Differential Immunohistochemical Staining Research Articles

Expression of CD 20 B-Lymphocyte in oral epithelial dysplasia and oral squamous cell carcinoma: A comparative immunohistochemistry study.

As the progressive trends in the field of immunotherapy, it is very favourable to reconsider the role played by B lymphocytes in the tumor microenvironment. Both the protumorogenic and antitumorogenic responses have to be evaluated to formulate an effective immunotherapeutic protocol. The study was primarily conducted to assess the qualitative expression of B lymphocytes in pretumorogenic (oral epithelial dysplasia) and tumorogenic environment (oral squamous cell carcinoma). The differential immunohistochemical staining of CD 20 immune marker was assessed in about 60 cases that included 30 cases of oral epithelial dysplasia and 30 cases of oral squamous cell carcinoma. The study found significant correlation between CD 20 IHC immune expression and histopathological diagnosis along with significant correlation between the subject's age group and histopathological diagnosis. Modulating the immune response in a precancerous state can be highly beneficial in implementing better immunotherapeutic strategies to treat or prevent malignancy at an early stage.

A Previously Unrecognized Monocytic Component of Pheochromocytoma and Paraganglioma.

We describe a consistently present, previously unrecognized, population of monocytes in pheochromocytomas and paragangliomas. Although sustentacular cells are generally recognized as a common component of these tumors, differential immunohistochemical staining for CD163 and S100 shows that monocytes can in factbe more numerous. These cells frequently resemble sustentacular cells topographically and cytologically, possibly explaining why they have not been previously noticed. They contribute to the tumor proteome and may have implications for tumor biology. No correlations were identifiable between the presence of these cells and any clinical characteristics of the tumors in the present study. A possible association with genotype is suggested by immunoblot showing high expression of CD163 protein in tumors with succinate dehydrogenase mutations.

Abstract B51: Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection

Abstract Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor of infants. We constructed a tissue microarray (TMA) including 47 diagnostic NB cases and performed immunohistochemical (IHC) staining for a number of biomarkers. Differential IHC staining was identified between duplicate tissue cores for a proportion of NB cases. Differential IHC staining occurs frequently in cancer specimens, and can be due to technical factors, tissue artefacts, or underlying biological differences between tumor sub-regions. As laser capture microdissection (LCM) allows isolation of cells from formalin-fixed paraffin-embedded (FFPE) tissue slides, we applied LCM to FFPE NB samples, to investigate the basis of differential IHC staining. Material and Methods: NSE, NB84, ALK, TPD52 and MGMT proteins were detected within diagnostic NB TMA cores and whole NB sections using IHC. Cells were isolated from FFPE sections using LCM, and genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Extracted genomic DNA was quantified using a Nanodrop spectrophotometer, and DNA quality was assessed by a Bioanalyzer 2100. All DNA samples were tested using PCR with TPD52 and ALK primers. Results including clinical and sample variables were analysed using SPSS version 19. Results: Of 47 NB cases within the TMA, 35 cases were represented by duplicate tissue cores, whereas the remaining 12 cases were represented by single cores. IHC analysis of 5 biomarkers showed visually different IHC staining in tumour tissue between duplicate tissue cores in 15/35 (43%) cases. Whole tissue sections from 6 of these 15 cases were then examined, which reproduced differential IHC staining. Isolation of NB cells by LCM was successfully performed after substantial optimisation. A total of 14 regions from 8 NB cases were microdissected, producing DNA yields ranging from 0.12 - 4.05 pg/ µm2 tissue. There were no significant associations between total yields of extracted DNA, and microdissected tissue areas, or tissue sample ages. All DNA samples were successfully amplified by TPD52 but not by ALK PCR primers. Conclusions: Genomic DNA can be reliably extracted from microdissected FFPE NB tissues, although only one of 2 genes could be successfully PCR amplified. Downstream assays of DNA extracted from FFPE samples may therefore have particular design requirements. Further application of this approach may help improve our understanding of the level of tumour heterogeneity present in NB samples at diagnosis. Citation Format: Le M. Thwe, Laurence C. Cantrill, Daniel R. Catchpoole, Loretta Lau, Jennifer A. Byrne. Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B51.

Prostate Cancer Cell Surface-Associated Keratin 8 and Its Implications for Enhanced Plasmin Activity

Serum PSA, Gleason score, pathological stage, and positive surgical margins are currently used as predictors for disease recurrence. However, these criteria are less than precise in predicting disease outcome, with only 10% specificity at the 90% sensitivity level. Keratins are intermediate filament proteins that are contained within normal epithelia. However, human prostate cancer tissue shows differential immunohistochemical staining of keratin 8 (K8) when compared to normal prostate tissue. Our immunofluorescence and flow cytometry data show that K8 is also present on the cell surface of transformed prostate cancer cell lines. K8 is expressed at high levels on the surfaces of DU-145 and PC-3 cells but is expressed at comparatively lower levels on the surfaces of LNCaP cells, BPH-1 cells, and RWPE-1 cells. We hypothesize that extracellular K8 (eK8) present on epithelial prostate cancer cells plays an integral role in migration and in vivo dissemination. We found that K8 increased the rate of activity of plasmin approximately fivefold over a 48-h period. Functionally, K8 also enhanced the plasmin-mediated proteolysis of vitronectin, an important component of the prostate extracellular matrix. Taken together, our data show that K8 enhances the proteolytic activity of the plasminogen activation system, indicating that eK8 may be an important distinguishing marker in prostate cancer and progression.

Expression of membrane-type 5 matrix metalloproteinase in human endometrium and endometriosis

Background. The metalloproteinases (MMPs) are a family of proteolytic enzymes involved in tissue remodeling and cell migration. Endometrial tissue remodeling proceeds during the menstrual cycle and requires a temporary and spatially balanced expression of several different MMPs. Various members of the MMPs also seem to play an important role in the invasion process of endometriosis; however, so far only a limited number of studies have focused on membrane-associated MMPs.Methods. The present study investigated the expression of membrane-type 5 metalloproteinase (MT5-MMP) in the human endometrium and endometriotic lesions by microarray hybridization, real-time polymerase chain reaction (PCR) and immunofluorescence.Results. Both the gene chip expression analyses as well as PCR indicated expression of MT5-MMP in normal human endometrium and strongly elevated transcript levels in most peritoneal endometriosis lesions analyzed. Moreover we detected enhanced MT5-MMP expression in the eutopic endometrium from patients suffering from endometriosis, further supporting a role of MT5-MMP in the formation of endometriosis. Immunohistochemical analysis was used to determine the intracellular localization and tissue distribution of MT5-MMP. While the MT5-MMP antigen expression could be clearly attributed to the membrane of epithelial cells, a highly complex differential immunohistochemical staining of MT5-MMP in the various compartments of endometrial tissue was observed. The strongest staining was seen in luminal epithelial cells, whereas endometrial glands frequently showed partial expression of MT5-MMP.Conclusion. Our microarray analysis and real-time PCR of MT5-MMP transcripts may point to an elevated tissue remodeling and cell migration in endometrium from endometriosis patients as implied by the function of related MMPs.

The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir?

Up to now, the localization of stem cells in human anagen hair follicle relied on three complementary approaches; namely, detection of slow cycling cells, detection of high colony forming cells, and differential immunohistochemical staining. These techniques, however, gave conflicting results since stem cells were localized either as long label retaining cells in the so-called bulge area or as high colony forming cells in the lower third of the follicle. In the present study we investigated the expression of cytokeratin 19, a marker for putative stem cell-containing epithelial compartments, in order to characterize stem cell distribution in the human hair follicle throughout the hair cycle. We found that anagen human hair follicles contain two distinct reservoirs for stem cells located in the upper and lower thirds of the follicle. These two reservoirs fuse during the catagen-telogen transition phase and individualize again in the newly forming anagen hair follicle.

Differential Immunohistochemical Staining for Retinoblastoma Protein with the Antibodies C15 and 1F8 in Malignant Mesothelioma

The results of an immunohistochemical study of retinoblastoma protein in human non-neoplastic mesothelium (40 cases) and in malignant mesothelioma (36 cases) using the antibodies 1F8 and C15 are reported. All cases of malignant mesothelioma and non-neoplastic mesothelium were immunoreactive in more than 75% of the mesothelial cells for retinoblastoma protein with the C15 polyclonal antibody. Immunoreactivity for retinoblastoma protein was virtually absent with the 1F8 monoclonal body in neoplastic cells of all investigated mesotheliomas. In contrast, stromal cells in these mesotheliomas and all cases with non-neoplastic mesothelium were immunoreactive with the monoclonal antibody 1F8 in more than 75% of the cells. These findings may be indicative for the presence of an abnormal retinoblastoma protein in malignant mesothelioma lacking an epitope coded for between exons 21-27.

Pigmented breast carcinoma. A clinical and histopathologic simulator of malignant melanoma

A case of infiltrating breast carcinoma presenting as a pigmented lesion of the areola and nipple is described. The pigmentation was found to be primarily due to interspersed melanocytes and melanophages within the tumor. The histologic criteria and the differential immunohistochemical staining characteristics of melanoma and breast carcinoma are presented. Also emphasized is the difficulty in differentiating pagetoid malignant melanoma and epidermotrophic breast carcinoma.