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Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.

Background: In patients with unexplained cytopenias, abnormal karyotyping studies can be found with inconclusive light microscopic findings. Multidimensional flow cytometry (FCM) can identify myelomonocytic cells with aberrant phenotypes often not seen by standard morphology. Methods: In 431 patients presenting with unexplained cytopenia(s) FCM results were compared to abnormal karyotyping and FISH results recognized as associated with myelodysplastic syndrome (MDS) in the 2008 WHO classification, to assess the degree of and types of phenotypic abnormalities observed using a previously reported flow cytometric scoring system (FCSS). Fluorescence activated cell sorting was also used to identify subpopulations of abnormal maturing myelomonocytic cells that carry the genotypic abnormality. Results: For marrows with complex (three or more karyotypic abnormalities), two abnormalities, isolated chromosome seven anomalies, del(5q) or del(13q), 100% of cases were positive when using a FCSS cutoff of ≥2. Trisomy 8, del(20q), and minus Y had flow scores ≥2 in 72, 60, and 18%, respectively, but in some cases the flow score was high, indicating myeloid dysplasia. Most patients (16/22) with high myeloid progenitor cells (MyPC) (>20%) also exhibited maturing myeloid cell abnormalities by FCM. Morphology was negative in the maturing myeloid cells in many cases with phenotypically abnormal myeloid cells. Conclusions: The high correlation between genotypic and phenotypic abnormalities suggests a possible increased utility of flow cytometry in the diagnosis of patients with unexplained cytopenias and may be useful in future clinical studies and in the classification by the WHO, using the FCSS rather than simple counting of flow cytometric abnormalities. © 2010 International Clinical Cytometry Society

Background: In this study we demonstrate the technical application of flow cytometry and cell sorting combined with gene-rearrangement clonality profiling to detect and confirm minimal disease in 2 leukemia and 2 lymphoma cases.Methods: Specimens with low percentages (0.05%–5%) of abnormal lymphoid populations were identified by flow cytometry. The abnormal lymphoid populations were sorted by flow cytometry, and the purified tumor populations along with unsorted fractions were subsequently analyzed for the presence of clonal gene rearrangements by PCR and fluorescence-based capillary electrophoresis fragment analysis.Results: In 3 cases, distinct clonality profiles could be detected in the purified tumor cell fraction, and suspicious amplicons of identical sizes were detected among the polyclonal backgrounds in the unsorted specimens. For 1 patient, a monoclonal signal was detected in the sorted tumor cell fraction but not in the unseparated bone marrow specimen containing 0.05% abnormal lymphoblasts. A subsequent bone marrow specimen containing 4.8% recurring leukemia cells tested positive with a clonality profile that matched the previous profile in the sorted cell population.Conclusions: The described method integrating 2 technologies allows genotypic confirmation of an aberrant population detected by immunophenotype to increase diagnostic certainty. This strategy provides a sensitive tool for disease monitoring without the need for patient-specific primer design and assay optimization required for quantitative PCR analysis.

Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.

Background: Myelodysplastic syndromes (MDS) are associated with cytogenetic clones. To follow the maturation sequence of original clones and evolved subclones with additional cytogenetic abnormalities, progenitor cells, immature and mature myeloid cells were sorted by flow cytometry and analyzed separately by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). Methods: Flow cytometry sorted cell fractions from bone marrows for sixteen patients with MDS-associated cytogenetic abnormalities were evaluated by FISH. Flow cytometric cell sorting was based on CD34+/low side scatter (SS) for progenitors, CD13+/CD16-/high SS for immature myeloid cells, and CD13+/CD16+/high SS for mature myeloid cells. Customized color labeling (spectrum orange, green and aqua) of FISH probe combinations were designed to detect and to analyze clonal evolution for each patient based on their known cytogenetic abnormalities and clonal evolution patterns. Three marrow aspirates were sorted for analysis by SNP/CGH microarray. Results: The 16 bone marrow specimens evaluated by FISH were categorized into three groups: (1) eight patients with a single, good-to-intermediate cytogenetic abnormality; (2) four patients with monosomy 7; and (3) four patients with more than one chromosome abnormality with evidence of clonal evolution by conventional cytogenetic analysis, excluding monosomy 7. The Group-1 abnormalities included deletion 20q (n=4), trisomy 8 (n=2), deletion 5q (n=1), and trisomy 11 (n=1). All specimens from this group showed FISH abnormalities in equal proportions in myeloid progenitors, immature and mature myeloid cells. Group-2 had four patients with monosomy 7. All four had monosomy 7 concentrated in the progenitor cells (45-79%) compared to immature and mature myeloid compartments (less than 9-36%). For Group-3, known original clones with single cytogenetic abnormalities (deletion 20q, monosomy 3, deletion 7q or 5q) were monitored by FISH analysis. Using customized FISH panels, the presence of subclones with additional cytogenetic aberrations (trisomy 8 in three patients and gain of a marker chromosome characterized by the centromere of chromosome 4 in a fourth patient) was assessed using single-cell resolution. The progenitor and immature myeloid compartments had the original founding clones containing a single cytogenetic abnormality at 15-34% and the subclones with the additional aberrations at 23-76%. In contrast, the mature myeloid cells were comprised of the original clone at 20-40%, but the subclones with additional aberrations were absent in the mature myeloid compartment in three patients and reduced by more than half in a fourth. For three patients sorted bone-marrow fractions were analyzed by SNP/CGH microarray. In one patient with trisomy 8 as the sole abnormality, the same aberration was observed in both the immature and mature myeloid compartments. In two other patients, additional abnormalities not seen in the mature myeloid cells were detected in the progenitor and immature myeloid compartments. Conclusions: These data show two main patterns for the distribution of clonal cytogenetic abnormalities among myeloid cells in MDS: 1) A continuous distribution at all stages of maturation was found for single aberrations with good-to-intermediate prognostic associations (Group 1) and 2) A disrupted distribution pattern with accumulation of the cytogenetic aberrations in the progenitor compartment as compared to the immature and mature myeloid compartments for specimens characterized by monosomy 7 (Group 2). Similarly, subclones characterized by additional cytogenetic abnormalities (Group 3) were sequestered in the progenitor and immature myeloid compartments while the original founding clone was evenly distributed throughout maturation. These data demonstrate that specific cytogenetic abnormalities associated with poor prognosis (e.g. monosomy 7) as well as acquired cytogenetic abnormalities as a result of clonal evolution can cause disruption of myeloid cell maturation in MDS. Disclosures Zehentner: HematoLogics Inc.: Employment, Equity Ownership. Hartmann:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Chapman:Hematologics Inc.: Employment. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Singleton:Hematologics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:Hematologics: Employment, Equity Ownership.

Background: Waldenström's macroglobulinemia (WM) and lymphoplasmacytic lymphoma (LPL) are lymphoproliferative disorders with bone marrow infiltration by clonal lymphoplasmacytic cells (Treon et al., 2003, 2005). The somatic point mutation L265P in the myeloid differentiation primary response gene 88 (MYD88) has been reported in more than 90% of WM patients (Treon et al., 2012). Therefore MYD88 mutation analysis has been implemented in clinical practice to support the diagnosis of LPL/WM. After the implementation of MYD88 L265P assays with increased detection sensitivity, a substantial portion of patients with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS) was also reported MYD88 L265P positive. Splenic marginal zone lymphoma (SMZL), B-cell chronic lymphoproliferative disorders (B-CLPD) and diffused large B-cell lymphoma (DLBCL) have been found positive with much lower incidence rates (Varettoni et al., 2013; Xu et al., 2013). Hence the remaining need to differentiate WM/LPL from other lymphoproliferative disorders with co-occurring plasma cells with high confidence. Patients and Methods: In this study, flow cytometric cell sorting was utilized to isolate clonal plasma and B lymphoid cell fractions as previously described (Zehentner et al., 2011). 69 patient specimen fractions with a clinical history of WM /LPL, multiple myeloma, CLL and lymphoma were analyzed for MYD88 L265P mutation using Sanger sequencing. Furthermore, the Biomed-2 primer sets for the immunoglobulin heavy (IgH) and/or the immunoglobulin kappa light chain region (IgK) were used to compare B cell clonality profiles in the lymphoid versus plasma cell compartments. Results: MYD88 L265P mutation was detected in all specimens with confirmed Waldenström's macroglobulinemia (17/17, 100%). Of these 16/17 (94%) revealed MYD88 L265P as well as identical monoclonality profile by gene rearrangement analysis in both the plasma and the B lymphoid cell collections. In 47% (8/17), the mutation was only detected in the plasma and B cell fractions, but not in the whole bone marrow specimens. 21 patient specimens with a known clinical history of LPL and co-occurring clonal plasma cells were tested. 9 of 21 (43%) were categorized with identical B cell clonality profile when comparing plasma and B lymphoid cells; whereas 12 (57%) had unrelated clonality profiles. 7 of the 9 (78%) specimens in the identical clonality group tested positive for MYD88 L265P in both the plasma and B lymphoid clone. None of the unrelated clonality group specimens carried the mutation in both cell fractions; for 7/12 (58%) MYD88 L265P was found in either the plasma (2) or the B-cell fraction (5) whereas 5/12 (42%) tested negative. 11 bone marrow aspirate specimen with known presence of lymphoma (including splenic marginal zone (SMZL), mantle cell and marginal zone) were analyzed. 10/11 (90%) tested negative for MYD88 L265P, with the exception of one SMZL specimen. Furthermore, 12 known myeloma, 5 CLL and 3 healthy donor specimens were analyzed, all tested negative. Conclusions: In this study, we developed and tested a novel approach to assess MYD88 L265P mutation status in order to assist WM/LPL diagnosis. Flow cytometric cell sorting for clonal plasma and B cell populations was combined with molecular analysis. Subsequently, MYD88 L265P mutation as well as B-cell clonality profile was compared in both cell fractions. Our study postulates a significant improvement in sensitivity and most importantly specificity when applying MYD88 L265P mutation status in combination with cell sorting for WM/LPL diagnostic decisions. 47% WM patients (8/17) and 44% LPL patients (4/9) were positive for the MYD88 mutation exclusively in both flow cytometry sorted cell fractions but not in whole bone marrow specimens. 94% (16/17) WM as well as 78% (7/9) LPL specimens with identical plasma and B-cell clonality profile revealed the presence of the MYD88 L265P mutation in both the plasma cell and the B lymphoid cell clones. Whereas LPL specimens with unrelated clonality profile of the plasma and lymphoid cell fractions as well as other control specimens (lymphoma, myeloma, CLL and healthy) either tested negative or positive only in one of the sorted cell fractions. We therefore conclude that confirming the presence of MYD88 L265P in both B-lymphoid and plasma cell fraction is an important prerequisite to distinguish LPL/WM from related disorders with high confidence. Disclosures Wang: HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Burnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Wentzel:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.