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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 93, Num. 4, 1998, pp. 439-440
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 93 (4), July/August 1998,
pp. 439-440
RESEARCH NOTE
Detection of Type 16 Human Papillomavirus DNA in Formalin-fixed Invasive
Squamous Cells from Laryngeal Cancers by Polymerase Chain Reaction
Yudira Soto^+, Odalys Valdes, Mayra Mune, Tamara Pimentel, Rosa Ramirez
Instituto de Medicina Tropical Pedro Kouri, Autopista Novia del Mediodia Km
6, Apartado Postal 601, Marianao 13, Ciudad de la Habana, Cuba
^+Corresponding author: Fax +53-7-336051
Received 9 July 1997; Accepted 29 April 1998
Code Number:OC98086
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Key words: human papillomavirus - laryngeal cancer - polymerase
chain reaction - Cuba
RESEARCH NOTE
To date approximately 77 distinct human papillomavirus (HPV) genotypes have
been described and the genomic sequences of most of them have been fully
analyzed. About 30 additional partial sequences have been obtained from
putative novel HPV types, suggesting that the total number of existing HPV
genotypes is in excess of 100 (H zur Hausen 1996 Biochim Biophis
Acta 1288: 55-78). HPV cause benign tumors in the respiratory
tract. Mounting evidence suggests that they also play a role in the
etiology of a subset of head and neck cancers. Carcinomas in patients with
a history of recurrent respiratory papillomatosis are caused clearly by
persistent HPV interacting with one or more carcinogenic agents (BM
Steinberg et al. 1996 Cancer and Metastasis Reviews 15: 91-112).
Previous studies have suggested squamous neoplasia of the larynx and may
also be HPV related (HAB Multhaupt et al. 1994 Hum Pathol 12:
1302-1305).
Current research also indicates that HPV may be involved in the development
of benign tumors and also squamous cell cancers of head and neck (O Arndt
1994 Laryngo-Rhino-Otol 73: 527-532). The first reports of the
presence of HPV in malignant tumors of the oral cavity appeared in 1985 (EM
de Villiers et al. 1985 Int J Cancer 36: 575-578). They were
followed by similar reports on HPV DNA in individual cases of cancer of the
larynx. Although subsequently, by using polymerase chain reaction (PCR)
analysis, the HPV detection data varied between 0 and 100% (zur Hausen
loc. cit.).
Studies performed with reliable techniques commonly came up with positivity
rates of between 10 to 20%. In these studies HPV16 was the most frequently
encountered genotype (PJF Snijders et al. 1994 J Gen Virol 75:
2769-2775). Regularly HPV16 DNA has been found in invasive squamous cell
cancers of the larynx in a relative high percentage (Arndt loc.
cit.).
In the present study the presence of HPV16 in 40 formalin-fixed and
paraffin-embedded tissues from laryngeal cancers, 20 glottic and 20
supraglottic, kindly supplied by The National Institute of Oncology and
Radiobiology in Havana City, was investigated. The studied samples were
selected from a group of patients aging from 34 to 58 years old who had
presented a history of aggressive carcinoma of the larynx. All material was
routinely fixed in 10% neutral-buffered formalin and paraffin-embedded.
Sections were mounted on glass slides for histological diagnosis (Multhaupt
loc. cit.) and the original histological diagnosis was confirmed.
DNA extraction from paraffin-embedded sections of laryngeal biopsies was
performed by treatment of the sections (4-5 mm) with xylol and four cycles
of ethanolic hydratation. After digestion with proteinase K solution and
protein extraction with phenol choroform, DNA was precipitated (DK Shibata
1988 J Exp Med 167: 225-230).
HPV16 DNA was detected using a Nested PCR. PCR was performed using two
specific modified primers to E6 region of HPV16. They are synthetic
oligonucleotides, one of which is biotinylated while the second one is
labeled with DNP hapten. Briefly, targed DNA in the sample was denatured
and underwent a first amplification step with two specific non-modified
primers. The product of this first amplification was diluted. Then a second
amplification was carried on using the two specific modified primers. The
product of this second amplification was immobilized on an Avidin coated
tube. After fixation DNA duplexes were revealed by use of an anti-DNP
monoclonal antibody conjugated to peroxidase (Amplicis HPV primers kit, Cis
bio International, France).
In this study 20 patients (50%) with laryngeal cancer were HPV16 DNA
positive. One retrospective study reviewed 36 tumor specimens, using PCR
amplification of paraffin sections followed by southern blot hybridization,
to test for the presence of HPV16 DNA. These workers found HPV16 in tumors
from 45% of the cases (DM Fliss et al. 1994 Laryngoscope 104:
146-152). A number of studies have shown that laryngeal carcinomas do
contain HPV16 DNA. The majority of them only examined a very small number
of cases but 75% to 100% of the lesions studied were positive for HPV16 or
HPV16 related DNA (Steinberg loc. cit.).
In the present study significantly more glottic cancers 14 of 20 (70%),
were positive to HPV16 than supraglottic carcinomas because only 6 of 20
(30%) were positive to HPV16. Arndt (loc. cit.) studied a total of
100 formalin-fixed and paraffin-embedded laryngeal cancer specimens, 41
glottic and 59 supraglottic by use of E6 specific PCR for HPV16 DNA.
They detected HPV16 in 26 of 41 glottic cancers (63.4%) but only 10.2% (6
of 59) of the supraglottic carcinomas. These results are comparable to ours
because we found HPV16 in 14 of 20 (70%) glottic cancers while only 6 of 20
(30%) of supraglottic cancers were positive for HPV16. There is a
predominance of HPV16 DNA in supraglottic carcinomas but this could be
explained because the level of some risk factors like nicotine and alcohol
was significantly higher in the patients studied with supraglottic tumors.
These patients had presented a history of alcohol consumption for more than
five years and 100% were smokers since they were teenagers. The presence of
both carcinogenic agents and HPV16 infection could be an important means by
which to analyze the pathogenesis of laryngeal cancers.
Copyright 1998 Fundacao Oswaldo Cruz - Fiocruz
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