Detection of human papillomavirus using hybrid capture 2 in oral brushings from patients with oropharyngeal squamous cell carcinoma

Detection of high-risk (HR) human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (SCC) has important prognostic implications; patients exhibit improved survival compared with patients with HPV- SCC. Oral brushing and rinsing samples were obtained from patients with oropharyngeal, oral cavity, or hypopharyngeal SCC and tested for HR-HPV using Hybrid Capture 2 (HC2; QIAGEN, Valencia, CA). HR-HPV in situ hybridization (ISH) was performed on biopsy tissue samples from the same patients. Oral cytologic samples from 16 SCCs were tested by HC2. Biopsy tissue samples were available for ISH in 11 cases. Five oropharyngeal SCCs were HR-HPV+ by ISH and HC2 (oral brushing). Of the oropharyngeal SCCs, 2 were positive by HC2 (oral brushing) and negative or equivocal by ISH. We found that 2 oral cavity carcinomas and 2 hypopharyngeal carcinomas were negative by HC2. One hypopharyngeal cancer was positive by ISH. All oral rinsing samples were negative by HC2. HC2 may be an effective method of determining HR-HPV status in patients with oropharyngeal SCC.     

Nuclear translocation of β‐catenin and decreased expression of epithelial cadherin in human papillomavirus‐positive tonsillar cancer: an early event in human papillomavirus‐related tumour progression?

Stenner M, Yosef B, Huebbers C U, Preuss S F, Dienes H‐P, Speel E‐J M, Odenthal M & Klussmann J P (2011) Histopathology 58 , 1117–1126 Nuclear translocation of β‐catenin and decreased expression of epithelial cadherin in human papillomavirus‐positive tonsillar cancer: an early event in human papillomavirus‐related tumour progression? Aims: High‐risk human papillomaviruses (HPVs) constitute an important risk factor for tonsillar cancer. This study describes changes in cell adhesion molecules during metastasis of HPV‐related and HPV‐unrelated tonsillar carcinomas. Methods and results: We examined 48 primary tonsillar carcinoma samples (25 HPV‐16 DNA‐positive, 23 HPV‐16 DNA‐negative) and their respective lymph node metastases for their HPV status and for the expression of p16, epithelial cadherin (E‐cadherin), β‐catenin, and vimentin. A positive HPV‐specific polymerase chain reaction finding correlated significantly with p16 overexpression in both primary tumours and their metastases (P < 0.0001 for both). In HPV‐unrelated carcinomas, the expression of E‐cadherin was significantly lower in metastases than in primary tumours (P < 0.001). In contrast, the expression of nuclear β‐catenin was significantly higher in metastases than in primary tumours (P = 0.016). In HPV‐related carcinomas, nuclear localization of β‐catenin expression was already apparent in primary tumours (P = 0.030). The expression of vimentin significantly correlated with the grading of the primary tumour (P = 0.021). Conclusions: Our data indicate that the down‐regulation of E‐cadherin and the up‐regulation of nuclear β‐catenin expression might be crucial steps during tumour progression of tonsillar carcinomas, being already present in primary tumours in HPV‐driven carcinomas, but becoming apparent in HPV‐unrelated tumours later in the process of metastasis.     

Incidence of human papillomavirus in oropharyngeal squamous cell carcinomas: now and 50 years ago

The increasing incidence of oropharyngeal squamous cell carcinomas is believed to be due to the growing proportion of human papillomavirus-positive oropharyngeal squamous cell carcinoma. To examine the time trend in the incidence of human papillomavirus, oropharyngeal squamous cell carcinomas diagnosed from 1956 to 1969 (n = 43) and from 2007 to 2009 (n = 54) were tested by p16 immunohistochemistry and in situ hybridization for human papillomavirus. Morphologically, in these 2 time periods, the predominant type of oropharyngeal squamous cell carcinoma changed from keratinizing to nonkeratinizing, with increase in nonkeratinizing cases from 28% to 67% (P < .001). Also, there was an increase in surgical resections versus biopsies (11% versus 46%; odds ratio, 6.6; P < .001) and base of tongue versus tonsillar location (20% versus 40%; odds ratio, 2.6; P = .04). The proportion of human papillomavirus-positive oropharyngeal squamous cell carcinoma increased from 35% to 72% (odds ratio, 4.9; P < .001). This increase was most apparent in men (odds ratio, 4.2; P < .001). This study provides the earliest historic baseline for human papillomavirus incidence in oropharyngeal squamous cell carcinoma and may serve as a reference point for evaluating the results of human papillomavirus infection preventive measures, such as human papillomavirus vaccination.     

Human papillomavirus DNA sequences and p53 over-expression in laryngeal squamous cell carcinomas in Northeast China

One-hundred-two patients with laryngeal squamous cell carcinomas in Northeast China were examined for human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR) coupled with Southern blot hybridization, and for p53 over-expression by immunohistochemical staining. HPV DNAs were found in 60 cases (58.8%). HPV-16, -18, -6, -11, and -33 DNAs were detected in 30 cases, 22 cases, 25 cases, two cases, and one case, respectively. In addition, coinfection either with HPV-6 and -16 or with HPV-6 and -18 was detected in 20 cases (33.3% of HPV DNA-positive cases). p53 over-expression was observed in 60 patients (58.8%). p53 was over-expressed significanty in the poorly-differentiated SCC and in patients with metastasis to lymph nodes (P < 0.05, respectively). Both HPV DNA and p53-expression were positive in 35 patients, and negative in 17 patients. Either HPV DNA or p53-expression were positive in 50 patients (25 cases each). Although p53 was detected in 35 (58.3%) of HPV-positive patients, there was no significant correlation between HPV infection and p53 over-expression in laryngeal squamous cell carcinomas of Northeast China. J. Med. Virol. 54:186–191, 1998. © 1998 Wiley-Liss, Inc.     

The relationship between p16 expression and high-risk human papillomavirus infection in squamous cell carcinomas from sites other than uterine cervix: a study of 137 cases

p16 is known to be an excellent surrogate marker of human papillomavirus infection in squamous cell carcinoma of the cervix. Recent studies have demonstrated a link between human papillomavirus infection and a subset of head and neck squamous cell carcinomas, especially from the oropharynx. The aims of this study were to determine the incidence of p16 expression in squamous cell carcinomas of noncervical origin and to assess its utility as a surrogate marker of human papillomavirus infection in various noncervical primary sites. One hundred thirty-seven squamous cell carcinomas from 5 primary sites, including 34 from the oropharynx (tonsil and base of tongue), 43 cases from nonoropharyngeal head and neck sites, and 20 cases each from the lung, esophagus, and skin, were retrieved from our surgical pathology archives. Immunohistochemistry for p16 was performed on each case. All p16-positive cases and 21 p16-negative cases were further tested for both high-risk and low-risk human papillomavirus by in situ hybridization. p16 expression was detected in 54 cases overall, including 25 (74%) of 34 oropharyngeal squamous cell carcinomas, 8 (19%) of 43 nonoropharyngeal head and neck squamous cell carcinomas including 3 of 4 from the sinonasal cavity, 6 (30%) of 20 esophageal squamous cell carcinomas, 7 (35%) of 20 lung squamous cell carcinomas, and 8 (40%) of 20 skin squamous cell carcinomas. Of the 54 p16-positive cases, 30 were positive for high-risk human papillomavirus, including 24 (96%) of 25 from the oropharynx, 5 (63%) of 8 from nonoropharyngeal head and neck sites, and 1 (17%) of 6 from the esophagus. All 7 lung and 8 skin cases tested were negative. All p16-positive cases were negative for low-risk human papillomavirus. In selected head and neck squamous cell carcinomas, mainly from the oropharynx and sinonasal cavity, p16 positivity correlates well with high-risk human papillomavirus infection. p16 is not a reliable indicator of high-risk human papillomavirus infection in squamous cell carcinomas of the lung, skin, and esophagus.     

Different numerical chromosomal aberrations detected by FISH in oropharyngeal, hypopharyngeal and laryngeal squamous cell carcinoma

AIMS: Little information is available about stage- or site-specific chromosomal aberrations in head and neck squamous cell carcinoma (HNSCC). We tried to identify whether different patterns of chromosomal gain or loss in squamous cell carcinomas were associated with different stages or head and neck sites. METHODS AND RESULTS: We investigated isolated interphase cells from paraffin sections of 5 3 squamous cell carcinomas of the head and neck region by fluorescence in situ hybridization techniques. We used centromeric DNA probes for chromosomes 1, 3, 4, 6, 7, 9, 10, 11, 12, 15, 17, 18, X and Y. The majority of tumour samples showed aneuploidy for most chromosomes analysed. We were able to find differences in the chromosomal aberrations between the tumour sites and stages of the HNSCC investigated. The main numerical chromosomal abnormalities were an under-representation of chromosomes 3 (26%), 6 (17%), 9 (26%), 10 (23%) and 18 (32%). The Y chromosome was lost in 53% of male tumours. A loss of chromosomes 3 and 10 was detected mostly in laryngeal SCC (39% and 30%, respectively); the under-representation of chromosome 9 was predominantly seen in oropharyngeal SCC (54%) and a copy number decrease of chromosomes 18 was found in 57% of hypopharyngeal tumours. CONCLUSION: Although the number of tumour samples investigated is rather low, our results suggest that the different tumour sites of HNSCC may also show different patterns of chromosomal changes.     

PROPERTIES OF HPV-POSITIVE AND HPV-NEGATIVE ANAL CARCINOMAS

Evidence of human papillomavirus (HPV) can be found in up to 85 per cent of anal carcinomas. In the vulva, a discrete subset of HPV-positive carcinomas which show koilocytic morphology and distinct clinical features has recently been identified (warty carcinoma). The morphological and prognostic features of HPV-positive and HPV-negative anal carcinomas were compared in this study of the tumour distribution of HPV DNA. Vulval and anal neoplasia are similar in many ways and we have also looked to see if their similarity extends to ‘warty’ morphology in relation to HPV status. Thirty-five resection specimens of anal carcinoma were examined with biotin-labelled probes for HPV 6, 11, 16, and 18 DNA, using a non-isotopic in situ hybridization (ISH) technique. No tumour was found to contain HPV 6, 11, or 18. Twenty-four (72 per cent) showed positivity for HPV 16 DNA. Staining was homogenous and independent of local squamous, basaloid, or ductal differentiation. The majority of tumours showed staining suggestive of episomal, non-productive HPV infection. HPV-positive tumours were more likely to occur in the anal canal than perianally and to show a mixed squamous and basaloid appearance. No difference between the two groups was found in patient age, presence of adjacent dysplasia, ductal differentiation, or prognosis. There was no correlation between condylomatous tumour morphology and HPV 16 DNA positivity; thus, a subset equivalent to vulval warty carcinoma could not be identified.     

Skin cancer and papillomaviruses in cattle

We examined proliferative lesions on the sun-exposed, unpigmented skin of 13 cattle. Ages of animals at first examination ranged from 4 to 15 years, and 4 were observed for from one to 3 years, during which time progression to malignancy occurred in 2 of them. Early lesions consisted of keratin scales and horns; histology showed underlying acanthosis and hyperkeratosis. Advanced lesions were either squamous cell carcinomas or basaloid tumours with sebaceous and/or squamous differentiation; some were locally invasive but no metastases were found in the 6 animals that were available for necropsy. All 3 types of lesion could occur on the same animal. In early lesions from 11 of 12 animals, there was evidence for the presence of papillomavirus, either virions or viral DNA, the latter detected by gel electrophoresis and/or molecular hybridization. Viral DNA was also detected in 3 basaloid tumours and 2 squamous cell carcinomas from 4 animals. The DNA bound to a probe of bovine papillomavirus type 1 DNA under conditions of low stringency. We suggest that both infection with papillomavirus and exposure to sunlight, possibly in conjunction with other factors such as a period of photosensitization, are involved in the production of this spectrum of proliferative lesions, which bear some resemblance to human skin cancer.     

The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma

Stankiewicz E, Prowse D M, Ktori E, Cuzick J, Ambroisine L, Zhang X, Kudahetti S, Watkin N, Corbishley C & Berney D M
(2011) Histopathology 58 , 433–439
The retinoblastoma protein/p16 ^INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma Aims: The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood. Human papillomavirus (HPV) may be involved in carcinogenesis, but few studies have compared cell‐cycle protein expression in HPV positive and negative cancers. The aim was to determine the extent of HPV infection in different histological subtypes of PSCC and its impact on the expression of key cell‐cycle proteins: p53, p21, p16^INK4A and retinoblastoma (RB) protein. Methods and results: One hundred and forty‐eight PSCC samples were examined immunohistochemically for RB, p16^INK4A, p53 and p21 protein expression. One hundred and two cases were typed for HPV by PCR. HPV DNA was detected in 56% of tumours, with HPV16 present in 81%. Basaloid tumours were related strongly to HPV infection (10 of 13), while verrucous were not (three of 13). Fifty‐nine per cent (38 of 64) of usual type SCCs had HPV infection. RB protein correlated negatively (P < 0.0001) and p16^INK4A (P < 0.0001) and p21 (P = 0.0002) correlated positively with HPV infection. p53 did not correlate with HPV infection. Conclusions: HPV infection is present in more than half of penile cancers and it is responsible for RB pathway disruption. However, no link between HPV and p53 immunodetection was found. Only basaloid and half of usual‐type PSSCs correlate with HPV infection, confirming possible separate aetiologies for those tumours.     

The prevalence of human papillomavirus in the oropharynx in healthy individuals in a Brazilian population

Oncogenic human papillomavirus (HPV), a causative agent of uterine cervical cancer, has also been detected in head and neck squamous cell cancers, especially in squamous cell carcinomas of the tonsils. However, the true HPV prevalence in normal and neoplasic oropharyngeal mucosa remains uncertain. To determine the prevalence of HPV DNA in normal oropharyngeal mucosa of cancer-free individuals, a study was carried out on 50 Brazilian subjects. PCR was performed to identify HPV DNA in samples from four sites in the oropharynx (tonsils, soft palate, base of the tongue, and back wall of the pharynx). For amplification of the HPV DNA, MY09/11 consensus primers were used, and specific genotypes were identified by dot-blot hybridization or cloning and sequencing. HPV DNA was present in 14.0% of the individuals, and the identified genotypes were 16, 18, 52, and 61. All these types are considered high-risk (HR) HPV. The tonsils and the soft palate were the sites with the highest HPV prevalence. This study shows the prevalence of HR HPV in the oropharynx of normal individuals. However, the prevalence of HPV is still unclear, and if HPV infection in a healthy it is not known individual predisposes to HPV-associated disease such as oropharyngeal cancer. Thus, it is important to assess the prevalence of HPV in cancer-free individuals, in order to compare it with the HPV prevalence in oropharyngeal carcinomas and to attempt to determine the true role of HPV in the development of head and neck squamous cell cancers.     

Dual-colour chromogenic in-situ hybridization is a potential alternative to fluorescence in-situ hybridization in HER2 testing

Hwang C‐C, Pintye M, Chang L‐C, Chen H‐Y, Yeh K‐Y, Chein H‐P, Lee N & Chen J‐R
(2011) Histopathology 59 , 984–992 Dual‐colour chromogenic in‐situ hybridization is a potential alternative to fluorescence in‐situ hybridization in HER2 testing Aim: Dual‐colour chromogenic in‐situ hybridization (dc‐CISH) is an emerging methodology for characterizing genomic alterations. This study was aimed at evaluating the performance of a dc‐CISH kit (ZytoVision) in determining human epidermal growth factor receptor 2 (HER2) status in breast cancer. Methods and results: Two hundred and twenty‐eight invasive breast carcinomas arranged in tissue microarrays were analysed in parallel with dc‐CISH, fluorescence in‐situ hybridization (FISH), and immunohistochemistry. Of 227 tumours with available FISH and dc‐CISH results, HER2 amplification and non‐amplification were detected in 49 (21.6%) and 178 (78.4%) tumours, respectively, by both assays. The concordance between dc‐CISH and FISH results showed 100% agreement (κ‐coefficient = 1.00). Immunohistochemically, 162 (71%), 25 (11.0%) and 41 (18%) tumours were scored 0/1+, 2+, and 3+, respectively. The corresponding results with both FISH and dc‐CISH demonstrated HER2 amplification in two (3.2%), nine (36%) and 38 (93%) tumours, respectively. Complete consensus among these three methods was observed in 197 cases, representing 98% of all 3+ and 0/1+ tumours (κ‐coefficient = 0.92). Confirmatory testing of 25 2+ tumours showed complete consensus between FISH and dc‐CISH. Conclusions: dc‐CISH is a promising alternative to FISH in HER2 testing, and the single‐institute incidence of HER2 amplification in breast cancer in Taiwan is 21.2%.     

Association between human papillomavirus infection and laryngeal squamous cell carcinoma

The aim of this study was to compare the prevalence of human papillomavirus (HPV) infection in laryngeal squamous cell carcinoma using two methods: PCR‐DNA enzyme immunoassay (PCR/DEIA) and immunohistochemistry (IHC) for detection of HPV in specimens of laryngeal squamous cell carcinoma and to correlate the presence of HPV with the epidemiological and clinicopathological features of recurrence and survival. HPV DNA was amplified from 93 paraffin‐embedded laryngeal squamous cell carcinoma tissue specimens by the short PCR fragment (SPF 10) primer set using PCR/DNA method. HPV detection using monoclonal anti‐human papilloma virus antibodies Clone K1H8 for IHC reaction was performed on 130 specimens. HPV was identified in 35.5% of patients with laryngeal squamous cell carcinoma using PCR/DEIA and 27.7% using IHC. There was no statistically significant association between the presence of HPV and the epidemiological and clinicopathological features and recurrence. There was no statistically significant association between the presence of HPV and overall survival nor disease specific survival. Statistically significant correlation between HPV detection using PCR/DEIA technique and IHC technique was found. The presence of HPV infection in 27.7% and 38.9% of the patients suggests a possible role in the etiology of laryngeal squamous cell carcinoma. The SPF₁₀ PCR/DEIA technique is the most accurate method for detection of HPV in laryngeal squamous cell carcinoma. J. Med. Virol. 82:1017–1023, 2010. © 2010 Wiley‐Liss, Inc.     

Human papillomavirus-positive tonsillar carcinomas: a different tumor entity?

Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyer's tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site ( P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per beta-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading ( P=0.008) and alcohol consumption ( P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.     

两种检测头颈部鳞状细胞癌HPV16/18感染状态方法的对比

 目的 比较荧光定量PCR法（RT-PCR）和原位杂交法检测头颈部鳞状细胞癌中HPV（HPV）16/18亚型感染的差异。方法 采用RT-PCR法和原位杂交法对78例头颈部鳞状细胞癌患者的肿瘤组织进行HPV感染状态的检测,评价两种方法的一致性。结果 RT-PCR法检测到62.8%的头颈部鳞癌组织中含有HPV16/18 DNA。原位杂交法检测到47.4%的肿瘤组织中有HPV16DNA。用RT-PCR方法,唇、口腔、口咽及下咽的HPV16/18DNA阳性率分别为33.3%、66.67%、70%和57.14%;用原位杂交方法,唇、口腔、口咽、下咽的HPV16 DNA阳性率分别为33.3%、43.8%、60.0%和57.1%。总体上,两种方法检测HPV16/18DNA具有较高一致性（Kappa=0.595,P<0.001）。 结论 RT-PCR和原位杂交法检测HPV16/18DNA结果的一致性较高。     

Adjuvant targeted therapy with trastuzumab may decrease metastatic capacity in specific group of oropharyngeal cancer patients: downregulation of E-cadherin-catenin complex by cooperative effect of erbB-2 and human papillomavirus type 16 E6/E7 protooncogenes

Human papillomavirus (HPV) type 16 is causally associated with a subset of oral cancers, predominantly those cancers arising in the oropharynx (OP). Increased HPV16 E6 and E7 oncogene expressions are responsible for the malignant transmission in these cancers. ErbB-2 is the family member most closely implicated in human cancer, where it is overexpressed in about 30% of carcinomas including head and neck squamous cell carcinoma. Coexpressions of E6/E7 and ErbB-2 downregulate E-cadherin and catenin expression, therefore induces metastatic process. Trastuzumab is a humanized monoclonal antibody that recognizes the ErbB-2 protein receptor and breakthrough in the treatment of metastatic breast cancer in combination with chemotherapeutic agents. This antibody is also in clinical testing for adjuvant treatment of breast cancer. We propose that trastuzumab as an adjuvant treatment may decrease process of tumor metastasis in oropharyngeal cancer patients who completed primary treatment (surgery and/or radiotherapy) and show expression of both HPV16 E6/E7 and erbB-2 oncoproteins. In vitro and in vivo studies with trastuzumab in these subgroup of patients may support our hypothesis.     

Expression of the pro-opiomelanocortin gene in lung neuroendocrine tumours: In situ hybridization and immunohistochemical studies

Neuroendocrine tumours of the lung may be associated with the ectopic adrenocorticotrophin (ACTH) syndrome and may synthesize and secrete ACTH-related peptides in the absence of the syndrome. However, immunocytochemical analysis may not confirm these biochemical findings, particularly in small cell carcinoma, which is poorly granulated. To investigate further the morphological evidence for expression of the pro-opiomelanocortin (POMC) gene in neuroendocrine lung tumours, we have examined a series of 46 small cell carcinomas and 13 carcinoid tumours of the lung by in situ hybridization for POMC mRNA using a digoxigenin-labelled oligoprobe. We have compared the findings with the immunocytochemical detection of ACTH and β-endorphin. In situ hybridization was positive in 15 of 46 small cell carcinomas (33 per cent) and in 8 of 13 carcinoid tumours (62 per cent). Immunocytochemical staining was positive in only one carcinoid tumour. These in situ hybridization studies have corroborated biochemical data indicating POMC gene expression in a high proportion of lung neuroendocrine tumours. This suggests that the low levels of expression detected by immunocytochemistry may be due to low levels of hormone storage. Multivariate analysis showed a weak negative association between POMC expression and survival in small cell carcinomas, although this did not reach statistical significance.     

p16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Klingenberg B, Hafkamp H C, Haesevoets A, Manni J J, Slootweg P J, Weissenborn S J, Klussmann J P & Speel E‐J M
(2010) Histopathology 56, 957–967
p16 ^INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV Aims: Oncogenic human papillomavirus (HPV) type 16 has been strongly associated with tonsillar squamous cell carcinoma (TSCC) and appears to be of prognostic significance. Because HPV+ TSCC also accumulates p16^INK4A, this cyclin‐dependent kinase inhibitor has been proposed as a potential biomarker for HPV in clinical diagnosis. The aim of this study was to determine the prevalence of HPV in tumour‐free tonsillar tissue and the value of p16^INK4A overexpression in predicting its presence. Methods and results: p16^INK4A overexpression was detected by immunohistochemistry in tissue sections of tumour‐free tonsils of 262 patients. They were treated for non‐oncological reasons (snoring or chronic/recurrent tonsillitis) consisting of tonsillectomy. Genomic DNA isolated from these tissues was subjected to HPV‐specific polymerase chain reaction (PCR) analysis. p16^INK4A immunoreactivity was detected in 28% of samples in both crypt epithelium (49/177) and lymphoid germinal centres (52/187), which correlated with each other (P < 0.0001). No reactivity was observed in superficial squamous cell epithelium. HPV16 and 18 were detected by PCR analysis in 2/195 cases (1%), which, however, were negative on fluorescence in situ hybridization analysis and discrepant on p16^INK4A immunostaining. Conclusions: No proof was found for the presence of HPV in tumour‐free tonsil tissue, despite increased p16^INK4A expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16^INK4A up‐regulation.     

Human papillomavirus DNA in oesophageal carcinomas in South Africa

Using morphological criteria, the presence of human papillomavirus (HPV) in oesophageal carcinomas has been inferred in patients from Finland and South Africa. However, studies to demonstrate the viral antigen in tissue sections of these tumours have proved disappointing. This study investigates 48 archival oesophageal carcinoma biopsies from South Africa for the presence of HPV DNA using non-isotopic in situ hybridization (NISH) with HPV DNA probes to HPV 6, 11, 16, 18, 31, and 33. HPV DNA sequences were detected in 25/48 (52 per cent) oesophageal cancers. HPV 16 was present in 84 per cent of the HPV-positive cancers. A NISH type 2 signal pattern (punctate/dot) was present in all HPV-positive tumours. This signal pattern was previously shown to represent integrated HPV DNA within host chromosome. Integrated HPV DNA in oesophageal cancers has also been demonstrated in patients from China and Japan. In addition, the prevalence of HPV DNA in oesophageal cancers from high-risk countries like South Africa (52 per cent) and China (49 per cent) would appear to be consistent.