An Introduction to the Performance of Immunohistochemistry.

Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope. A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (Lin F, Chen Z. Arch Pathol Lab Med 138:1564-1577, 2014) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (Taylor CR, Shi S-R, Barr NJ. Techniques of immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ (ed) Diagnostic immunohistochemistry: theranostic and genomic applications, 3rd edn. Saunders, Philadelphia, 2010; Shi SR, Key ME, Kalra KL. J Histochem Cytochem 39:741-748, 1991) and automated methods for high volume processing with reproducibility (Prichard J, Hicks D, Hammond E. Automated immunohistochemistry overview. In: Fan L, Jeffrey P (eds) Handbook of practical immunohistochemistry: frequently asked questions, 2nd edn. Springer, New York, 2015). IHC is frequently utilized to assist in the classification of neoplasms, determination of a metastatic tumor's site of origin and detection of tiny foci of tumor cells inconspicuous on routine hematoxylin and eosin (H&E) staining. Furthermore, it is increasingly being used to provide predictive and prognostic information, such as in testing for HER2 amplification in breast cancer (Wolff AC, Hammond MEH, Hicks DG et al. Arch Pathol Lab Med 138:241-256, 2014) in addition to serving as surrogate markers for molecular alterations in neoplasms, including IDH1 and ATRX mutations in brain tumors (Appin CL, Brat DJ. Mol Aspects Med. 45:87-96, 2015). In this chapter we describe the basic methods of immunohistochemical staining which has become an essential tool in the daily practice of anatomic pathology worldwide.

Tissue microarray analysis for epithelial membrane protein-2 as a novel biomarker for gliomas.

Epithelial membrane protein-2 (EMP2) expression is noted in many human cancers. We evaluated EMP2 as a biomarker in gliomas. A large tissue microarray of lower grade glioma (WHO grades II-III, n = 19 patients) and glioblastoma (GBM) (WHO grade IV, n = 50 patients) was stained for EMP2. EMP2 expression was dichotomized to low or high expression scores and correlated with clinical data. The mean EMP2 expression was 1.68 in lower grade gliomas versus 2.20 in GBMs (P = 0.01). The percentage of samples with high EMP2 expression was greater in GBMs than lower grade gliomas (90.0 vs. 52.6%, P = 0.001). No significant difference was found between median survival among patients with GBM tumors exhibiting high EMP2 expression and survival of those with low EMP2 expression (8.38 vs. 10.98 months, P = 0.39). However, EMP2 expression ≥2 correlated with decreased survival (r = -0.39, P = 0.001). The EMP2 expression level also correlated with Ki-67 positivity (r = 0.34, P = 0.008). The mortality hazard ratio for GBM patients with EMP2 score of 3 or higher was 1.92 (CI 0.69-5.30). Our findings suggest that elevated EMP2 expression is associated with GBM. With other biomarkers, EMP2 may have use as a molecular target for the diagnosis and treatment of gliomas.

Early experience with formalin-fixed paraffin-embedded (FFPE) based commercial clinical genomic profiling of gliomas-robust and informative with caveats.

BACKGROUND: Commercial targeted genomic profiling with next generation sequencing using formalin-fixed paraffin embedded (FFPE) tissue has recently entered into clinical use for diagnosis and for the guiding of therapy. However, there is limited independent data regarding the accuracy or robustness of commercial genomic profiling in gliomas. METHODS: As part of patient care, FFPE samples of gliomas from 71 patients were submitted for targeted genomic profiling to one commonly used commercial vendor, Foundation Medicine. Genomic alterations were determined for the following grades or groups of gliomas; Grade I/II, Grade III, primary glioblastomas (GBMs), recurrent primary GBMs, and secondary GBMs. In addition, FFPE samples from the same patients were independently assessed with conventional methods such as immunohistochemistry (IHC), Quantitative real-time PCR (qRT-PCR), or Fluorescence in situ hybridization (FISH) for three genetic alterations: IDH1 mutations, EGFR amplification, and EGFRvIII expression. RESULTS: A total of 100 altered genes were detected by the aforementioned targeted genomic profiling assay. The number of different genomic alterations was significantly different between the five groups of gliomas and consistent with the literature. CDKN2A/B, TP53, and TERT were the most common genomic alterations seen in primary GBMs, whereas IDH1, TP53, and PIK3CA were the most common in secondary GBMs. Targeted genomic profiling demonstrated 92.3%-100% concordance with conventional methods. The targeted genomic profiling report provided an average of 5.5 drugs, and listed an average of 8.4 clinical trials for the 71 glioma patients studied but only a third of the trials were appropriate for glioma patients. CONCLUSIONS: In this limited comparison study, this commercial next generation sequencing based-targeted genomic profiling showed a high concordance rate with conventional methods for the 3 genetic alterations and identified mutations expected for the type of glioma. While it may not be feasible to exhaustively independently validate a commercial genomic profiling assay, examination of a few markers provides some reassurance of its robustness. While potential targeted drugs are recommended based on genetic alterations, to date most targeted therapies have failed in glioblasomas so the usefulness of such recommendations will increase with development of novel and efficacious drugs.