VS38 staining contributes to a novel gating strategy in flow cytometry for small B cell lymphoma, especially in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

BACKGROUND: Multi-parametric flow cytometry (MFC) is a helpful tool for detecting neoplastic cells in malignant lymphoma; however, lymphoma cells can be difficult to detect when characteristic immunophenotypic abnormalities are not evident. We evaluated the stainability of VS38, which is used for multiple myeloma, in normal and abnormal B cells using MFC to develop a new strategy for detecting lymphoma cells. METHODS: We compared the median fluorescence intensity of VS38 staining in lymphocytes from patients without hematopoietic neoplasms and in B cells from 26 patients with B cell lymphoma (BCL). To evaluate the performance of VS38 gating, we compared VS38-positive B cells with the percentages of BCL cells, and with the mutation ratios of MYD88 L265P measured by droplet digital PCR in patients with lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM). RESULTS: CD27-positive memory B cells were stained with VS38, whereas normal lymphocytes were faintly stained. Lymphoma cells were stained with VS38 in 11 of 12 patients with LPL/WM, 3 of 3 with chronic lymphocytic leukemia, 3 of 5 with mantle cell lymphoma, 2 of 4 with follicular lymphoma, and 1 of 1 with splenic marginal zone lymphoma. The percentages of VS38-positive B cells in VS38-positive BCL were equivalent to those of lymphoma cells and the mutation ratios of MYD88 L265P in LPL/WM. CONCLUSIONS: VS38 identified neoplastic cells in plasma cell disorders and BCL. This might improve the accuracy of BCL diagnosis, especially in patients with LPL/WM.

Sensitive detection of GATA1 mutations using complementary DNA-based analysis for transient abnormal myelopoiesis associated with the Down syndrome.

INTRODUCTION: GATA1 mutation plays an important role in initiating transient abnormal myelopoiesis (TAM) and in the clonal evolution towards acute megakaryoblastic leukaemia (AMKL) associated with Down syndrome (DS). This study aimed to develop and validate the clinical utility of a complementary DNA (cDNA) analysis in parallel with the conventional genomic DNA (gDNA) Sanger sequencing (Ss), as an initial screening test for GATA1 mutations. METHODS: GATA1 mutations were evaluated using both gDNA and cDNA in 14 DS patients using Ss and fragment analysis (FA), respectively. RESULTS: The detection sensitivity of conventional gDNA sequencing was limited in low blast percentage TAM (LBP-TAM); however, cDNA-based Ss readily detected all the pathognomonic GATA1 mutations. The cDNA-based FA readily detected GATA1 frameshift mutation with a reliable sensitivity ranging from 0.005% to 0.01% of clonal cells. CONCLUSIONS: GATA1 mutations are heterogeneous; therefore, we would like to propose a dual cDNA and gDNA analysis as a standard diagnostic approach, especially for LBP-TAM. cDNA-based FA promises an excellent sensitivity for detecting frameshift GATA1 mutations in the longitudinal clonal evolution towards AMKL without using a patient specific primer.

cDNA-Based Mutation Screening Using a Combination of High-Resolution Melting Curve and Fragment Analysis Facilitates Efficient CCR4 Mutation Analysis in Adult T-Cell Leukemia/Lymphoma.

OBJECTIVES: C-C chemokine receptor type 4 (CCR4) proteins are expressed on the neoplastic cells of adult T-cell leukemia/lymphoma (ATLL). As the mutation status of CCR4 gene is reported to correlate with significant clinical information such as prognosis and response to mogamulizumab, we aimed to establish a screening method that is suitable for clinical laboratory tests. METHODS: In 34 patients with ATLL, CCR4 mutation analysis, high-resolution melting (HRM) analysis, fragment analysis, and direct sequencing were performed using both genomic DNA and complementary DNA (cDNA). Furthermore, 38 cases of asymptomatic carriers of human T-cell leukemia virus type 1 (HTLV-1) were screened for CCR4 mutation. RESULTS: Mutation analysis by direct sequencing of 34 ATLL clinical samples detected CCR4 mutation in four genomic DNA samples and seven cDNA samples, and two novel mutations were identified. All CCR4 mutations detected by direct sequencing were positive for HRM analysis and/or fragment analysis. CCR4 mutation was not detected in the asymptomatic carriers of HTLV-1. CONCLUSIONS: CCR4 mutation screening by a combination of HRM and fragment analysis using cDNA is a simple and practical method, and it will contribute to better decision making for a therapeutic strategy, providing a rapid CCR4 mutational status to clinicians.

VS38 as a promising CD38 substitute antibody for flow cytometric detection of plasma cells in the daratumumab era.

The development of effective therapies has enabled long-term survival for many patients with multiple myeloma (MM). However, the administration of antibody drugs, such as daratumumab, which bind to plasma cell (PC) surface proteins, may prevent PC detection by flow cytometry. We propose VS38 as an alternative antibody for CD38. VS38 recognizes cytoskeleton-linking membrane protein 63 (CLIMP-63) on the rough endoplasmic reticulum, and this protein may be expressed in secretory cells. We investigated VS38 staining in normal hematopoietic cells from five control samples, as well as PCs from 21 patients with plasma cell disorder (PCD). In normal hematopoietic cells, although VS38-stained monocytes, myeloid cells, and a subpopulation of B cells, PCs were significantly and brightly stained by VS38. There was no significant difference in VS38 staining between normal and abnormal PCs obtained from five patients with monoclonal gammopathy of undetermined significance. Furthermore, PCs in 21 PCD cases were clearly identified by VS38 in all cases, in contrast to CD38, even in daratumumab-administered patients whose CD38 epitopes on PCs were masked. These results suggest that the use of the VS38 antibody in flow cytometry contributes to PC detection, independent of therapeutic treatment.