New MYC IHC Classifier Integrating Quantitative Architecture Parameters to Predict MYC Gene Translocation in Diffuse Large B-Cell Lymphoma.

A new automated MYC IHC classifier based on bivariate logistic regression is presented. The predictor relies on image analysis developed with the open-source ImageJ platform. From a histologic section immunostained for MYC protein, 2 dimensionless quantitative variables are extracted: (a) relative distance between nuclei positive for MYC IHC based on euclidean minimum spanning tree graph and (b) coefficient of variation of the MYC IHC stain intensity among MYC IHC-positive nuclei. Distance between positive nuclei is suggested to inversely correlate MYC gene rearrangement status, whereas coefficient of variation is suggested to inversely correlate physiological regulation of MYC protein expression. The bivariate classifier was compared with 2 other MYC IHC classifiers (based on percentage of MYC IHC positive nuclei), all tested on 113 lymphomas including mostly diffuse large B-cell lymphomas with known MYC fluorescent in situ hybridization (FISH) status. The bivariate classifier strongly outperformed the "percentage of MYC IHC-positive nuclei" methods to predict MYC+ FISH status with 100% sensitivity (95% confidence interval, 94-100) associated with 80% specificity. The test is rapidly performed and might at a minimum provide primary IHC screening for MYC gene rearrangement status in diffuse large B-cell lymphomas. Furthermore, as this bivariate classifier actually predicts "permanent overexpressed MYC protein status," it might identify nontranslocation-related chromosomal anomalies missed by FISH.

New Robust and Reproducible Stereological IHC Ki67 Breast Cancer Proliferative Assessment to Replace Traditional Biased Labeling Index.

There is a pressing need for an objective decision tool to guide therapy for breast cancer patients that are estrogen receptor positive and HER2/neu negative. This subset of patients contains a mixture of luminal A and B tumors with good and bad outcomes, respectively. The 2 main current tools are on the basis of immunohistochemistry (IHC) or gene expression, both of which rely on the expression of distinct molecular groups that reflect hormone receptors, HER2/neu status, and most importantly, proliferation. Despite the success of a proprietary molecular test, definitive superiority of any method has not yet been demonstrated. Ki67 IHC scoring assessments have been shown to be poorly reproducible, whereas molecular testing is costly with a longer turnaround time. This work proposes an objective Ki67 index using image analysis that addresses the existing methodological issues of Ki67 quantitation using IHC on paraffin-embedded tissue. Intrinsic bias related to numerical assessment performed on IHC is discussed as well as the sampling issue related to the "peel effect" of tiny objects within a thin section. A new nonbiased stereological parameter (VV) based on the Cavalieri method is suggested for use on a double-stained Ki67/cytokeratin IHC slide. The assessment is performed with open-source ImageJ software with interobserver concordance between 3 pathologists being high at 93.5%. Furthermore, VV was found to be a superior method to predict an outcome in a small subset of breast cancer patients when compared with other image analysis methods being used to determine the Ki67 labeling index. Calibration methodology is also discussed to further this IHC approach.

Canadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee for High Complexity Testing/Immunohistochemistry: guidelines for the preparation, release, and storage of unstained archived diagnostic tissue sections for immunohistochemistry.

OBJECTIVES: Formalin-fixed, paraffin-embedded unstained archived diagnostic tissue sections are frequently exchanged between clinical laboratories for immunohistochemical staining. The manner in which such sections are prepared represents a type of preanalytical variable that must be taken into account given the growing importance of immunohistochemical assays, especially predictive and prognostic tests, in personalized medicine. METHODS: Recommendations were derived from review of the literature and expert consensus of the Canadian Association of Pathologists-Association canadienne des pathologists National Standards Committee for High Complexity Testing/Immunohistochemistry. RESULTS: Relevant considerations include the type of glass slide on which to mount the unstained sections; the thickness of the tissue sections; the time from slide preparation to testing; the environment, particularly the temperature at which the unstained sections will be maintained prior to testing; the inclusion of on-slide positive control tissue where possible; and whether patient identifier(s) should be included on slide labels. CONCLUSIONS: Clear communication between requesting and releasing laboratories will facilitate the proper preparation of unstained sections and also ensure that applicable privacy considerations are addressed.