Jon M. Bible

Evaluation of human papillomavirus testing for squamous cell carcinoma of the tonsil in clinical practice

Background Oncogenic human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC) is a subtype of head-and-neck cancer with a distinct clinical and prognostic profile. While there are calls to undertake HPV testing for oropharyngeal SCCs within the diagnostic setting and for clinical trials, there are currently no internationally accepted standards. Methods 142 tonsil SCCs were tested using p16 immunohistochemistry (IHC), high-risk HPV DNA in situ hybridisation (ISH) and HPV DNA polymerase chain reaction (PCR; GP5+/6+ primers). Results There were high levels of agreement between pathologists for p16 IHC and HPV ISH scoring; however, around 10% of HPV ISH cases showed some interobserver discrepancy that was resolved by slide review. The combination of p16 IHC and HPV ISH classified 53% of the samples as HPV-positive, whereas the combination of p16 IHC and HPV PCR classified 61% of the samples as HPV-positive. By employing a three-tiered, staged algorithm (p16 IHC/HPV ISH/HPV PCR), the authors were able to classify 98% of the cases as either HPV-positive (p16 IHC+/HPV DNA+; 62%) or HPV-negative (p16 IHC−/HPV DNA−; 35%). Conclusions The current study suggests that using a combination of p16 IHC/HPV ISH/HPV PCR, in a three-tiered, staged algorithm, in conjunction with consensus reporting of HPV ISH, leads to less equivocal molecular classification. In order to ensure consistent reporting of this emerging disease, it is increasingly important for the head-and-neck oncology community to define the minimum requirements for assigning a diagnosis of ‘HPV-related’ oropharyngeal SCC in order to inform prognosis and for stratification in clinical trials.

High prevalence of human papillomavirus type 16 infection among children

Infection with high‐risk human papillomaviruses (HPV), is the most significant risk factor for cervical cancer and it may be possible to prevent this malignancy by immunisation. Before immunisation programmes can be designed, however, it is necessary to know the age of acquisition and all routes of infection for these viruses. Sexual transmission is well documented and vertical transmission has also been demonstrated, although the frequency of transmission remains controversial. We previously showed that vertical transmission frequently results in persistent infection, and now present data on the prevalence of HPV‐16 DNA (the most prevalent high‐risk HPV type) in healthy children. Buccal samples from 267 healthy children aged 3–11 years were tested for HPV DNA by generic PCR (MY09/MY11), and a HPV‐16 specific nested PCR. Reverse transcriptase (RT)‐PCR was used to determine the prevalence of transcriptionally active HPV‐16 infection in a subset of children. HPV‐16 DNA was detected by nested PCR in 138 of 267 (51.7%) samples, whereas HPV DNA was detected in only 45 (16.8%) specimens by generic PCR, that has a lower analytical sensitivity. There were no significant differences in prevalence according to age or sex. Early region mRNA was detected by RT‐PCR in six (11.3%) of 53 HPV‐16 E5 DNA positive samples. HPV‐16 E5 DNA sequences from 10 children confirmed the identity of the sequences detected and identified 13 HPV‐16 variants. J. Med. Virol. 61:70–75, 2000.

Lineages, Sub-Lineages and Variants of Enterovirus 68 in Recent Outbreaks

Enterovirus 68 (EV68) was first isolated in 1962. Very few cases of EV68 infection were described over the ensuing 40 years. However, in the past few years, an increase in severe respiratory tract infections associated with EV68 has been reported. We identified two clusters of EV68 infection in South London, UK, one each in the autumn/winters of 2009 and 2010. Sequence comparison showed significant homology of the UK strains with those from other countries including the Netherlands, Japan and the Philippines, which reported EV68 outbreaks between 2008 and 2010. Phylogenetic analysis of all available VP1 sequences indicated the presence of two modern EV68 lineages. The 2010 UK strains belonged to lineage 2. Lineage 1 could be further divided into two sub-lineages: some Japanese and Dutch strains collected between 2004 and 2010 form a distinct sub-lineages (sub-lineage 1.1), whereas other strains from the UK, Japan, Netherlands and Philippines collected between 2008 and 2010 represent sub-lineage 1.2. The UK 2009 strains together with several Dutch and Japanese strains from 2009/2010 represents one variant (1.2.1), whereas those from the Philippines a second variant (1.2.2). Based on specific deletions and substitutions, we suggest rules for the assignment of lineages and sub-lineages. Molecular epidemiological analysis indicates rapid recent evolution of EV68 and this may explain the recent findings of a global resurgence of EV68. Continuous global monitoring of the clinical and molecular epidemiology of EV68 is recommended.

Cervical lesions are associated with human papillomavirus type 16 intratypic variants that have high transcriptional activity and increased usage of common mammalian codons.

Human papillomavirus type 16 (HPV-16) is a major cause of cervical neoplasia, but only a minority of HPV-16 infections result in cancer. Whether particular HPV-16 variants are associated with cervical disease has not yet been clearly established. An investigation of whether cervical neoplasia is associated with infection with HPV-16 intratypic variants was undertaken by using RFLP analyses in a study of 100 HPV-16 DNA-positive women with or without neoplasia. RFLP variant 2 was positively associated [odds ratio (OR)=2.57] and variant 5 was negatively associated with disease (OR=0.2). Variant 1, which resembles the reference isolate of HPV-16, was found at a similar prevalence among those with and without neoplasia. Variants 1 and 2 were also more likely to be associated with detectable viral mRNA than variant 5 (respectively P=0.03 and P=0.00). When HPV-16 E5 ORFs in 50 clones from 36 clinical samples were sequenced, 19 variant HPV-16 E5 DNA sequences were identified. Twelve of these DNA sequences encoded variant E5 amino acid sequences, 10 of which were novel. Whilst the associations between HPV-16 E5 RFLP variants and neoplasia could not be attributed to differences in amino acid sequences, correlation was observed in codon usage. DNA sequences of RFLP variant 2 (associated with greatest OR for neoplasia) had a significantly greater usage of common mammalian codons compared with RFLP pattern 1 variants.

Patient characteristics and severity of human rhinovirus infections in children

Abstract Background It is increasingly recognized that human rhinoviruses (HRV) can be associated with severe infections. However, conflicting results have been reported on the relative prevalence and severity of the three HRV species. Objectives The relative prevalence and clinical characteristics of HRV-A, B and C, in children attending a South London teaching hospital were investigated retrospectively. Study design Children aged <16 years with episodes of respiratory tract infections and detectable entero/rhinovirus RNA in respiratory samples between November 2009 and December 2010 were investigated. Retrospective case review was performed and patients’ characteristics recorded. Results Entero/rhinoviruses were the commonest viral pathogens (498/2316; 21.5%). Amongst 204 infection episodes associated with entero/rhinovirus, 167 were typed HRV, HRV-C was the most prevalent (99/167, 59.3%) followed by HRV-A (60/167; 35.9%) and HRV-B (8/167, 4.8%). The severity spectrum of HRV-A and HRV-C infections were similar and affected all parts of the respiratory tract. Co-pathogens were observed in 54 (26.5%) episodes. Severity was increased in patients with non-viral co-pathogens and those with an underlying respiratory condition. Univariate and multiple regression analyses of potential prognostic variables including age, co-pathogens and underlying respiratory illnesses showed that mono-infection with HRV-C, as compared with other HRV species, was associated with more severe disease in young children <3 years. Conclusions HRV-C was the most prevalent species and on its own was associated with severe disease in children <3 years. The association between infection with HRV species and clinical presentation is complex and affected by many confounding factors.

Analytic sensitivities of hybrid-capture, consensus and type-specific polymerase chain reactions for the detection of human papillomavirus type 16 DNA

Human papillomavirus type 16 (HPV‐16) DNA is detected commonly in cervical carcinomas; in this study, we have determined the analytical sensitivities of Hybrid Capture, HPV‐consensus PCR, and three HPV‐16‐specific polymerase chain reactions (PCRs) for the detection of HPV‐16 DNA. Samples investigated included a cervical cancer cell line, cervical scrapes from 20 patients attending colposcopy clinics, and buccal swabs from eight immunosuppressed children. HPV‐16 E7 and E5‐nested PCRs [Cavuslu et al. (1996): Journal of Virological Methods, in press] produced positive signals from samples containing fewer than ten HPV‐16 genomes per reaction. HPV‐consensus PCR [Manos et al. (1989): Cancer Cells 7:209–214] and HPV‐16 PCR using primers of van den Brule et al. [(1990): Journal of Clinical Microbiology 25:2739–2743] were of intermediate sensitivity (i.e., produced positive signals from samples containing 250 and 2,500 HPV‐16 genoms/reaction, respectively) and Hybrid Capture could detect just 50,000 HPV‐16 genomes/reaction. Highest rates of positivity for cervical samples were detected with HPV‐16 E7 or E5‐nested PCRs [50% (10 of 20 samples) and 60% (12 of 20 samples) positive, respectively], intermediate rates with HPV‐consensus PCR and PCRs using the primers of van den Brule et al. [both 35% (7 of 20 samples)], and lowest rates of positivity [25% (5 of 20 samples)] with Hybrid Capture. None of eight buccal swab samples from immunosuppressed children were positive by Hybrid Capture, yet three (37.5%) were positive by HPV‐16 E5‐nested PCR. These data indicate that HPV‐16 type‐specific PCRs should be used for the investigation of specimens that may contain low amounts of HPV‐16 DNA.

IGHV1 , IGHV5 and IGHV7 subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.

Proliferative T-cell responses to human papillomavirus type 16 E5 are decreased amongst women with high-grade neoplasia

Proliferative responses to human papillomavirus type 16 (HPV-16) E5 peptides were determined for short-term cell lines derived from peripheral blood mononuclear cells of 75 women. Cell lines from 16 of the 75 women proliferated in response to stimulation with pooled E5 peptides; this was most common for patients with low-grade squamous cervical intraepithelial lesions (LSIL; 6 of 15 patients, 40%) and less frequent for asymptomatic women with no cervical lesions (4 of 20, 20%), those with high-grade squamous intraepithelial lesions(HSIL; 5 of 33, 15%) and others with cervical cancer (1 of 7, 14%, P = 0.027). Amongst these patients, proliferative responses were exclusive to those that were positive for HPV-16 DNA (12 of 41, 29%; c.f. none of 13 HPV-16 DNA-negative subjects exhibited a proliferative response; P= 0023) and were again most prevalent amongst HPV-16 DNA-positive LSIL (6 of 14, 43%), as compared with HPV-16 DNA-positive HSIL (5 of 23, 22%) or HPV-16 DNA-positive cervical cancer patients (1 of 4, 25%, P > 0.05). In contrast, for asymptomatic women, responsiveness was statistically independent of HPV-16 DNA status, i.e. responsiveness in HPV-16 DNA-positive and DNA-negative subjects was observed in 3 of 15 (20%) and 1 of 5 (20%) cases, respectively (P > 0.05). There were no associations between detection of HPV-16 mRNA and proliferative responses (P> 0.05). These data suggest that HPV-16 E5-specific T-helper activity is depressed amongst women with HSIL lesions.

Human papillomavirus detection in dysplastic and malignant oral verrucous lesions

The role of human papillomaviruses (HPV) in dysplastic and malignant oral verrucous lesions is controversial since there is a wide range in the incidence of virus detection. This study used a multi-tiered method of HPV detection using DNA in-situ hybridisation (ISH) for low- and high-risk subtypes, consensus PCR, and HPV genotype analysis in archival tissue from 20 cases of dysplastic and malignant oral verrucous lesions. The biological significance of HPV DNA detection was assessed by p16 immunohistochemistry (IHC). While 1/7 carcinomas and 5/13 dysplasias contained HPV DNA by consensus PCR and genotype analysis, all specimens were negative for low- and high-risk HPV ISH and negative for p16 IHC. Results show that although high-risk HPV DNA is detectable in a subset of these lesions, the lack of p16 overexpression suggests that the oncogenic process is not driven by HPV oncoproteins.

Immunoglobulin light-chain genes in the rhesus macaque I: kappa light-chain germline sequences for subgroups IGKV1, IGKV and IGKV3

The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and six IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported, but only one light-chain germline gene has been published so far. Here we report the isolation of new macaque IGKV genes by polymerase chain reaction (PCR) amplification from macaque genomic DNA using primers based on the human sequences. Twenty-eight IGKV1, 22 IGKV2 and 12 IGKV3 germline genes for the macaque were found, the open reading frames of which exhibit high homology to their human counterparts (>96, >99 and >96%, respectively).