Unmasking the mysteries of antigen or epitope retrieval and formalin fixation.
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Epitopes↗
Biomedical subjects
Allen M Gown
Publications and source records attributed to Allen M Gown.
Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis.
The goal of this study was to discover novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes, and to examine new interactions through which perlecan may influence cell behavior. We employed the yeast two-hybrid system and used perlecan domain V as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, we isolated a approximately 1.6-kb cDNA insert that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a novel splice variant that we name ECM1c. The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants. The C terminus of ECM1 interacted specifically with the epidermal growth factor-like modules flanking the LG2 subdomain of perlecan domain V. Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia. ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells, and induce angiogenesis. Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro- and anti-angiogenic activities. Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression.
Amino Acid Sequence↗
Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain.
Identification of myoepithelial cells using antibodies to cytoskeletal proteins, such as smooth muscle myosin heavy chain (SMM-HC) and calponin, can play an important role in distinguishing invasive carcinoma from its histologic mimics. However, antibodies to these proteins may also cross-react with stromal myofibroblasts and vascular smooth muscle cells. It has recently been demonstrated that myoepithelial cells express the nuclear protein, p63, a member of the p53 gene family. We compared the patterns of reactivity of antibodies with p63, calponin, and SMM-HC on 85 breast lesions, including 11 cases of sclerosing adenosis, 33 cases of ductal carcinoma in situ, including 10 that showed microinvasion, 6 cases of lobular carcinoma in situ, and 35 cases of infiltrating ductal carcinoma. All three antibodies were positive on the vast majority of myoepithelial cells in all cases. A small minority of cases showed focal gaps in the revealed myoepithelial cell layer, reflected in discontinuous positive immunostaining around noninvasive epithelial nests (including ductal carcinoma in situ). No case showed p63 expression by myofibroblasts or vascular smooth muscle cells, whereas myofibroblasts expressed, in 8% and 76% of cases, SMM-HC and calponin, respectively. Although no tumor cell reactivity was noted with antibodies to calponin or SMM-HC, tumor cells in 11% of cases showed at least focal p63 expression. And although antibodies to p63 offer excellent sensitivity and increased specificity for myoepithelial detection relative to antibodies to calponin and SMM-HC, they have the following diagnostic limitations: 1) they occasionally demonstrate an apparently discontinuous myoepithelial layer, particularly around ductal carcinoma in situ, and 2) they react with a small but significant subset of breast carcinoma tumor cells. p63 may represent a myoepithelial marker that can complement or replace SMM-HC and/or calponin in the analysis of difficult breast lesions.
Biomarkers, Tumor↗
CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas.
CDX2 is a recently cloned homeobox gene that encodes an intestine-specific transcription factor, expressed in the nuclei of epithelial cells throughout the intestine, from duodenum to rectum. While expression of CDX2 protein in primary and metastatic colorectal carcinomas has been previously documented, neither the sensitivity nor the specificity of CDX2 expression, as determined by immunohistochemistry, for colorectal adenocarcinoma has been determined. We performed an immunohistochemical survey of 476 tumors with a monoclonal antibody, CDX2-88, including 89 tumors from the colon and duodenum and 95 tumors from other gastrointestinal sites, including the esophagus, stomach, pancreatobiliary system, gastrointestinal carcinoids, and liver. CDX2 was expressed uniformly (that is, in 76-100% of tumor cells) in all but one of the evaluated colorectal and duodenal tumors. High-level expression of CDX2 was also found, however, in mucinous ovarian carcinomas and adenocarcinomas primary to the urinary bladder of which 64% and 100% were positive, respectively. Gastric, gastroesophageal, and pancreatic adenocarcinomas and cholangiocarcinomas all showed similar, heterogeneous patterns of CDX2 expression. Most tumors in each group showed CDX2 expression by a minority of cells, whereas a substantial minority of cases in each group was completely negative and a smaller minority was uniformly positive. Gastrointestinal carcinoids gave similarly varied results, but the majority (58%) was negative. Hepatocellular carcinomas showed no expression of CDX2. Only very rare examples of carcinomas of the genitourinary and gynecologic tracts, breast, lung, and head and neck showed significant levels of CDX2 expression. In this study of primary and metastatic epithelial tumors, uniform CDX2 expression is demonstrated to be an exquisitely sensitive and highly, but incompletely, specific marker of intestinal adenocarcinomas. Compared with villin, a previously described marker of GI adenocarcinomas, CDX2 demonstrated superior sensitivity and comparable specificity. CDX2 expression can be seen, however, in selected non-GI adenocarcinomas such as mucinous ovarian carcinomas and adenocarcinomas of the urinary bladder.
Adenocarcinoma↗
Immunohistochemical markers of melanocytic tumors.
While historically detection of premelanosomes by electron microscopic studies was the only means possible of confirming melanocytic lineage of a neoplastic process, advances in the field of immunohistochemistry have allowed for accurate and reliable diagnosis using antibodies to 1 of a number of melanocyte-restricted proteins. S-100 was the first such marker exploited by immunohistochemistry; subsequently, the HMB45 monoclonal antibody to gp100 became widely used as a sensitive and specific melanocytic marker. More recently, antibodies to other melanocytic proteins have become available, inciuding the MART-1 gene product and microphthalmia transcription factor. This article provides a brief overview of these markers in terms of their specificity and sensitivity and offers a discussion of tumors with partial melanocytic differentiation.
Biomarkers, Tumor↗
Pharmacologically regulated in vivo selection in a large animal.
The inefficiency of gene transfer has greatly hindered gene therapy. In vivo selection may increase the frequency of genetically modified cells, thereby circumventing this critical limitation. Here we demonstrate regulated in vivo selection in a large animal. CD34(+) cells from 2 dogs were engineered to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Activation of the receptor through administration of a dimerizing drug, AP20187, produced reversible, drug-dependent rises in genetically modified red cells, white cells, and platelets in both animals, with minimal side effects. Cell growth switches could greatly enhance the efficacy and applicability of gene and cell therapy.
Animals↗
Testing for HER-2/neu in breast cancer: is fluorescence in situ hybridization superior in predicting outcome?
Testing for alterations in HER-2/neu in breast cancer has become increasingly popular in recent years, particularly with the recent development of a humanized antiHER-2/neu monoclonal antibody, trastuzumab, which is currently being employed in conjunction with cytotoxic chemotherapy to treat metastatic breast cancer in patients whose tumors exhibit this HER-2/neu alteration. Controversy exists not only on the optimal method of laboratory testing for this HER-2/neu alteration (i.e., fluorescence in situ hybridization (FISH) versus immunohistochemistry (IHC) versus others), but also on the type of reagents used for a given method. A plethora of published studies on tissue-based HER-2/neu testing has recently appeared in many peer-reviewed journals; many have concluded that IHC could be used as a first-line screening test, with the recommendation of FISH to confirm indeterminate results. In contrast to these studies, a recent study by Pauletti et al. showed that HER-2/neu testing by IHC does not predict clinical outcome as accurately as does FISH. This commentary discusses the findings of this study, within a broader review of critical issues relating to HER-2/neu testing in breast cancer.
Antibodies, Monoclonal↗
Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3.
Apoptosis has gained central importance in the study of many biological processes, including neoplasia, neurodegenerative diseases, and development. One of the limitations of many studies is the difficulty of specifically identifying individual apoptotic cells. Of the many specific methods developed to detect apoptotic cells, most are not applicable to histological sections of archival paraffin-embedded tissues. Recently, advances in the understanding of the molecular events in apoptosis have led to the realization that caspase activation is by far the most specific indicator of this cell suicide mechanism. Several publications have reported the development of antibodies directed at neoepitopes that are generated in various substrates through the action of caspases. One of these is that present on activated caspase 3, a ubiquitously distributed caspase that is a main effector caspase of the apoptotic cascade within cells. This study demonstrates the utility of using a recently commercially available antibody to cleaved caspase 3 in archival paraffin sections, suggesting that this may be a highly specific and sensitive method generally applicable to many studies of archival material.
Antibodies↗
Controversies and guidelines in tissue-based HER-2/neu testing in breast cancer.
While it is tempting to think of the results of laboratory tests as quantitative, reproducible and accurate, many tests in fact have embedded variables that could lead to inconsistency and sometimes false reporting of results. Tissue-based testing of HER-2/neu in breast cancer represents an example of the latter. This article discusses the available technologies for HER-2/neu testing with some of the sources of testing errors.
Breast Neoplasms↗