JOURNAL OF PATHOLOGY, VOL.

165: 243-246 (I 99 1)

HUMAN PAPILLOMAVIRUS 6, 11, AND 16 IN LARYNGEAL PAPILLOMAS PAUL DICKENS, GOPESH SRIVASTAVA, SHEE LOONG LOKE AND SIEPIE LARKIN

Department of Pathology, University of Hong Kong, Hong Kong Received 13 February 1991 Accepced 16 May 1991

SUMMARY Twenty-seven cases of benign laryngeal papillomas, both single and multiple variants, were analysed for human papillomavirus (HPV) by DNA slot-blot hybridization chiefly to determine the pattern of infection in Hong Kong Chinese. DNA was extracted from paraffin blocks of formalin-fixed tissue and probed separately for HPV 6,11, 16, and 18. Sixteen cases (59 per cent) showed the presence of at least one of these four HPV genomes. Thirteen cases (48 per cent) werepositivefor HPV 1 1 only. Threeothercases(I1 percent)showed triplepositivityforHPV6,l ],and 16. None were positive for HPV 18. The predominance of HPV 1 1 infection contrasts with other series which have shown either an almost equal distribution of HPV 6 and 11 or a predominance of HPV 6. The finding of HPV 16 in three cases was unexpected. Using the polymerase chain reaction (PCR) with primers complementary to the upstream regulatory region of the HPV 16 viral DNA, the presence of HPV 16 genome was confirmed in all three cases. As the number of HPV 16-positive cases in this study is small, analysis of more cases using fresh biopsy material and a wider range of HPV type-specific PCR primers is warranted to determine the relative incidence of HPV subtypes in these benign laryngeal papillomas. KEY

WoRDs-Human

papillomavirus, laryngeal papilloma, slot blot, polymerase chain reaction.

INTRODUCTION The presence of HPV genotypes in laryngeal papillomas has been established by immunohistochemical' and DNA hybridi~ation~-~ techniques. The purpose of our study was to delineate the pattern of HPV types in laryngeal papillomas in Hong Kong Chinese. MATERIALS AND METHODS

Selection of cases Twenty-nine cases of laryngeal papilloma, both adult and juvenile types, were obtained from the files of the Department of Pathology, University of Hong Kong over the period 1978-1988. Two cases were found to have insufficient tissue left in the blocks for DNA analysis by slot blotting and were thus excluded. The slides were reviewed and the diagnoses confirmed prior to DNA analysis. Addressee for correspondence: Dr P. Dickens, Department of Pathology, University of Hong Kong, Queen Mary Hospital, Hong Kong.

0022-3417/9 l / 1 1024344 $05.00 0 1991 by John Wiley & Sons, Ltd.

H P V D N A probes The plasmid DNA of HPV 6, 1 1, 16, and 18 was provided by Professor H. zur Hausen and Dr L. Gissmann (German Cancer Research Centre, Heidelberg, Germany).'@I3 HPV probes were prepared by oligo-labelling of the HPV DNA inserts using "P-dCTP according to the supplier's conditions (Bresatec, Australia). DNA slot-blot hybridization analysis Five to twenty 16 pm thick sections were cut from each case and deparaffinized. DNA was extracted using proteinase KI4and denatured DNA ( 5 pg) was slot blotted on a nylon membrane (Genescreen Plus@, Dupont, NEN Products, Boston, U.S.A.). The hybridization conditions were identical to those previously reported. l 4

Control D N A DNA from cell lines was used as controls to determine the specificity of the HPV 16 and HPV 18 probes. They were (i) CaSki (400-500 copies of HPV

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P. DICKENS ET A L .

Table I-Sequences

of oligonucleotide primers used for the amplification of HPV 16 DNA

Size of amplified product (base pairs)

Sequence (5’ > 3‘) A: AAG GCC AAC TAA ATG TCA C (19-mers) B : GCG GAT CCT GTC TGC TTT TAT ACT AA (26-mers)

16 per cell), (ii) SiHa (1-2 copies of HPV 16 per cell), and (iii) HeLa (10-50 copies of HPV 18 per cell). Hence, HeLa was used as a positive control for HPV 18 but as a negative control for HPV 16 and so on. Since no cell lines are known to contain HPV 6 or HPV 1 1 DNA, no positive cell-line control could be used. CaSki, SiHa, and HeLa cell lines were obtained from American Type Culture Collection, U.S.A. High molecular weight DNA was extractedt4and the results of probing of the slot-blotted DNA showed that the HPV 16 probe specifically hybridized to CaSki (high positive) and SiHa (low positive); and the HPV 18 probe hybridized to HeLa. The hybridization conditions could detect signals for HPV 16 from 1 ng of CaSki genomic DNA, 10 ng of SiHa DNA, and for HPV 18 from 10 ng of HeLa DNA. Polymerase chain reaction HPV sequences were amplified by the PCR using the oligonucleotide primers specific for HPV 16 which were complementary to the upstream regulatory region” as shown in Table I. This region was chosen because it is almost always retained even in the HPV DNA integrated in tumours. The oligonucleotide primers were commercially synthesized by Bresatec Pty Ltd. (Australia). For the three selected cases, one to ten 10pm thick sections were cut from the paraffin-embedded tissues, deparaffinized, and amplification of HPV 16 DNA sequence was carried out” using a DNA amplification kit (Perkin Elmer Cetus). The amplified products were analysed by gel electrophoresis using 4 per cent agarose, followed by ethidium bromide staining. The specificity of the amplified product was confirmed by Southern blotting and hybridization to 32P-labelled HPV 16 genomic probe using conditions identical to those reported p r e v i ~ u s l y . ’ ~ RESULTS The resultsofslot-blot hybridizationare tabulated in Table 11. In the 27 cases analysed, HPV 11

228

predominated, being detected in 16 (59 per cent). In contrast, only three cases (1 1 per cent) showed HPV 6 and in these same three cases HPV 1 1 and 16 were also detected. HPV 18 was not detected in any of the cases. Following slot-blot hybridization, the three cases showing positivity for HPV 16 were selected for the PCR to further substantiate that HPV 16 was present. In two of these cases (Table 11, cases 1 1 and 12), there was insufficient residual DNA for the PCR but ample for case 13. The ethidium bromidestained electrophoretic gel following PCR on the residual DNA from this case clearly indicated a band at the expected position for HPV 16 DNA. To confirm further this finding for case 13, and also for cases 1 1 and 12, one section for case 13 and five to ten sections for cases 11 and 12 (because of the very small amount of residual tissue) were cut from the remaining tissue in the paraffin blocks and the PCR was performed. Although no distinct bands of expected size of the PCR products were visible after ethidium bromide staining of the gel in the three cases, strong positive hybridization signals were obtained for all three after probing for HPV 16 DNA, thus confirming the presence of HPV 16 genome in these cases. DISCUSSION The marked predominance of HPV 11 (16/27 cases) in the laryngeal papillomas that we have studied is at variance with previous studies which have shown either an almost equal distribution of HPV 6 and 1 12.’ or a predominance of HPV 6.’,16We have only three cases positive for HPV 6. However, all these three cases are also positive for HPV 1 1 and thus we cannot rule out the possibility of crosshybridization between types 6 and 11. In this study, there are two few juvenile papillomas to draw any conclusions concerning different patterns of infection between juveniles and adults, but the overall infection pattern may indicate a difference in the Hong Kong Chinese population. This report is also

245

H P V 16 IN LARYNGEAL PAPILLOMAS

Table 11-Results Case No.

Age (years)

I 2 3 4 5

8 28 32 4

6

26 20 64 21 89 53 8 55 30 11 8 32 61 31 13 59 50 61 63 55 5 60

I 8 9 10 11

12 13 14 15 16 17 18

19 20 21 22 23 24 25 26 21

1

of DNA slot-blot hybridization

Sex

M M F F M M M M M F F M F M M M M M M M M M F M M

F F

Path. diagnosis

6

HPV type 11 16

18

Juvenile papillomatosis Single papilloma Single papilloma Juvenile papillomatosis Juvenile papillomatosis Single papilloma Single papilloma Single papilloma Single papilloma Single papilloma Single papilloma Single papilloma Multiple papillomas Multiple papillomas Single papilloma Juvenile papillomatosis Single papilloma Single papilloma Single papilloma Juvenile papillomatosis Single papilloma Single papilloma Single papilloma Single papilloma Single papilloma Juvenile papillomatosis Single papilloma

the first record of HPV 16 detection in laryngeal papillomas. Interestingly, these three HPV 16positive cases are also positive for HPV 6/11. Other w ~ r k e r s , ~ - ~using . ' ~ ~ ' 'various molecular biology methods including PCR16 have demonstrated only types 6/11 in juvenile or adult laryngeal papillomas. Moreover, two recent r e ~ i e w s ' ' ~do ' ~ not include laryngeal papilloma as being associated with HPV 16. However, HPV and a related type 3OZ5 have been demonstrated in laryngeal squamous carcinomas. The significance of the finding of HPV 16 in our series of benign laryngeal lesions is not clear. The number of such cases in this study is small and therefore warrants the analysis of more cases using fresh biopsy material and a wider range of HPV type-specific PCR primers to determine the relative incidence of HPV subtypes in these papillomas. Histological examination of the three HPV 16-positive papillomas showed no evidence of dysplasia or malignancy, only focal koilocytosis.

However, in the light of the association of HPV 16 with squamous carcinomas of the larynx and cervix,26we suggest close follow-up of such patients, although the finding of HPV 16 in the normal cervix by Southern blotting27and PCR'5,2'-30has cast some doubt on its oncogenic role. There is little information available concerning the frequency of latent HPV infection in the normal larynx and it would be interesting to study this in order to define more clearly the role of HPV in the pathogenesis of benign and malignant laryngeal neoplasms. ACKNOWLEDGEMENTS

We wish to thank Professor H. zur Hausen and D r L. Gissmann for the HPV-6/11/16/18 plasmid DNA. This research project was supported by a grant from the Department ofPathology, University of Hong Kong and from UPGC (Hong Kong) grant No. 337/046/0010.

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P. D I C K E N S E T A L .

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