Peter J.F. Snijders

Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.

A recent report that 93 per cent of invasive cervical cancers worldwide contain human papillomavirus (HPV) may be an underestimate, due to sample inadequacy or integration events affecting the HPV L1 gene, which is the target of the polymerase chain reaction (PCR)‐based test which was used. The formerly HPV‐negative cases from this study have therefore been reanalysed for HPV serum antibodies and HPV DNA. Serology for HPV 16 VLPs, E6, and E7 antibodies was performed on 49 of the 66 cases which were HPV‐negative and a sample of 48 of the 866 cases which were HPV‐positive in the original study. Moreover, 55 of the 66 formerly HPV‐negative biopsies were also reanalysed by a sandwich procedure in which the outer sections in a series of sections are used for histological review, while the inner sections are assayed by three different HPV PCR assays targeting different open reading frames (ORFs). No significant difference was found in serology for HPV 16 proteins between the cases that were originally HPV PCR‐negative and ‐positive. Type‐specific E7 PCR for 14 high‐risk HPV types detected HPV DNA in 38 (69 per cent) of the 55 originally HPV‐negative and amplifiable specimens. The HPV types detected were 16, 18, 31, 33, 39, 45, 52, and 58. Two (4 per cent) additional cases were only HPV DNA‐positive by E1 and/or L1 consensus PCR. Histological analysis of the 55 specimens revealed that 21 were qualitatively inadequate. Only two of the 34 adequate samples were HPV‐negative on all PCR tests, as against 13 of the 21 that were inadequate ( p< 0·001). Combining the data from this and the previous study and excluding inadequate specimens, the worldwide HPV prevalence in cervical carcinomas is 99·7 per cent. The presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer. The extreme rarity of HPV‐negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening. Copyright

WORLDWIDE DISTRIBUTION OF HUMAN PAPILLOMAVIRUS TYPES IN CYTOLOGICALLY NORMAL WOMEN IN THE INTERNATIONAL AGENCY FOR RESEARCH ON CANCER HPV PREVALENCE SURVEYS: A POOLED ANALYSIS

BACKGROUND The proportion of women infected with human papillomavirus (HPV) varies greatly across populations, as might the distribution of HPV types. We aimed to compare HPV-type distribution in representative samples of women from different world regions. METHODS Women were randomly selected from the general population of 13 areas from 11 countries (Nigeria, India, Vietnam, Thailand, Korea, Colombia, Argentina, Chile, the Netherlands, Italy, and Spain). A standardised protocol was used for cervical specimen collection. All HPV testing was by GP5+/6+ PCR-based EIA. The proportion of HPV-positive women infected with different HPV types was compared by study area and between pooled regions with age-adjusted odds ratios (ORs) with corresponding 95% floating CIs. FINDINGS 15 613 women aged 15-74 years without cytological abnormalities were included in a pooled analysis. Age-standardised HPV prevalence varied nearly 20 times between populations, from 1.4% (95% CI 0.5-2.2) in Spain to 25.6% (22.4-28.8) in Nigeria. Although both overall HPV prevalence and HPV16 prevalence were highest in sub-Saharan Africa, HPV-positive women in Europe were significantly more likely to be infected with HPV16 than were those in sub-Saharan Africa (OR 2.64, p=0.0002), and were significantly less likely to be infected with high-risk HPV types other than HPV16 (OR 0.57, p=0.004) and/or low-risk HPV types (OR 0.44. p=0.0002). Women from South America had HPV-type distribution in between those from sub-Saharan Africa and Europe. Heterogeneity between areas of Asia was significant. INTERPRETATION Heterogeneity in HPV type distribution among women from different populations should be taken into account when developing screening tests for the virus and predicting the effect of vaccines on the incidence of infection.

Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: Variation by geographical region, histological type and year of publication

Pooled data on human papillomavirus (HPV) type distribution in invasive cervical cancer (ICC) can help to predict the potential impact of HPV type‐specific vaccines and screening tests, and to understand the carcinogenicity of HPV types. We performed a meta‐analysis of HPV type‐specific prevalence data published from 1990 to 2010, including a total of 243 studies and 30,848 ICC. The proportion of ICC associated with HPV16 and/or 18 (HPV16/18) was between 70 and 76% in all world regions except Asia. In Western/Central Asia, 82% of ICC was HPV16/18‐associated compared to only 68% in Eastern Asia. The 12 most common HPV types identified, in order of decreasing prevalence, were HPV16 (57%), 18 (16%), 58, 33, 45, 31, 52, 35, 59, 39, 51 and 56. The prevalence of other types, phylogenetically related to those above, ranged from <0.1% for HPV85 to 0.6% for HPV68. Overall HPV prevalence increased significantly from 85.9% in studies published from 1990 to 1999 to 92.9% in studies published from 2006 to 2010. Prevalence increases were large for multiple infections (from 4.0 to 15.7%) and for HPV16 (from 51.8 to 60.0%, including HPV16 alone or in multiple infections). Smaller but significant increases in prevalence were also seen for HPV39, 53 and 58. A large amount of recently published data has improved the understanding of the contribution of a broad range of HPV types to ICC in different world regions. However, estimating the fraction of ICC attributable to different types is increasingly complicated by the detection of multiple HPV infections in ICC.

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial.

BACKGROUND Tests for the DNA of high-risk types of human papillomavirus (HPV) have a higher sensitivity for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) than does cytological testing, but the necessity of such testing in cervical screening has been debated. Our aim was to determine whether the effectiveness of cervical screening improves when HPV DNA testing is implemented. METHODS Women aged 29-56 years who were participating in the regular cervical screening programme in the Netherlands were randomly assigned to combined cytological and HPV DNA testing or to conventional cytological testing only. After 5 years, combined cytological and HPV DNA testing were done in both groups. The primary outcome measure was the number of CIN3+ lesions detected. Analyses were done by intention to treat. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN20781131. FINDINGS 8575 women in the intervention group and 8580 in the control group were recruited, followed up for sufficient time (> or =6.5 years), and met eligibility criteria for our analyses. More CIN3+ lesions were detected at baseline in the intervention group than in the control group (68/8575 vs 40/8580, 70% increase, 95% CI 15-151; p=0.007). The number of CIN3+ lesions detected in the subsequent round was lower in the intervention group than in the control group (24/8413 vs 54/8456, 55% decrease, 95% CI 28-72; p=0.001). The number of CIN3+ lesions over the two rounds did not differ between groups. INTERPRETATION The implementation of HPV DNA testing in cervical screening leads to earlier detection of CIN3+ lesions. Earlier detection of such lesions could permit an extension of the screening interval.

A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen.

Human papillomavirus type 16 (HPV16) plays a role in the development of a subgroup of head and neck squamous cell carcinomas (HNSCC). However, uncertainty exists about the true impact of HPV in this tumor type as conflicting reports have been published with prevalence rates from 0 to 100%. We aimed to find a detection algorithm of a biologically and thus clinically meaningful infection, applicable for high‐throughput screening of frozen and formalin‐fixed paraffin embedded (FFPE) specimens. By considering detection of HPV E6 oncogene expression in frozen biopsies as gold standard for a meaningful HPV infection, the value of several assays was evaluated on FFPE tumor specimens and sera of 48 HNSCC patients. The following assays were evaluated on FFPE tissue samples: HPV DNA general primer (GP)5+/6+ PCR, viral load analysis, HPV16 DNA FISH detection, HPV16 E6 mRNA RT‐PCR, p16 immunostaining, and on corresponding serum samples detection of antibodies against the HPV16 proteins L1, E6 and E7. Comparing single assays on FFPE tissue samples detection of E6 expression by RT‐PCR was superior, but application remains at present limited to HPV16 detection. Most suitable algorithm with 100% sensitivity and specificity appeared p16 immunostaining followed by GP5+/6+ PCR on the p16‐positive cases. We show that clinically meaningful viral HPV infections can be more reliably measured in FFPE HNSCC samples in a standard and high throughput manner, paving the way for prognostic and experimental vaccination studies, regarding not only HNSCC, but possibly also cancer types with HPV involvement in subgroups such as penile and anal cancer.

GP5+/6+ PCR followed by Reverse Line Blot Analysis Enables Rapid and High-Throughput Identification of Human Papillomavirus Genotypes

ABSTRACT In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.

Bead-Based Multiplex Genotyping of Human Papillomaviruses

ABSTRACT Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.

Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older

Given the strong etiologic link between high‐risk HPV infection and cervical cancer high‐risk HPV testing is now being considered as an alternative for cytology‐based cervical cancer screening. Many test systems have been developed that can detect the broad spectrum of hrHPV types in one assay. However, for screening purposes the detection of high‐risk HPV is not inherently useful unless it is informative for the presence of high‐grade cervical intraepithelial neoplasia (CIN 2/3) or cancer. Candidate high‐risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high‐grade CIN and cervical cancer to minimize redundant or excessive follow‐up procedures for high‐risk HPV positive women without cervical lesions. Data from various large screening studies have shown that high‐risk HPV testing by hybrid capture 2 and GP5+/6+‐PCR yields considerably better results in the detection of CIN 2/3 than cytology. The data from these studies can be used to guide the translation of high‐risk HPV testing into clinical practice by setting standards of test performance and characteristics. On the basis of these data we have developed guidelines for high‐risk HPV test requirements for primary cervical screening and validation guidelines for candidate HPV assays.

HPV-mediated cervical carcinogenesis : concepts and clinical implications

Persistent infection with a high‐risk human papillomavirus (hrHPV) is generally accepted as a necessary cause of cervical cancer. However, cervical cancer is a rare complication of an hrHPV infection since most such infections are transient, not even giving rise to cervical lesions. On average, it takes 12–15 years before a persistent hrHPV infection may ultimately, via consecutive premalignant stages (ie CIN lesions), lead to an overt cervical carcinoma. This argues that HPV‐induced cervical carcinogenesis is multi‐step in nature. In this review, the data from hrHPV‐mediated in vitro transformation studies and those obtained from analysis of clinical specimens have been merged into a cervical cancer progression model. According to this model, a crucial decision maker in the early stages following infection involves individual susceptibility for certain HPV types depending on the genetic make‐up of immune surveillance determinants. Once a CIN lesion has developed, altered transcriptional regulation of the viral E6/E7 oncogenes, resulting in genomic instability and distinguishing the process of cell transformation from a productive viral infection, probably provides the subsequent important step towards malignancy. The additional (epi)genetic alterations that subsequently accumulate in high‐grade CIN lesions may result in overt malignancy via immortality and growth conditions that gradually become less sensitive to growth‐modulating influences mediated by cytokines and cell–cell and cell–matrix adhesions. The potential implications of hrHPV testing and some other biomarkers deduced from this model for cervical screening and the clinical management of CIN disease are also discussed. Copyright