Winifred Trotman

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.

Cell adhesion molecule 1: a novel risk factor for venous thrombosis

Protein C (PC) deficiency increases the risk of venous thrombosis (VT) among members of Kindred Vermont II but fails to fully account for the inheritance pattern. A genome scan of the pedigree supported the presence of a prothrombotic gene on chromosome 11q23 (nominal P < .0001), with weaker support on chromosomes 10p12 (P < .0003) and 18p11.2-q11 (P < .0007). Resequencing of 109 genes in the linkage regions identified 5030 variants in a sample of 20 kindred members. Of 16 single nucleotide polymorphisms in 6 genes tested in the larger family set, only single nucleotide polymorphisms in cell adhesion molecule 1 (CADM1) associated with VT. Among the 8 CADM1 single nucleotide polymorphisms genotyped in the complete sample, rs6589488 was most strongly supported (P < .000007), but the association was limited to the PC-deficient subset of the sample (P < .000001). Haplotype analysis narrowed the region containing the causative variant to the coding region of the CADM1 gene. CADM1 gene expression analyzed in blood outgrowth endothelial cells cultured from family members was decreased compared with control subjects, lending phenotypic support to this conclusion. Finally, we have for the first time demonstrated CADM1 in endothelial cells, where it appears to be selectively involved in endothelial cell migration, suggesting a role in endothelial barrier repair.

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.

Screening for Human Papillomavirus in Basaloid Squamous Carcinoma: Utility of p16INK4a, CISH, and PCR

This study compares p16 INK4a immunohistochemistry (IHC), HPV chromogenic in situ hybridization (ISH), and HPV polymerase chain reaction (PCR) genotyping for detection of HPV infection in basaloid squamous carcinoma (BSCC). A retrospective histopathological analysis of 40 BSCC from a single institution was carried out. p16 IHC, HPV DNA extraction and ISH, and HPV PCR genotyping were performed, and there was excellent agreement between all 3 methods of HPV detection. Analysis of variance yielded no significant differences between the results of the 3 tests ( P = .354) and Pearson product—moment correlation coefficients calculated for each pair of tests demonstrated direct correlation (r = .61 for PCR and IHC, r = .61 for PCR and ISH, and r = 1.00 for ISH and IHC). This supports the use of p16INK4a IHC as an initial screening tool for HPV infection in BSCC, while definitive evidence of HPV DNA can be sought subsequently with PCR or CISH.

Multifluorescence Confocal Microscopy: Application for a Quantitative Analysis of Hemostatic Proteins in Human Venous Valves

Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276-1279, 2009 and Histochem Cell Biol 135:141-152, 2011).

A Comparison of p16INK4A Immunohistochemistry, Chromogenic in situ Hybridization, and Polymerase Chain Reaction Genotyping for Screening for Human Papillomavirus in Basaloid Squamous Carcinoma

Human papillomaviruses (HPV) can promote tumorigenesis at various anatomical sites in the body. Screening for dysplastic changes caused by HPV has decreased the morbidity and mortality due to invasive cervical cancer. Over the last two decades, HPV detection has included molecular methods as an adjunct to traditional Papanicolaou test for cervical cancer screening. HPV-positive HNSCC, specifically high risk HPV-16, have been recognized as a distinct molecular, behavioral and clinical subtype than HPV-negative tumors and therefore the importance of HPV detection in the head and neck region is becoming clear. On the molecular level, oncogenic HPV has two viral genes that synergistically cause deregulation of the cell cycle and malignant transformation. Specifically, viral proteins E6 and E7 act to degrade p53 and retinoblastoma gene family proteins (Rb), respectively. Inhibition of these tumor suppressor genes provokes a cascade of events that causes two points of interest: cell proliferation that escapes normal DNA check points and p16INK4A over-expression. Physiologically, p16INK4A is a cyclin-dependent kinase inhibitor that acts to offset the downstream actions of E6. With increase E6 activity, there is increase p16INK4A expression. Through another mechanism, with the degradation of Rb by E7, the transcription factor E2F is released and this also promotes p16INK4A synthesis. The purpose of this study was to evaluate correlation of HPV detection when using polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH) or p16 immunohistochemistry (IHC), to determine p16INK4A could be used as a surrogate marker for HPV detection. Figure 1: Focal p16 IHC pattern (A) vs. diffuse p16 IHC pattern (B). Both at 40x magnification.