Human papillomavirus infections in the respiratory tract

Human Papillomavirus Infections in the Respiratory Tract FUJU CHANG, MD, PHD, LIJUAN WANG, BA, STINA SYRJANEN, AND KARI SYRJANEN, MD, PHD

The papillomaviruses are small DNA viruses that belong to the family Papovaviridae. They are ubiquitous and affect both man and animals, including dogs, cats, cattle, sheep, rabbits, and other species.le4 The virion consists of a central core of circular, doublestranded DNA with a nucleotide length of about 7.9 kb, enclosed within an outer capsid of viral proteins.le3 The viral capsid is composed of 72 subunits (capsomeres) arranged in a symmetrical, 20-sided (icosahedral) pattern that gives the individual virion an almost spherical shape on electron microscopy. Although the papillomaviruses share groupspecific antigens, they can be classified according to their host range and the degree of homology of their nucleic acids. Any new isolate that exhibits less than 50% homology (tested by reassociation in the liquid phase] with existing members is designated as a new type and numbered in order of discovery. If the homology is higher than 5O%, they are considered subtypes.le3 So far, more than 66 types of human papillomaviruses (HPV) have been identified,5 and the number is likely to increase in the future. Papillomaviruses are epitheliotrophic and primarily affect the skin and mucous membranes of the genitourinary and upper aerodigestive tracts, inducing hyperplastic, papilloFrom the Department of Pathology, Kuopio Cancer Research Centre, University of Kuopio, Kuopio, Finland; Department of Precancerous Studies, Henan Medical University, Zhengzhou, Henan, China; A.I. Virtanen Institute, Kuopio, Finland. Original studies included in this review have been supported in part by research grants from the International Union Against Cancer (UICC) (for FC) and from the Finnish Cancer Society, a research contract (Grant No. 1041051) from the Medical Research Council of the Academy of Finland, and a joint research grant from the British American Tobacco Company and Fabrique de Tabac Reunie. Address reorint requests to Fuiu Chano, MD, PhD, Department of Pathology, University of Kuopio, POB 1627, SF-7021 1 Kuopio, Finland. Copyright 0 1992 by W.B. Saunders Company 0196-0709/92/l 304-0003$5.00/O 210

American

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of Otolaryngology,

DDS, PHD,

matous, and verrucous lesions of the squamous epithelium.4~“-8 HPV infections have been reported in a number of body sites, including the anogenital tract, urethra, skin, larynx, tracheobronchial mucosa, nasal cavity/ paranasal sinuses, oral cavity, esophagus, and conjunctiva.6-25 HPV involvement in a variety of benign lesions of the aerodigestive tract, including those of the oral mucosa (squamous cell papilloma/condyloma, verruca, focal epithelial hyperplasia, lichen planus, and leukoplakia) as well as inverted papilloma of the nasal cavity and paranasal sinuses, and respiratory papillomatosis (eg, laryngeal papillomas), has been demonstrated by histopathological, ultrastructural, immunohistochemical, and DNA hybridization studies.7-25 Of the known HPV types, HPV 1,2,4,6, 7,11,13,16, 18, 30, 32, 40, and 57 have been detected in these lesions.7-25 Of these, HPV 11 was originally cloned from a recurrent lesion of laryngeal papillomatosis,21 HPV 13 and 32 from focal epithelial hyperplasia of the oral cavity, 22,23 HPV 30 from a laryngeal carcinoma,z4 and HPV 57 from an inverted papilloma of the maxillary sinus.25 In addition to these benign squamous cell lesions, strong evidence has also been accumulated in the past few years implicating an etiological role for specific genotypes of HPV (eg, HPV 16, 18, 31, and 33) in the development of squamous precancer lesions and carcinomas, including those of the anogenital tract, those associated with epidermodysplasia verruciformis (EV) lesions and warts in immunocompromised patients, verrucous carcinomas, and Bowen’s disease, as well as carcinomas arising in the upper aerodigestive tract 6-926-33 The evidence on HPV involvement in neoplasia of the oral cavity and esophagus has been reviewed recently.11*17*1g.34 This article summarizes the current evidence on HPV infections in the respiratory tract with special attention to the possible transmission routes

Vol 13, No 4 (July-August),

1992: pp 210-225

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of HPV as well as the pathogenesis of a variety of HPV-induced squamous cell lesions in the airways.

HPV INFECTIONS IN THE NASAL CAVITY AND PARANASAL SINUSES The mucous membrane of the nasal cavity and paranasal sinuses, also known as the schneiderian membrane, is thought to be formed from the neuroectoderm of the olfactory placode.35 Inverted papilloma, emerging from the schneiderian membrane, is an uncommon benign tumor of the nasal cavities and the paranasal sinuses.35-43 The frequency of this lesion has been calculated as 1 in 25 to 1 in 50 of that reported for the common inflammatory p01yps.~’ In general hospitals the incidence varies from 0.4% to 4.7% of all tumors of the nasal cavities.3g.40 Clinically, nasal-inverted papilloma has been observed in patients of almost all age groups. However, the lesion is much more common in patients in their fifth to seventh decades.35-43 In almost all series reported, males are affected more commonly than women, the sex ratio ranging from 3:l to 10:1.35m43 More than 20 different names have been used to describe this lesion, including schneiderian papilloma, inverted papilloma, inverting papilloma, inverted schneiderian papilloma, transitional papilloma, transitional cell papilloma, cylindrical cell papilloma, Ewing’s papilloma, papillary sinusitis, polyp with inverting metaplasia, squamous cell papilloma, squamous papillary epithelioma, benign transitional cell growth, epithelial papilloma, soft papilloma, papillary fibroma, papillomatosis, villiform cancer, and cylindrical cell carcinoma.35-43 Today, the three most generally accepted names to describe this entity are the inverted papilloma, transitional papilloma, and schneiderian papilloma. On gross appearance, these lesions tend to be firm, polypoid, quite bulky, and more vascular than the inflammatory polyps. These tumors form frond-like extensions that are usually pale pink or grey in color, but in some areas may be red.35-43 They often fill the entire nasal cavity, extending from the nares to the nasopharynx. Histologically, the most characteristic feature of this tumor is penetration of the neoplastic epithelium into the underlying

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stroma, rather than proliferation outwards. Thus, in 1938 Ringertz35 coined the term inverted papilloma. The neoplastic epithelium may be of respiratory, transitional, or squamous type, with orderly maturation and minimal mitosis and atypia generally present. Microcysts containing mucin are occasionally trapped beneath the surface, and a basement membrane is present that separates the inverted epithelium from the underlying stroma. The normal respiratory epithelium undergoes metaplastic transformation from pseudostratified ciliated columnar epithelium to stratified columnar or squamous epithelium. Cellular atypia and mitoses are frequently observed, sometimes concomitantly with foci of invasive squamous cell carcinoma.35-48. Despite its rarity, inverted papilloma of the nasal cavity and paranasal sinuses has recently attracted increasing attention. This interest has been prompted by the observations on its tendency for multiple recurrences and the frequent association with malignant tumors, especially squamous cell carcinoma. Recurrence rates from 28% to 67% have been reported in larger series,4”s47 even after adequate therapy, and, more importantly, the rates of malignant conversion range from 3% to 32%.47f48 The cause of inverted papillomas is still not clear. Possible factors include allergic nasal polyposis, chronic inflammatory reaction, viral infection, as well as environmental toxins.35-40*45-4g One of the most popular theories is that HPVs are involved in the development of these tumors. The clinical behavior of the nasal inverted papillomas, particularly frequent recurrence and infrequent progression to malignancy, are similar to those documented for the recurrent respiratory papillomatosis in the larynx and suggest an infectious (HPV) cause for these lesions. As early as in 1970, Kusiak and Hudson” reported 40 to 50 nm intranuclear bodies on electron micrographs of 2/4inverted papillomas. The hypothesis on HPV involvement was first suggested by histopathological observations demonstrating HPVsuggestive changes, eg, papillomatosis, koilocytosis, or binucleation or multinucleation, and dyskeratosis in nasal inverted papillomas.51‘53 Syrjanen et al (1983)51 examined one such papilloma (with extensive squamous cell

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component) of a paranasal sinus and found HPV capsid antigen in the nuclei of a few koilocytotic cells and dyskeratotic cells. Later, the same group demonstrated HPV-structural antigens in 50% (7/14) of such lesions.52 Convincing evidence for an HPV cause of inverted papilloma comes from demonstration of HPV DNA sequences in a high percentage of such lesions.4g,52*53 Using DNA in situ hybridization technique, Syrjanen et a152 detected HPV DNA sequences in 10 of 14 cases (71.4%); HPV type 11 alone was found in 5 cases, HPV type 16 in 5 cases, and HPV types 11 and 16 together in 3 cases. Similarly, Weber et a153 identified HPV DNA in 76% (16/21) of inverted papillomas using hybridization probes for HPV 6b and HPV 11 under stringent conditions. These findings were further confirmed by other investigators, who isolated and characterized HPV DNA sequences from papilloma lesions. Respler et al (1987)54 and Pater et al (1988)55 isolated and cloned a papillomavirus from a nasal-inverted papilloma. Restriction mapping and DNA sequencing showed that the genome of this virus was closely related to HPV 11. Later, de Villiers et al in 198gz5 cloned another HPV, tentatively designated as HPV 57, from an inverted papilloma of the maxillary sinus. After sequencing the viral genome, an 83% nucleotide homology to HPV 2 DNA was identified. So far, HPV types 2, 6, 11, 16, 18, and 57 DNA sequences have been detected in nasal-inverted papillomas, of which HPV 11 DNA seems to be the most prevalent (57°?&760h).4g,52-61 In addition to benign lesions of the nasal cavity and paranasal sinuses, HPV DNA sequences have been also detected in lesions associated with malignant conversion.4gv52*58*60.“1 by in situ hySyrjanen et al 52 demonstrated bridization the presence of HPV 16 in two inverted papillomas that were associated with invasive cancer. Similarly, Brandwein et a15’ found HPV 16/18 in two cases of inverted papillomas, one with mild dysplasia and one associated with invasive squamous cell carcinoma. So far, HPV types 11, 16, and 18 DNA sequences have been detected in nasal precancer lesions and cancer, HPV 16 being most frequently found in papillomas associated with malignancies.4g~52~53.60.61 Malignant transformation of inverted papillomas seems to be most consistently associ-

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ated with the following features: bilateral papilloma, a predominance of mature squamous epithelium, the presence of all three epithelial types (metaplastic squamous, mature squamous, and cylindrical), severe hyperkeratosis, a mitotic index ~2 per high-power field, absence of concomitant inflammatory polyps, an abundance of plasma cells, and an absence of neutrophils.45 In a flow cytometric analysis, Klemi et a16’ found that the aneuploid lesions associated with HPV 16 infection were shown to be more closely associated with malignant transformation. According to the current concepts of HPV-mediated oncogenesis,26-33 it may be postulated that HPV infection present in the nasal cavity and paranasal sinuses induces cell immortalization with restricted noninvasive epithelial proliferation, and that subsequent malignant transformation depends on other cofactors. These factors may include chemical carcinogens, genetic predisposition, or infection with other oncogenic viruses (Epstein-Barr virus [EBV], adenovirus).

HPV INFECTIONS IN THE PHARYNX Inverted papillomas, morphologically resembling those arising in the nasal cavity and paranasal sinuses, can be occasionally identified at other sites such as the nasolacrimal duct,62 nasal septum,63 nasopharynx,“4s”5 oropharynx,66 and hypopharynx,67 either as a result of widespread growth of nasal papillomas or occurring independently as solitary lesions. Furthermore, HPV-induced respiratory papillomatosis occur mainly in the larynx, as will be discussed later. However, these papillomas may also extend upwards to epiglottis, pharyngeal walls, and even to soft palate. Undoubtedly, the established HPV etiology of the nasal-inverted papillomas and laryngeal papillomatosis may also be applicable to these similar lesions arising in the pharynx. Recently, Debiec-Rychter et a1”8 established two immortalized cell lines (NW-l and NPCN) by in vitro transfection of human nasal and nasopharyngeal epithelial cells with HPV 16. In situ hybridization with an HPV 16 DNA probe demonstrated that the virus was integrated in the host cell genome in both cell lines. Like the HPV-immortalized keratinocytes of other body sites, NW-l and NPC-N

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did not show tumorigenicity in nude mice, although both cell lines grow indefinitely in methylcellulose in vitro. These results indicate that pharyngeal epithelial cells may also be suitable targets for HPV infection, and like other sites of the respiratory mucosa, HPV infection may be also involved in pharyngeal carcinogenesis. This hypothesis was further supported by the recent discovery of HPV 16 and 18 DNA sequences in 12.5% (l/8) and 18% (2/11) of pharyngeal squamous cell carcinomas.6g,70 It is well known that nasopharyngeal carcinomas are associated with EBV infection. Over 90% of anaplastic nasopharyngeal carcinomas show evidence of EBV infection.32 Noteworthy was the recent demonstration of HPV 6 and/or 11 DNA sequences in 9 of 14 (64.3%) histologically normal nasopharyngeal mucosa.71 The role of HPV infection and the possible synergistic action between HPV and EBV in nasopharyngeal carcinogenesis, however, await further investigation. HPV INFECTIONS Laryngeal

IN THE LARYNX

Papilloma

Laryngeal papillomas are benign epithelial neoplasms consisting of a central fibrous connective tissue core covered by squamous epithelium. The true cords of the larynx are the primary site most often affected. Lesions may also occur in the nasal vestibule, nasopharynx, all sites within the larynx, trachea (particularly at the site of prior tracheostomy), and in the lower bronchial tree.72-75 Clinically, laryngeal papillomas can produce serious clinical problems because of their potential for airway obstruction, frequent recurrences, and tendency to spread throughout the respiratory tract. Increased risk of malignancy has been reported as we11.73,75 Laryngeal papillomas show a biphasic age distribution, occurring in both young children and adults, and therefore being distinguished as juvenileand adult-onset types.73-75 According to their clinical presentation, eg, male to female ratio and multiple versus solitary papillomas, the papillomas can be further classified into four groups: (1) multiple juvenile papillomas, (2) solitary juvenile papillo-

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mas, (3) multiple, and (4) solitary adult papillomas.74 Children under 5 years of age are at the highest risk of developing this disease. In a series of 76 children, 21 (28%) presented between birth and 6 months of age and 43 (57%) appeared before 2 years of age.75 Many patients with the juvenile-onset form continue to have the disease throughout their life, although spontaneous regression of papillomatosis has been described at puberty. The age distribution of the adult-onset papillomas usually shows the peak between the ages 20 and 40 years in most studies. Males are affected more frequently than females, accounting for 55% to 60% of cases.73’74 Viral origin has been suspected for decades. As early as 1923, Ullman76 reported that homogenized laryngeal papilloma tissue from a 6-year-old boy injected into his own arm produced wart-like lesions 90 days later. Cell-free extracts from the same tissue inoculated in his arm and in an assistant’s arm yielded similar lesions after the same delay and then successfully implanted this in canine vaginal mucosa. This study was duplicated by Ishikawa in 1936,77 but many others have failed to reproduce it.78 Resler and Snow7’ were able to transplant the growths via a cell-free filtrate from human laryngeal papillomas to dog vaginal mucosa and skin but not to the larynx or oral mucosa. Several investigators have been successful in the unequivocal demonstration of virus particles in these lesions.80-83 For examples, Dmochowski et ala0 found papillomavirus particles in 5 of 6 cases, Boyle et al” in 2 of 27 biopsies, Spoendlin and Kistler8’ in 1 of 12 biopsies, and Incze et a183 in 3 of 17 biopsies. These results were confirmed by immunohistochemical studies. By using HPVcommon capsid antibodies, Lack et ala4 found HPV antigens in 26 of 35 juvenile-onset cases, and Lancaster and Jensona5 in 3 of 4 cases. Similarly, Costa et al’” found HPV antigens in 11 of 19 juvenile-onset cases but in none of 5 adult-onset cases, and Braun et a187 in 7 of 15 juvenile-onset papillomas but in none of 6 adult-onset cases. Later, Mounts et a18’ found immunoreactivity in 2 of 12 juvenile-onset cases and in 2 of 8 adult-onset cases, and Strauss and Jensonsg in 3 of 8 juvenile-onset cases and in 4 of 8 adult-onset cases.

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However, convincing evidence for HPV origin of laryngeal papillomas was provided by molecular hybridization techniques. In 1983, Gissmann et alzl isolated and characterized a papillomavirus, assignated as HPV 11, from a recurrent lesion of juvenile laryngeal papillomatosis in a E&year-old girl. With different hybridization techniques, HPV DNA or RNA sequences have been demonstrated in 50% to 100% of juvenile- and adult-onset laryngeal papillomas.gO-gg HPV 6, 11, 16,and 18 DNA sequences have been detected in laryngeal papillomas, of which HPV 6 and 11 are the two most frequent types associated with laryngeal papillomas, accounting for more than 80% of cases.gO-gg In general, the HPV DNA detection rates in adult-onset laryngeal papillomas are considerably lower than those of juvenile papillomas.go-g8 This could be due to either (1) the involvement of different causative agents in these two diseases or (2) the different copy numbers of the virus in these lesions. By using a more sensitive method polymerase chain reaction (PCR), Levi et algg were able to substantially increase the rate of HPV-positive specimens; all the juvenile- and adult-onset papillomas were specifically amplified with HPV primers homologous to the viral late genomic region. This strongly suggests that adult papillomas contain lower viral copy numbers as compared with the juvenile papillomas, and therefore may exhibit a lower detection rate by conventional hybridization methods. Children and adults appear to be infected by the same HPV types, which would explain the development of papillomas that are quite similar clinically and histologically. Verruca Vulgaris Verruca vulgaris (common wart) is a common skin lesion caused by HPVs. Clinically, skin warts present as single or multiple small, firm, elevated, nontender growths and can appear anywhere on the skin.loO Occasionally, warts can also arise on mucous membranes, particularly the lips, rarely in the oral cavin the larity, 101,102 and exceptionally ynx. lo3,104Based on the previous case reports, laryngeal verruca vulgaris occurs mainly in older age groups (average age, 56), and has

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thus far been limited to the true vocal cords of the larynx.103*104This lesion appears white on laryngoscopic examination, and it closely resembles laryngeal papillomas on light microscopy, except for its usual heavy keratinization and abundance of keratohyaline granules. Cutaneous common warts are known to be induced by HPV types 2 and 4,1°0 whereas that of the mucous membranes of the lips and oral cavity is associated with HPV type 2 and only rarely with HPV type 4.l” There is no doubt about the HPV cause of laryngeal verruca vulgaris as well. Two (67%) of the three cases of laryngeal verruca vulgaris were positive for HPV structural antigens.103S104This is consistent with the 55% to 65% positivity rate in verruca vulgaris of the lips and oral muInterestingly, in contrast to identicosa. 101~102 fication of cutaneous HPV types HPV 2 and 4 in the skin and oral cavity, laryngeal verruca vulgaris seems to be associated with HPV 6 and 11 infections.104 Laryngeal Carcinoma Malignant conversion of laryngeal papillomas has been reported, although it is an uncommon event.18*105S115 A review of the available case reports shows that malignant neoplasms are more likely to occur in cases of severe papillomatosis with long duration and where the lesions spread throughout the reTherefore, malignant spiratory tract. 18~105-107 conversion seems to be more common in cases of juvenile-onset papillomas, but it has also been reported in adult-onset disease.18.105-107 Malignant transformation has been associated with irradiation, and the risks of this treatment are generally recognized.108-“1 Majoros et al reported that up to 14% of cases receiving irradiation exhibited malignant transformation later in life.ll’ Radiotherapeutic management of these lesions is therefore no longer appropriate. Some authors now report the occurrence of malignant conversion even in the absence of irradiation.107*‘12 To emphasize the role of HPV, viral DNA sequences have been detected both in the benign and malignant lesions.113-115 Zarod et a1113 reported a patient with laryngeal papilloma associated with HPV 6 infection, with subsequent development of a squamous cell carcinoma 5 years

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later. Byrne et a1114 reported a case of laryngotracheobronchial papillomatosis with lung carcinoma and hepatic metastasis and detected HPV 11 DNA in all tissues, including the primary papilloma, lung carcinoma, and metastatic lesions. The predominantly solitary adult papillomas appear to represent a clearly premalignant lesion.26*116 Although recurrence is rather frequent after surgical removal, these tumors usually do not spread to other sites due to mechanical factors. They occur twice as frequently in males as in females. Intraepithelial neoplasia ranging from mild dysplasia to squamous carcinoma frequently accompany these lesions. The rate of malignant transition is high, above 20% in longitudinal studies. Males are particularly prone to develop carcinomas within solitary laryngeal papillomas, and heavy smoking seems to promote the malignant transition.2”,116 In such cases, the possible synergistic effects between HPV and other carcinogens must be clearly considered. In addition to an increased risk for malignant transformation of the HPV-associated laryngotracheobronchial papillomatosis, evidence is also accumulating to suggest an association of HPV infection with laryngeal precancer lesions and carcinomas even in the absence of preceding papillomatosis. Of the 166 laryngeal squamous cell carcinomas screened by Syrjanen and Syrjanen,‘20 30% showed histological changes consistent with HPV infections, similar to those found in the genital tract. Thus, changes resembling papillomatous condyloma (31%), flat condyloma (19%), and inverted condyloma (51%) were distinguished. In a series of 36 laryngeal squamous cell carcinomas showing histological features consistent with HPV infection, HPV capsid antigens were detectable in 42% of the papillomatous lesions, in 33% of the flat lesions, and in 33% of the inverted lesions.“’ The HPV cause of laryngeal carcinoma in situ has been assessed using an immunohistochemical technique to detect viral capsid antigens by Kashima et a1.1z2 In a series of 60 biopsies collected from 20 patients, HPV antigens were demonstrated in 14 cases. Nuclei expressing the HPV capsid antigens were found in dysplastic cells as well as in areas adjacent or overlying the dysplastic cells. In two addi-

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tional cases not shown to be HPV-positive, histological evidence suggestive of a viral effect (ie, koilocytosis, parakeratosis, and multinucleation) was identified.122 These observations were further supported by the demonstration of HPV DNA sequences in laryngeal carcinomas.24,g1~123-128 In 1986, Kahn et alz4 isolated and cloned a new HPV sequence known as HPV 30 from a laryngeal carcinoma, but they failed to demonstrate this HPV type in other laryngeal carcinomas. Using the in situ hybridization technique for HPV 6, 11, 16, and 30, Syrjtinen et algl analyzed 116 cases of laryngeal squamous cell carcinomas and demonstrated HPV DNA of at least one type in 12.9% of the tumors (HPV 11 in 7.8% HPV 16 in 5.2% and HPV 6 in 4.3%). More recently, using a more sensitive DNAdetecting technique (PCR), Kiyabu et a1123 could detect HPV 16 DNA sequences in 40% (4/10) of laryngeal carcinomas. Similarly, Perez-Ayala et a1124 found HPV 16 DNA sequences in 54% (26148) of laryngeal squamous cell carcinomas. In addition to primary laryngeal carcinomas, the HPV genomes have also been detected in their metastases. Dekmezian et all” disclosed HPV 11 in the lymph node metastases of laryngeal carcinomas by in situ hybridization, and Hoshikawa et al’*” reported HPV 16 DNA in 6 of 34 metastatic laryngeal carcinomas (17.6%) by PCR. They found that laryngeal carcinomas of the glottic origin were HPV 16 DNA positive more frequently (44.4%) than those of the supraglottic origin (8.7%). The detection of HPV DNA in laryngeal carcinomas appears to be of substantial interest. Laryngeal carcinomas are known to be closely associated with chemical factors such as cigarette smoking [only 5% of the tumors occur in nonsmokers), alcohol abuse, occupational exposures, and irradiation. Specific HPV types are known to interact with chemical or physical carcinogens (initiators) in malignant conversion of infected cells. Therefore, laryngeal carcinogenesis may share common features with genital tract carcinogenesis. A synergistic interaction of HPV with chemical factors, especially with cigarette smoking, must be seriously considered in the development of laryngeal carcinoma, as suggested in carcinogenesis of the uterine cervix as well.

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Verrucous Carcinoma of the Larynx Verrucous carcinoma is a distinct variant of well-differentiated squamous cell carcinoma with a gray-white, exophytic, warty gross appearance, and characterized by slow growth, local invasion, and minimal metastatic potential, clinically resembling laryngeal papilloma more than true carcinoma.‘zg*130 Of 105 cases reported by Krauss and Perez-Mesa,“’ 11% occurred in the larynx and 73% appeared in the oral cavity. Another 4% were present in the nasal fossa, and the remainder were found on the glans penis, with scattered cases in the vulva, vagina, scrotum, and perineum. Lesions have also been described in the pyriform sinus, nose, paranasal sinuses, esophagus, external and middle ear, and skin. Laryngeal verrucous carcinoma most frequently originates from the true vocal cords. The incidence of this tumor is 1% to 2% of all the malignant neoplasms in the larynx, with the peak occurrence between 40 and 69 years of age and with a clear male preponderance.‘2g~130 Risk factors associated with verrucous carcinomas of the aerodigestive tract include cigarette smoking and alcohol intake. It has been proposed that HPV may have a causative role in the development of verrucous carcinomas, analogous to its role in the genital tract neoplasms.13’ Morphological features showing papillomatosis, dyskeratosis, and koilocytosis suggest an HPV origin.131 This hypothesis is further supported by demonstration of HPV DNA sequences in a substantial proportion of laryngeal lesions studied so far.132,‘33 Abramson et alJ3’ examined biopsies from five verrucous carcinomas (with histological changes of HPV infection), and HPV 16-related sequences were detected in all cases. Similarly, Brandsma et a1133 reported HPV Is-related DNA sequences not only in all six verrucous tumors but also in four biopsies taken adjacent to the margins of these tumors. PerezAyala et alIz recently detected HPV 16 DNA sequences in 3 of the 3 verrucous carcinomas studied. HPV INFECTIONS IN THE TRACHEOBRONCHIAL MUCOSA Tracheobronchial papillomas are rare benign tumors that may occur with or without

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concomitant or preceding laryngeal papillomatosis.134 The frequency of tracheal papillomatosis was reported as being similar for both the juvenile-onset (26%) and adult-onset disease (25%) by Weiss and Kashima.135 Clinically, there is a tendency for papilloma to spread into previously uninvolved areas of the respiratory tract, including the lungs. Tracheal extension of the disease from the larynx has been reported to occur in 2% to 36% of the cases. 18,134-137Extension into the bronchi and lungs is far less common (
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smears of HPV-infected women.138*13g Indeed, characteristic koilocytosis, binucleation or multinucleation, and dyskeratosis, similar to cervical condylomas and laryngeal papillomatosis, could be readily identified in the biopsy specimens derived from tracheobronchial papillomatosis.140-143 Their HPV etiology has been also confirmed by identification of HPV particles and demonstration of HPV DNA sequences in these lesions.140-144 HPV types 6 and 11 account for the most frequent types associated with tracheobronchial papillomatosis. One of HPV 6 subtypes (HPV 6c) has been identified in more than half of the cases, and it has been associated with more severe disease as indicated by frequent spread of lesions throughout the respiratory tract and the high frequency of surgery needed to maintain an open airway.143 As in laryngeal papillomatosis, reports have been published in which bronchial squamous cell carcinomas had developed from preexisting tracheobronchial papillomatosis, even without radiation therapy, as well as during interferon therapy.145-150 In a series of 14 patients with pulmonary parenchymal extension of papillomas, three developed bronchogenic carcinoma without prior radiation.‘50 Byrne et al”* examined a bronchial squamous cell carcinoma arising in a patient with HPV ll-positive laryngotracheobronchial papillomatosis, in which episomal HPV 11 DNA sequences were detected in large copy numbers both in the primary tumor and its metastases. Bejui-Thivolet et a1151 described a case of papillary squamous cell carcinoma of the lung developed from a condylomatous papilloma, in which HPV 11 DNA sequences were found. Recently, Hording et al ‘*’ reported a bronchoesophageal papillomatosis showing a clinically malignant course, in which HPV 11 DNA sequences were demonstrated. A number of morphological and DNA hybridization studies suggest that HPV infections exist in bronchogenic precancer lesions and carcinomas even in the absence of papillomatosis.152‘15* HPV-suggestive lesions were first reported by Syrjanen et al in 1979,152 who observed a case of bronchial squamous cell cancer, where epithelium adjacent to the malignant lesion contained areas identical to

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those previously described in genital HPV lesions. In a subsequent survey of 104 bronchial squamous cell carcinomas, morphologically HPV-suggestive lesions were found in 36 (34.6%) cases.153 These observations were verified in another series of 220 bronchial squamous cell carcinomas, in which morphological evidence for HPV involvement was found in 67 (30.Ei”b)cases.15* Similar observations were recently reported by Bejui-Thivolet et a1,155 who demonstrated condylomatouslike changes in 12 of 33 (36%) welldifferentiated bronchial squamous cell carcinomas. Additional evidence was recently presented to substantiate these morphological findings when HPV 16 DNA was disclosed in anaplastic lung carcinoma.15” In a series of 99 bronchial squamous cell carcinomas, Syrjanen and Syrjanen157 detected HPV DNA in five tumors (5%) using DNA in situ hybridization. Recently, Bejui-Thivolet et a1155tested a series of 43 paraffin-embedded bronchial samples (10 squamous metaplasia and 33 epidermoid carcinoma) for histological evidence of HPV and HPV DNA by in situ hybridization with biotinylated probes. Fourteen of these lesions (32.5%) showed typical condylomatous histological changes. HPV DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one bronchial metaplasia case. Six carcinomas (18%) contained HPV DNA: HPV 18 (3 cases), HPV 16 (1 case), HPV 11 (1 case), HPV 6 (1 case), and HPV 16 and 18 (1 case). Thus, HPV types 6, 11, 16, and 18 DNA sequences have been detected in bronchogenic squamous cell carcinomas, of which HPV 16 and 18 seem to be the most frequent.155-158 By in vitro transfection of normal human bronchial epithelial cells with cloned fulllength HPV 16 or HPV 18, Willey et a115’ recently established two HPV 18-immortalized cell lines (BEPl and BEP2) and one HPV 16transformed cell line (BEP3). Consonant with the HPV-immortalized human keratinocytes originated elsewhere, these cell lines have sustained growth in vitro but remained nontumorigenic in vivo. HPV DNA sequences were demonstrated to be integrated in their chromosomes and HPV E6-E7 open reading frames were highly transcribed and expressed.

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In respiratory carcinogenesis, a broad range of environmental agents have been identified or suspected as causative agents. These include tobacco products, asbestos, polycyclic aromatic hydrocarbons, nickle, chromium, and arsenic, as well as a large number of pollutants encountered in the workplace and in the general environment. It is well known that the major cause of bronchogenic carcinoma is cigarette smoking. However, other cofactors may be involved in the multistage respiratory carcinogenesis. In this connection, synergistic actions between cigarette smoking and HPV infection should be considered. MODES OF TRANSMISSION HPV infections of the anogenital tract are known to be a sexually transmitted disease (STD).6-8 However, the route by which HPV reaches the respiratory mucosa is not known with certainty. Evidently, three possibilities could exist for respiratory HPV infections in neonates (18): (1)intrapartum during the passage through the infected birth canal, (2) transmission in utero before birth either by viremia or ascending infection, and (3) postnatally from an infected mother or other infected individual. One generally accepted mode of transmission of HPVs to neonates is infection during the fetal passage through the infected genital tract of the mother or during mucous withdrawal from the child nasopharynx. This can easily account for the development of juvenile-onset laryngeal papillomatosis. This concept is supported by the fact that approximately 68% of children with laryngeal papillomatosis were born to mothers with genital condylomas during pregnancy or at parturition. 16’*161Furthermore, the existence of HPV types 6 and 11 in both the anogenital condylomas and respiratory papillomatosis, the findings of HPV DNA in the foreskin of a normal newborn,16’ and the high percentage of oropharyngeal mucosa in neonates vaginally delivered from HPV-infected mothers as well as in the amniotic fluid,163,‘64 all favor the mechanisms for HPV transmission at birth, either by intrapartum or transplacental route. The modes by which adult-onset respiratory papillomatosis is acquired are not clear. It

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is possible that some adult-onset papillomas are acquired through infection at birth as well, but for unknown reasons the virus remains latent or subclinical for several decades. Accordingly, the development of adult-onset respiratory papillomatosis may represent a reactivation of a quiescent virus acquired at birth. Postpartum infection by contact with an HPV-infected individual is also a possibility. Both the juvenile- and adult-onset form are characterized by recurrence of the disease. Recurrences are multiple, sometimes occurring every 2 weeks. The fact that the HPV type in recurrences of the individual patients almost invariably remains unchanged suggests an endogenous reactivation of a latent virus rather than a reinfection. Further support to this hypothesis comes from the work of Steinberg et HPV DNA in biopal, 165 who demonstrated sies from patients whose disease was in remission, as well as in grossly normal tissue sites of two patients with active disease. The discovery of HPV DNA sequences in normal laryngeal tissues adjacent to the cancer lesion is of special interest with regard to the possible latency of HPV infections at these sites. Latent HPV genomes present in apparently normal epithelium are potential reservoirs for new lesions after a seemingly successful removal of a manifest lesion. This could also explain why recurrence is common after surgical removal of all abnormal-appearing laryngeal tissues. Furthermore, it is possible that many more children may be infected with HPVs at birth than actually develop the disease afterwards. The triggers for reactivation of a latent infection are currently unknown. Local chronic mechanical irritation may represent one of the triggering factors, as recently indicated by the observed reactivation of latent papillomavirus infection and formation of papillomas by scratching the skin with glasspaper in a laboratory strain of Mastomys natalensis.16” Alternatively, postpartum infection by direct contact with infected individuals is also possible. Viral particles may be transmitted late in a person’s life by oral contact with infected genitalia or other mucosal sites. Moreover, intact HPV has been recently demonstrated in the CO,-laser plume from plantar or mosaic warts,167 anogenital condylomas,168 as well as laryngeal papillomas.16’ Additionally,

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Sawchuk et a117’ observed infectious bovine papillomavirus in vapor from bovine warts. Therefore, the risk of infection for the laser team during the removal of any virally induced neoplasm should be regarded as more than a theoretical possibility. This is supported by a recent case report of laryngeal papillomatosis in a surgeon who had repeatedly inhaled the laser plume during treatment of anogenital condylomas.‘71

PATHOGENESIS OF HPV-INDUCED AIRWAY LESIONS The mechanisms by which HPVs induce benign and malignant squamous lesions are not known in detail yet. The major difficulty in elucidating the role of HPV in the pathogenesis of squamous lesions has been the lack of an appropriate in vitro culture system that would permit the growth of the virus and allow an analysis of its transforming properties.1-4*6‘8 It is generally believed that basal cells are the target for papillomavirus infection.43”8 The viruses may get direct access to the basal cells of the respiratory mucosa when exposed in a wound or an abrasion caused by mucous withdrawal from the oral cavity and nasopharynx of the children at birth or later in life. Soon after the initial infection, the viral DNA may undergo a limited replication leading to a few copies of viral DNA in each of the infected cells. Also, certain viral proteins that are necessary for the replication of viral functions, encoded in the early genes of the virus, are produced. Once replication of the virus is established in the cell, it has two outcomes: latent or productive infections. In the latent state, the virus may remain silent for a long time and may produce RNA at very low levels. Alternatively, the early and late viral genes may be extensively transcribed and expressed, leading to active replication of the virus. A successful establishment of infection depends on whether the host cells are permissive or nonpermissive for virus replication. Permissive cells allow the synthesis of viral DNA, RNA, and proteins, leading to the assembly and release of infectious viral particles, whereas nonpermissive cells do not pro-

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vide appropriate conditions for viral replication, thus leading to aborted infection. It is known that productive functions of papillomaviruses, including vegetative viral DNA synthesis and expression of late viral genes that encode capsid proteins, are closely associated with differentiation of the squamous epithelial cells, Only the differentiating cells of the stratum spinosum and stratum granulosum permit productive virus replication.1‘4 Mature viral particles are absent in the basal stem cells, which only contain viral DNA. This indicates that the stages of viral life cycle may require specific factors only provided by the sequentially differentiating permissive keratinocytes. Therefore, papillomavirus infections have a specific tropism for the squamous epithelium. This fact may also account for the major difficulty in establishing an in vitro cell culture system for the propagation of these viruses. A common feature of all HPV-induced lesions is hyperplasia of the squamous epithelial cells, leading to the thickening of the epithelium, especially the intermediate layers and to formation of papillomatous lesions. This change is assumed to occur through an abnormal metabolism and proliferation of the infected cells and it is likely to be accompanied by the expression of certain viral gene products. In the more superficial layers, there is nuclear degeneration and in some cells, cytoplasmic vacuolization referred to as koilocytosis.1‘4 Occasionally, the papillomaviruses may induce premalignant and malignant lesions. This event presumably requires both the cellular mutations and changes in the regulation of HPVs. Infections with certain specific genotypes of HPVs, eg, HPV 16, 18, 31, and 33, have been shown to have a higher risk for the development of anogenital malignancies, whereas the others, such as HPV 6 and 11, are more frequently associated with benign anogenital lesions.26-33 Viral DNA is always present in episomal form in benign lesions, whereas in carcinomas the viral DNA is almost always integrated in the cellular genomes suggesting that the viral DNA integration may represent a necessary event for HPVmediated oncogenesis.2”W33 As in anogenital HPV infections, current data suggest that HPV

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6 and 11 are the two most frequent HPV types associated with benign papillomas of the respiratory tract, and HPV 16 and 18 are frequently present in carcinomas. However, such a division is not as strict as in anogenital HPV infections, because a number of reports are available on the detection of HPV 6 and 11 DNA sequences in respiratory tract malignancies 52~59~ll3~115~141~152 Recent evidence suggests that the transforming activity of HPVs depends on the expression of E6 and E7 ORFS.‘“-~~ New insight into the possible mechanisms of HPVassociated transformation has derived from the recent findings that these transforming proteins can interact with some cellular tumor suppressor genes, eg, Rb and ~53, presumably leading to inactivation of the latter.172*173Although the E7 protein of all HPV types is capable of binding to Rb, the E7 protein of HPV types associated with carcinomas shows a higher affinity than the E7 gene product of HPV types found in benign lesions.‘72*173 Similarly, it was recently demonstrated that the other transforming protein of HPV types 16 and 18,the E6 protein, can bind the p53 protein, whereas the HPV 6iE6 and ll/E6protein appears to bind these host-cell proteins less actively. Thus, the interaction of high-risk E6-E7 gene products with cellular Rb and p53 proteins may represent an important pathway for HPV-mediated oncogenesis.172*173 LOSS of wild type Rb and p53 functions can be achieved by at least two different mechanisms, either somatic mutation within the Rb and p53 loci, which are frequently caused by mutagenic exogenous factors, or by the binding of these cell proteins to viral oncoproteins. 174*175Recent data suggest that mutation of one allele of the Rb gene, caused by other factors, could certainly enhance the ability of E7 protein to complex all Rb proteins.172*173 This sheds further light on the molecular basis of synergistic action between HPVs and other carcinogenic agents. It is known that respiratory mucosa is exposed to high levels of a large number of environmental agents such as cigarette smoke, alcohol abuse, occupational exposures, numerous pollutants encountered in the occupational and general environment, as well as various microorganisms, which may act syn-

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ergistically with HPV leading to the development of carcinomas. Many identified human carcinogens are known to elicit DNA-base substitutions and cause gene mutations either in bacteria and mammalian cells in vitro or in experimental animals in vivo. Mutated or deleted Rb and p53 genes have been found in a variety of malignant tumors, including carcinomas of the colon, lung, esophagus, breast, liver, brain, bone, reticuloepithelial tissues, and hematopoietic tissues.‘749175 According to this concept, the difference in the carcinogenic potential between the low-risk (eg, HPV 6 and 11) and high-risk HPV (HPV 16 and 18) types may well be due to the different degree of dependence on the synergistic exogenous factors needed for the final malignant conversion.32*33The malignant progression of benign lesions induced by the low-risk HPV types may largely depend on such exogenous carcinogens, whereas the lesions associated with high-risk HPV types could progress to malignancies without such factors by the virusgenerated endogenous events.32s33 This could help explain the relatively common occurrence of HPV 6 and 11 associated malignancies in the respiratory tract. Therefore, respiratory carcinoma seems to be a suitable model for the study of HPV-mediated oncogenesis and the synergistic actions of other carcinogenic agents. CONCLUSIONS Based on the evidence of transmission experiments of laryngeal papillomas to man and animals, the demonstration of HPV-suggestive changes, identification of papillomavirus particles and antigens, as well as detection of HPV genomes in a high proportion (50% to 100%) of respiratory papillomas or papillomatosis, it appears beyond a doubt that these lesions are induced by HPV infection. Furthermore, up to 54% of squamous cell carcinomas of the respiratory tract and some 10% of the head and neck cancer cell lines176 tested thus far were also demonstrated to contain HPV DNA sequences. Among the 66 identified HPV types, HPV types 1, 2,4,6,11,13, 16,18,30,32,40,and 57 have been detected in a variety of proliferative lesions of the aerodigestive tract, and HPV 11,13,30,32,and 57

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have been originally isolated from benign or malignant lesions of that area. As with anogenital HPV infections, current data suggest that HPV 6 and 11 are the two most frequent HPV types associated with benign papillomatous lesions of the respiratory tract, and HPV 16 and 18 are often present in respiratory tract carcinomas. However, this division is not as strict as in anogenital HPV infections, because a number of reports have been published on the detection of HPV 6 and 11 DNA sequences in aerodigestive malignancies. So far, however, the series of biopsies from malignant respiratory tract lesions containing any of the known HPV types have been small. Thus, there is an urgent need for further studies on a larger series of samples. Similarly, the large number of negative results obtained when these lesions have been tested for the specific HPV DNA sequences may point to the fact that the HPV types responsible for the development of respiratory tract carcinomas have not been identified. Extensive further investigations should be performed to identify and characterize these eventual new HPV types. Many similarities apparently exist in the cellular nature and early neoplastic changes between the respiratory mucosa and that of the uterine cervix. Therefore, the established HPV-mediated oncogenesis models in the cervix are easily applied to the respiratory tract as well. Current data suggest that specific genotypes of HPVs are necessary but are insufficient in malignant transformation, synergistic actions with other initiating events being evidently required. This fits well with the fact that respiratory mucosa is continuously exposed to high levels of a large number of environmental agents such as cigarette smoke, alcohol, occupational exposures, numerous pollutants encountered in the general and occupational environment, as well as to various microorganisms that may act synergistically with HPV leading to the development of carcinomas. Respiratory carcinoma seems to be a suitable model for the study of oncogenesis either by HPV itself or in synergistic collaboration with other carcinogenic agents. Further studies on viral types, integrity, expression, and the physical state of HPVs in respiratory lesions should be of critical importance in un-

raveling the causative lesions.

role of the virus in these

ACKNOWLEDGMENT The authors gratefully acknowledge the skillful assistance of Pirkko Karttunen, Kaarina Hoffren, Helena Kemilainen, Eija Sedergren-Varis, and Ritva Sormunen. The authors also want to extend their special thanks to Prof. Dr. Lutz Gissmann, Prof. Dr. Harald zur Hausen, DKFZ, Heidelberg, Germany, and Prof. Gerard Orth, Pasteur Institute, Paris, France, for providing the HPV DNA probes at our disposal.

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