Johannes Bogers

Syndecan‐1 expression in malignant mesothelioma: correlation with cell differentiation, WT1 expression, and clinical outcome

Syndecan‐1 binds basic fibroblast growth factor (bFGF), modulates neovascularization, plays a role in epithelial differentiation and is up‐regulated by WT1. Malignant mesothelioma of the pleura is one of the most aggressive tumours known and expresses high levels of angiogenic growth factors. This study has analysed syndecan‐1 expression in mesothelioma tumours and cell lines by immunohistochemistry and immunoblotting, using anti‐syndecan‐1 antibody directed against the core protein, and has examined its relation to morphology, bFGF, WT1, and intra‐tumoural microvascular density (IMD). Shedding of syndecan‐1 in the conditioned medium of mesothelioma cell lines was detected in variable amounts. These studies indicate that (1) there is no correlation of syndecan‐1 with either bFGF expression or IMD in mesotheliomas in vivo; (2) syndecan‐1 is strongly expressed in the epithelial type of mesothelioma and in the epithelial component of biphasic mesotheliomas and the expression is reduced or lost in sarcomatoid differentiation; together with the finding that (3) syndecan‐1 correlates with WT1 immuno‐expression, this suggests that syndecan‐1 might relate to the differentiation state of mesothelial/mesothelioma cells; and (4) syndecan‐1‐positive tumours are associated with a longer survival (p =0·02) than mesotheliomas with no or little syndecan‐1 expression, on univariate analysis. These findings therefore indicate that syndecan‐1 can be an important prognostic indicator in mesotheliomas and its loss may be important in the epithelial–mesenchymal transformation of mesothelioma cells. Copyright

Screening for Cervical Cancer Precursors With p16/Ki-67 Dual-Stained Cytology: Results of the PALMS Study

Background Pap cytology is known to be more specific but less sensitive than testing for human papillomavirus (HPV) for the detection of high-grade cervical intraepithelial neoplasia (CIN2+). We assessed whether p16/Ki-67 dual-stained cytology, a biomarker combination indicative of transforming HPV infections, can provide high sensitivity for CIN2+ in screening while maintaining high specificity. Results were compared with Pap cytology and HPV testing. Methods A total of 27349 women 18 years or older attending routine cervical cancer screening were prospectively enrolled in five European countries. Pap cytology, p16/Ki-67 immunostaining, and HPV testing were performed on all women. Positive test results triggered colposcopy referral, except for women younger than 30 years with only positive HPV test results. Presence of CIN2+ on adjudicated histology was used as the reference standard. Two-sided bias-corrected McNemar P values were determined. Results The p16/Ki-67 dual-stained cytology positivity rates were comparable with the prevalence of abnormal Pap cytology results and less than 50% of the positivity rates observed for HPV testing. In women of all ages, dual-stained cytology was more sensitive than Pap cytology (86.7% vs 68.5%; P < .001) for detecting CIN2+, with comparable specificity (95.2% vs 95.4%; P = .15). The relative performance of the tests was similar in both groups of women: younger than age 30 and 30 years or older. HPV testing in women 30 years or older was more sensitive than dual-stained cytology (93.3% vs 84.7%; P = .03) but less specific (93.0% vs 96.2%; P < .001). Conclusions The p16/Ki-67 dual-stained cytology combines superior sensitivity and noninferior specificity over Pap cytology for detecting CIN2+. It suggests a potential role of dual-stained cytology in screening, especially in younger women where HPV testing has its limitations.

WT1 MUTATION IN MALIGNANT MESOTHELIOMA AND WT1 IMMUNOREACTIVITY IN RELATION TO p53 AND GROWTH FACTOR RECEPTOR EXPRESSION, CELL‐TYPE TRANSITION, AND PROGNOSIS

The Wilms tumour 1 (WT1) gene is believed to contribute to the growth and differentiation of certain tissues, including mesothelium. This study assessed WT1 gene status by mutational screening in 42 malignant mesotheliomas (MMs) and 3 MM cell lines and detected two tumours with identical heterozygous single nucleotide deletions in intron 7, with no apparent consequence for WT1 function. Furthermore, the expression pattern of the WT1 gene was studied in MMs and related lesions using three anti‐WT1 monoclonal antibodies (MAbs). Strong to moderate nuclear immunoreactivity was noted in MM in situ (54/56), cultured mesothelioma cells (4/5), and hyperplastic and normal pleural (non‐neoplastic, NNM) specimens. WT1 immunoreactivity was absent in all primary tumours of lung and in pleural metastases from adenocarcinomas of breast and colon; immunoreactivity was present in pleural metastases from renal carcinomas, melanomas, and papillary carcinomas of the ovary. Expression of the WT1 protein in MM was not correlated with survival. Coordinate expression of the WT1 protein and its putative transcriptional target genes was determined by correlating WT1 immunostaining with epidermal growth factor receptor (EGF‐R) and insulin‐like growth factor 1 receptor (IGF‐1R) expression on MM and NNM; no significant correlation was found, irrespective of p53 expression status. Finally, the putative involvement of WT1 in cell‐type transition was supported by this study, in that epithelial mesothelioma showed the strongest WT1 immunoreactivity while sarcomatous mesothelioma showed the least.

Platelets and vascular endothelial growth factor (VEGF): A morphological and functional study

The growth of primary tumours beyond a critical mass is dependent on angiogenesis. The switch to the angiogenic phenotype involves changes in the local equilibrium of cytokines with either pro- or anti-angiogenic properties. Vascular Endothelial Growth Factor (VEGF) is one of the major positive regulators of tumour angiogenesis. Serum VEGF is, in cancer patients, correlated with worse prognosis. Recent evidence suggests that platelets are the main contributors of serum VEGF. We demonstrate, ultrastructurally and with immunofluorescence techniques, the alpha granule and membranous localisation of VEGF and provide further evidence for the role of platelets, both in healthy individuals as in patients with locally and advanced breast cancer, in the storage of circulating VEGF. We also demonstrate that, linear with tumoural progression, platelets accumulate more VEGF. Enhanced production in bone marrow platelet progenitors as well as endocytosis of circulating VEGF by platelets and/or megakaryocytes could explain the higher VEGF load in platelets from advanced cancer patients. This study provides further evidence for a role of platelets in transporting VEGF.

P16INK4a as an adjunct marker in liquid‐based cervical cytology

Cytological screening for cervical cancer is hampered by high false negative rates. Inter‐observer reproducibility needs optimizing. The potential of p16INK4a as a biomarker for cervical lesions was examined in a study of liquid‐based cytology (LBC), HPV DNA testing by MY09/MY11 consensus PCR and type‐specific PCRs and p16INK4a immunocytochemistry on a series of 291 patients selected from routine screening. Comparison of the number of p16INK4a immunoreactive cells/1,000 cells exhibited a significantly higher mean count in HSIL (8.80 ± 1.13) than other cytological groups. The mean count of LSIL (1.09 ± 0.18) was significantly higher than that of the negative group (0.82 ± 0.40). ASC‐H and HSIL combined showed a significantly higher mean count (6.46 ± 1.17) than negative, ASC, ASC‐US and LSIL. The mean count of immunoreactive cells/1,000 cells was significantly higher in HPV16 positive samples (3.22 ± 0.72) than in samples containing infections with types of unknown malignant potential (0.83 ± 0.26) or HPV negative samples (1.17 ± 0.41). The mean count in infections with other high‐risk HPV types (2.55 ± 0.52) was significantly higher than that in HPV negative samples. Receiver‐operating characteristic curves yielded a test accuracy (area under curve) of 0.76, 0.79, 0.88 and 0.95 for ASCUS, LSIL, ASC‐H/HSIL and HSIL, respectively. Thresholds for 95% sensitivity were at 0.005, 0.007, 0.098 and 0.445 immunopositive cells/1,000 cells for ASCUS, LSIL, ASC‐H/HSIL and HSIL, respectively. The 95% specificity threshold for the detection of HSIL was at 1.87 immunopositive cells/1,000 cells. P16INK4a immunocytochemistry can be used as an adjunct to LBC in cervical screening, because it has a good diagnostic accuracy to discriminate HSIL and ASC‐H from other lesions. It could be used as a surrogate marker of high‐risk HPV infections.

Detection of human papillomavirus DNA in urine. A review of the literature

The detection of human papillomavirus (HPV) DNA in urine, a specimen easily obtained by a non-invasive self-sampling method, has been the subject of a considerable number of studies. This review provides an overview of 41 published studies; assesses how different methods and settings may contribute to the sometimes contradictory outcomes; and discusses the potential relevance of using urine samples in vaccine trials, disease surveillance, epidemiological studies, and specific settings of cervical cancer screening. Urine sampling, storage conditions, sample preparation, DNA extraction, and DNA amplification may all have an important impact on HPV DNA detection and the form of viral DNA that is detected. Possible trends in HPV DNA prevalence in urine could be inferred from the presence of risk factors or the diagnosis of cervical lesions. HPV DNA detection in urine is feasible and may become a useful tool but necessitates further improvement and standardization.

Immunocytochemistry in liquid-based cervical cytology: analysis of clinical use following a cross-sectional study

Cytological screening for cervical cancer is hampered by imperfect sensitivity and low inter‐observer reproducibility. Human papillomavirus (HPV) testing lacks specificity as a primary screening method. Studies indicate that immunocytochemical detection of alterations caused by HPV in the host cells can optimise screening. Here, the potential of p16INK4a (cyclin‐dependent kinase inhibitor p16) and MIB‐1 (Ki‐67 proliferation marker) as adjunct molecular markers for cervical lesions was investigated in a prospective, cross‐sectional study of 500 samples in the framework of opportunistic screening in Flanders, Belgium. A consecutive series of 200 samples and 100 samples from the cytological categories ASC, LSIL and HSIL were investigated. Surepath samples were interpreted according to the Bethesda 2001 reporting system. HPV testing was done with MY09/MY11 consensus PCR. Immunocytochemistry for p16INK4a and MIB‐1 was performed with an automated staining protocol. The number of immunoreactive cells/1,000 cervical cells was assessed. There was a higher mean number of p16INK4A and MIB‐1 immunoreactive cells/1,000 cells in HSIL (4.06 ± 1.93 and 11.13 ± 2.83, respectively) compared to other cytological categories. Both markers showed a large spread in counts, for all categories. In cases of HSIL without immunoreactive cells for either marker, low cellularity and long‐term storage in water were often the cause of false negativity. This study confirms that positive staining for p16INK4a and MIB‐1 is highly correlated with presence of high‐grade lesions. These markers could be used as adjuncts to increase the sensitivity of cytological screening as well as the specificity of the HPV test. However, clear methodological standards are needed for optimal performance of immunocytochemistry in a clinical setting.

Prospective evaluation of p16/Ki-67 dual-stained cytology for managing women with abnormal Papanicolaou cytology: PALMS study results

Testing for the presence of the human papillomavirus (HPV) is widely accepted for triaging Papanicolaou cytology results categorized as atypical squamous cells of undetermined significance (ASC‐US). In contrast, HPV testing has limited use in triaging cytological low‐grade squamous intraepithelial lesions (LSILs) due to prevalence rates of typically >80%. In the current study, the authors assessed the diagnostic performance of p16/Ki‐67 dual‐stained cytology in triaging ASC‐US and LSIL cases within the prospective, multicentric Primary ASC‐US LSIL Marker Study (PALMS).

Prior knowledge of HPV status improves detection of CIN2+ by cytology screening

OBJECTIVE The objective of the study was to investigate whether knowledge of human papillomavirus (HPV) deoxyribonucleic acid test results increases sensitivity of guided cytology screening for the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical lesions. STUDY DESIGN This was a prospective colposcopy-controlled study of 2905 BD SurePath samples to identify cases with CIN2+ within a 24 month follow-up period. Sensitivity and specificity to detect CIN2+ was evaluated, comparing guided cytology screening with and without prior knowledge of HPV status. RESULTS Prior knowledge of HPV status resulted in significantly higher detection rate of CIN2+ compared with screening blinded to HPV status (P = .005) with limited loss of specificity (P = .026). Gain in sensitivity is higher in older women (43.8%, P = .008) vs in younger women (10.2%, P = .317), whereas loss of specificity is more pronounced in younger women (P < .001) vs older women (P = .729). CONCLUSION Guided cytological screening performed with prior knowledge of HPV status results in an improved detection of CIN2 or higher-grade lesions.

Optimization of HPV DNA detection in urine by improving collection, storage, and extraction

The benefits of using urine for the detection of human papillomavirus (HPV) DNA have been evaluated in disease surveillance, epidemiological studies, and screening for cervical cancers in specific subgroups. HPV DNA testing in urine is being considered for important purposes, notably the monitoring of HPV vaccination in adolescent girls and young women who do not wish to have a vaginal examination. The need to optimize and standardize sampling, storage, and processing has been reported.In this paper, we examined the impact of a DNA-conservation buffer, the extraction method, and urine sampling on the detection of HPV DNA and human DNA in urine provided by 44 women with a cytologically normal but HPV DNA-positive cervical sample. Ten women provided first-void and midstream urine samples. DNA analysis was performed using real-time PCR to allow quantification of HPV and human DNA.The results showed that an optimized method for HPV DNA detection in urine should (a) prevent DNA degradation during extraction and storage, (b) recover cell-free HPV DNA in addition to cell-associated DNA, (c) process a sufficient volume of urine, and (d) use a first-void sample.In addition, we found that detectable human DNA in urine may not be a good internal control for sample validity. HPV prevalence data that are based on urine samples collected, stored, and/or processed under suboptimal conditions may underestimate infection rates.