Elena Kasamatsu

Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study

BACKGROUND Knowledge about the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. We aimed to provide novel and comprehensive data about the worldwide genotype distribution in patients with invasive cervical cancer. METHODS Paraffin-embedded samples of histologically confirmed cases of invasive cervical cancer were collected from 38 countries in Europe, North America, central South America, Africa, Asia, and Oceania. Inclusion criteria were a pathological confirmation of a primary invasive cervical cancer of epithelial origin in the tissue sample selected for analysis of HPV DNA, and information about the year of diagnosis. HPV detection was done by use of PCR with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridisation line probe assay. Sequence analysis was done to characterise HPV-positive samples with unknown HPV types. Data analyses included algorithms of multiple infections to estimate type-specific relative contributions. FINDINGS 22,661 paraffin-embedded samples were obtained from 14,249 women. 10,575 cases of invasive cervical cancer were included in the study, and 8977 (85%) of these were positive for HPV DNA. The most common HPV types were 16, 18, 31, 33, 35, 45, 52, and 58 with a combined worldwide relative contribution of 8196 of 8977 (91%, 95% CI 90-92). HPV types 16 and 18 were detected in 6357 of 8977 of cases (71%, 70-72) of invasive cervical cancer. HPV types 16, 18, and 45 were detected in 443 of 470 cases (94%, 92-96) of cervical adenocarcinomas. Unknown HPV types that were identified with sequence analysis were 26, 30, 61, 67, 69, 82, and 91 in 103 (1%) of 8977 cases of invasive cervical cancer. Women with invasive cervical cancers related to HPV types 16, 18, or 45 presented at a younger mean age than did those with other HPV types (50·0 years [49·6-50·4], 48·2 years [47·3-49·2], 46·8 years [46·6-48·1], and 55·5 years [54·9-56·1], respectively). INTERPRETATION To our knowledge, this study is the largest assessment of HPV genotypes to date. HPV types 16, 18, 31, 33, 35, 45, 52, and 58 should be given priority when the cross-protective effects of current vaccines are assessed, and for formulation of recommendations for the use of second-generation polyvalent HPV vaccines. Our results also suggest that type-specific high-risk HPV-DNA-based screening tests and protocols should focus on HPV types 16, 18, and 45.

Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva

BACKGROUND Human papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions. METHODS Histologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16(INK4a) by immunohistochemistry (CINtec histology kit, ROCHE). An IVC was considered HPV driven if both HPV-DNA and p16(INK4a) overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI). RESULTS Of 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16(INK4a) positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC_WB) (N=326) were more likely to be HPV and p16(INK4a) positive (AP=69.5%, CI=63.6-74.8) versus KSCC (AP=11.5%, CI=9.7-13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold). CONCLUSION Combined data from HPV-DNA and p16(INK4a) testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide.

HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients

BACKGROUND We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation. METHODS Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results. RESULTS A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America. CONCLUSIONS HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs.

Human papillomavirus DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.

Value of p16INK4a in the Pathology of Invasive Penile Squamous Cell Carcinomas: A Report of 202 Cases

BackgroundOne third to one half of penile squamous cell carcinomas (SCCs) are related to human papillomavirus (HPV) infection. Viral detection is usually carried out by polymerase chain reaction (PCR) or other molecular methods. In this study, we evaluated p16INK4a immunohistochemical expression, which is simpler and less costly, as a potential marker of high-risk HPV (HR-HPV) infection in penile SCC. Design and MethodsWe pathologically classified 202 invasive penile carcinomas and performed HPV genotyping by short PCR fragment (SPF)10 PCR and p16INK4a immunohistochemistry. We also evaluated HPV and p16INK4a according to the histologic subtypes of penile SCC. Tumors depicting continuous p16INK4a immunostain in all neoplastic cells were considered positive. HPV and p16INK4a status were compared using classifier performances and concordance indexes. ResultsEvidence of HPV (low-risk and high-risk genotypes) was found in 63 cases (31%) by PCR. Fifty-three p16INK4a-positive cases were identified (26%). Overexpression of p16INK4a had a sensitivity of 67% and a specificity of 91% for defining the HPV status. Concordance indexes between p16INK4a and HPV status were high (≥78%) in general cases and in all histologic subtypes of penile SCC. The stain was useful in the differential diagnosis of basaloid and low-grade warty carcinomas. Low-risk HPV genotypes were found in 5 tumors, 4 of which were p16INK4a negative. Basaloid and nonbasaloid high-grade (grade 3) SCCs were more likely to be HR-HPV positive when compared with grades 1 to 2 tumors (P<0.000001 and 0.0417, respectively). Conclusionsp16INK4a overexpression was found to be a reliable marker for HR-HPV and a helpful tool in the differential diagnosis of low-grade verruciform and high-grade solid penile tumors. SCC variants depicting basaloid features were more likely to be HPV and p16INK4a positive than low-grade, keratinizing lesions. We also observed a tendency toward HPV positivity in high-grade nonbasaloid tumors. Our results indicated a concordance between HPV and p16INK4a status and this observation may have diagnostic and prognostic implications.

Time trends of human papillomavirus types in invasive cervical cancer, from 1940 to 2007.

Contribution over time of human papillomavirus (HPV) types in human cancers has been poorly documented. Such data is fundamental to measure current HPV vaccines impact in the years to come. We estimated the HPV type‐specific distribution in a large international series of invasive cervical cancer (ICC) over 70 years prior to vaccination. Paraffin embedded ICC cases diagnosed between 1940 and 2007 were retrieved from eleven countries in Central‐South America, Asia and Europe. Included countries reported to have low‐medium cervical cancer screening uptake. Information on age at and year of diagnosis was collected from medical records. After histological confirmation, HPV DNA detection was performed by SPF‐10/DEIA/LiPA25 (version1). Logistic regression models were used for estimating the adjusted relative contributions (RC) of HPV16 and of HPV18 over time. Among 4,771 HPV DNA positive ICC cases, HPV16 and HPV18 were the two most common HPVs in all the decades with no statistically significant variations of their adjusted‐RC from 1940–59 to 2000–07 (HPV16—from 61.5 to 62.1%, and HPV18—from 6.9 to 7.2%). As well, the RC of other HPV types did not varied over time. In the stratified analysis by histology, HPV16 adjusted‐RC significantly increased across decades in adenocarcinomas. Regarding age, cases associated to either HPV16, 18 or 45 were younger than those with other HPV types in all the evaluated decades. The observed stability on the HPV type distribution predicts a high and stable impact of HPV vaccination in reducing the cervical cancer burden in future vaccinated generations.

Susceptibilidad a antibióticos de cepas paraguayas de Helicobacter pylori aisladas de pacientes con enfermedad gastro-duodenal

BACKGROUND Antimicrobial resistance is one of the main obstacles for an effective eradication of H. pylori infection. AIM To determine the susceptibility of H. pylori strains obtained from gastric biopsies to metronidazole, clarithromycin and amoxicillin. MATERIAL AND METHODS Susceptibility to metronidazole, clarithromycin and amoxicillin was determined using E-test in 46 isolates of H. pylori obtained from gastric biopsies of 54 adult patients. RESULTS Thirty three percent of isolates were resistant to metronidazole and 2% were resistant to clarithromycin and amoxicillin. One isolate was resistant to metronidazole and clarithromycin. CONCLUSIONS The antimicrobial susceptibility of these strains of H. pylori obtained from Paraguayan patients, may help to decide the initial therapy to eradicate this infection.

Local and systemic cytokine expression during experimental chronic Trypanosoma cruzi infection in a Cebus monkey model.

Cebus apella is an acceptable model for chronic chagasic cardiomyopathy (CCC), since it is possible to experimentally induce cardiac lesions after 1 year of Trypanosoma cruzi infection. The T. cruzi Y strain, shown previously to produce CCC in C. apella monkeys, was used to experimentally infect 10 monkeys. Parasitological, serological and clinical parameters were monitored during a 19‐month follow‐up, and systemic cytokine responses were assessed sequentially in five monkeys selected according to the differential parasitemia pattern exhibited. Ten additional monkeys, infected with the same strain for 5, 10 and 12 years, were analysed cross‐sectionally. Three monkeys/time point and one uninfected control animal were sacrificed for gross pathology, histology, presence of parasites, and local cytokine gene expression. Elevated expression of interleukin (IL)‐4 was observed throughout the study in monkeys that had persistent, high parasitemias, whereas a high level of interferon (IFN)‐γ was seen in monkeys that controlled parasitemias soon after infection. Chronically infected monkeys expressed a nonpolarized, Th0‐type response. Cardiac tissue collected from a monkey that succumbed to acute infection had elevated levels of proinflammatory cytokine [IL‐1β, IL‐6, tumour necrosis factor‐α] and interstitial cell adhesion molecule (ICAM)‐1, platelet‐derived growth factor (PDGF)‐α, transforming growth factor (TGF)‐β and IL‐10 transcripts. Cytokine production in cardiac tissue of chronically infected monkeys was also characterized by elevated expression of ICAM‐1, PDGF‐α and TGF‐β, which correlated with the detection of T. cruzi DNA by polymerase chain reaction.

Type-specific human papillomavirus distribution in invasive cervical carcinomas in Paraguay. A study of 432 cases

Cervical carcinoma is the most common malignant tumor among woman in Paraguay. Cytological screening programs have not been successful and a plan for human papillomavirus (HPV) based‐screening program and/or vaccination is under evaluation. This study aimed to identify the contribution of HPV genotypes in invasive cervical cancer in Paraguay to provide essential background data to guide and assess the introduction and impact of new preventive strategies based on HPV. Four hundred thirty two histologically confirmed cases (1960–2004) were analyzed. HPV detection in paraffin blocks was performed at the Catalan Institute of Oncology using PCR with SPF‐10 broad spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe analysis. The majority of cases were squamous cell carcinoma (92.8%). Mean patients age was 48 years old. HPV DNA was detected in 73.1% of the cases and single infections were predominant (97.8%). The most common HPV single types were 16, 18, 45, 33, 31, 52, 35, and 39. 73.1% of HPV positive cases had an HPV 16, 18 as single infection. HPV16 was frequent in SCC whereas HPV 18 and 45 were prevalent in glandular tumors. Significant decrease of HPV 16 with age groups (P‐trend = 0.022) and increase in other HPV types (P‐trend > 0.001) were observed. The potential impact of HPV 16 and 18 for a vaccination program was 73.1%. The study provide a profile of the HPV situation in the country, with robust clinical, pathological and virological data which would permit a better cervical cancer screening and vaccination programs. J. Med. Virol. 84:1628–1635, 2012.

Distribution of human papillomavirus genotypes in Paraguayan women according to the severity of the cervical lesion.

The incidence of cervical cancer in Paraguay is among the highest in the world. This study aimed to determine the distribution of human papillomavirus (HPV) genotypes in Paraguayan women, according to the severity of the cervical lesion. This cross‐sectional study included 207 women without a squamous intraepithelial lesion, 164 with low‐grade squamous intraepithelial lesions, 74 with high‐grade squamous intraepithelial lesions, and 41 with cervical cancer. Type‐specific HPV was determined by the polymerase chain reaction with MY9/11 L1 and GP5+/GP6+ L1 primers, followed by restriction fragment length polymorphism and reverse line blotting hybridization, respectively. In total, 12 high‐risk and 24 low‐risk HPVs types were detected. HPV 16 was the most prevalent, followed by HPV 18 in cervical cancer (14.6%), HPV 31 in high‐grade squamous intraepithelial lesions (14.9%), HPVs 58/42 in low‐grade squamous intraepithelial lesions (9.1% each), and HPVs 31/58 (2.4% each) in women without squamous intraepithelial lesions. Among 285 positive samples, 24.2% harbored multiple HPV types, being this more prevalent in women with squamous intraepithelial lesions (30.8% in low‐grade squamous intraepithelial lesions, 22.5% in high‐grade squamous intraepithelial lesions, and 22.0% in cervical cancer) than in women without lesions (9.3%). The higher prevalence of HPV 16 and other high‐risk HPVs in women both with and without cervical lesions may explain the high incidence of cervical cancer in Paraguay. This information may be of importance for local decision makers to improve prevention strategies. In addition, these results may be useful as baseline pre‐vaccination data for a future virological surveillance in Paraguay. J. Med. Virol. 83:1351–1357, 2011.