Correlation of Multiparameter Flow Cytometry and Fluorescence In Situ Hybridization in Quantifying Malignant Cells at Low Frequencies in B-Cell Lymphoproliferative Diseases.

Diagnostic and follow-up evaluation of patients with B-cell lymphoproliferative diseases includes the assessment of peripheral blood and bone marrow samples by both multiparameter flow cytometry (MFC) and fluorescence in situ hybridization (FISH) for the detection and quantification of malignant cells. Depending on the type of aberrant immunophenotype and on the probes applied for FISH sensitivity may vary particularly if small populations of malignant cells are present. We evaluated 61 peripheral blood (n=20) and bone marrow (n=41) samples from patients with B-cell lymphoproliferative diseases in which malignant cell populations as quantified by MFC were <15% and in which chromosomal aberrations were identified by FISH analysis. The diagnoses included CLL and CLL/PL (n=23), follicular lymphoma (FL, n=16), mantle cell lymphoma (MCL, n=10), and other B-cell lymphoproliferative diseases comprizing mainly marginal zone lymphomas and lymphoplasmacytic lymphomas (B-NHL, n=12). A standardized panel was used for MFC including antibodies against CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD25, CD38, CD45, CD79b, CD103, FMC7, IgM, Kappa, and Lambda. FISH was performed including probes for del(6q), del(11q), trisomy 12, del(13q), del(17p), t(11;14), t(14;18), IGH, CMYC, and BCL6. The median number of malignant cells as quantified by MFC amounted to 8% (range, 2% to 14%). The median number of malignant cells as quantified by FISH amounted to 9% (range, 1% to 42%). Overall, there was a high degree of correlation between both methods (r=0.544, p<0.0001). In particular, there were good correlations within the subgroups CLL (r=0.402, p=0.057) and MCL (r=0.876, p=0.001). Accordingly, correlations were significant for results obtained by FISH probes for trisomy 12 (r=0.649, p=0.005) and for t(11;14) (r=0.693, p=0.009) while the correlation for cases with t(14;18) were less strong (r=0.411, p=0.101). The present results confirm that in patients with B-cell lymphoproliferative diseases malignant cells may be detected and quantified by both MFC and FISH with good correlation even if present at very low frequencies only. So far our data are not in favor of either MFC or FISH for analyzing low percentages of lymphoma infiltration. Future studies should assess the clinical relevance of low level infiltration of peripheral blood and bone marrow and the respective roles of MFC and FISH in their detection.