J. M. M. Walboomers

The Use of General Primers in the Polymerase Chain Reaction Permits the Detection of a Broad Spectrum of Human Papillomavirus Genotypes

A novel polymerase chain reaction (PCR) method was developed that permits the detection of 11 different human papillomavirus (HPV) genotypes using two general primer sets. By computer-assisted sequence analysis, two pairs of general primers were selected from the conserved L1 open reading frame and tested in the PCR on a set of cloned HPV genotypes. Experimental analysis showed that up to three mismatches between primers and target DNA did not influence the efficiency of the assay. The use of these primers in the PCR enabled the detection of HPV genotypes HPV-1a, -6, -8, -11, -13, -16, -18, -30, -31, -32 and -33, and was also successfully applied to well characterized cervical carcinoma cell lines and clinical samples. For the HPV types tested sub-picogram amounts of cloned DNA could be detected after general primer-mediated PCR and subsequent hybridization. The specificity of the amplification products was confirmed by blot hybridization procedures and RsaI restriction enzyme digestion. The results indicate that this PCR method can be a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas suspected of having an HPV aetiology.

Age-dependence of human papillomavirus DNA presence in oral squamous cell carcinomas

The aetiology of oral cancer is thought to be multifactorial. Apart from the two known major risk factors (tobacco and alcohol), a viral aetiology has been proposed, with special reference to human papillomavirus (HPV). 35 cases of oral squamous cell carcinoma (OSCC), seen at the Departments of Oral & Maxillofacial Surgery and Oral Pathology and Otolaryngology of the Free University of Amsterdam, were analysed as well as 12 biopsies of clinically and histologically normal gingival mucosa collected from healthy individuals after tooth extractions, using the polymerase chain reaction (PCR) and two different sets of primers that are able to detect a broad spectrum of HPV types. An overall HPV positivity of 54.3% in OSCC was found, the majority of positive cases (78.9%) harbouring HPV type 16. In contrast, no positivity for HPV was detected in the clinically normal oral mucosal samples analysed. Furthermore, a significant association between HPV presence and age was found: patients older than 60 years showed a lower prevalence of the virus (29.4%) compared with patients below this age (77.8%) (P < 0.05). The results from the present study suggest an association between HPV and OSCC, particularly in patients under the seventh decade.

Differences in MHC and TAP-1 expression in cervical cancer lymph node metastases as compared with the primary tumours.

In previous studies we have shown down-regulation of class I major histocompatibility complex (MHC) expression in a significant proportion of primary cervical carcinomas, which was found to be strongly correlated with loss of expression of the transporter associated with antigen presentation (TAP). By contrast, class II MHC expression was frequently up-regulated on neoplastic keratinocytes in these malignancies. In order to investigate whether these changes are associated with biological behaviour of the tumours, 20 cervical carcinomas were analyzed for MHC (HLA-A, HLA-B/C, HLA-DR) and TAP-1 expression in the primary tumours and in lymph node metastases by immunohistochemistry. The results showed a significant increase in the prevalence of HLA-A and HLA-B/C down-regulation in metastasised neoplastic cells as compared with the primary tumour (P = 0.01). In all cases this was accompanied by loss of TAP-1 expression. Up-regulated HLA-DR expression was found exclusively in primary tumours and was absent in the corresponding metastases (P = 0.002). These data are consistent with the hypothesis that loss of TAP-1 and the consequent down-regulation of class I MHC expression provides a selective advantage for neoplastic cervical cells during metastasis. Furthermore, the lack of class II MHC expression in metastasised cells either reflects a different local lymphokine production or indicates that these cells may have escaped CD4+ cytotoxic T-lymphocyte (CTL)-mediated killing.

Analysis of MHC class I and II expression in relation to presence of HPV genotypes in premalignant and malignant cervical lesions

Cervical intraepithelial neoplasia (CIN) grades I to III lesions (n = 94) and squamous cell carcinomas of the uterine cervix (n = 27) were analysed for MHC class I and II expression and presence of HPV genotypes. MHC class I and II expression was studied by immunohistochemistry and HPV typing was performed by general primer- and type-specific primer mediated PCR (GP/TS PCR). Both techniques were performed on paraffin embedded tissue sections. Results show disturbed MHC class I heavy chain expression in CIN I to CIN III, as well as in cervical carcinomas. Upregulated MHC class II expression on dysplastic epithelial cells was also found in the different CIN groups and carcinomas. Prevalence of HPV genotypes increased with the severity of the lesion, mainly due to the contribution of the HPV types 16 and 18. No correlation could be established between the presence of specific HPV genotypes and any MHC expression pattern in the different CIN groups or cervical carcinomas. In some cases these data were confirmed by RNA in situ hybridisation showing HPV 16 E7 transcripts in the same dysplastic/neoplastic cells from which MHC status was determined. The results indicate that local differences may exist in the type of cellular immune response to HPV induced lesions.

Immune responses against human papillomavirus (HPV) type 16 virus-like particles in a cohort study of women with cervical intraepithelial neoplasia. I. Differential T-helper and IgG responses in relation to HPV infection and disease outcome.

T-helper (Th) cell-dependent IL-2 production and plasma IgG responses to virus-like particles consisting of the human papillomavirus type 16 (HPV-16) major capsid protein L1 (L1-VLP) were determined in patients with cytological evidence of cervical intraepithelial neoplasia (CIN) participating in a non-intervention prospective cohort study. IgG responses were associated with HPV-16 persistence and high-grade CIN lesions, while high frequencies of Th responses were observed in patients with both virus clearance and virus persistence, irrespective of CIN grade. The IgG response was found in conjunction with an IL-2 response to L1-VLP in 87% of the patients. Recognition of the HPV-16 L1 Th epitope (amino acids 311-335) was found to be more closely associated than recognition of L1-VLP as a whole to HPV exposure and CIN development. Among the HPV-16+ patients included in this study, those showing a Th response to amino acids 311-335 were more likely to carry the HLA DRB1*11/DQB1*0301 haplotype, while those showing an IgG response to L1-VLP were more likely to carry DRB1*0101/DQB1*0501. However, neither cell-mediated nor humoral immune responses against HPV-16 L1 appear to be sufficient for the natural control of HPV infection and CIN development.

Human papillomavirus DNA and genotypes: prognostic factors for progression of cervical intraepithelial neoplasia.

A retrospective study of 227 patients presenting with abnormal cervical cytology was conducted to investigate the relationship between human papillomavirus (HPV) and progression of untreated cervical intraepithelial neoplasia (CIN) lesions. All patients had colposcopically directed biopsies for histologic diagnosis. The patients were followed cytologically and colposcopically for a mean of 19 months (range 6–42 months). Progression of a cervical lesion was defined as progression to a higher CIN grade confirmed histologically by directed biopsy. HPV DNA detection was done on material remaining from the cervical swabs by the general primer polymerase chain reaction (PCR) and type-specific PCR method, which made the detection of HPV types 6, 11, 16, 18, 31, 33 and not yet sequenced DNA types (X) possible. The presence of HPV DNA increased with the severity of the lesion (P < 0.001). In CIN III, a 100% HPV DNA prevalence was found, with HPV type 16 being the most prevalent type in 75%. Progression was significantly related to the presence of HPV DNA, in particular HPV type 16. The percentage of progressive disease was 21% in the case of HPV DNA positive lesions (n = 130) and 29% in the presence of HPV type 16, whereas HPV DNA negative lesions (n = 97) showed no progression. The detection of HPV DNA and HPV genotype can be used to identify patients with high-risk cervical lesions, since the presence of HPV DNA and genotype 16 in particular are closely related to CIN progression.

High risk human papillomavirus in women with normal cervical cytology prior to the development of abnormal cytology and colposcopy

Objective To study the significance of the presence of high risk human papillomavirus (HPV) in women with initially normal cervical cytology for the development of abnormal cytology and an abnormal colposcopic impression.

Papillomaviruses and Cancer of the Upper Digestive and Respiratory Tracts

At present human papillomaviruses (HPVs) are linked to up to 10% of the worldwide cancer burden, mainly due to their involvement in anogenital cancer. Accumulating experimental data on cervical squamous cell carcinoma strongly support the assumption that certain HPV types have carcinogenic potential and are causally related to cervical cancer (zur Hausen 1991). H PV-16 and HPV-18, the major types associated with cervical cancer, exhibit transforming and immortalizing functions in primary rodent cells and human keratinocytes, respectively (Matlashewski et al. 1987; Pecoraro et al. 1989; Pirisi et al. 1987), and the viral E6 and E7 open reading frames (ORFs) have been found to encode the HPV-transforming proteins (Barbosa and Schlegel 1989; Hawley-Nelson et al. 1989; Munger et al. 1989a). The HPV-16 and HPV-18 E6 and E7 proteins can form complexes with the tumor suppressor gene products p53 and pRB, respectively (Werness et al. 1990; Munger et al. 1989b), which at least in part may explain at what level these HPV types interfere with cell cycle control mechanisms. However, viral E6–E7 functions are not sufficient for the development of malignant growth, and the additional modification of host cell genes by chemical factors has been considered to be important as well (zur Hausen 1982, 1989). Owing to this hypothesis, interest has been provoked for a role of HPV in the pathogenesis of carcinomas within the upper digestive and respiratory tracts since these tumors are known to be related to chemical factors such as tobacco and alcohol. Worldwide, squamous cell carcinoma of the ororespiratory tract, especially that of the lung, is the major form of cancer in humans. Therefore, efforts to study the relationship of specific HPV types with carcinomas of this tract are justified.

HPV typing and testing in gynaecological pathology: has the time come?

Herrington and Wells in the October 1997 issue of Histopathology present an interesting overview of the potential value of human papillomavirus (HPV) typing in order to predict the behaviour of cervical epithelial neoplasia (CIN). They reach the conclusion ‘that current evidence does not support the introduction of routine HPV typing in histopathology or cytology’ and that ‘the association of HPV infection with significant cervical disease warrants further investigation.’ We have a more optimistic view on HPV testing in gynaecological pathology. Results from recent casecontrol studies show (1) a close association between high risk type HPV infection and the development of cervical cancer with relative risks that range from 60– 130 and (2) that the risk for women to develop cervical cancer does not differ significantly for the different high risk HPV types. This indicates that individual HPV typing, as discussed extensively by Herrington and Wells, is not necessary and not useful. Therefore, we advocate ‘high risk HPV testing’, i.e. the application of a general high risk HPV polymerase chain reaction (PCR) test that identifies the complete spectrum of the high risk HPV group. In our opinion, much of the debate about whether HPV testing should be used in histopathology and cytopathology depends on whether the HPV test performed has enough analytical sensitivity to detect high grade CIN and carcinomas. If the sensitivity does not reach the femtogram level, at least 20% of the high grade cervical lesions and the cervical carcinomas will be missed. Techniques which can be used on a large scale and that do reach this sensitivity are PCR-based tests and probably also the latest version of the hybrid capture assay (not yet available for routine screening). Using PCR-based techniques the following findings are important for the clinical relevance of high risk HPV detection in cervical smears and biopsies.

Prevalence of Epstein-Barr virus in oral squamous cell carcinomas, premalignant lesions and normal mucosa : a study using the polymerase chain reaction

Epstein-Barr virus (EBV) prevalence was assessed in 12 clinically normal oral mucosas, nine premalignant lesions, 36 oral squamous cell carcinomas (OSCCs) and a human papillomavirus (HPV) 16 positive cell line, derived from an OSCC studied. The polymerase chain reaction (PCR) with two pairs of primers with different sensitivities was used. With primers specific for the BamHIW repeat, EBV was found in 100% of OSCCs, in 77.8% of premalignant lesions and in 8.3% of clinically normal oral mucosas (P = 0.0001). Using primers specific for the single copy BNLF-1 gene, EBV was detected in 50% of OSCC and in none of the premalignant lesions. No statistically significant associations could be established among EBV presence and clinico-pathological data of OSCC. The cell line derived from an HPV/EBV-positive carcinoma did not reveal EBV DNA. The higher prevalence of EBV in OSCCs and premalignant lesions may be due to increased EBV shedding, possibly due to associated immunodepression in these patients, rather than its clonal presence in the neoplastic cells.