Multicolor Flow Cytometry Detects Monoclonal Plasma Cells In the Vast Majority of Marrows From Patients with Plasma Cell Disorders Shown to Have Genetic Abnormalities by Fluorescence In Situ Hybridization (FISH)

Abstract 1896

Flow cytometric analysis (FCM) is now considered an essential tool for the identification of malignant hemopoietic and lymphoid cells, and it is particularly useful in evaluating plasma cell clonality to differentiate normal from neoplastic plasma cells (PC). Neoplastic PC typically show loss of CD19 and aberrant expression of markers such as CD56 and CD117, but the demonstration of an expansion of PC containing a single immunoglobulin light chain (monotypic or clonal expansion) is critical for determining the nature of a plasma cell population. Genetic abnormalities are becoming essential markers for prognosis of plasma cell disorders (PCD). Because of the relatively low proliferation or low representation of PC in the sample, FISH rather than conventional cytogenetics is used routinely or the detection of karyotypic abnormalities in PCD and the use of FISH in plasma cell-enriched preparations has improved the sensitivity of detection of genetic abnormalities in these disorders. We wished to determine how often FCM would detect monoclonal cells in marrows with genetic abnormalities observed by FISH. For this purpose, we analyzed marrow samples that were either non-enriched or enriched for PC using immunomagnetic beads for cell separation. From February 2009 until present, we studied marrows sent to our laboratory for diagnostic purposes from 158 patients who either had a clinical or laboratory abnormality suggestive of or consistent with PCD, or their marrows demonstrated immunophenotypic abnormalities of PC. The FCM consisted of a simultaneous determination of surface CD45, CD19, CD38, CD56 and intracytoplasmic kappa and lambda immunoglobulin (Ig) light chains in a single tube, using a Canto II cytometer (BD). Between 10⁵ and 3×10⁵ cells were analyzed in each case. Clonal expansions were detected based on the Ig light chain expression in cells selected as normal or abnormal plasma cells by their surface phenotype. FISH analysis was performed using Abbott probes for CEP 7, CEP 9, CCND1/IgH, RB1, and p53 on both enriched and non-enriched interphase cells. FISH samples were processed according to standard procedures and 200 cells were analyzed for each probe. Plasma cell enrichment was performed by incubating marrow cells with CD138-coated immunomagnetic beads, and isolating them on Miltenyi separation columns using an auto MACS Pro Separator. Of the 158 samples studied, 52 underwent plasma cell enrichment. Enrichment was performed on samples with less than 8% PC as assessed by FCM. The fraction of PC in enriched samples was approximately 10 times greater than those in non-enriched samples. FISH abnormalities were detected in enriched samples from cases that contained as low as 0.04% PC prior to enrichment. The majority of the enriched samples that showed FISH abnormalities exhibited multiple FISH alterations, the most common being additional copies of CEP 7, CEP 9 and CCND1 DNA sequences, and deletion of RB1 and p53. Of the 158 samples, 46 (29%) were found to be polyclonal by FCM and normal by FISH. These included 8 samples enriched for PC. Of the 69 non-enriched samples only 33 (48%) demonstrated FISH abnormalities. Of these abnormal samples, 32 (97%) were monoclonal by FCM. Among the 43 samples that underwent plasma cell enrichment, 37 (86%) demonstrated FISH abnormalities. Of these enriched abnormal samples, 34 (92%) were shown to be monoclonal by FCM. Our results showed that, confirming previous observations, targeted FISH analysis using plasma cell enrichment increases the detection frequency of abnormalities in PCD, which can be found in the majority of cases. This study also showed that FCM detected monoclonality in the vast majority of samples of PCD that demonstrated FISH abnormalities, whether or not they were enriched for PC, validating the clinical utility of FCM in the routine diagnostic evaluation of patients with PCD.