Václav Mandys

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.

Large contribution of human papillomavirus in vaginal neoplastic lesions: A worldwide study in 597 samples

AIM This work describes the human papillomavirus (HPV) prevalence and the HPV type distribution in a large series of vaginal intraepithelial neoplasia (VAIN) grades 2/3 and vaginal cancer worldwide. METHODS We analysed 189 VAIN 2/3 and 408 invasive vaginal cancer cases collected from 31 countries from 1986 to 2011. After histopathological evaluation of sectioned formalin-fixed paraffin-embedded samples, HPV DNA detection and typing was performed using the SPF-10/DNA enzyme immunoassay (DEIA)/LiPA25 system (version 1). A subset of 146 vaginal cancers was tested for p16(INK4a) expression, a cellular surrogate marker for HPV transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance. RESULTS HPV DNA was detected in 74% (95% confidence interval (CI): 70-78%) of invasive cancers and in 96% (95% CI: 92-98%) of VAIN 2/3. Among cancers, the highest detection rates were observed in warty-basaloid subtype of squamous cell carcinomas, and in younger ages. Concerning the type-specific distribution, HPV16 was the most frequently type detected in both precancerous and cancerous lesions (59%). p16(INK4a) overexpression was found in 87% of HPV DNA positive vaginal cancer cases. CONCLUSIONS HPV was identified in a large proportion of invasive vaginal cancers and in almost all VAIN 2/3. HPV16 was the most common type detected. A large impact in the reduction of the burden of vaginal neoplastic lesions is expected among vaccinated cohorts.

Intercellular adhesion molecule‐1 (ICAM‐1) deficiency protects mice against severe forms of experimentally induced colitis

ICAM‐1 (CD54), the ligand for LFA‐1 and Mac‐1, is up‐regulated during inflammatory reaction on the activated vascular endothelium. To determine its role in intestinal inflammation, we induced acute experimental colitis in mice with a deleted ICAM‐1 gene, by feeding them with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. Chronic colitis was elicited by DSS similarly, followed by 2 weeks with water. In the acute phase of inflammation, ICAM‐1‐deficient mice exhibited a significantly lower mortality rate (5%) than control C57Bl/6J mice (35%). Control animals, but not the ICAM‐1‐deficient mice, exhibited diarrhoea and rectal bleeding. Histological examination of large‐bowel samples evaluated the intensity of inflammatory changes, and type and extent of mucosal lesions. In the acute phase, 33.3% of samples from ICAM‐1‐deficient mice exhibited mucosal defects (flat and fissural ulcers), predominantly mild to moderate inflammatory infiltrate within the lamina propria mucosae and lower grades of mucosal lesions. Much stronger inflammatory changes were present in control animals, flat ulcers (sometimes multiple) and fissural ulcers being observed in 62.5% of samples. Mucosal inflammatory infiltrate was moderate to severe, typically with higher grades of mucosal lesions. In chronic colitis, smaller inflammatory changes were found in the large bowel. The two mouse strains differed, the chronic colitis being accompanied by an increased serum level of anti‐epithelial IgA autoantibodies in C57Bl/6 control mice but not in ICAM‐1‐deficient mice. These findings provide direct evidence of the participation of ICAM‐1 molecule in the development of experimentally induced intestinal inflammation.

Comparison of morphological, immunohistochemical, and molecular genetic features of inflammatory fibroid polyps (Vanek´s tumors)

Vanek´s tumor (inflammatory fibroid polyp) is a rare benign lesion occurring throughout the digestive tract. Histologically, two patterns can be recognized. Classical Vanek´s tumor contains concentric formations of proliferating spindle cells which are CD34 positive. Atypical, inflammatory pseudotumor-like Vanek´s tumor lacks concentric formations and is CD34 negative. Recently, mutations in platelet-derived growth factor receptor alpha (PDGFRA) were reported in gastric and small intestinal Vanek´s tumors. In this study, KIT exons 9, 11, 13, and 17, PDGFRA exons 12, 14, and 18, and a part of exon 15 BRAF for point mutation V600E were screened in 23 cases of Vanek´s tumor, both classical (n = 16) and inflammatory pseudotumor-like (n = 7). No mutations in all analyzed exons of KIT and BRAF and in exon 14 of PDGFRA were detected. Six Vanek´s tumors harbored activating mutations in PDGFRA exons 12 (n = 5) and 18 (n = 1), respectively: S566_E571delinsK (n = 1), S566_E571delinsR (n = 4), and D842 del (n = 1). The mutations were detected in the classical (n = 5), as well as inflammatory pseudotumor-like (n = 1) Vanek´s tumors. The results of this study suggest that the two morphological patterns of Vanek´s tumor more probably represent only variants of one type of tumor than two different lesions. Furthermore, BRAF mutations were not shown to drive growth of PDGFRA wild-type Vanek´s tumors.

Cisplatin-induced apoptosis in cultures of human Schwann cells.

To investigate the sensitivity of human Schwann cells to cisplatin (cis-DDP), different approaches to estimate DNA damage were used: the comet assay, morphological evaluation of the granular condensation of nuclear chromatin and the terminal transferase-mediated dUTP nick-end-labelling (TUNEL) method. The number of micronuclei (MNi), as a sign of cisplatin-induced genotoxicity, was counted. DNA damage assessed by the comet assay was already evident after 1.5 microM cisplatin treatment at all exposure times (24, 48, and 72 h). Initial morphological changes characterised by the granular condensation of nuclear chromatin were detectable after 24 h exposure to 25 microM cis-DDP, while an increased number of apoptotic cells, determined by the TUNEL method, was noted after 48 h exposure to the same concentration. The first significant increase in the number of MNi was observed in cells treated with 75 microM cis-DDP for 24 h. We demonstrate that the comet assay is a highly sensitive method for measuring cisplatin induced DNA damage. Morphological observation revealed advanced as well as less prominent alterations in the nuclear chromatin. In contrast, the TUNEL method detected only those cells with advanced DNA fragmentation.

Cytokeratins 8 and 18 in adult human corneal endothelium.

The aim of this study was to determine if cytokeratins (CKs) 8 and 18--typical epithelial cell markers--are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.

Expression of CD44s and CD44v6 in transitional cell carcinomas of the urinary bladder: comparison with tumour grade, proliferative activity and p53 immunoreactivity of tumour cells.

Alterations of CD44 glycoproteins have been shown to play an important role in progression of various malignancies, including urothelial cancer. We investigated expression patterns of CD44s and CD44v6 in transitional cell carcinoma (TCC) of the urinary bladder in relation to tumour grade, proliferative activity, and immunoreactivity for p53. The selected markers were detected immunohistochemically in 122 samples of TCC. We found a close relationship between CD44s and CD44v6 expression and tumour grade. The extension of positive staining for CD44s and CD44v6 towards the luminal surface was a predominant feature of differentiated carcinomas (grades 1 and 2), suggesting deranged maturation of cancer cells related to their neoplastic transformation. Heterogeneous expression of CD44s and CD44v6 predominated in poorly differentiated tumours (G3–4). However, areas of squamous differentiation within the high‐grade tumours displayed strong immunoreactivity for both CD44s and CD44v6. The proliferative activity and p53 overexpression increased with the dedifferentiation of the tumour. The results of this study are discussed in relation to the significance of CD44 expression in TCC and to the explanation for controversial results reported in previous studies on the relationship between CD44 expression and the biological behaviour of urothelial cells.

Comparison between degree of carotid stenosis observed at angiography and in histological examination

SummaryBackground. The generally accepted indications for carotid endarterectomy are the clinical picture and degree of per cent stenosis of the carotid artery. Despite the fact that stenosis measurement is defined, the methods vary considerably. The correlation of particular methods, especially angiography and duplex sonography, has been repeatedly demonstrated. However, the correlation between any technique and true anatomical stenosis, as evaluated on the surgical specimen, has been only anecdotally reported. Method. During carotid endarterectomy, the atherosclerotic plaque was removed in one piece and subsequently stored and histologically processed. The histological slides were evaluated under an optical microscope, scanned and the slide with maximum stenosis was determined using a planimetric program. Both the minimal lumen area and the area of the whole plaque were measured. The stenosis was calculated using the planimetric method. On the maximum stenosis slice, the minimal diameter and the diameter of the whole plaque were also measured. Angiographic images were scanned and the per cent stenoses were remeasured, according to the NASCET and ECST criteria. In total, of 147 cases, all above-mentioned parameters were obtained. Student’s t tests for paired samples were used to evaluate the results. Findings. The t-tests indicated significant differences between the per cent stenosis as measured on the anatomical specimen and on the angiogram (p<0.05). The results indicate that the angiographic measurement underestimates the degree of in-situ anatomical stenosis. The underestimation was more marked the less the degree of stenosis. Conclusions. Our study finds that per cent stenosis measurement obtained by angiography with NASCET or ECST methods does not reliably reflect the anatomical degree of per cent stenosis, which makes questionable the rigorous following of percentage stenosis using angiography as the sole indicator for carotid endarterectomy in all cases.