The online version of this article contains a supplementary appendix. BackgroundThe diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts. Design and MethodsWe designed a flow cytometry protocol applicable in many laboratories and verified its diagnostic utility in patients without those diagnostic markers. The cardinal parameters, analyzable from one cell aliquot, were myeloblasts (%), B-cell progenitors (%), myeloblast CD45 expression, and channel number of side scatter where the maximum number of granulocytes occurs. The adjunctive parameters were CD11b, CD15, and CD56 expression (%) on myeloblasts. Marrow samples from 106 control patients with cytopenia and 134 low-grade myelodysplastic syndromes patients, including 81 lacking both ringed sideroblasts and cytogenetic aberrations, were prospectively analyzed in Japan and Italy. ResultsData outside the predetermined reference range in 2 or more parameters (multiple abnormalities) were common in myelodysplastic syndromes patients. In those lacking ringed sideroblasts and cytogenetic aberrations, multiple abnormalities were observed in 8/26 Japanese (30.8%) and 37/55 Italians (67.3%) when the cardinal parameters alone were considered, and in 17/26 Japanese (65.4%) and 42/47 Italians (89.4%) when all parameters were taken into account. Multiple abnormalities were rare in controls. When data from all parameters were used, the diagnostic sensitivities were 65% and 89%, specificities were 98% and 90%, and likelihood ratios were 28.1 and 8.5 for the Japanese and Italian cohorts, respectively. ConclusionsThis protocol can be used in the diagnostic work-up of low-grade myelodysplastic syndromes patients who lack specific diagnostic markers, although further improvement in diagnostic power is desirable.Key words: myelodysplastic syndromes, flow cytometry, diagnosis.Citation: Ogata K, Della Porta MG, Malcovati L, Picone C, Yokose N, Matsuda A, Yamashita T, Tamura H, Tsukada J, and Dan K. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study. Haematologica 2009;94:1066-1074. doi:10.3324/haematol.2009 This is an open-access paper. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study
were analyzed using 4-color flow cytometry (FCM). We objectively determined reference ranges of 13 parameters related to CD34 ؉ cells with data from controls. InLGw/oRS patients, various abnormalities of CD34 ؉ cells-eg, decrease in CD34 ؉ B-cell precursors, aberrant expression or overexpression of various antigens on CD34 ؉ myeloblasts-were observed. We constructed a reproducible, flow cytometric scoring system for LGw/oRS diagnosis. High scores were observed in 16 of 27LGw/oRS patients, regardless of the presence or absence of chromosomal aberrations, but not in any of the 90 controls. Among LGw/oRS patients with chromosomal aberrations, patients with trisomy 8 or del20(q) had low FCM scores (P ؍ .002). As a result, most LGw/oRS patients were identified based on high FCM score, chromosomal aberration, or both.
During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-␥ (IFN␥) and tumor necrosis factor-␣ (TNF␣) on MDS blasts. This induction was associated with the activation of nuclear factor-B (NF-B) and nearly completely blocked by an NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1 ؉ MDS blasts had greater intrinsic proliferative capacity than B7-H1 ؊ MDS blasts when examined in various assays. Furthermore, B7-H1 ؉ blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken IntroductionB7-H1 (CD274), which was identified by us as a costimulatory molecule, plays a crucial role in T-cell regulation in various immune responses. 1,2 B7-H1 molecules deliver a costimulatory signal through an unknown receptor on naive T cells. [1][2][3] They also deliver an inhibitory signal to activated T cells through programmed cell death 1 (PD-1) molecules, 4 which are a type I transmembrane protein belonging to the CD28 receptor family and were originally identified in T cells undergoing apoptosis. 5 B7-H1 expression is detected not only on antigenpresenting cells but also on activated T cells and some tumor cells (ie, renal cell, colon, breast, and lung carcinoma, and Hodgkin lymphoma). [6][7][8][9][10] Rodent data suggest that B7-H1 molecules on tumor cells deliver negative signals through PD-1 and other receptors on tumorspecific cytotoxic T lymphocytes and inhibit antitumor immune responses. 11,12 Consistent with those data, it was reported that in patients with renal cell carcinoma and breast cancer, patients whose tumor cells expressed B7-H1 had a poor prognosis. 9,13 In a mouse leukemia model in which mice were immunized with irradiated DA1-3b leukemia cells and then challenged with live DA1-3b cells, only leukemia cells expressing high levels of B7-H1 survived for a long period. Moreover, these cells gained tolerance to specific cytotoxic T lymphocytemediated killing. 14 Therefore, B7-H1 molecules on leukemia cells may be associated with immune evasion in this model.Myelodysplastic syndromes (MDS) are clonal hematologic stem cell disorders characterized by cytopenias, excessive apoptosis of hematopoietic cells, and a high risk of progression to acute myeloid leukemia (AML). In MDS, various immune abnormalities, including lymphopenia and T-cell dysfunction, have been reported, 15-17 although data on B7-related molecules, in particular B7-H1, are lacking. With disease progression, that is, with i...
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