Detection of mucosal and cutaneous human papillomaviruses in oesophagitis, squamous cell carcinoma and adenocarcinoma of the oesophagus

Study design HPV DNA has been searched by PCR and characterized by nucleotide sequence analysis in paraffin-embedded biopsies from Italian patients with oesophageal squamous cell carcinoma ( n = 36), sarcomatoid cell carcinoma ( n = 1), adenocarcinoma ( n = 20) and oesophagitis lesions ( n = 27). Results A broad spectrum of HPVs, primarily cutaneous types was demonstrated in 27.8% (10/36) of squamous cell carcinomas with a significantly higher frequency in well (G1) and moderately (G2) differentiated grades (47.3%, 9/19) compared to poorly (G3) differentiated (5.9%, 1/17) squamous cell carcinoma ( p = 0.008), and in 10% (2/20) of adenocarcinomas and in 29.6% (8/27) of oesophagitis. HPV types detected included mucosal types HPV 6 and 16, cutaneous types HPV 8, 15, 20 and 25; and the putative new HPV types X14, X15, DL473, PPHL1FR and CJ198. Conclusions There is no evidence of any association between mucosal HPVs and oesophageal neoplasia. The cutaneous HPVs are detected at low frequency in adenocarcinoma and poorly differentiated squamous cell carcinoma, while they are frequently detected in oesophagitis and in well and moderately differentiated squamous cell carcinoma suggesting their tropism for keratinized tissue, although a significant association with such neoplasias cannot be drawn. Abbreviations HPV human papillomavirus SCC squamous cell carcinoma AC adenocarcinoma Keywords Mucosal human papillomaviruses Cutaneous human papillomaviruses Oesophageal adenocarcinoma Oesophageal squamous cell carcinoma Oesophagitis Italy 1 Introduction Human oesophageal cancer is the eighth most common malignancy in the world. 1 The age-standardized rates exceed 15 per 100,000 individuals in geographic regions like Eastern Asia (China, Mongolia and Japan), Eastern-Southern Africa and South Central Asia (Turkmenistan, Kazakhstan and Iran). 2 The great majority of oesophageal cancers (over 95%) are either squamous cell carcinomas, more frequent in Black and Asian populations, or adenocarcinomas, affecting predominantly white populations in United States and Western Europe. 3,4 Moreover these cancer types have distinct etiological and pathological characteristics. The squamous cell carcinoma develops from squamous epithelium mostly of the middle third of the oesophagus according to a classical progressive sequence from mild to severe dysplasia, carcinoma in situ and invasive carcinoma. 5 The adenocarcinoma mostly occurs within the distal third of the oesophagus and is preceded by a well-defined preneoplastic lesion called gastro-oesophageal reflux disease and Barrett oesophagus. 6–8 The risk factors involved in the aetiology of the oesophageal cancer and the reasons for the geographical variation are poorly understood, but it is known that several factors that cause inflammation and chronic irritation, such as dietary and cultural habits, nutritional deficiencies, excessive use of tobacco and alcohol, microbial infection and infection with certain DNA tumour viruses, have been associated with this malignancy. 9,10 Human papillomaviruses (HPVs) have been considered the most likely viral candidate in the aetiology of oesophageal squamous cell carcinoma since the description of the resemblance of the precursor lesions to those of cervical carcinoma and the detection of HPV DNA sequences in both benign and malignant squamous cell lesions of the oesophagus. 11,12 The association of HPV infection with oesophageal cancer is, however, still controversial with a number of epidemiological studies reporting prevalence of mucosal HPV DNA ranging from 0 to 70%, depending on the tumour histology, virus detection methods and geographical origin. 13–17 The majority of studies were designed to investigate for the presence of mucosal HPV, mainly HPV 16 and 18 genotypes, and only few studies have searched for mucosal as well as cutaneous HPV sequences in oesophageal lesions. 18,19 The present study reports on the analysis of a case series of squamous cell carcinoma and adenocarcinoma at different stages of differentiation (from well differentiated [G1] to poorly differentiated [G3]), along with tissue sections diagnosed with oesophagitis, in order to determine the prevalence of mucosal and cutaneous HPV types in such lesions. 2 Methods Cases of oesophageal carcinoma diagnosed between December 1999 and April 2003 were retrieved from the files of the Pathology Department at the “A. Cardarelli” Hospital in Naples, Italy. A total of 66 paraffin-embedded tumour cases, including squamous cell carcinoma ( n = 35), sarcomatoid carcinoma ( n = 1), adenocarcinoma ( n = 30) and 35 non-neoplastic oesophageal mucosa samples (oesophagitis) were identified. All cases were re-reviewed to confirm the diagnosis and sub-classification into the histological subtypes. From each paraffin block, four 10-μm thick sections were cut with disposable microtome knives and placed into sterile Eppendorf tubes. To control for possible human and viral DNA cross-contamination between samples both an empty paraffin block and paraffin-embedded uterine leiomyoma biopsy were cut in between oesophageal samples and used as a PCR control for contamination during microtome use. Genomic DNA was extracted according to published procedures. 20 In particular tissue sections were extracted twice with 1 ml of xylenes, for paraffin removal, and twice with 500 μl of 100% ethanol, for organic solvents removal, and digested at 60 °C for 30 min with Proteinase K in 100 μl of lysis buffer (10 mM Tris–HCl pH 7.6, 5 mM EDTA, 150 mM NaCl, 1% SDS). DNA was further purified by phenol and phenol–chloroform–isoamyl alcohol (25:24:1) extraction and ethanol precipitation in 0.3 M sodium acetate (pH 4.6). All samples were evaluated for nucleic acid degradation by PCR amplification with specific oligoprimers targeting a 133 bp fragment of the exon 7 within the TP 53 gene. 21 These tests provided 57 (86.4%) oesophageal carcinoma and 27 (77.1%) oesophagitis cases suitable for subsequent amplifications. The mean age of oesophageal cancer and oesophagitis patients at diagnosis was 61.3 (±11.0) and 46.2 (±10.7) years, respectively. HPV detection was carried out by general-primer-mediated PCR, as shown in Table 1 , using the following primer pairs: (a) MY09/MY11 22 followed by GP5+/GP6+, 23 for the nested amplification of mucosal HPVs 24 ; (b) CP65/CP70 followed by CP66/CP69 for the amplification of so-called Epidermodysplasia Verruciformis (EV)-related HPV types 25 ; and (c) FAP59/FAP64 for the amplification of 75 different types of mucosal, cutaneous and EV-related HPVs. 26 In addition, we used type-specific PCR targeting E6 genes of individual HPV 16 and HPV 38 types. 27,28 Serial dilutions (from 1 to 1000 copies) of eleven plasmid clones, containing HPV genomes representative of mucosal (HPV 6, 11, 16, 18, 31, 33) cutaneous (HPV 10), muco-cutaneous (HPV 2) and EV-related (HPV 5, 8, 38) HPV types, were used as positive controls. Reaction mixtures without template DNA, included in every set of 5 clinical specimens, represented the negative controls. HPV genotypes were identified by nucleotide sequencing of amplified products using the PCR oligoprimers in accordance to protocols described previously. 24 Briefly, amplified products were purified by precipitation at 37 °C for 15 min with 10% polyethylene glycol (PEG 6000) in 1.25 M NaCl. Aliquots of 30 ng to 100 ng of DNA were denatured at 95 °C, in presence of 10% DMSO and 20 pmol of the oligoprimer, cooled immediately in liquid nitrogen and subsequently sequenced with the Sequenase 2.0 kit according to manufacturer's instructions (GE Healthcare) modified in the labelling step (3 min on ice). All DNA samples ( n = 4) showing sequence patterns compatible with multiple infections have been subcloned in SmaI pBS-minus vector (Stratagene), as described previously, 29 and subjected to sequence analysis using the 17-mer universal and the 16-mer reverse M13 sequencing primers with the Sequenase 2.0 kit according to manufacturer's instructions (GE Healthcare). All HPV-positive samples were amplified and analyzed in duplicate to identify point mutations possibly originated by the PCR reaction. HPV type identification was performed by alignments of HPV sequences with those present in the GenBank database using the BLASTn software ( http://www.ncbi.nlm.nih.gov/blast/html ). The data were analyzed with Epi Info 6 Statistical Analysis System Software (Version 6.04b, 1997, Centers for Disease Control and Prevention, USA). Unpaired t -test was used for comparisons of continuous variables (i.e. age); χ 2 test, Yates-corrected χ 2 test and, where appropriate, two-sided Fisher's exact test were used for comparison of categorical data. Differences were considered to be statistically significant when p -values were less than 0.05. 3 Results The histology of the 57 oesophageal tumour cases included in the study identified 36 (63.2%) as squamous cell carcinomas ( n = 6 well differentiated [G1]; n = 13 moderately differentiated [G2]; n = 17 poorly differentiated [G3]), one (1.7%) as sarcomatoid carcinoma and 20 (35.1%) lesions as adenocarcinomas ( n = 3 well differentiated [G1]; n = 10 moderately differentiated [G2]; n = 7 poorly differentiated [G3]) ( Table 2 ). All samples have been analyzed by PCR using six primer sets to amplify mucosal and cutaneous HPV types. Combining the results obtained with MY09/MY11-GP5+/GP6+, CP65/CP70-CP66/CP69 and HPV 16-specific primer pairs 20 samples were positive for the presence of HPVs. No viral sequences were detected in either cancer cases or oesophagitis lesions with both FAP59/FAP64 and HPV 38-specific primer pairs. In particular, HPV sequences were amplified in 21.1% (12/57) of the oesophageal carcinomas and 29.6% (8/27) of the oesophagitis lesions ( p = 0.557). The detection frequencies were 27.8% (10/36) and 10% (2/20) in squamous cell carcinoma and in adenocarcinoma cases, respectively, ( p = 0.178). The results of HPV DNA detection in the different histological categories are summarized in Table 3 . HPV sequences have been identified in 50% (3 out of 6) and 0% (0 out of 3) of well differentiated (G1), in 46.1% (6 out of 13) and in 10% (1 out 10) of moderately differentiated (G2), in 5.9% (1 out 17) and in 14.3% (1 out of 7) of poorly differentiated (G3) squamous cell carcinomas and adenocarcinomas, respectively. A statistical significant high frequency of HPV infection is observed in well (G1) and moderately differentiated grades (G2) (47.3%, 9/19) compared to poorly differentiated (G3) (5.9%, 1/17) squamous cell carcinoma ( p = 0.008), while this difference is not statistical significant compared to oesophagitis lesions ( p = 0.359). Sequence analysis of the HPV amplimers obtained by the nested PCR with GP5+/GP6+ and CP66/CP69 allowed the identification of two mucosal HPV types (6 and 16), four cutaneous HPVs (8, 19, 20, 25) and five putative new cutaneous HPV-related sequences (CJ198, 30 DL473 [AJ010825], PPHL1FR, 31 X14 and X15 25 ). The two HPV-positive adenocarcinoma samples harboured HPV 8, 20 and 25 as well as the putative new HPV types X15, CJ198 and PPHL1FR. The ten HPV-positive squamous cell carcinoma samples contained HPV 6, 8, and 20 as well as the putative new HPV type DL473, PPHL1FR and X14. The HPV types 19, 20, and 25 along with the putative new HPV types CJ198, DL473, and PPHL1FR were demonstrated in the oesophagitis samples. Each of the putative new HPV types is related to one of the so-called Epidermodysplasia Verruciformis (EV)-related HPV types. CJ198 is related to HPV 36 (90% sequence homology), DL473 is related to HPV 15 (80%), PPHL1FR is related to HPV 23 (83%), X14 is related to HPV 17b (82%) and X15 is related to HPV 36 (83%) ( Table 4 ). Three multiple HPV infections have been detected. These included HPV 8/25/CJ198/X15 and HPV 20/PPHL1FR in two adenocarcinoma samples, and HPV 19/20 in one oesophagitis sample. No viral sequences were detected in the negative controls with all primer pairs used in this study. 4 Discussion HPVs are a large and heterogeneous group of viruses classified into higher order “genera” such as genus alpha, including HPVs infecting mucosa, and genera beta and gamma, comprising cutaneous HPVs 32 . More than 15 high-risk alpha HPV genotypes have been causally related with virtually all cases of cervical cancer, 33 and with a variable fraction (3–90%) of other genital (penis, vulva, vagina, anus) and non-genital (mouth, orophanrynx) squamous cell carcinomas 34 . Several cutaneous types (HPV 5, 8, 9, 12,14, 15, 17, and 19–25) have been associated with cutaneous squamous cell carcinomas in immunosuppressed individuals, such as patients with the Epidermodysplasia Verruciformis disease 35,36 or with organ transplantation 25,37–42 , however, their implication in the pathogenesis of other human malignancies remains controversial. 16 In this study the search for mucosal HPV genotypes, performed by broad spectrum nested PCR 24 and HPV type 16 E6 specific PCR, 27 showed a very low HPV prevalence both in oesophageal cancers (3.5%) and in oesophagitis lesions (0%) with HPV type 16 identified in only one moderately differentiated squamous cell carcinoma and HPV type 6 in only one-differentiated squamous cell carcinoma. These results confirm previous observations reporting a lack of association between mucosal HPV infection and oesophageal squamous cell carcinomas in Italy, 43 France, 44 Germany, 45 the Netherlands 46 and Slovenia 47 . A different picture emerged from the PCR analysis of cutaneous HPV in oesophageal lesions. Using three distinct sets of primers, 25,26,48 an overall HPV detection rate of 22.2% in squamous cell carcinomas, 10% in adenocarcinomas and 25.9% in oesophagitis lesions was observed. The absence of a statistically significant difference of detection rate of cutaneous HPVs in well and moderately squamous cell carcinomas versus oesophagitis lesions does not allow any conclusion on the role of cutaneous HPV infection in the pathogenesis of oesophageal cancer. Conversely the significantly higher ( p < 0.05) HPV detection rate in well-differentiated and moderately differentiated squamous cell carcinomas (47.3%) compared to poorly differentiated tumours (5.9%) suggest a specific tropism of the virus with a productive viral replication mainly in keratinized tissue. Genotype characterization allowed the identification of four cutaneous HPVs and five putative new cutaneous HPV-related sequences, all belonging to species 1 and 2 of beta genus. The HPV types 8, 20, 25 and PPHL1FR have been previously identified in oesophageal squamous carcinoma samples. 18,31 The HPV types 19, DL473 (De Villiers EM, unpublished), X14 and X15 have been previously identified in squamous cell carcinoma of the skin, 25,49 while the HPV CJ198 has been previous identified in conjunctival neoplasia. 50 These data are consistent with one previous study reporting a broad spectrum of different HPVs, including also cutaneous types 9, 20, 24 and 25; and the putative new HPV types DL231, DL428 and DL436, in 34.5% and 26.4% of the squamous-cell carcinomas from China and South Africa, respectively. 18,19 The cutaneous HPV type 38, previously identified in 66% of oral squamous cell carcinoma specimens, 51 was not detected in the oesophageal samples neither with broad spectrum nor with HPV 38 E6 specific primers. If the HPV 38 association with oral cancer would be confirmed also in Caucasian patients, its different distribution in various upper gastro-intestinal cancers would raise questions on tissue tropism and viral pathogenesis. Although no high-risk HPV types within the genus beta have been specifically associated with human tumours it has been postulated that cutaneous HPVs, in synergy with other cofactors such as UV exposure, smoking, alcohol, etc., could have a role in tumour initiation and progression with a “hit-and-run” mechanism of carcinogenesis. 35 Several in vitro models have shown that E6 proteins from diverse cutaneous HPV types influence both growth and differentiation of primary human keratinocytes in organotypic raft cultures 52 and inhibit apoptosis in response to UV irradiation. 53–55 Furthermore, E6 genes of HPV types 12, 14, 15, 24 36, and 49 have been shown to have a significant transforming potential in vitro in cooperation with activated ras. 56 All these results provide molecular support for an indirect role of these viruses (i.e. by facilitating UV-related carcinogenesis via preventing UV-induced apoptosis or impairing DNA repair) in the development of certain human malignancies. This and several other mechanisms of cutaneous HPV potentially involved in the pathogenesis of squamous cell carcinoma have been recently reviewed by Feltkamp et al. 57 aiming to foresee their role in cutaneous carcinogenesis. In conclusion the present study, although with limitations due to the modest sample size and to the absence of information about cancer cofactors (smoking, drinking habits, immune status and occupational history/exposure to carcinogens), is the first study reporting an unexpectedly high prevalence of cutaneous HPV types in oesophagitis lesions and in the subset of differentiated oesophageal squamous cell carcinoma. The presence, however, of different HPV types in squamous cell carcinoma, with type distribution and frequency rate similar to that observed in oesophagitis lesions, does not support the existence of a specific high-risk cutaneous HPV type involved in the pathogenesis of oesophageal neoplasia. 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