Petrus Josephus Ferdi Snijders

Frequency of down-regulation of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1 expression

The development of cervical carcinoma is strongly associated with specific types of human papillomaviruses (HPVs). A role for cellular immunity in cervical disease is supported by the increased occurrence of HPV-associated lesions in immunosuppressed individuals. Upon viral infection or malignant transformation, ensuing alterations in gene expression result in the generation of novel sets of peptides which can form complexes with specific HLA class I heavy chains and beta 2-microglobulin. These are then expressed at the cell surface as potential targets for specific T cells. In this study of 100 carcinomas HLA-A and -B class I expression by the tumour cells was down-regulated at one or more alleles in at least 73% of cervical carcinomas. Interference with the transporter associated with antigen presentation (TAP), which translocates cytosolic peptides from endogenously synthesised proteins (e.g. viral) into the lumen of the endoplasmic reticulum was found in 38% of the HLA class I down-regulated tumours. Loss of expression for common HLA class I alleles ranged from 36% to 71%, and such changes might be expected to influence specific immunogenic peptide presentation and consequent immune recognition. These results underline the importance of single as well as multiple allelic loss in cervical neoplasia and have important implications for attempts to intervene immunologically in cervical cancer.

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.

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.

Specific p53 immunostaining patterns are associated with smoking habits in patients with oral squamous cell carcinomas

Aims: To identify immunostaining patterns that are predictive for p53 mutations and to investigate whether p53 mutations are associated with established risk factors for oral squamous cell carcinoma (OSCC). Methods: Fifty five OSCCs were investigated for p53 protein expression by immunohistochemistry (IHC). Ten of these cases, including five p53 immunopositive and five p53 immunonegative cases, were subjected to microdissection of representative tumour areas followed by sequence analysis for the detection of TP53 mutations. Results: Paired IHC and sequence analysis revealed that p53 immunoexpression in more than 25% of tumour cells was indicative of TP53 mutations, whereas p53 immunonegativity was not informative. Therefore, for p53 immunohistochemical interpretation, p53 immunonegative cases were excluded from the analysis and the cut off value for p53 immunoexpression was set at 25%. Of the OSCCs showing any p53 immunoexpression, 64% revealed staining in more than 25% of the tumour cells. p53 immunoexpression in more than 25% of the neoplastic cells was significantly associated with smoking but not with alcohol consumption. No significant association with smoking habits was found when OSCCs were dichotomised into p53 immunonegative and p53 immunopositive. Conclusions: In OSCCs the following conclusions can be made: (1) p53 immunonegativity is not informative for TP53 mutations; (2) 25% p53 immunopositive cells appears to be a good cut off value to predict TP53 mutations; (3) p53 immunostaining patterns that appeared to be predictive for TP53 mutations were associated with the smoking habits of the patients.

No direct role for Epstein-Barr virus in oral carcinogenesis: a study at the DNA, RNA and protein levels

Reports on the association of EBV with oral squamous‐cell carcinomas (OSCCs) are scarce and inconclusive. To determine the potential role of EBV in oral carcinogenesis, we investigated 36 EBV DNA PCR‐positive OSCCs for the expression of EBV transcripts and proteins. From these EBV DNA‐positive OSCCs, 13 were analysed for the presence of EBV products, either at RNA and/or protein level. EBER transcripts were investigated by RNA in situ hybridisation. EBNA‐1, EBNA‐2, LMP‐1, LMP‐2, BHRF1 and BARF0 transcripts were investigated by RT‐PCR and/or NASBA. EBNA‐1, LMP‐1 and ZEBRA protein expressions were investigated by immunohistochemistry. All 36 OSCCs were positive for EBV DNA, using the highly sensitive BamHI W PCR, and 18 of these (50%) were positive using the less‐sensitive PCR, which targets BNLF‐1. However, virtually all OSCCs tested failed to reveal EBV transcripts, including EBERs and EBNA‐1 transcripts. No ZEBRA and LMP‐1 proteins were found in the neoplastic or any other cells of the OSCCs investigated. Immunohistochemistry using a monoclonal antibody (MAb) raised against EBNA‐1 (2B4) resulted in positive staining in some cases of OSCCs, but these results were non‐specific, since EBV‐negative epithelial tissues showed extensive non‐specific staining and no EBNA‐1‐specific transcripts were detected by RT‐PCR or NASBA. The absence of expression of EBV encoded transcripts and proteins indicate that, with the present knowledge on EBV, an active role in oral carcinogenesis for this virus is unlikely. Int. J. Cancer 86:356–361, 2000.

Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and β2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of β2-m on the cell surface. The absence of β2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal β2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response.