Cell cycle modulation by hematopoietic growth factors in myelodysplastic syndromes: analysis by three-color flow cytometry.

Investigations of the effects of hematopoietic growth factors (HGFs) on the cell cycle of cells from myelodysplastic syndrome (MDS) have been hampered by technical difficulties. In this study, using a recently established flow cytometric method that enables detailed analysis of the cell cycle (Gzero-, G1-, S-, and G2/M-phases) of target cells in a heterogeneous cell population, we examined the effects of granulocyte colony-stimulating factor (G-CSF) and other HGFs on the cell cycle of CD13-positive cells (blasts and other malignant myelocytic and monocytic cells) in MDS. The cell cycle response to G-CSF (decrease in Gzero-phase cells and increase in S-phase cells) was heterogeneous among MDS cases. When the data for 13 MDS cases and 15 de novo AML cases were compared statistically, the magnitude of cell cycle activation by G-CSF was weaker for the cells from the MDS cases. Stem cell factor, interleukin-3, or a combination of these HGFs with G-CSF reduced the Gzero-phase cell percentage in all examined MDS cases whose cell cycle was unresponsive to G-CSF alone. When cytosine arabinoside was added to cells with or without stimulation by HGFs, the viable G0-phase cell count was reduced in HGF-stimulated cells compared with unstimulated cells in seven of eight cases. The present results suggest that G-CSF-induced cell cycle stimulation of malignant cells can be expected in a fraction of MDS patients and that even in MDS patients whose cells do not respond to G-CSF, employment of other HGFs and their combination with G-CSF is worth consideration. The results also suggest that a well-designed therapy using HGFs and chemotherapeutic drugs may reduce the quiescent (Gzero) cell count in MDS, which is assumed to be responsible for drug resistance derived from cell kinetics.

Effects of interleukin 2 on myelodysplastic syndromes.

To evaluate the clinical usefulness of interleukin 2 (IL-2) on myelodysplastic syndromes (MDS), the serum IL-2 level, the effect of IL-2 on the proliferation of blasts, and the cell-mediated cytotoxic effect of IL-2 on blasts were examined in MDS patients. Of 18 patients, 2 patients had an increased serum IL-2 level. Although the proliferation of blasts in most cases, including the two patients having a high serum IL-2 level, was not stimulated by IL-2, the blasts of one case apparently proliferated in response to IL-2. It was also clearly shown that IL-2-stimulated normal peripheral blood mononuclear cells (PBMNC) showed cytotoxicity against MDS blasts, whereas the PBMNC of the advanced stages of MDS were usually defective in regard to this IL-2-dependent cytotoxicity. The therapeutic usefulness of IL-2 or lymphokine-activated killer cells for MDS was not established by the present study.

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.

Aplastic anemia with circulating erythroblasts.

Since the presence of erythroblasts (Ebl) in the peripheral blood of patients suspected to have aplastic anemia (AA) has been thought to suggest an error in diagnosis, such patients may not receive appropriate therapy promptly, with potentially fatal results. However, we recently experienced patients who had the typical features of AA except for circulating Ebl, and responded well to therapy for AA. In this study, we examined 55 untreated AA patients for the presence of circulating Ebl. Seven patients (12.7%) had circulating Ebl (at least one Ebl per 200 leukocytes counted). These seven patients showed the typical features of AA. There were no significant differences in age or hematologic parameters, including the morphology of bone marrow Ebl, between the AA patients having circulating Ebl (Ebl(+) AA) and the AA patients without Ebl. All but one of the Ebl(+) AA patients responded well to the therapy for AA. During 12.0-110.4 months of follow-up, transformation to other diseases did not occur, and all seven patients were alive. We conclude that circulating Ebl are not rare in patients who have otherwise typical features of AA, and that if such Ebl(+) AA patients are severely cytopenic, they should receive treatment for AA without delay.

Plasma soluble interleukin-2 receptors in patients with myelodysplastic syndromes.

We examined the plasma soluble interleukin-2 receptor (sIL-2R) level in 80 subjects with myelodysplastic syndromes (MDS) and analyzed its correlation with hematologic/immunologic parameters and the subsequent clinical course. Compared with low-risk MDS (refractory anemia (RA) and RA with ringed sideroblasts) and normal individuals, the plasma sIL-2R level was significantly elevated in high-risk MDS (three other MDS subtypes and acute leukemia following MDS) patients. There was a significant negative correlation between the plasma sIL-2R level and the absolute counts of T and natural killer cells. Furthermore, the plasma sIL-2R level showed a significant positive correlation with the total cell mass and blast mass in particular, in the marrow, but not with the absolute count of IL-2Ralpha-chain-positive lymphocytes in the circulation. Fourteen of our 40 low-risk MDS subjects developed at least one of the following events during the follow-up period: erythrocyte transfusion dependence, infections requiring hospitalization, disease progression or MDS-related death. The plasma sIL-2R level was significantly higher in these patients than in event-free low-risk cases. By logistic regression analysis of various parameters in the 40 low-risk subjects, the plasma sIL-2R level was identified as a valuable independent parameter for predicting the development of events. Based on these findings, we hypothesize that the sIL-2R plays a role in the development of morbidity and mortality in MDS by inducing immunologic dysfunction.

Interleukin-2 therapy for myelodysplastic syndrome: does it work?

Recent clinical studies suggested that interleukin-2 (IL-2) has therapeutic potential for some hematologic malignancies, but the therapeutic role of IL-2 for myelodysplastic syndrome (MDS) is still unclear. MDS is a clonal malignant disorder which often involves a variety of immunologic abnormalities. Examination of the effects of IL-2 on MDS in vitro yielded the following results: (1) IL-2 did not induce the proliferation of blasts in most MDS cases. (2) The cytotoxicity of IL-2-induced lymphokine-activated killer (LAK) cells for cell lines and MDS blasts was reduced in the high-risk MDS group (refractory anemia with excess blasts (RAEB), RAEB in transformation and MDS transformed to acute leukemia), but it was still preserved in the low-risk MDS group (refractory anemia (RA) and RA with ringed sideroblasts). However, considerable variation in LAK cell cytotoxicity was noted in each group. (3) The reduced LAK cell cytotoxicity observed in MDS was explained, at least in part, by the presence of a reduced of number of natural killer (NK) cells amongst the LAK cells. (4) MDS patients who have a high blood soluble IL-2 receptor (sIL-2R) level often had defects in NK and CD8+ T cells. These in vitro findings suggest that the response to IL-2 is heterogeneous in MDS patients, and those who have a low-risk MDS subtype and/or a low blood sIL-2R level, may be prone to respond to IL-2 therapy. Clinical trials are mandatory in order to elucidate the efficacy of IL-2 therapy in the treatment of MDS.

Acute gastric mucosal lesions associated with cytomegalovirus infection in a non-immunocompromised host.

A 75-year-old woman with epigastric pain and tarry stool was admitted to our hospital, where upper gastrointestinal endoscopic study revealed multiple gastric ulcers. The endoscopic biopsy specimens obtained on the seventh hospital day disclosed a few typical intranuclear cytomegalovirus inclusions. Cytomegalovirus-DNA was detected using polymerase chain reaction in a biopsy specimen. No immunologic abnormalities were demonstrated by any laboratory tests. While only a few cases of cytomegalovirus-associated gastric ulcer in non-immunocompromised hosts have been reported, this entity may be more frequently detected when careful histological examination is performed in the active stage rather than postponed until after healing of the ulcer.

[Two cases of acute leukemia with t(6;9) (p23;q34)].

Two cases of acute leukemia with a t (6;9) (p23;34) chromosome abnormality are reported. The first case was a 34-year-old female who was hospitalized in October 1989. A diagnosis of FAB-M1 was made. Chromosomal analysis of the bone marrow cells showed a 46, XX, t (6;9) (p23;q34). Complete remission was achieved after two courses of BHAC-DMP therapy. In September 1991, at the time of relapse, chromosomal analysis revealed two abnormal clones consisting of a 46, XX, t (6;9) (p23;q34), -12, -17, +der (12) t (12;17) (p11.2;q11.2) with a residual normal clone. She died in February 1992. The second case was a 42-year-old male who was hospitalized in January 1990. He was diagnosed as having RAEB. Chromosomal analysis of the bone marrow cells showed 46, XY, t (6;9) (p23;q34). Three months later, the disease progressed to acute leukemia accompanied by leg ulceration with leukemic cell infiltration. Small-dose ara-C therapy was given, but with no effect. After two subsequent courses of therapy with low-dose etoposide, complete remission was achieved. Four months later, relapse occurred, and the patient died of sepsis in February 1991. In the literature, 31 cases of myeloproliferative disorders with t (6;9) have been reported.

Lineage-unrestricted hematologic response to granulocyte colony-stimulating factor in a patient with refractory anemia with excess blasts.

We report a patient with refractory anemia with excess blasts who showed a lineage-unrestricted hematologic response to granulocyte colony-stimulating factor (G-CSF). After 17 months of a stable disease state, the patient developed pneumonia, progression of cytopenia, and reduced cellularity and blast mass in the bone marrow. He was given G-CSF to overcome the pneumonia. Not only the neutrophil count, but also the platelet count increased soon after initiation of the G-CSF therapy; both counts became normal on the fifth day of the G-CSF therapy. Additionally, the anemia improved gradually. The neutrophil and platelet counts were maintained in the normal range for 3 months after cessation of the G-CSF. In vitro studies showed that G-CSF alone stimulated megakaryocyte colony formation from bone marrow mononuclear cells (BMMNC), and accessory cells in the BMMNC were necessary for expression of this G-CSF-induced in vitro megakaryocytopoiesis. These results suggest that, in coordination with accessory cells, G-CSF stimulated megakaryocytopoiesis in the patient. This case provides valuable information for understanding the mechanisms of a lineage-unrestricted hematologic response to G-CSF, which is very rarely observed in MDS.

Natural killer cells in the late decades of human life.

We examined the lymphocyte subsets and indices of natural killer (NK) cell activity (lytic unit (LU), index of absolute NK cell activity in vivo (ALU), and NK cell activity on a per-cell basis (PCNK)) in 82 people (age, 30-99 years) who were immunologically normal. Although the number of NK cells was maintained throughout the examined age range, the ALU and PCNK values correlated negatively with age. We then examined whether any of the various immunologic parameters, including the function and cell counts of NK cells, T cells, and neutrophils, related to past infectious episodes and death in the follow-up period in 44 elderly subjects (age, 63-98 years). Only low ALU and PCNK values correlated with a past history of severe infection, while low LU, ALU, and PCNK values were the only parameters which correlated with death due to infection during the follow-up period. We propose that human NK cells do not escape the aging process and that a low NK cell function relates to the development of severe infections, which may be fatal, in elderly subjects.

Effect of thrombopoietin on proliferation of blasts from patients with myelodysplastic syndromes.

Thrombopoietin (TPO), a major cytokine involved in megakaryocytopoiesis/thrombopoiesis, may be effective for treatment of the thrombocytopenia associated with myelodysplastic syndromes (MDS). However, it has been unclear whether TPO stimulates proliferation of MDS blasts, as observed in de novo acute myeloid leukemia. This study examined this concern. When marrow cells from 37 MDS cases were cultured with or without recombinant human PEGylated TPO, TPO increased the blast number (stimulation index > or =1.5) in 9 of 16 high-risk MDS cases (refractory anemia with excess blasts [RAEB] and RAEB in transformation) and 4 of 10 cases with MDS transformed to acute leukemia (MDS-AL), but none of 11 cases with low-risk MDS (RA and RA with ringed sideroblasts). When the cell cycle of cultured cells was determined by three-color flow cytometry, TPO activated the cell cycle of MDS cells (causing a decrease in G(0)-phase cells) in most of the cases whose blast number increased in response to TPO. Reverse transcriptase-polymerase chain reaction analysis detected TPO receptor messenger RNA in purified blasts from all six cases examined, irrespective of the response of their blasts to TPO in culture. Analysis of the patients' characteristics identified a high-serum lactate dehydrogenase (LDH) value as being associated with blast proliferation in high-risk MDS cases (p = 0.0036). We conclude that TPO stimulates in vitro proliferation of blasts from a fraction of MDS patients. High-risk MDS patients, especially those who have a high-serum LDH value, and MDS-AL patients should be monitored with particular care in clinical trials of TPO for MDS.

A novel megakaryocyte potentiator produced by MC-1 human lung cancer cell line.

The hematopoietic stimulating activity of a human lung cancer cell line, MC-1, was investigated. The protein fraction (MC-1 protein) was prepared from the serum-free culture supernatant of MC-1 cells using hydroxyapatite and concanavalin A-agarose columns. In serum-containing cultures, MC-1 protein stimulated colony formation by megakaryocyte colony-forming units (CFU), erythroid burst-forming units and granulocyte/macrophage (GM) CFU. The stimulating effect was strongest for megakaryocyte CFU. The factor having megakaryocyte colony-stimulating activity was shown to be a protein whose molecular weight was determined to be 23,000 daltons by gel filtration. By various analyses, this protein was shown to be molecularly different from the heretofore-identified cytokines that may affect megakaryocytopoiesis, i.e., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-7, IL-11, granulocyte colony-stimulating factor (CSF), macrophage CSF, GM-CSF, leukemia inhibitory factor, stem cell factor and tumor necrosis factor. Under serum-free conditions, MC-1 protein augmented murine megakaryocyte colony formation in the presence of murine IL-3 and increased the acetylcholinesterase activity of purified murine megakaryocytes. It was also shown that MC-1 protein stimulated human megakaryocyte colony formation. It was concluded that MC-1 cells produce a megakaryocyte potentiator which is molecularly different from any heretofore-identified cytokines.

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

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) gamma 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-Hodgkin's 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.

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-Hodgkin's 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.

Repeated cycles of G-CSF-combined postremission chemotherapy for acute myeloid leukemia in a first complete remission: a pilot study.

The cure rate of acute myeloid leukemia might increase if G-CSF were given concurrently with repeated postremission chemotherapy. However, this therapy might cause severe complications, including depletion of normal hematopoietic progenitors as a long-term toxicity. Thus, we conducted a pilot study of this strategy. Twenty-six acute myeloid leukemia patients in a first complete remission (CR) were treated with two courses of consolidation chemotherapy (10-day BHAC-DMP, consisting of behenoyl cytosine arabinoside, daunorubicin, 6-mercaptopurine and prednisolone) and repeated maintenance-intensification therapy including eight cycles of six-day BHAC-DMP. G-CSF (filgrastim) was administered concurrently with these BHAC-DMP therapies. Toxicity during the therapeutic period was not significant in the study group compared with the historical control, treated with the same regimen without G-CSF. Neutrophil recovery after the consolidation therapy was more rapid in the study group than in the historical control (p = 0.066 and 0.024 for the first and second consolidation courses, respectively). Long-term toxicity, such as cytopenia, has not been seen in eight patients who have remained in CR for a long period (range: 39-58 months). At a median follow-up of 39 months, the predicted rate of 42-month CR duration for these 26 patients was 50% (95% confidence limits: 30% to 71%). We conclude that G-CSF-combined repeated BHAC-DMP postremission therapy is feasible. Full elucidation of the clinical benefit of this strategy will require further study.

Autoimmune hemolytic anemia in patients with de novo acute myelocytic leukemia.

Autoantibody against erythrocytes has occasionally been observed in patients with de novo acute myelocytic leukemia (AML). However, it is not clear whether this autoantibody in AML patients induces frank hemolysis (autoimmune hemolytic anemia, AIHA), as seen in lymphoid neoplasms. We present two de novo AML patients who showed hemolysis due to antiglobulin test-positive and test-negative AIHA, respectively. AIHA should be considered as one cause of anemia in de novo AML patients, and blood transfusions should be given carefully in such cases to avoid harmful hemolysis.

Association between natural killer cell activity and infection in immunologically normal elderly people.

Congenital patients who lack natural killer (NK) cell activity experience repeated polymicrobial infections. NK cell activity varies significantly among normal people, but it is unknown whether this variation influences their ability to fight infections. This study examined this concern. NK cell activity and other variables, i.e. age, sex, performance status (PS), serum albumin value, lymphocyte and neutrophil counts, various lymphocyte subsets, etc. were determined for 108 immunologically normal elderly subjects who were in nursing homes due to an impaired PS. We analysed for correlations between these variables and the follow-up results of the subjects. Forty-eight subjects developed infection(s) during the first year of follow-up. A low NK cell activity was associated with the development of infection (P = 0.0105, multivariate logistic regression analysis). The relative risk for the development of infection increased in accordance with the decrease in the NK cell activity. Eleven subjects died of infection during the study period. A low NK cell activity was associated with short survival due to infection (P = 0.0056, multivariate Cox's proportional-hazards regression analysis). Our data indicate that low NK cell activity is associated with development of infections and death due to infection in immunologically normal elderly subjects with an impaired PS.

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.

Assessment of therapeutic potential of interleukin 2 for myelodysplastic syndromes.

The therapeutic potential of interleukin 2 (IL-2) for myelodsplastic syndromes (MDS) was evaluated in vitro. IL-2-induced lymphokine-activated killer (LAK) cells were prepared from 38 MDS patients and 20 normal subjects. The cytotoxicity of LAK cells against K562 and Raji cell lines and MDS blasts was significantly reduced in high-risk MDS (refractory anaemia with excess blasts (RAEB), RAEB in transformation, and leukaemic transformation of MDS), but was relatively well-preserved in low-risk MDS (refractory anaemia (RA) and RA with ringed sideroblasts). Examination of the immunophenotypes of freshly-isolated lymphocytes showed that the percentage of CD4+ cells in low-risk MDS and the percentage of CD3+, CD4+ and CD8+ cell populations in high-risk MDS was significantly reduced compared with these populations in normal subjects. After cultivation with IL-2, these three cell populations were still reduced in the corresponding MDS groups and the percentage of CD3-CD56+ cells were significantly reduced in high-risk MDS. There was a positive correlation between the percentage of K562 cells lysed by MDS LAK cells and the percentage of CD3-CD56+ lymphocytes in MDS LAK cells. These aberrant lymphocyte subpopulations appeared to explain, at least in part, the reduced LAK cell cytotoxicity in MDS. These results present a possibility that IL-2 and LAK therapies are ineffective for most high-risk MDS patients, whereas they have potential value for low-risk MDS patients whose lymphocyte cytotoxicity is usually preserved.

Defective natural killer (NK) cell-mediated cytotoxicity does not imply clonal involvement of NK cells in myelodysplastic syndromes.

The clonality of purified cells was examined in 10 myelodysplastic syndromes (MDS) patients by analysing the restriction fragment length polymorphism and methylation pattern of the phosphoglycerate-kinase gene. Natural killer (NK) cell-mediated cytotoxicity was also examined. The granulocytes and monocytes were monoclonal or oligoclonal in all cases, except for the monocytes in one case. Conversely, the NK and T cells had a polyclonal pattern in most cases, including all cases who had defective NK cell-mediated cytotoxicity. The hypothesis that reduced NK cell-mediated cytotoxicity in MDS is caused by a clonal involvement of NK cells was not supported by the present study.