Mark F. Evans

p16INK4A immunoexpression and HPV in situ hybridization signal patterns: potential markers of high-grade cervical intraepithelial neoplasia.

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16INK4a, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16INK4a immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16INK4a immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P < 0.001). p16INK4a immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P < 0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16INK4a staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16INK4a staining showed no evidence of viral integration. Both increased p16INK4a immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16INK4a immunostaining in CIN 1 appears independent of HPV punctate signal type.

Biotinyl-Tyramide-Based In Situ Hybridization Signal Patterns Distinguish Human Papillomavirus Type and Grade of Cervical Intraepithelial Neoplasia

In this study, the prevalence of human papillomavirus integration in cervical intraepithelial neoplasia Grades I, II, and III has been investigated using a highly sensitive biotinyl-tyramide–based in situ hybridization methodology. This method is able to demonstrate integrated viral DNA by punctate signals within the nucleus and episomal viral DNA by a diffuse signal throughout the nucleus. Fifteen viral types were identified by General Primer 5+/6+ polymerase chain reaction assay among 26 Grade I and 22 Grade II/III lesions. High-risk human papillomavirus (Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) was found in 20 (77%) Grade I and in 22 (100%) Grade II/III lesions (P =.025). Human papillomavirus Type 16 was identified in 2 (7%) Grade I and in 15 (68%) Grade II/III samples (P <.0001) and was distinguished from other high-risk types by its demonstration in both Grade I and Grade II/III lesions as frequent punctate signals, detectable at all levels of the epithelium including the basal layer. In contrast, punctate signals, when detected among Grade I lesions that were positive for other high-risk types, did not involve the basal layer and were restricted to occasional cells in the superficial layers. However, Grade II/III lesions positive for high-risk types other than human papillomavirus Type 16 demonstrated frequent punctate signals throughout the epithelium. Overall, punctate signals were detected in 22 (100%) high-risk human papillomavirus–positive Grade II/III lesions and in 5 (25%) high-risk positive Grade I lesions (P <.0001). These data are consistent with human papillomavirus Type 16 possessing a high potential for integration, which may explain its frequent association with cervical intraepithelial neoplasia Grade III and carcinomas. Acquisition of the punctate correlate, especially in the basal layer, is also indicated as important in the development of Grade II/III lesions. The data illustrate the unique potential of biotinyl-tyramide–based in situ hybridization to address key issues concerning the biology of cervical intraepithelial neoplasia.

Glypican-3 Immunocytochemistry in Liver Fine-needle Aspirates A Novel Stain to Assist in the Differentiation of Benign and Malignant Liver Lesions

Glypican‐3 (GPC3) is a heparan sulfate proteoglycan which is elevated in the serum of patients with hepatocellular carcinoma (HCC), but not in healthy blood donors, or patients with benign liver disease. GPC3 immunohistochemistry (IHC) is a promising marker of HCC in surgical pathology. This study explores the value of GPC3 expression in liver fine‐needle aspirates (FNAs) by immunocytochemistry (ICC), and compares its sensitivity and staining intensity with that of IHC.

Valves of the deep venous system: an overlooked risk factor

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.

Human papillomavirus integration: detection by in situ hybridization and potential clinical application

Human papillomaviruses (HPVs) are accepted as a necessary cause of cervical neoplasia. However, the benefits of testing simply for high‐risk HPV types are limited because of their high prevalence in intraepithelial lesions of all grades, the majority of which regress if left untreated. One factor considered to be of key importance for the progression of intraepithelial lesions to invasive disease is integration of HPV into the host cell genome. Although questions remain about the prevalence of integration amongst pre‐invasive lesions, sensitive in situ hybridization techniques utilizing tyramide reagents may aid determination of the significance of HPV infection by enabling routine detection of both high‐risk HPV and its physical status. This will provide important data relevant not only to our understanding of the biology of HPV‐associated neoplasia, but also potentially to clinical testing for HPV. Copyright

Sarcomatoid renal cell carcinoma is an example of epithelial–mesenchymal transition

Background Sarcomatoid renal cell carcinomas (SRCC) are composed of two cell populations, a sarcomatous component (SC) and a carcinomatous component (CC). SRCC are particularly aggressive and often present at an advanced stage at diagnosis. Epithelial–mesenchymal transition (EMT) has been proposed as a mechanism for the development of SC from CC. Aims and methods E- to N-cadherin switching, localisation of β-catenin, and expression of Snail and secreted protein acidic and rich in cysteine (SPARC) (markers of EMT) were studied to determine whether SRCC is an example of EMT. Expression of these markers was analysed by immunohistochemistry on 21 cases of SRCC that had both SC and CC and scored according to intensity and extent. Results E-cadherin expression was decreased in SC (Wilcoxon signed-rank test, p=0.0004) while N-cadherin expression was high in both components (p=0.46). Membranous β-catenin expression was decreased in SC (p<0.0001) while cytoplasmic expression was increased (p=0.0002). Snail and SPARC had higher expression in SC (p=0.002 and p<0.0001, respectively). When the scores were dichotomised into low and high expression levels, the results using McNemars test substantiated the above results. Conclusions E- to N-cadherin switching, dissociation of β-catenin from the membrane, and increased expression of Snail and SPARC in SC indicate that SRCC is an example of EMT. High expression of N-cadherin and Snail in CC suggest early involvement in initiating EMT. Once EMT is established, loss of E-cadherin, release of β-catenin into the cytoplasm, and expression of SPARC correspond with mesenchymal phenotypic expression.

Detection of Sarcocystis Parasites in Retail Beef : A Regional Survey Combining Histological and Genetic Detection Methods

Sarcocystis spp. are parasitic protists acquired when undercooked, cyst-laden meat is consumed. While both Sarcocystis hominis and S. cruzi encyst in beef, only S. hominis is pathogenic to humans. In this study, we used histological methods and novel molecular techniques to determine the regional prevalence and identity of Sarcocystis spp. in retail beef. Of 110 samples, 60 supported amplification of parasite rRNA by PCR. All 41 sequenced representatives were identified as S. cruzi. To compare detection methods, 48 samples were then examined in parallel by histology and PCR, and 16 and 26 samples, respectively, were positive. Five samples positive by initial histologic sections were not amplified by PCR. Fifteen PCR-positive samples did not contain sarcocysts on initial histologic section, but additional sections from these samples revealed sarcocysts in an additional 12 samples. When combined, histology with additional sections and PCR detected 31 positive specimens of the 48 total specimens. We found no evidence of human pathogen S. hominis and confirm that cattle pathogen S. cruzi is highly prevalent in this regional sample. PCR assays may increase the detection sensitivity of Sarcocystis spp. and contribute diagnostic precision.

Human papillomavirus is detectable in Barrett's esophagus and esophageal carcinoma but is unlikely to be of any etiologic significance

UNLABELLED Barretts esophagus (BE), a known precursor of esophageal adenocarcinoma has recently been associated with human papillomavirus (HPV). p16(INK4a) expression is a recognized surrogate marker of HPV infection in the cervix. OBJECTIVES This study has assessed the possible role of human papillomavirus (HPV) infection in BE and esophageal adenocarcinoma, in the North American population by screening esophageal tissues for HPV by a combination of assays. STUDY DESIGN Formalin-fixed, paraffin-embedded blocks from cases of Barretts esophagus (n=84), esophageal adenocarcinoma (n=36) and normal gastro-esophageal junction (n=29) were examined for HPV by PCR, chromogenic in situ hybridization, and p16(INK4a) immunohistochemistry. RESULTS HPV DNA was detected by PCR in 23 of 84 (27.4%) BE cases, 11 of 36 (31%) cases of adenocarcinoma and in 7 of 29 (24%) normal control cases (p=0.82). p16(INK4a) staining was positive in 10 (12%) cases of BE, 15 (42%) cases of adenocarcinoma and 6 (21%) cases of the control group. Positive p16(INK4a) staining was not statistically different between the three groups whether positive or negative for HPV DNA (p=0.91 and p=0.91 respectively). Similarly, negative p16(INK4a) staining did not show a difference between the three groups for whether positive or negative for HPV DNA (p=0.50 and p=0.28, respectively). HPV was not detected by CISH in the adenocarcinomas while in BE and control groups, CISH was non-contributory. CONCLUSIONS These data suggest that while HPV is detectable in a subset of esophageal lesions and tumors, the HPV detected is unlikely to be of etiologic significance or a factor accounting for the increase in BE and esophageal adenocarcinoma cases in the United States.

Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem

BackgroundViral myocarditis is a major cause of sudden unexpected death in children and young adults. Until recently, coxsackievirus B3 (CVB3) has been the most commonly implicated virus in myocarditis. At present, no standard diagnosis is generally accepted due to the insensitivity of traditional diagnostic tests. This has prompted health professionals to seek new diagnostic approaches, which resulted in the emergence of new molecular pathological tests and a more detailed immunohistochemical and histopathological analysis. When supplemented with immunohistochemistry and molecular pathology, conventional histopathology may provide important clues regarding myocarditis underlying etiology.MethodsThis study is based on post-mortem samples from sudden unexpected death victims and controls who were investigated prospectively. Immunohistochemical investigations for the detection of the enteroviral capsid protein VP1 and the characterization and quantification of myocardial inflammatory reactions as well as molecular pathological methods for enteroviral genome detection were performed.ResultsOverall, 48 sudden unexpected death victims were enrolled. As for controls, 37 cases of unnatural traffic accident victims were studied. Enterovirus was detected in 6 sudden unexpected death cases (12.5 %). The control samples were completely enterovirus negative. Furthermore, the enteroviral capsid protein VP1 in the myocardium was detected in enterovirus-positive cases revealed by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike control samples, immunohistochemical investigations showed a significant presence of T and B lymphocytes in sudden unexpected death victims.ConclusionsOur findings demonstrate clearly a higher prevalence of viral myocarditis in cases of sudden unexpected death compared to control subjects, suggesting that coxsackie B enterovirus may contribute to myocarditis pathogenesis significantly.

Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16 INK4a Immunohistochemistry

Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16INK4a immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16INK4a staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16INK4a negative and two HPV negative samples were p16INK4a positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16INK4a positive, PCR and CISH HPV positive/p16 INK4a negative, or PCR HPV positive/p16 INK4a and CISH negative, also requires further investigation.