Immunohistochemistry for the Novel Markers Glypican 3, PAX8, and p40 (ΔNp63) in Squamous Cell and Urothelial Carcinoma

Dept of Pathology, University of Iowa Hospitals and Clinics, University of Iowa Carver College of Medicine, Iowa City, IA 52242
American Journal of Clinical Pathology (Impact Factor: 2.51). 12/2013; 140(6):872-80. DOI: 10.1309/AJCP4NSKW5TLGTDS
Source: PubMed


To examine squamous cell carcinomas (SCCs) from diverse anatomic sites and invasive urothelial carcinomas (UCs) for expression of the oncofetal antigen glypican 3 (GPC3), the paired box transcription factor PAX8, and the ΔN isoform of p63 (p40).

Immunohistochemistry for GPC3, PAX8, and p40 was performed on whole sections of 107 SCCs from 11 anatomic sites and 49 UCs; evaluation included extent and intensity of staining.

GPC3 was detected in 20% of SCCs and 12% of UCs and PAX8 in 3% of SCCs, limited to the uterine cervix, and 10% of UCs. p40 Was found in 99% of SCCs and 96% of UCs.

GPC3 expression is frequent in SCC/UC, awareness of which should guard against an incorrect diagnosis of hepatocellular carcinoma, while PAX8, limited in distribution, may have some use in suggesting a cervical or urothelial tract origin in a metastatic squamotransitional carcinoma of unknown primary. There is no drop-off in sensitivity for the diagnoses of SCC or UC with ΔNp63-specific immunohistochemistry, and if this performance can be extended to other applications, p40 may supplant the dominant "pan-p63" antibody clone.

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    • "Added to this is the use of antibodies that are poorly characterized (it is often uncertain which isoforms are recognized and which not) and that may even show cross-reaction with other family members, such as the cross-reactivity of 4A4 (by far the most widely used anti-p63 antibody) and other p63 antibodies with p73, and of anti-p73 antibodies with p63 (Rosenbluth et al., 2009; Nekulova et al., 2013). Fortunately, novel antibodies with improved selectivity and/or isoform-specificity are beginning to become available (Nenutil et al., 2003; Rosenbluth et al., 2009; Karni-Schmidt et al., 2011; Romano et al., 2012; Nekulova et al., 2013; Veselska et al., 2013; Tacha et al., 2014) (Fig. 6) and are being more widely used for both research and clinical studies as they demonstrate the improved data they provide (Karni- Schmidt et al., 2011; Bishop et al., 2012; Butnor and Burchette, 2013; Gailey and Bellizzi, 2013; Nekulova et al., 2013; Rossi et al., 2013; Sailer et al., 2013; Alomari et al., 2014; Vogt et al., 2014). We can also expect that the more detailed analysis of tumour material for p63 and p73 isoforms will lead to a better understanding of their roles in tumour progression and their ability to act as predictive biomarkers and will be a step further in the targeted therapy of cancer. "
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    ABSTRACT: p63 and p73, the two other members of the p53 family, were identified almost 15 years ago. Here, we review their potential use for diagnosis, prognosis and prediction of response to therapy in various cancers. The two genes show distinct expression patterns in both normal and cancer tissues and each gene gives rise to multiple protein isoforms with different activities, including those with tumour-suppressor or oncogenic effects. Despite such complexity, some common themes emerge; p63 is commonly over-expressed as the ΔNp63 isoform and sometimes associated with TP63 amplification, whereas p73 is often reduced (by methylation or gene loss), or there is an increase in the ratio of ΔNp73 to TAp73. These generalisations do not apply universally; TAp63 is overexpressed in haematological malignancies, TP63 mis-sense mutations have been reported in squamous cancers and TP63 translocations occur in lymphomas and some lung adenocarcinomas. There are associations with disease prognosis and response to specific therapies in individual cancer types for both p63 and p73, making their analysis of clinical relevance. We also discuss their utility for aiding in differential diagnosis, which has been demonstrated for p63, but not yet for p73. Throughout, we highlight the discrepant nature of many studies due to the variable methodologies employed, the lack of systematic evaluation of isoforms and the problems of poor antibody characterization and cross-reactions within the p63/p73 family. Finally, we emphasize the value of recently developed isoform-specific reagents that have clear advantages for the study of p63 and p73 experimentally and clinically. (247 words).
    No preview · Article · Dec 2014 · Histology and histopathology
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    ABSTRACT: BACKGROUNDGATA3 (GATA-binding protein 3) expression in urothelial carcinoma (UC) and mammary carcinomas has been recently reported. However, to the authors' knowledge, studies examining GATA3 staining of metastatic UC (MUC) in cytology specimens are lacking. Delta Np63 (p40) has been shown to be expressed highly selectively in squamous cell carcinomas (SCCs) but the literature concerning the expression of p40 in UC is limited and controversial. In the current study, the authors evaluated the usefulness of GATA3 and p40 in the diagnosis of MUC in cytology specimens.METHODS Thirty-two MUC cytology cases and 44 controls (22 UC cases and 22 SCC cases) were stained for GATA3, p40, and p63 and nuclear staining intensity and the percentage of positive cells were recorded and compared.RESULTSMUC cytology cases stained positive for GATA3, p40, and p63 in 78.13%, 80.65%, and 61.29% of cases, respectively, with moderate/strong staining intensity. MUC cases had a significantly higher percentage of GATA3 positivity compared with SCC controls (P < .001), but GATA3 positivity was not found to be significantly different from UC controls (90.91%) (P = .28). For p40 positivity, there was no significant difference observed between MUC cases, UC controls (95.45%), and SCC controls (90.91%) (P = .29). p63 positivity was found to be significantly lower in MUC cases compared with UC controls (95.45%) and SCC controls (95.45%) (P < .01).CONCLUSIONS The results of the current study demonstrate that GATA3 is useful in confirming the diagnosis of MUC in cytology specimens and in distinguishing between MUC and SCC. p40 is a valuable adjunct to GATA3 in the diagnosis of MUC in cytology specimens, especially when SCC is not part of the clinical differential diagnosis. Cancer (Cancer Cytopathol) 2014. © 2014 American Cancer Society.
    Preview · Article · Jun 2014 · Cancer Cytopathology
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    ABSTRACT: Background Glypican 3 (GPC3) is a member of the family of glypican heparan sulfate proteoglycans (HSPGs). The GPC3 gene may play a role in controlling cell migration, negatively regulating cell growth and inducing apoptosis. GPC3 is downregulated in several cancers, which can result in uncontrolled cell growth and can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma. Methods Five clear cell renal cell carcinoma cell lines and carcinoma samples were used to analyze GPC3 mRNA expression (qRT-PCR). Then, representative cell lines, one primary renal carcinoma (786-O) and one metastatic renal carcinoma (ACHN), were chosen to carry out functional studies. We constructed a GPC3 expression vector and transfected the renal carcinoma cell lines, 786-O and ACHN. GPC3 overexpression was analyzed using qRT-PCR and immunocytochemistry. We evaluated cell proliferation using MTT and colony formation assays. Flow cytometry was used to evaluate apoptosis and perform cell cycle analyses. Results We observed that GPC3 is downregulated in clear cell renal cell carcinoma samples and cell lines compared with normal renal samples. GPC3 mRNA expression and protein levels in 786-O and ACHN cell lines increased after transfection with the GPC3 expression construct, and the cell proliferation rate decreased in both cell lines following overexpression of GPC3. Further, apoptosis was not induced in the renal cell carcinoma cell lines overexpressing GPC3, and there was an increase in the cell population during the G1 phase in the cell cycle. Conclusion We suggest that the GPC3 gene reduces the rate of cell proliferation through cell cycle arrest during the G1 phase in renal cell carcinoma.
    Full-text · Article · Aug 2014 · BMC Cancer
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