Chromogenic In Situ Hybridization Methods for microRNA Biomarker Monitoring of Drug Safety and Efficacy.

Disease research and treatment development have turned to the impact and utility of microRNA. The dynamic and highly specific expression of these molecular regulators can be used to predict and monitor disease progression as well as therapeutic treatment efficacy and safety, thus aiding decisions in patient care. In situ hybridization (ISH) of biopsy material has become a routine clinical pathology procedure for monitoring gene structure, expression, and sample characterization. For ribonucleic acid (RNA), determining cell source and level of expression of these biomarkers gives insight into the cellular function and physiopathology. Identification and monitoring of microRNA biomarkers are made possible through locked nucleic acid (LNA)™-based detection probes. LNA™ enhances the sensitivity and specificity of target binding, most profoundly so for the short, highly similar, microRNA sequences. We present a robust 1-day ISH protocol for formalin-fixed, paraffin-embedded (FFPE) tissue sections based on microRNA-specific LNA™ detection probes which can be labeled with digoxigenin (DIG) or 6-carboxyfluorescein (FAM) and detected through enzyme-linked specific antibodies that catalyze substrates into deposited chromogen products at the target RNA site. The variety of haptens and detection reagents in combination with LNA™ chemistry offer flexibility and ease to multiple target assessment of therapeutic response.

Multiparametric flow cytometry profiling of neoplastic plasma cells in multiple myeloma.

BACKGROUND AND AIM: The clinical impact of multiparametric flow cytometry (MFC) in multiple myeloma (MM) is still unclear and under evaluation. Further progress relies on multiparametric profiling of the neoplastic plasma cell (PC) compartment to provide an accurate image of the stage of differentiation. The primary aim of this study was to perform global analysis of CD expression on the PC compartment and subsequently to evaluate the prognostic impact. Secondary aims were to study the diagnostic and predictive impact. DESIGN AND METHODS: The design included a retrospective analysis of MFC data generated from diagnostic bone marrow (BM) samples of 109 Nordic patients included in clinical trials within NMSG. Whole marrow were analyzed by MFC for identification of end-stage CD45(-) /CD38(++) neoplastic PC and registered the relative numbers of events and mean fluorescence intensity (MFI) staining for CD19, CD20, CD27, CD28, CD38, CD44, CD45, CD56, and isotypes for cluster analysis. RESULTS: The median MFC-PC number was 15%, and the median light microscopy (LM)-PC number was 35%. However, the numbers were significant correlated and the prognostic value with an increased relative risk (95% CI) of 3.1 (1.7-5.5) and 2.9 (1.4-6.2), P < 0.0003 and P < 0.004 of MFC-PC and LM-PC counts, respectively. Unsupervised clustering based on global MFI assessment on PC revealed two clusters based on CD expression profiling. Cluster I with high intensity for CD56, CD38, CD45, right-angle light-scatter signal (SSC), forward-angle light-scatter signal (FSC), and low for CD28, CD19, and a Cluster II, with low intensity of CD56, CD38, CD45, SSC, FSC, and high for CD28, CD19 with a median survival of 39 months and 19 months, respectively (P = 0.02). CONCLUSIONS: The MFC analysis of MM BM samples produces diagnostic, prognostic, and predictive information useful in clinical practice, which will be prospectively validated within the European Myeloma Network (EMN). © 2010 International Clinical Cytometry Society.

Regulation of the CD56 promoter and its association with proliferation, anti-apoptosis and clinical factors in multiple myeloma.

Multiple myeloma (MM) is an incurable B-cell malignancy characterised by uncontrolled growth and accumulation of malignant plasma cells in the bone marrow. Aberrant expression of CD56 in patients with MM is thought to contribute to a worsened disease course and metastasis. We therefore investigated the regulation of the CD56 promoter in relation to typical clinical factors. We used qPCR and FACS to measure the expression levels of CD56, and potential regulatory factors in patients with MM and related these with MM progression/prognosis. The transcription factors BTBD3, Pax5, RUNX1 and MMSET were positively associated with CD56 expression, as was CYCLIN D1, which is involved in disease progression, anti-apoptosis and proliferation. RUNX1 was negatively associated with the survival of stem-cell transplanted patients. Our findings propose four potential activators of the CD56 promoter and for CD56 to be involved in proliferation and anti-apoptosis, leading to disease progression in MM.