Eight-color multiparameter flow cytometry (EuroFlow-NGF) is as sensitive as next-generation sequencing in detecting minimal/measurable residual disease in autografts of patients with multiple myeloma.

The prognostic value of minimal/measurable residual disease (MRD) detection in autografts of patients with multiple myeloma (MM) in an autologous stem-cell transplantation setting has been reported. Next-generation flow (NGF) cytometry has lower sensitivity (2 × 10-6) to detect MRD than next-generation sequencing (NGS) (<10-6). We compared the clinical value of high-sensitivity NGF (cutoff: <10-6) and NGS (cutoff: 10-6) for the detection of MRD in the cryopreserved autografts of 49 patients with newly diagnosed MM. The sensitivity test using frozen/thawed autografts revealed a strong correlation among MRD levels of 5 × 10-7 and 1 × 10-4 (r = 0.9997, p < 0.0001) when an adequate number of cells were analyzed. Autograft MRD levels determined using NGF and NGS were highly correlated (r = 0.811, p < 0.0001). MRD-negative patients identified with NGF (cutoff: <10-6) showed significantly longer progression-free survival (PFS) than MRD-positive patients (p = 0.026). The PFS of MRD-negative patients determined by NGS (cutoff: 10-6) was similar to that determined by NGF. These results show that the high-sensitivity NGF method can assess MRD in frozen/thawed autografts, and its prognostic value is comparable to that of NGS.

Frequent HLA-DR loss on hematopoietic stem progenitor cells in patients with cyclosporine-dependent aplastic anemia carrying HLA-DR15.

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA), we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-γ for 72 h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g., CD48, CD74, and CD86) in DR(-) cells, which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-ß.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-ß co-receptor and inhibits TGF-ß signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-ß. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-ß to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-ß signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-ß, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.