Presence of Human Papillomavirus Type 16 Related Sequences in Verrucous Carcinoma of the Larynx'

Janet L. Brandsma,2 Bettie M. Steinberg, Allan L. Abramson, and Barbara Winkler Departments ofOtola,yngology (I. L B., B. M. S., A. L. A.J and Pathology (B. W.J, Long Is!andjewish Medical Center, New Hyde Park, New York 11042

ABSTRACT according to the method of Batsakis et a!. (3). The presence of histo logical features associated with human papillomavirus infection of the Verrucous carcinoma of the larynx clinically resembles laryngeal squamous epithelium including koilocytosis, hyperplasia, hyperkera papilloma in that both are wart-like masses on the vocalcords and may tosis, dyskeratosis, giant cells, and bi- or multinucleate cells (9) was be characterized by multifocalityand recurrence. Human papillomavirus recorded. Tissue sections werestained for papillomavirusgroup-specific (HPV) infectionis an etiologicalfactor in laryngealpapilloma,and recent antigen using an antibody produced in rabbits to HPV particles cx evidenceimplicates HPV in squamousneoplasias.To determinewhether . tracted from plantar warts and purified through two cesium chloride HPV is also associated with verrucous carcinoma of the larynx, we gradients. The avidin:biotinylated horseradish peroxidase complex pro analyzedtissue specimensfrom six patients with verrucouscarcinomaof cedure (10) (Vector Laboratories) was used for detection. Sections of the larynx by Southern and DNA dot blot hybridizationfor HPV DNA. skin warts were used as positive controls in each assay. From three patients, specimensof normal laryngealepitheliumwerealso Molecular HybridizationStudies. Biopsyspecimens or excised tissue studied. All tissues showed evidence of HPV sequences related but not samples were removed during direct laryngoscopy or following laryn identical to HPV-16. They were negative for HPV-I 1. In contrast, four gectomy and stored in liquid nitrogen. Tissues were processed without squamouscellcarcinomasofthe larynxand three normallaryngealtissues thawing to extract and purify total DNA (11). For Southern blots, 10 were negativefor HPV DNA. Histological sections of the six verrucous Mgofcellular DNA or standard reconstructions representing 1, 10, and lesions were found to contain koilocytosis. Immunoperoxidasestaining 100 copies per cell of HPV DNA were digested with PstI using condi for HPV capsid antigens was negative in all these cases. The consistent tions recommended by the supplier (New England Biolabs) (100 mr.@i and specific association of HPV with the verrucous carcinomas in this NaCl:10 mM Tris (pH 7.5):bovine serum albumin (100 zg/ml)J; electro report suggests the possibility of a pathogenic involvement. phoresed through a 1% agarose gel; and transferred to Gene Screen Plus (New England Nuclear Research Products, Boston, MA), accord ing to the procedure of Southern (12). Gene Screen Plus was chosen INTRODUCTION because its high strength and binding properties allow multiple rehy Verrucous carcinoma of the larynx is a distinct variant of bridizations without noticeable loss ofsignal (13). For DNA dot blots, 1 @gofundigested cellular DNA was applied to well-differentiated squamous cell carcinoma characterized by a Gene Screen Plus membrane usinga manifold (Seleicherand Schuell). low incidence (1â€”2%of all carcinomas of the larynx), male For standards, HPV inserts were separated from appropriately digested preponderance, average age over 60, and local aggressiveness plasmid DNAs (7, 8, 14) by agarose gel electrophoresis and recovered without potential to metastasize. First described by Ackerman by electroelution. Probes were cloned HPV types 18, 16, and 11 (7, 8, (1) in the oropharynx, verrucous carcinomas have been reported 14)nick-translated with all four nucleotide [a-32Pjtriphosphates(15) to in the vulva, vagina, glans penis, esophagus, and tracheobron specificactivitiesof0.2â€”0.9x10@cpm/@g.Southern and dot blots were cial tree as well as in the larynx (2â€”4).Verrucous carcinomas hybridized sequentially with the probes of choice in 5x SSC (lx SSC are generally exophytic with a markedly hyperkeratotic, papil = 0.15 M NaC1:0.015 M sodium citrate), 50 mr@i sodium phosphate (pH lary surface. They bear a number of similarities to laryngeal 6.8), 1% sodium dodecyl sulfate, 30% formamide, and 10% dextran sulfate at 42C overnight (Tm â€”34C).Blots were first washed in 2x papilloma. Macroscopically, both appear as warty masses, with SSC:l% sodium dodecyl sulfate at 50C (Tm â€”41C,â€œnonstringentâ€•) notable papillary projections of the epithelial surface. Micro and sometimes rewashed in 0.lx SSC at 60T (Tm â€”9C,â€œstringentâ€•). scopically, both are composed of cytologically bland thickened Between hybridizations, the probe was eluted as described (16). The squamous epithelium. Clinically, both types of lesions may be effectivenessof the elution was verified autoradiographically. For au characterized by multifocality and have the tendency to recur toradiography, wet filters were wrapped in Saran Wrap and exposed to locally following endoscopic surgical removal. Because HPV3 Kodak XAR5 film with DuPont Lightening Plus intensifying screens infection is known to be an important etiological factor in for varying lengths of time (see Legends to Figs. 1â€”3). laryngeal papilloma (5, 6), and because recent evidence impli cates the involvement of HPV in the pathogenesis of squamous neoplasia in a variety of body sites (7, 8), we have examined RESULTS verrucous carcinoma of the larynx for the presence of HPV. [email protected],malesaged51â€”59,pre Our findings indicate that HPV-16 related DNA is associated sented with a common history of smoking and persistent with verrucous carcinoma of the larynx. hoarseness. At direct laryngoscopy all the lesions were diag nosed as verrucous carcinoma based on their clinical appear MATERIALS AND METHODS ance. Each was circumscribed, papillary, and leukoplakic and involved one or both vocal cords. Histology and Immunohistochemistry. Histological sections from for Histology and Immunohistochemistry. Table 1 summarizes maIm-fixed, paraffin-embedded tissues were reviewed and classified the major histological findings of the six patients. Four were diagnosed as verrucous carcinoma, having thickened processes Received 8/9/85; revised 12/12/85; accepted I 2/18/85. The costs of publication of this article were defrayed in part by the payment of cytologically bland squamous epithelium extending into the of page charges. This article must therefore be hereby marked advertisement in underlying connective tissue and broad invading pushing mar accordance with 18 U.S.C. Section 1734 solely to indicate this fact. gins (17). Two (Patients 1 and 6) showed no penetration of the I This investigation was supported by Grant 5 F32 A106643-02 from the National Institute of Allergy and Infectious Disease and by Grant P01 NS19214 basement membrane in the sections examined but were other from the National Institute of Neurologic and Communicative Disorders. wise consistent with verrucous carcinOma. Koilocytosis, char 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: HPV. human papillomavirus; SSC, 0.15 M acteristic of HPV infection, was identified in all cases. NaCl:0.0l5 M sodium citrate, pH 7.0; Tm, melting temperature ofduplex DNA. Adjacent tissues in four of the six patients showed focal 2185

Table 1 The presence ofkoilocytosis, papillomavirus group-specific antigen, and larynxKoilocytosishuman papillomavirus DNA in verrucous carcinoma ofthe E @i E@1 fl (14) was graded to indicate the relative abundance of koilocytotic 1233 12345 sss 61 cells. The presence of HPV capsid antigen was determined by use of a rabbit antibody to human plantar wart virus (see â€œMaterialsand Methodsâ€•). The presence of HPV DNA was assessed underconditions.HPV nonstringent capsid PatientDNA1 Koilocytosis antigen HPV +2 2+ â€” +3 1-2+ â€” +4 Focal-l+ â€” +5 1+ â€” +6 1+ â€” 1+ â€” + @ O

1@ U 1 II 1233 12345 sss 67 @. @. .,@

. . . ,@. .:â€˜.

L@4.

Fig. 2. Detectionof HPV-l I related DNA in verrucousand other tissues by Southern blot hybridizationunder nonstringentconditions.See Fig. 1for details. This autoradiogram was exposed for 3 days.

four (Patients 2â€”5)andprobably all five ofthe verrucous lesions (Fig. 1). Differences in the restriction pattern of the DNAs relative to the cloned HPV-16 are noted. Very faint hybridiza tion to HPV-18 was also detected (data not shown). The hy bridization patterns were similar with both probes, but stronger with HPV-16. In addition, the DNAs from visually normal sites adjacent to the tumors of three patients (Fig. 1) were also positive for HPV DNA. It is especially important to note that the adjacent site from Patient 1 is definitely positive. Despite this patient's lesion being borderline, his larynx does harbor Fig. I. Detection of HPV-l6 related DNA in verrucous and other tissues by HPV-16 related sequences. It is of interest to note that the Southern blot hybridization under nonstringent conditions. Lanes marked â€œ.5â€•amount of detectable HPV-16 related DNA in the lesion did are standards representing I, 10, and 100 copies per cell of HPV-l6; these are bracketed by two blank lanes. The other lanes are numbered according to patient: not correlate with the extent of koilocytosis (Table 1). Stringent Patients 1â€”5hadverrucous carcinoma of the larynx; and Patients 6 and 7 had washing of this blot reduced but did not totally remove signals oropharyngeal and inverted nasal papillomas, respectively. The four lanes at the from the verrucous tissue DNAs, whereas it made hybridization extreme left of the blot contain DNA from visually normal laryngeal epithelium taken from outside the margins of the verrucous tumors. The other numbered to the inverted nasal papilloma DNA no longer detectable. lanes contain DNA from the lesions. This autoradiogram was exposed for 3 days. Following moderately nonstringent hybridization to the HPV-1 1 probe, no hybridization to the DNA of the verrucous nonspecific hyperplasia without koilocytosis. The laryngectomy tissues was seen (Fig. 2). HPV-l I and HPV-16 are only dis specimen in one case (Patient 4) also contained a typical squa tantly related, and cross-hybridization is not detectable even mous papilloma. under nonstringent conditions, except in the case of extremely Immunoperoxidase staining for HPV capsid antigens was high HPV copy numbers. The two bands seen in the standards uniformly negative in all cases. This result indicates the absence in Fig. 2 are pBR DNA. These bands had equal intensity to of productive infection, since the antibody used cross-reacts those in Fig. 1 when exposed for equivalent time. Longer with capsid antigens of all known types of HPVs. exposures revealed increased nonspecific background binding, Molecular Hybridization Studies. Moderately nonstringent but, again, no bands were visualized in any of the nine lanes hybridization conditions were used to permit the detection of containing the verrucous DNAs. The inverted nasal papilloma, related as well as homologous types ofHPV (18). The Southern however, was strongly positive, and little or no probe washed blot in Figs. I and 2 were hybridized sequentially in the follow off in the subsequent stringent wash. ing order to probes containing HPV-16, HPV-l 1, HPV-18, DNAs from several other specimens (Fig. 3A) were then HPV-16, and HPV-1 I. Results obtained with the HPV-16 and extracted and tested by DNA dot blot hybridization. Histolog HPV-l 1 probes were reproducible on rehybridization. ical review of these specimens was not done. Of all the tissues Hybridization of tissue DNAs to HPV-16 (Fig. 1) revealed studied on this blot, the only one positive for HPV-16 related definite evidence of the presence of related sequences in at least sequences (Fig. 3, B and C) was the sixth verrucous carcinoma 2186

and 12 of 14 tested under stringent conditions were positive for vv 4 HPV-6 or HPV-1 1 (6). In the present study, one papilloma of five tested with HPV-16 appeared to hybridize better to HPV

NN 16 than to HPV-l I. In addition, one vocal cord polyp proved positive for HPV-16 related sequences. Among the other head and neck tissues examined, two additional positives were iden Nâ€”C tified. One of these was a recurrent inverted nasal papilloma in a 54-yr-old man. The other was a particularly aggressive pap

CC illoma of the conjunctiva in a 12-yr-old boy.

DISCUSSION This study demonstrates a strong correlation between the presence ofHPV-16 related sequences and verrucous carcinoma of the larynx in our six patients. Indeed, analysis of the DNA ABC D E hybridizationresultsindicatesthepresenceofHPV-16related sequences not only in all six verrucous tumors but also in the Fig. 3. Detection of HPV-l6 related DNA in verrucous and other tissues by dot blot hybridization. A shows a diagram of the tissues analyzed: verrucous four tissues outside the margins of these tumors that were carcinoma (JJ); normal epithelium (N); squamous cell carcinoma (C); and papil studied. In addition, the laryngeal specimens ofall six verrucous loma (P). All tissues are laryngeal except the left-hand V. which is a verrucous patients were reviewed and found to contain koilocytosis, a carcinoma of the mandible, and the lower P. which is an inverted papilloma of the nose (Patient 7 in Fig. 1). B and C show the signals obtained following well-established marker of HPV infection (9). The absence of hybridization to HPV-16 probe: nonstringent wash (B) and stringent rewash (C). immunohistochemically detectable HPV antigens is not sur D and E show the signals obtained following hybridization to HPV-l 1 probe: prising, since peroxidase positivity for HPV antigens has been nonstringent wash (D) and stringent rewash (E). Autoradiograms (B) and (D) were exposed for 3 days; C and E were exposed for 1 wk. shown to be extremely low in squamous intraepithelial neopla sia and virtually absent in squamous cell carcinoma (10, 19). TableneckThe 2 The presence ofHPV DNA in various diseases ofthe head and Immunohistochemistry is limited to the detection of productive presence of type-specific HPV DNAs was assessed under stringent con viral infections. DNA hybridization, on the other hand, has the ditions by Southern and/or dot blot analysis.HPV- advantage of being able to detect latent and transforming as HPV 16LarynxVerrucousPathology 6/Il well as productive infections, which renders it potentially more sensitive. 6/6Squamouscarcinoma 0/6 The Southern and dot blot analyses presented here clearly 0/4Papillomacell carcinoma O/@l@ demonstrate that HPV-16 related sequences are present in 1/5Polyp l2/l4a 1/1Normal 0/3 verrucous carcinoma of the larynx. HPV-16 and -18 have been 0/30/3Other implicated in human genital squamous carcinomas and its precursors, and HPV-1 1 is known to be involved in both neckVerrucous head and carcinomaMandible laryngeal papilloma and cervical condyloma (5, 6, 20, 21). The 0/1PapillomaNasal 0/1 one blot presented here does not permit definitive conclusions 1/2Eye 0/2 to be drawn regarding the type of HPV DNA present in the 1/1Palate 0/I verrucous tissues. However, among the three types of HPV 0/1Lip 0/1 studied, the strongest â€¢signalswere detected using HPV-16 0/1Adenoid 0/1 0/1 0/1 DNA, indicating that the sequence in the verrucous tissues are aThree squamouscellcarcinomasand 14laryngealpapillomaswerepreviously more closely related to HPV-16 than to either of the other two analyzed under stringent conditions to HPV-6/HPV-l 1 DNA (18). types tested. This finding was obtained twice during the series of sequential hybridizations performed in this study. ofthe larynx. The laryngeal papilloma which was positive under The hybridization signals detected in the verrucous DNAs nonstringent conditions did not contain an HPV-16 related are specific to HPV-16and not the result of cross-hybridization virus, because the probe eluted under stringent conditions. All to cellular sequences. Under the moderately nonstringent hy other tissues were negative, including the four squamous cell bridization conditions used, sequences which are only distantly carcinomas of the larynx and the vemicous carcinoma of the related do not cross-hybridize, as evidenced by the fact that the mandible. HPV-16 viral-specific DNA fragments on the Southern blot in When this blot was rehybridized with the HPV-1 1 probe (Fig. Fig. 2 are not appreciably detected by the HPV-1 1 probe. (The 3, D and L), both of the papillomas showed continued strong two fragments which are detected are the plasmid fragments.) hybridization following stringent washing. In contrast, the ver Note, too, that the pattern observed in the verrucous DNAs is rucous carcinoma did not contain HPV-1 1, because the initial not found in the lane containing DNA from a human orophar weak hybridization was nearly totally abrogated following the yngeal papilloma (Fig. 1, Patient 6) which was negative for all stringent wash. the HPVs tested. Moreover, HPV-negative human DNAs hy We also screened additional samples to examine the preva bridized in our laboratory on other Southern blots under con lence of HPV-16 related sequences in other lesions. Table 2 ditions identical to those used in this study have never produced summarizes the composite of our findings. In the larynx, ver hybridization patterns such as those detected here in the ver rucous carcinomas were consistently found to harbor HPV-1 6 rucous tissue DNAs. related sequences. In contrast, no HPV DNA was identified in The differences in restriction patterns between the verrucous squamous cell carcinomas. This laboratory has previously re tissue and cloned HPV-16 DNAs could be explained either by ported testing 20 papillomas for HPV-6 or HPV-l 1 DNA. All the presence of a different but related type of HPV or by 20 hybridized to HPV DNA under nonstringent conditions, incomplete enzyme digestion. We suspect the presence of a new 2187

type, because the hybridization was weak and because some of REFERENCES the probe eluted during the stringent wash (data not shown); I. Ackerman, L. V. Verrucouscarcinoma of oral cavity. Surgery (St. Louis), also, the DNA from the inverted nasal papilloma (Fig. 1, 23:670â€”678, 1948. Patient 7) was cleaved to completion. 2. Kraus, F. T., and Perez-Mesa, C. Verrucous carcinoma. Clinical and patho logic study of 105 cases involving oral cavity, larynx, and genitalia. Cancer In addition to the findings of HPV-16 related sequences in (Phila.), 19: 26â€”38,1966. the verrucous lesions, we also found similarly related sequences 3. Batsakis, J. G., Hybels, R., Crissman, J. D., and Rice, D. H. The pathology of head and neck tumors: verrucouscarcinoma. Part 15. Head Neck Surg., in four â€œnormalâ€•laryngealtissues of three of these patients 5:29â€”38,1982. taken from sites well outside the margins of the tumor. As in 4. Ferlito, A., and Recher,G. Ackerman'stumor (verrucouscarcinoma)of the laryngeal papilloma (6), the presence of a latent viral infection larynx. Cancer (Phila.), 46: 1617â€”1630,1980. 5. Mounts, P., Shah, K. V., and Kashima, H. Viral etiology of juvenile-onset in adjacent tissues of verrucous carcinoma could certainly be and adult-onset squamous papilloma of the larynx. Proc. NatI. Acad. Sci. important in explaining the recurrent and multifocal nature of USA, 79: 5425â€”5429,1982. the disease. 6. Steinberg, B. M., Topp, W. C., Schneider, P. S., and Abramson, A. L. Laryngeal papillomavirus infection during clinical remission. N. Engl. J. No specific hybridization to DNAs from the verrucous tissues Med.,308:1261â€”1264,1983. was observed to the HPV-1 1 probe. The latter finding is partic 7. Durst, M., Gissman, L., Ikenberg, H., and zur Hausen, H. A papillomavirus ularly important for two reasons. (a) It indicates that the virus DNA from a cervical carcinoma and its prevalence in cancer biopsies from different geographic regions. Proc. NatI. Acad. Sci. USA, 80: 3812â€”3815, associated with the verrucous lesions is distinct from the HPVs 1983. found in laryngeal papillomas. (b) The absence of hybridization 8. Boshart, M., Gissmann, L., Ikenberg, H., Uleinheinz, A., Scheurlen, W., and zur Hausen, H. A new type of papillomavirus DNA, its presence in genital to the HPV-1 1 probe indicates that the hybridization to the cancer biopsies and in cell lines derived from cervical cancer. EMBO J., 3: HPV-16 probe is not due to cross-hybridization to bacterial 1151â€”1157,1984. plasmids contaminating the human tissues. (All probes con 9. Meisels, A., Morin, C., and Casas-Cordero, M. Human papillomavirus infection ofthe uterine cervix. Int. J. Gynecol. Pathol., 1: 75â€”94,1982. tamed pBR DNA.) Unfortunately, further characterization of 10. Kurman,R. J., Jensen, A. B.,Sinclair,C. F., and Lancaster,W. D. Detection the DNAs from these six patients is precluded by the lack of of human papillomaviruses by immunocytochemistry. In: R. A. DeLellis sufficient material for further analysis. Whether HPV-16 re (ed.),Advancesin Immunohistochemistry,pp. 201â€”221.NewYork: Masson Publishing, Inc., 1984. lated DNA will be found in all cases of verrucous carcinoma of I 1. Krieg,P., Amtmann, E., and Sauer, G. The simultaneousextraction of high the larynx is not known. As additional verrucous specimens are molecular-weight DNA and ofRNA from solid tumors. Anal. Biochem., 134: 288â€”294,1983. made available to us, attempts will be made to identify, clone, 12. Southern, E. M. Detection of specific sequences among DNA fragments and characterize the HPV sequences associated with verrucous separatedby gelelectrophoresis.J. Mol. Biol.,98: 503â€”517,1975. carcinoma ofthe larynx. Potentially, HPV typing may be useful 13. Gatti, R. A., Concannon, C., and Salser, W. Multiple use of Southern blots. BioTechniques,2:148â€”155,1984. in predicting the biological behavior of laryngeal squamous 14. Gissman, L., Diehl, V., Schultz-Coulon, H.-J., and zur Hausen, H. Molecular lesions, as has been shown in the female genital tract, and may cloning and characterization of human papilloma virus DNA derived from a serve as a marker for patients at high risk for the development laryngeal papilloma. J. Virol., 44: 393â€”400,1982. 15. Rigby, P. W., Dieckmann, M., Rhodes, C., and Berg, P. Labeling deoxyri of carcinoma (20, 21). bonucleic acid to high specific activity in vitro by nick translation with DNA What role, if any, the HPV-16 related virus plays in the polymerase I. J. Mol. Biol., 113: 237â€”251,1977. 16. Van Doren, K., Hanahan, D., and Gluzman, Y. Infectionof eukaryoticcells generation of verrucous carcinoma of the larynx is not clear. by helper-independent recombinant adenoviruses: early Region I is not Although the possibility of a passenger virus cannot be ruled obligatory for integration of viral DNA. J. Virol., 50: 606â€”614,1984. out at this time, the consistent and specific association with the 17. Maw, A. R., Cullen, R. J., and Bradfield, J. W. B. Verrucous carcinoma of the larynx.Clin. Otolaryngol.,7:305â€”311,1982. verrucous lesions in this paper clearly suggests the possibility 18. Howley, P. M., Law, M.-F., Heilman, C., Engel, L., Alonso, M. C., Isreal, of a pathogenic involvement. M. A. Lowy, D. R., and Lancaster, W. D. Viruses in naturally occurring cancers. Cold Spring Harbor Conf. Cell Prolif., 7: 233â€”249,1980. 19. Syrjanen, K., Syrjanen, S., and Pyrrhonen, S. Human papilloma virus (HPV) antigens in lesions of laryngeal squamous cell carcinomas. J. Otorhinolar yngeal Relat. Spec., 44: 323â€”334,1982. ACKNOWLEDGMENTS 20. Gissman, L., Boshart, M., Durst, M., Ikenberg, H., Wagner, D., and zur Hausen, H. Presence of human papillomavirus in genital tumors. J. Invest. We are indebted to L. Taichman for antibody to plantar warts (HPV Dermatol.,83: 265â€”288,1984. 21. Crum, C. P., Ikenberg, H., Richart, R. M., and Gissman, L. Human papil 1), to L. Gissman for cloned HPV DNAs, and to R. Hamaker for tissue lomavirus type 16 and early cervical neoplasia. N. EngI. J. Med., 310: 880â€” from the verrucous carcinoma of the mandible. 883, 1984.

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Janet L. Brandsma, Bettie M. Steinberg, Allan L. Abramson, et al.

Cancer Res 1986;46:2185-2188.

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