Marianna Dávid

Multiplex Ligation-Dependent Probe Amplification and Fluorescence In Situ Hybridization are Complementary Techniques to Detect Cytogenetic Abnormalities in Multiple Myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i‐FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation‐dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32‐31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i‐FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy‐chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i‐FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra‐chromosomal and intra‐clonal rates of loss or gain. Our results suggest that MLPA is a reliable high‐throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i‐FISH should be applied as complementary techniques in diagnostic pathology.

New diagnostic tool for differentiation of idiopathic hypereosinophilic syndrome (HES) and secondary eosinophilic states

The hypereosinophilic syndrome (HES) is a very rare disease, characterized by persistent eosinophilia with tissue involvement and organ dysfunction which often precedes a subsequent T cell lymphoma. Interleukin-5 secreted by a T lymphocyte subpopulation has been described in previous reports as the most important factor responsible for the prolonged lifespan of the eosinophils. The goal of the present study was to describe a fast, simple diagnostic method for the differentiation of HES and secondary eosinophilic states. Beside the surface marker analysis of peripheral blood mononu-clear cells (PBMC) we measured surface bound IgE molecules on lymphocytes and eosinophil cells, intracellular cytokines (IL-5, INFγ) in CD4+ lymphocytes and eosinophil major basic protein (MBP) in eosinophils using flow cytometric detection method. The appearance of an IL-5 producing cell population with a decreased number of INFγ positive lymphocytes was characteristic for the blood samples of HES patients. Predominance of Th2 cells with the appearance of a CD8+/CD3-/CD56+ cell population was restricted for the HES cases and could not be detected in secondary eosinophilic individuals. Our flow cytometric cytokine detection method (with parallel cell surface marker analysis) does not require cell separation or long term cell culture steps previously described for the detection of IL-5 producing cells. Therefore it seems to be a more appropriate approach for the differential diagnosis of primary and secondary eosinophilic states.