Laryngeal cancer and human papillomavirus: HPV is absent in the majority of laryngeal carcinomas

Cancer Letters 146 (1999) 9±13 www.elsevier.com/locate/canlet

Laryngeal cancer and human papillomavirus: HPV is absent in the majority of laryngeal carcinomas Henning Lindeberg a,*, Annelise Krogdahl b a

Department of Maxillo-Facial Surgery and Oral Pathology, Royal Dental College, Aarhus University, DK-800 Aarhus C, Denmark b Institute of Pathology, Aalborg Hospital, Aalborg, Denmark Received 30 March 1999; accepted 21 May 1999

Abstract Thirty laryngeal carcinomas from patients without pre-existing laryngeal papillomatosis were examined by PCR for the presence of HPV DNA. The utmost care was taken during sectioning of the tissue blocks and DNA-extraction in order to avoid false positive results. Three pairs of consensus primers were used: MY9/MY11, GP51/GP61 and CPI/CPII. HPV was detected in 1/30 carcinomas. The HPV type present could not be determined, but it was not type 6, 11, 13, 16, 18, 30, 31, 33, 35 or 45. In other studies the reported frequency of HPV in laryngeal carcinomas, as estimated by PCR, varies between 3±85%. The reasons for this unacceptable variation in reported results are discussed. The present results indicate that HPV DNA does not have a major role in malignant tumours of the larynx in patients without pre-existing recurrent laryngeal papillomatosis. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HPV; Larynx; Cancer

1. Introduction Almost all epithelial cancers of the uterine cervix and their precursors harbour HPV DNA, mainly types 16 and 18, which are considered high risk HPV types, or types 31, 33, 35, 45 and others, which are considered medium risk HPV types. While the relationship between anogenital cancer and HPV has been clearly demonstrated in numerous studies, a putative relationship between HPV and head and neck carcinomas is still controversial. In 1986 HPV type 30 was cloned from a laryngeal carcinoma [1], and HPV DNA has been demonstrated in carcinomas arising from preexisting laryngeal papillomas [2±4]. However, regard-

ing the majority of laryngeal carcinomas con¯icting evidence have accumulated, demonstrating HPV (mainly type 16) in 3±85% of the tumours, estimated by PCR. Thus, there is an unacceptable variation in different reports on the frequency of HPV in laryngeal carcinomas. Moreover, the PCR methods employed would have overlooked HPV type 30 if present. In an attempt to circumvent these problems we recently examined laryngeal precancers and demonstrated HPV DNA in only 1/30 [5]. However, Gorgoulis [6] reported HPV DNA in 7/40 (28%) laryngeal SCC and in 0/28 dysplastic laryngeal lesions, using type speci®c primers targeting the E6 ORF of HPV types 6,11,16 and 18. This prompted us to extend our study to include invasive carcinomas.

* Corresponding author. Fax 145-8619-5559. E-mail address: [email protected] (H. Lindeberg) 0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00210-4

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Table 1 PCR-conditions for the different HPV consensus primers a Primers

PCR-conditions

Cycling conditions

GP51/GP61

50 mM KCl, 10 mM Tris pH 8.5 3.5 mM MgCl2, 200 mM of each dNTP, 10 pmol of each primer, 1.25 U/50 ml Taq2000 (Stratagene w) As above, but with 2.5 mM MgC12 As above, but with 3.0 mM MgCl2

948C, 1 min; 408C, 2 min; 728C, 1.5 min

MY9/MY11 CPI/CPII

the GP primers will amplify HPV type 30 (Tables 1 and 2). The ampli®ed samples were evaluated by gelelectrophoresis and photographed under UV transillumination. The GP51/GP61 ampli®ed samples were further analysed by dot-blot hybridisation with DIGtailed oligoprobes for HPV types 6/11, 16, 18, 30 31, 33 and 35 [12]. The sequence of the HPV 30 probe was 5 0 CACAAACGTTATCCACATATAATTCAAGCC. 3. Results

948C, 1 min; 558C, 1 min; 728C, 1.5 min 948C, 1 min; 558C, 1 min; 728C, 1 min

a

PCR was performed in a total volume of 50 ml. Five microlitres of the sample DNA solution was used in each reaction. Number of cycles were 40. Cycling was preceded by an initial denaturation step (958C, 3 min). Before cooling, an elongation step was performed (728C, 7 min).

2. Methods Tissue blocks from 30 patients (23 males, 7 females, age 47±86 years, mean age 64 years) with laryngeal carcinomas were selected. Blocks with sparse malignant tissue were omitted, but in all other aspect the tumours represent a consecutive series. Three sections from each block were cut for PCR. Control sections before and after sectioning for PCR were cut and stained with H&E in order to ensure that the sections for PCR did contain tumour tissue. The sections for PCR were placed in a 1.5 ml tube and 200 ml digestion buffer (10 mM Tris pH7.0, 1 mM EDTA, proteinase K 200 mg/ml) was added. The sections were digested at 658C for 3 h, spinned, and the aqueous phase transferred to a new tube. Proteinase K was inactivated by boiling for 10 min. The samples, together with appropriate positive and negative controls, were ampli®ed with primers targeting a 288 bp fragment of the single copy betaglobin gene in order to ensure the integrity of the DNA [7]. Finally, PCR was performed with the universally accepted consensus primers GP51/ GP61 [8], CPI/CPII [9,10] and MY9/MY11 [11], respectively. These primers will amplify a great number of the known mucosatropic HPV types, and

Of the 30 carcinomas 15 were located in the glottic and 15 were located in the supraglottic areas. Histological examination revealed that eight tumours were squamous carcinomas grade I, eight tumours were classi®ed as grade II, and 13 were grade III. The remaining tumour was an undifferentiated carcinoma. The betaglobin fragment was ampli®ed from all blocks, indicating the presence of ampli®able DNA in all samples. HPV was not detected by PCR with GP51/GP61 primers and subsequent dot blot hybridisation, and PCR with MY9/MY11-primers also failed to demonstrate any HPV DNA. All positive controls worked as expected. With the CPI/CPII primers one sample showed a band of the expected Table 2 HPV-types which can be ampli®ed by the three different consensus primers. The table is based on published results and on analysis of the reported HPV frequencies Primers

Target

HPV types

GP51/GP61

L1, , 145 bp

MY9/MY11

L1, , 450 bp

CPI/CPII

El, , 188 bp

6, 7, 10, 11, 13, 14D, 16, 18, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 54, 55, 56, 58, 59, 61, 62, 66, 72, 73 2A, 6, 7, 8, 9, 10, 11, 13, 15, 16, 17, 18, 21, 22, 23, 24, 25, 28, 29, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 45, 48, 49, 50, 5 1, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 70, 72, 73 2A, 4, 5, 6, 7, 8, l0, 11, 16, 17, 18, 21, 24, 31, 33, 34, 35, 36, 44, 45, 47, 51, 52, 53, 54, 55, 56, 58, 59, 66, 68

H. Lindeberg, A. Krogdahl / Cancer Letters 146 (1999) 9±13

size (188 bp), indicating the presence of HPV in 1/30 samples. Restriction fragment length polymorphism of the ampli®ed DNA fragment with the restriction enzymes HaeIII, PstII and RsaI was performed [13], but the DNA fragment did not contain restriction sites for these enzymes. 4. Discussion In this study HPV was demonstrated in only one out of 30 carcinomas. The actual HPV type found could not be determined, but the presence of a band of the expected size (188 bp) and the absence of other bands strongly indicates that HPV of an undetermined type was present. HPV type 16, which is by far the most common reported HPV type in these lesions, was not detected at all, and neither was HPV type 30, con®rming the results of Kahn et al. (1986) [1], which were based on the most sensitive technique of that time, Southern blotting. A number of authors have addressed the question of HPV and laryngeal carcinomas by PCR with various

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primers (Table 3). The range of reported frequency of HPV in these tumours is 3±85%, which is unacceptable and cannot be explained by the primers used ± most authors used type speci®c primers and/or MY9/MY11 consensus primers. Brandwein [14] discussed the reasons for the incredible range in the published results and concluded that it could only be explained by a high frequency of false positive results in many of these reports, and we support her view. False positive results can originate from dif®culties in interpretation of the results of ®lter hybridisation or ± more likely ± from true HPV-negative samples, which have been contaminated with HPV DNA during collection of samples, during sectioning of paraf®n embedded tissue blocks, during DNA-extraction or when adding DNA-samples to PCR buffers, etc. The contaminating DNA may originate from other HPVpositive samples, from positive control samples or from HPV genomic probes used for in situ hybridisation in the same laboratory. Carry-over of ampli®ed DNA from samples processed previously is another well-known possibility. Contamination is likely to occur if the different steps in the process of preparing

Table 3 The reported frequencies of HPV DNA in laryngeal carcinomas as determined by PCR Author

Year

Primers

No. cases

No. HPV positive

% HPV positive

Kiyabu et al. [15] Hoshikawa et al. [16] Perez-Ayala et al. [15] Watts et al. [18] Morgan et al. [19] Ogura et al. [20] Kasperbauer et al. [21] Tyan et al. [22] Anwar et al. [23] Brandwein et al. [14] Gorgoulis et al.[6] Fliss et al. a [24] Clayman et al. b [25] Arndt et al. [26] Salam et al. [27] Shen et al. [2] Almadori et al. [28] Lie et al. [29]

1989 1990 1990 1991 1991 1991 1993 1993 1993 1993 1994 1994 1994 1994 1995 1996 1996 1996

10 34 48 4 16 28 20 10 22 40 40 29 65 100 36 30 45 39

4 7 26 3 7 3 17 2 16 3 7 13 30 32 8 1c 9 3

40 21 54 75 75 11 85 20 72 8 18 45 46 32 22 3.3 20 8

Paz et al.[30]

1997

16,18 6,16 11,16 6,11,16,18 6,11,16,18,33 16,18 MY9/MY11 6,11,16,18,33 ? MY9/MY11 6,11,16,18 6,11,16,18 MY9/MY11 6,11,16,18 MY9/MY11 MY9/MY11,16,18 (E7) 6,11,16,18 GP51/GP61, MY9/ MY11, CPI/CPII MY9/MY11, IU/IWDO

28

1

4

a

Verrucous carcinomas. Laryngeal and hypopharyngeal carcinomas. c Two cases with a history of laryngeal papillomatosis has been excluded. b

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material for PCR are not strictly separated by using three to ®ve different rooms in the laboratory, each of which is used solely for its speci®c purpose. In addition, each of the rooms which makes up the PCR-lab should be equipped with special ®lter-tipped or positive displacement pipettes, and pipettes should never leave the individual rooms. Such precautions are mandatory in a diagnostic PCR laboratory ± due to the extreme sensitivity of PCR minute amounts of contaminating HPV DNA may also be ampli®ed, changing true negative samples into false positive ones. There is no doubt that HPV is present in at least some laryngeal carcinomas, but in our opinion it is not a common event. Based on the present study as well as our previous study on laryngeal precancer [5], which was examined by three different pairs of universally accepted consensus primers, we feel con®dent to claim that laryngeal carcinomas are certainly not related to HPV in the same way that certain HPV types and anogenital cancers are related. This, however, does not exclude the possibility of a causal relation in a minority of the tumours. Recently, Lie [29] examined frozen samples from 39 patients with laryngeal carcinomas, using the same primers (although with somewhat different cycling parameters) and found three samples (8%) to contain HPV, supporting our ®ndings of low prevalence of HPV in laryngeal carcinomas. In addition, a low prevalence has recently been published by Shen [17,25] and by Paz [21] as well. The ultimate conclusions are that reports of the frequency of HPV in head and neck lesions, determined by PCR-based methods, should be interpreted with great care, and that HPV is not a general cause for laryngeal carcinomas.

Acknowledgements This study was supported by a grant from The Obel Family Foundation.

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