Abstract
Multiple myeloma (MM) is a B cell malignancy characterized by clonal expansion of plasma cells. Many MM patients achieve a complete remission by conventional criteria; yet most patients relapse as a consequence of residual disease. Current approaches for the measurement of minimal residual disease (MRD) in bone marrow (BM) are based on morphologic assessment of BM aspirate and biopsy, flow cytometry, immunohistochemistry, molecular (PCR) studies, conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) analyses. Morphologic assessment of MRD is often difficult due to the fact that normal plasma cells may also be present in the BM. Genetic factors have emerged as significant prognostic factors in MM; however, CC studies are hampered by the low proliferative nature of the malignant cells. FISH analyses have detected clonal abnormalities of -13/del(13q), 14q32/IGH, del(17p), and hyperdiploidy (+5,+9,+15) in >80% of newly diagnosed MM cases; yet, detection of these abnormalities post treatment by the standard FISH approach has proven to be very difficult in samples with less than 20% BM involvement. PCR-based approaches are sensitive but suffer a critically high false-negative rate. In this study, we investigated 137 post treatment samples collected from 101 MM patients (31 patients with multiple studies), all showing < 20% BM involvement, using a sequential May-Grünwald Giemsa (MGG) (morphology)/FISH approach to determine the plasma cell genotype (target or T-FISH). Cytospin slides were made using 200 μl of BM and stained with MGG for morphologic classification on a Duet™ Image Analyzer (Bioview Ltd., Rehovot, Israel). After identifying and mapping the plasma cells, the slides were destained and hybridized with one of four FISH probe sets corresponding to the chromosome aberrations listed above. The T-FISH results were correlated with CC, BM pathology, which quantified the percentage of plasma cells in the BM aspirate, and BIOMED-2 PCR analysis for IGH (FR1, 2 and 3) and IGK gene rearrangements (InVivoScribe Technologies, San Diego, CA). T-FISH identified MM aberrations in 123 of 137 (89.8%) samples, a finding significantly higher than the 50/83 (60.2%) positive cases detected by combined molecular IGH and IGK PCR studies (two-sided Fisher’s Exact p < 0.0001). T-FISH aberrations observed were IGH in 77 samples, del(13q)/-13 in 48, hyperdiploidy in 37, hypodiploidy in 6, and del(17p) in 4, with 42 samples showing more than one abnormality. Only 10 samples showed clonal karyotypic aberrations by CC; an additional 3 samples showed a “presumed” stemline with only one abnormal cell (9.5%). A comparison with the percentage of plasma cells in the BM smears showed T-FISH detected residual disease in all 48 samples with ≥6 % plasma cells, 14 of 15 hemodilute samples, one smoldering MM sample and 82.2% (60/73) of the samples with 1–5% plasma cells. Our data indicate T-FISH is a quick, universally applicable, and robust assay to quantitate neoplastic plasma cells regardless of treatment status, making it the most sensitive molecular assay currently available to monitor a patient’s clinical course. Furthermore, the T-FISH molecular cytogenetics strategy provides a novel approach to monitor both traditional and targeted therapies in low proliferative malignancies by their underlying genetic abnormalities.
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