Norio Yokose

Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study.

The diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts. This article proposes a flow cytometry protocol that can be used in the diagnostic work-up of low-grade myelodysplastic syndrome patients who lack specific diagnostic markers. See related perspective article on page 1041. Background The 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 Methods We 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. Results Data 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. Conclusions This 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.

CD20-positive T cell leukemia/lymphoma: case report and review of the literature

Abstract. We report on a case of CD20-positive peripheral T cell lymphoma. The lymphoma cell was positive for CD20 and T cell lineage markers such as cytoplasmic CD3, CD4, and CD5 and had a monoclonal rearrangement of the T cell receptor (TCR) γ chain gene. The clinical characteristics resembled angioimmunoblastic lymphadenopathy: spontaneous regression of lymphadenopathy and immunological abnormalities such as polyclonal hypergammaglobulinemia, positive results of direct and indirect antiglobulin tests, and a high antinuclear antibody titer. We reviewed seven cases of CD20-positive T cell malignancies including the present case. Three were immature T cell malignancies (acute lymphoblastic leukemia) and four were peripheral T cell malignancies (non-Hodgkins lymphoma and chronic lymphocytic leukemia). Hepatomegaly and/or splenomegaly were common features. Further cases must be evaluated to understand the clinical significance of the CD20 expression on the surface of T cell malignancies.

Reappraisal of the clinical significance of CD7 expression in association with cytogenetics in de novo acute myeloid leukaemia

Debate exists over whether CD7 expression indicates an unfavourable prognosis in de novo acute myeloid leukaemia (AML). Meanwhile, the type of cytogenetics is a strong prognostic factor in AML. We analysed 256 de novo adult AML cases and found that the proportion of CD7+ cases increased stepwise from the cases with favourable cytogenetics to the cases with intermediate and unfavourable cytogenetics (3 out of 69 cases, 51 out of 140 cases and 25 out of 47 cases respectively, P < 0·0001). CD7‐positivity adversely affected the survival only in the cases with unfavourable cytogenetics (P < 0·03). We recommend that CD7 expression in AML be interpreted in association with the cytogenetics.

Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes

Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO‐R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using 125I‐TPO. The MDS patients had higher plasma TPO levels (7.0 ± 9.3 fmol/ml) than 52 normal subjects (P < 0.0001). Refractory anaemia (RA) patients (n = 39) had higher plasma TPO levels than patients (n = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB‐t) (P = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients (P = 0.0027) but not in RAEB and RAEB‐t patients (P = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB‐t. The number of TPO‐R per platelet was significantly smaller in 19 MDS patients (17.5 ± 13.3) than in normals (P = 0.0014), but similar between RA patients and patients with RAEB and RAEB‐t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB‐t. Thus, in addition to thrombocytopenia, a reduced platelet TPO‐R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO‐R and marrow megakaryocytes, such as blasts expressing TPO‐R, may operate in RAEB/RAEB‐t.

Pulmonary toxicity after granulocyte colony-stimulating factor-combined chemotherapy for non-Hodgkin's lymphoma.

Sporadic cases have developed pulmonary toxicity after receiving chemotherapy and granulocyte colony-stimulating factor (G-CSF). However, because such cases received chemotherapy that alone frequently causes pulmonary toxicity, the role of G-CSF in this toxicity has been unclear. CHOP therapy (cyclophosphamide, doxorubicin, vincristine and prednisolone) only slightly induces pulmonary toxicity. However, we observed a considerable incidence of this toxicity in non-Hodgkins lymphoma subjects receiving CHOP therapy and G-CSF (6 out of 52 subjects, 11.5%). In this cohort, among various characteristics, including the dose and interval of CHOP therapy, only the mean peak leucocyte count (MPLC) with each therapy cycle was associated with development of this toxicity (MPLC > or = 23.0 x 10(9) l(-1), 6 out of 29 cases; MPLC < 23.0 x 10(9) l(-1), 0 out of 23 cases; P = 0.020). These findings suggest that the effect of G-CSF is the main determinant of the pulmonary toxicity in these cases. Because the toxicity was associated with a large MPLC and did not recur in cases readministered G-CSF, an idiosyncratic reaction to G-CSF is unlikely to be the pathogenesis of this toxicity. Thus, lowering the G-CSF dose seems to be useful in the prevention of this toxicity. In all six cases, the time course of manifestation of the toxicity was the same, and early application of high-dose corticosteroid led to cure. This knowledge will be helpful in the care of similar cases.

Chemotherapy for minimally differentiated acute myeloid leukemia (AML-M0) : a report on five cases and review of the literature

SummaryWith the objective of establishing the optimal therapy for minimally differentiated acute myeloid leukemia (AML-M0), we examined the therapeutic results of five AML-M0 cases and reviewed the literature. In a series of 63 patients with newly diagnosed acute leukemia who were admitted to the Main Hospital of Nippon Medical School, five patients fit the criteria for AML-M0: negative myeloperoxidase (MPO) and Sudan black B reaction by light microscopy, negative for B- and T-lineage markers, and positive for myeloid markers. They were treated by means of AdVP [adriamycin, vincristine, and prednisolone (PSL)] therapy and/or BHAC-DMP [behenoylcytosine arabinoside (BHAC), daunorubicin (DNR), 6-mercaptopurine (6-MP), and PSL] therapy. The AdVP therapy was unsuccessful in the two patients who received it, while a complete remission (CR) was achieved with the BHAC-DMP therapy in three of four patients. Although one patient treated with BHAC-DMP did not achieve CR, his blasts were apparently sensitive to the therapy. In assessable cases in the literature where leukemic blasts were MPO-negative, myeloid marker-positive and B- and T-lineage marker-negative, CR was achieved in 54.5% and 44.4% with anti-acute myeloid leukemia therapy and anti-acute lymphocytic leukemia therapy, respectively. Five cases in the literature were treated with a chemotherapeutic regimen containing BHAC [or cytosine arabinoside (Ara-C)], DNR, and 6-MP, and all achieved CR. The regimen containing BHAC (or Ara-C), DNR, and 6-MP may be useful as induction chemotherapy for AML-MO.

Prognostic significance of WT1 mRNA and anti-WT1 antibody levels in peripheral blood in patients with myelodysplastic syndromes

Wilms tumor gene (WT1) mRNA expression in peripheral blood cells was examined in 80 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) transformed from MDS. Serum anti-WT1 antibody titers were also determined in 45 patients. Their long-term follow-up showed that the survival rate became worse as the WT1 mRNA level increased. In particular, a high WT1 mRNA level was a strong predictor of a short time to AML transformation even if adjusted by the International Prognostic Scoring System category. Moreover, high values of anti-WT1 antibody were an independent predictor of longer survival. These data may justify therapeutic strategies targeting WT1 molecules in MDS.

Effects of interleukin‐12 on natural killer cell cytotoxicity and the production of interferon‐7 and tumour necrosis factor‐a in patients with myelodysplastic syndromes

The effects of Interleukin 12 (IL‐12) on natural killer (NK) cell cytotoxicity and on the production of interferon‐7 (IFN‐7) and tumour necrosis factor‐a (TNF‐a) were examined in 15 patients with myelodysplastic syndromes (MDS), which are well known to have immunologic defects, and in 11 normal subjects. The NK cell cytotoxicity of all of the normal subjects was augmented by incubation with IL‐12 alone, and co‐incubation with interleukin 2 (IL‐2) further augmented it (type A response). The MDS patients showed varied responses to IL‐12/IL‐2. Seven patients showed the type A response, resulting in augmented NK cell cytotoxicity which was similar to that in the normal subjects. In five other patients the cytotoxicity was not increased by IL‐12 alone, but the combination of IL‐12 and IL‐2 did augment the cytotoxicity (type B response). The augmented cytotoxicity in these type B patients was lower than that in the normal subjects. In the final three MDS patients the cytotoxicity was low and not affected by IL‐ 12 and/or IL‐2 (type C response). AH patients with refractory anaemia with excess blasts (RAEB) and patients with RAEB in transformation showed a type B or C response. Conversely, six of eight refractory anaemia patients showed a type A response. In MDS patients there was a positive correlation between the percentage of CD3CD56+ cells in pre‐incubated cells and the cytotoxicity of cells incubated with IL‐12/IL‐2. The combination of IL‐12 and IL‐2 augmented IFN‐7 and TNF‐Q production by nonadherent mononuclear cells in a synergistic or cumulative manner, respectively, in most patients. These results suggest that IL‐12, alone or with IL‐2, may modulate these important immunologic functions in most MDS patients.

Plasma soluble interleukin‐2 receptor level in patients with primary myelodysplastic syndromes: a relationship with disease subtype and clinical outcome

To assess the hypothesis that the plasma soluble interleukin‐2 receptor (sIL‐2R) level may have predictive value for morbidity/mortality in patients with myelodysplastic syndromes (MDS), we determined the plasma sIL‐2R level of 80 MDS patients and examined their subsequent clinical course. Compared with low‐risk MDS (refractory anaemia (RA) and RA with ringed sideroblasts) patients and normal subjects, the plasma sIL‐2R level was significantly elevated in high‐risk MDS (three other MDS subtypes and acute leukaemia following MDS) patients (high‐risk MDS versus low‐risk MDS, P < 0.01; high‐risk MDS versus normal subjects, P < 0.01). 14/40 low‐risk MDS patients developed at least one of the following during the follow‐up period: erythrocyte transfusion dependence, infections requiring hospitalization, disease progression or MDS‐related death. The plasma sIL‐2R level was higher in these eventful subjects than in event‐free low‐risk subjects (P < 0.0001), and all of 10 low‐risk subjects with a plasma sIL‐2R level > 540 U/ml experienced at least one event. By logistic regression analysis of various parameters in these 40 low‐risk subjects, the plasma sIL‐2R level was identified as the strongest independent parameter for predicting eventful subjects (P < 0.0047). The plasma sIL‐2R level did not show a predictive value in high‐risk MDS. This study revealed that the plasma sIL‐2R level is significantly elevated in high‐risk MDS and suggested that the plasma sIL‐2R level is a valuable predictive factor for the clinical outcome in low‐risk MDS.

Elevated plasma soluble interleukin 2 receptor level correlates with defective natural killer and CD8+ T-cells in myelodysplastic syndromes

The plasma soluble interleukin 2 receptor (sIL-2R) level and its relationships with haematologic and immunologic data were examined in 40 patients with myelodysplastic syndromes (MDS). The plasma sIL-2R level was significantly higher in the high-risk MDS group (refractory anaemia with excess blasts (RAEB), RAEB in transformation and chronic myelomonocytic leukaemia) than in the low-risk MDS group (refractory anaemia (RA) and RA with ringed sideroblasts) or in normal subjects, although there was considerable variation in the plasma sIL-2R level within each MDS group. The plasma sIL-2R level correlated positively with the bone marrow cellularity and bone marrow blast mass, but not with the absolute number of CD25+ lymphocytes. This may support the idea that plasma sIL-2R is derived from malignant MDS cells in the bone marrow. The plasma sIL-2R level correlated negatively with the absolute numbers of the CD8+, CD3-CD16+, and CD3-CD56+ cell populations in freshly isolated lymphocytes, the percentage of CD3-CD56+ cells in lymphokine (interleukin 2)-activated killer (LAK) cells, and the cytotoxicity of LAK cells. We conclude that MDS patients having a high plasma sIL-2R level often have a defect in natural killer and CD8+ T-cells.