Human Papillomavirus Associated with Papillary Squamous Cell

Infection

Case Report

Human Papillomavirus Associated with Papillary Squamous Cell Carcinoma of the Oropharynx in a Renal Transplant Recipient F. Cobo, C. García, P. Talavera, J. Bravo, C. Cabrera, A. Concha

Abstract The papillary squamous cell carcinoma (PSCC) is a rare variant of the head and neck squamous cell carcinoma. Established etiological factors can include tobacco smoking and heavy alcohol abuse. Moreover, human papillomavirus infection can be involved in the pathogenesis of PSCC. This tumor is more frequent in patients with immunosuppression including those who have received a transplant. Most of the cases are produced by genotype HPV-6 and HPV-16, although there is a possibility of infection by other HPV subtypes. We present a case report of a PSCC and papilloma with oropharyngeal location in which high-risk HPV type 16 and low-risk HPV type 6, respectively, were identified by PCR in a renal transplant patient. Infection 2006; 34: 176–180 DOI 10.1007/s15010-006-5026-7

Introduction Human papillomavirus (HPV) is generally associated with squamous cell carcinoma of a different origin (e.g., the female genital tract), but also includes the head and neck (HNSCC), because viral DNA, mostly of HPV type 16, has been found in tumor tissue [1]. Other HPV types (including low-risk genotypes) have been identified in squamous cell carcinomas. Estimates of HPV prevalence in HNSCC tumors vary with the detection method used and the nature of tissue preservation [1]. Papillary squamous cell carcinoma (PSCC) is a rare variant tumor of HNSCC recently described in the upper aerodigestive tract [2] and was accepted as a clinicopathologically distinct neoplasm in 1999 [3]. The tumor is an entity, with few cases published in the literature, which is mainly located in the supraglotic region [4, 5]. Like the rest of HNSCC, a relationship between PSCC with HPV infection has been published recently [6]. HPV infection is involved in the pathogenesis of PSCC in a similar way to that of other squamous cell carcinomas of the head and neck mucosa. We presented a case report of a PSCC with oropharyngeal location in which high-risk HPV type 16 was identified in an immunodeficient patient with renal transplant

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without other risk factors. Curiously, an adjacent squamous papilloma lesion was observed associated with a low-risk HPV type 6.

Case Report The patient is a 49-year-old man who has suffered chronic renal insufficiency of unknown origin and had hemodialysis since 1995. The patient has no known tobacco or alcohol exposure, although he was immunocompromised due to a renal transplant of a cadaver donor in December 1998. The immunosuppressive treatment was carried out with polyclonal antibodies-timoglobuline (500 mg/day/12 days), mophetil micophenolate (MMF) (2 g/day), prednisone (250 mg/day, progressively decreasing to 5 mg/day) and cyclosporine A (CsA) dosis to reach concentrations of 150–250 pg/ml. In October 2003, he came to our hospital with symptomatology consisting of the sensation of a strange body in the throat without other accompanying symptoms. In the physical examination of the oropharynx a palatine soft tumor was observed inside the right tonsil which measured 2 × 3 cm, with a granulomatous and exophytic nonulcerated appearance (Figure 1). There were no cervical lymph nodes. In the tomography (CAT) study of the neck, the presence of soft weave thickening was observed in the right margin of the soft palate that extended to the right tonsil. Furthermore, there was a discrete thickening on the left tonsil. A biopsy was performed with a diagnosis of PSCC without invasion in the submitted tissue. Prior to these findings, a progressive decrease of the immunosuppressive treatment was begun and surgery consisting of a tumor excision with an extension of safety margins at the edge of the tumor was performed. In November 2003, treatment with rapamicine began (due to antiproliferative and antiangiogenic properties) and the CsA was diminished progressively until being suspended in January 2004. At present the patient is asymptomatic and is following treatment with rapamicine (3 mg/day, to reach 6–11 pg/ml concentrations) and prednisone (5 mg/day).

F. Cobo (corresponding author), C. García, P. Talavera, C. Cabrera, A. Concha Infectious Pathology Unit (Dept. of Pathology), Hospital Universitario Virgen de las Nieves, Avenida Fuerzas Armadas, 2, 18014 Granada, Spain; Phone: (+34) 95802-0692, Fax: -0132, e-mail: [email protected] J. Bravo Dept. of Nephrology, Hospital Universitario Virgen de las Nieves, Granada, Spain Received: March 7, 2005 • Revision accepted: June 23, 2005

Infection 34 · 2006 · No. 3 © URBAN & VOGEL

F. Cobo et al. Papillary Carcinoma in Transplant Recipient

membrane; however, foci of microinvasion in the lamina propria were demonstrated in serial sections (Figure 3B). The epithelial cells showed nuclear atypia, mitotic activity and crowded disposition, and the papillae were composed of a thin fibrovascular axis with stroma made up of fibroblasts and mononuclear inflammatory infiltrate. Furthermore, the lesions demonstrated a focal socalled “koilocytic atypia” (Figure 3C), similar to those observed in SCC of the female genital tract associated with HPV, as seen by the presence of hyperchromatic and crenated nuclei surrounded by a clear halo of cytoplasm and an accentuated cell border. The small lesion adjacent to the main tumor shows characteristics of squamous cell papilloma without atypia or malignant features (Figure 3D).

Microbiological Diagnosis

Figure 1. Tumor in the right tonsil with a granulomatous and exophytic appearance.

Pathologic Findings Macroscopic Characteristics The main lesion consisted of a polypoid lesion of exophytic and mamelonade appearance, of pink coloration and friable, measuring about 2.8 × 2.4 cm, solid and without apparent areas of necrosis that had implant pedicles of 1.3 cm located in the right tonsil and with papillary architecture (Figure 2). Moreover, a second polypoid lesion that measured 0.7 × 0.6 cm with similar characteristics was observed 2 cm from the main tumor.

Histological Features Microscopic examination showed a tumor proliferation of nonkeratinized squamous cells with papillary architecture can be observed (Figure 3A) growing above the basal

The paraffin-embedded microdissected oropharyngeal tissue corresponding to the malignant lesion and adjacent papilloma was processed as follows: the DNA extraction was carried out by means of the Qiagen mini kit 50 (Qiagen) following the manufacturer’s instructions. Briefly, standard PCR were carried out in 50 µl containing 200 µM each deoxynucleoside triphosphate, 50 pmol each of the SPF10 primers, 25 mM MgCl2, PCR Buffer II 1 X, AmpliTaq Gold 1.5 U.I., and sterile water. A 9-min initial denaturation step at 94 °C, 40 cycles of 30 seg at 94 °C, 45 seg at 52 °C and 45 seg at 72 °C, and a chain final elongation step at 72 °C for 5 min and temperature held at 4 °C. Subsequently, aliquots (10 µl) of PCR product were layered on 3% agarose gels (Figure 4, left), and 10 µl of positive amplified DNA was used for viral typing for hybridization with the kit INNO-LiPA HPV (Figure 4, right) by means of the AUTO-LiPA method (INNOGENETICS DIAGNOSTIC, Belgium), following the manufacturer’s indications. In our case the identification showed HPV type 16 in PSCC and HPV type 6 in a papilloma. These results were corroborated in the Microbiology National Reference Center (Centro Nacional de Microbiología, Madrid, Spain).

Discussion

Figure 2. Polypoid lesion without areas of necrosis.


The PSCC is a peculiar subtype of squamous cell carcinoma [2]. The tumor appears macroscopically as an exophytic papillary proliferation, and microscopically is composed of thin papillae covered with overtly malignant nonkeratinized squamous cell [7] epithelium like high grade SIL or with histological characteristics similar to transitional cell neoplasm of the urogenital tract. These tumors show a predilection in the urogenital tract or upper aerodigestive tract. In the latter, the larynx is the most frequent localization, followed by the oropharynx and nasopharynx [6, 8], although the sinonasal localization was the most lethal. The relationship of PSCC to HPV has been reported, although comparison of HPV DNA detection rates

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Figure 3. A) Tumor proliferation of nonkeratinized squamous cells with papillary architecture, growing above the basal membrane. B) Foci of microinvasion in the lamina propria. C) Koilocytic atypia: presence of hyperchromatic and crenated nuclei surrounded by a clear halo of cytoplasma. D) Squamous cell papilloma without atypia or malignant features.

between various studies is difficult because of differences in the sample size, sensitivities of the various molecular biological techniques and detection protocols used, patient populations, and anatomic sites of the carcinomas. Furthermore, in some cases the presence of HPV in the lesions [5, 9, 10] has been not studied. The association of HPV with head and neck cancer dates to 1985, when HPV-16 DNA was first detected in an invasive squamous cell carcinoma by Southern blot hybridization [11]. We have also demonstrated the existence of integration of sequences of HPV 16/18 (high-risk types) in a large percentage of precursory, malignant primary and metastasic lesions of the larynx [12] by means of Southern blot techniques. Overall, HPV is associated with approximately 40% of head and neck squamous cell carcinomas [1], although there are differences between several reports. Gillison et al. [13] observed that HPV DNA in microdissected frozen specimens evaluated by PCR, Southern blot viral DNA sequencing and in situ hybridization was found overall in 62 of 253 cases (24.5%). High-risk HPV16 was identified in the majority (90%) of these tumors, according to McKaig et al. [1] and Zur Hausen [14] who

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observed that HPV-16 is the most dominant of the HPV subtypes, followed by HPV-18. In other reports [15], the authors observed that 15 of the 160 tumor specimens (9%) were positive for HPV16 DNA according to PCR. Most of the tumors positive for HPV-16 DNA were oropharyngeal tumors. Eighteen specimens (11.2%) contained DNA of other HPV types. Finally, Ringström et al. [16] with a PCR-based technique with restriction fragment length polymorphism analysis detected HPV in 18 (20%) of the 89 patients. HPV 16 was detected in 64% of tonsil tumors, 52% oropharyngeal tumors, and 5% oral cavity tumors. In these studies, different association rates of HPV to HNSCC were observed. Nevertheless, they emphasize that most of the cases are produced by HPV-16. In PSCC there is also some disagreement. Crissman et al. [17] reported that all PSCCs were uniformly negative for HPV DNA by in situ hybridization. With the same technique, Thompson et al. [8] reported only 1 of 41 cases tested demonstrated reactivity to identify HPV types 6/11. Other subtypes of HPV (like low-risk HPV type 6) were found by PCR in a single case of PSCC [18]. Suarez et al. [6] demonstrated the presence of HPV in 4 of 14 (29%)



Figure 4. Agarose gel showing two positive bands at 65 bp corresponding to DNA of PSCC and papilloma (left). Nitrocellulose strips showing reactive line 3 corresponding to HPV genotype 16 in PSCC (strip 1) and line 1 corresponding to HPV genotype 6 in the papilloma sample (strip 2) (right).

of PSCC by in situ hybridization and in 5 of 14 (43%) of PSCC by PCR. In most of these studies the presence of HPV in the tumoral tissue, although in a very variable proportion, is observed. However, HPV DNA presence in tumor specimens, although necessary, is not sufficient evidence for an etiologic role for the virus in the pathogenesis of this disease. In our opinion, a prospective seroepidemiologic study of HPV exposure and possible squamous cell carcinoma development will have to be performed. On the other hand, it is apparent that there are substantial differences with respect to the sensitivity of the different diagnostic methods which should also be assessed. The accomplishment of a comparative study of the different methods from detection of HPV would be advisable in a high population of patients and with the most homogeneous conditions possible. Established etiological factors include tobacco smoking and heavy alcohol abuse, although 15–20% of HNSCC patients have no known tobacco or alcohol exposure [13]. However, it is an evident fact that in immunocompromised patients an increase of neoplasia takes place, many of them associated with transforming infections by viruses like lymphomas associated with the Epstein-Barr virus. Likewise, the HPV infections can produce tumoral transformation in the patients with renal transplant due to a pharmacologic immunosuppression. For that reason


and faced with immunocompromised patients, especially those with renal transplant, we have to be alert to this possibility because there is an obvious risk of developing these lesions. To conclude, the PSCC is a rare variant of squamous cell carcinoma, although it is not clear that the biological behavior is different from that of the conventional squamous cell carcinoma. This tumor, like the squamous carcinoma of the head and the neck, seems to be associated with the infection by HPV and is more frequent in patients with immunosuppression, although the magnitude of this association and its influence in the pathogenesis, as well as the choice diagnosis method is still not exactly known. In our case it can be observed that within the same environment two biologically and histologically different lesions with the same conditioning factors coexist in a very reduced space. The malignant lesion is infected by a high power transforming genotype and curiously, the benign lesion is infected by a low power viral type, in which it has been described that malignant lesions can be produced. We would like to point out the increase in the prevalence of HPV low-risk genotypes documented in the literature (but not in our case), and also the need to demonstrate the presence of other HPV genotypes in order to design epidemiological and therapeutical strategies in patients. Likewise, we think that new pharmacological products can be used when dealing with immunocompromised patients

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to lower the risk of developing these tumors or to treat them if they have appeared.

Acknowledgments Ms. Angela Barnie for the translation and English correction of the manuscript.

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