Follicular lymphoma: validation of a novel approach for the detection of clonality and microRNA expression profiling

1. APPLICATION OF MICROFLUIDIC TECHNOLOGY TO THE BIOMED-2 PROTOCOL FOR DETECTION OF B-CELL CLONALITY The BIOMED-2 protocol is the successful result of a multicenter effort for standardizing and optimizing detection of clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions that are analyzed by either capillary electrophoresis or heteroduplex analysis on polyacrylamide gel-electrophoresis. We tested a commercially available microfluidic chip-electrophoresis apparatus (Agilent 2100 Bioanalyzer) as an alternative to capillary electrophoresis and heteroduplex analysis for the analysis of multiplex BIOMED-2 PCRs. We used the protocol for detection of B-cell clonality that requires 5 PCR reactions, three for immunoglobulin heavy and two for kappa light chain genes. We tested 68 B-cell lymphomas: 33 follicular and 35 non-follicular (18 chronic lymphocytic leukemia, 7 difuse large B-Cell lymphoma, 1 splenic marginal zone lymphoma, 1 extranodal marginal zone lymphoma, 1 lymphoplasmacytic lymphoma, 1 hairy cell leukemia, 1 Hodgkin lymphoma, 3 unclassified and 2 cases with no histology available), and 16 reactive lymph nodes. Chip electrophoresis was conclusive for monoclonality in 62/68 samples: 30/33 (91%) follicular lymphoma and 32/35 (91%) nonfollicular lymphoma samples. The interpretation of clonality was concordant for 19 of 22 samples (86%) analyzed by Southern blot. Chip electrophoresis was compared with capillary electrophoresis using 26 samples. Concordant results were obtained in 68 of 78 heavy chain and 48/52 kappa chain gene rearrangements. The concordance between the chip electrophoresis and capillary electrophoresis in the interpretation of clonality was 96%. We conclude that the chip-based apparatus is suitable for the analysis of B-cell clonality using the BIOMED-2 protocol. 2. EVALUATION OF microRNA INVOLVEMENT IN FOLLICULAR LYMPHOMA DEVELOPMENT BY EXPRESSION PROFILING Follicular lymphoma (FL) is an indolent lymphoma which accounts for 20%-30% of non-Hodgkin’s lymphomas. FL appears as a lymph node (LN) infiltrated by proliferating germinal center B-cells (GC B-cells). MicroRNAs are a class of small non-coding RNAs that play key roles in many cellular pathways during normal development and cellular homeostasis. Aberrant expression of microRNAs is a common feature of cancer and also of the haematological ones, suggesting they carry out an important role in the development of these pathologies. Since little is known on microRNA role in FL, we analyzed expression profile of 26 FL. As controls we profiled 12 LN and 10 purified GC Bcells samples. Many microRNA were differentially expressed when FL was compared to LN and to GC B-cells. Interestingly some of differentially expressed microRNA showed opposite trends in the two comparisons. The expression of miR-9/miR-9*, miR- 30 family, miR-15-16 family, miR-34a, cluster-17~92 and miR-28 was high in FL as compared to reactive LN, while the expression was lower when FL was compared to GC B-cells. Altogether these results suggested that these microRNAs may belong to a specific signature of GC B-cells and, therefore, that FL maintain the characteristics of the normal B-cells that they are derived from. Differently miR-21 and miR-219 were up-regulated with a high significance in FL compared to both FL and GC B-cells indicating their possible association with follicular lymphoma pathogenesis and progression.