Analysis of myeloid and lymphoid markers on the surface and in the cytoplasm of mononuclear bone marrow cells in patients with myelodysplastic syndrome

Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.     

Drug abuse in Japan

OBJECTIVE: Cis-platinum and 13-cis-retinoic acid have received much attention in the treatment of head and neck squamous cell cancer. Even though they have different mechanisms of action, little information is available on their interaction. This paper reviews experimental evidence for retinoic acid-cis-platinum synergy and presents toxicity data from patients with stage IV head and neck squamous cell cancer participating in a phase I trial combining 13-cis-retinoic acid and cis-platinum. METHODS: Patients were given 13-cis-retinoic acid orally daily for 7 days before and daily during high-dose (150 mg/m2 per week for 4 weeks) intraarterial cis-platinum treatment with concurrent radiation. Toxicity was scored with use of the cancer and leukemia group B scale. RESULTS: In the phase I clinical trial, 15 patients were treated to determine a maximum tolerated dosage for 13-cis-retinoic acid of 20 mg/day. Grade 4 hematologic toxicity was dose limiting in 3 of 8 patients treated with 40 mg/day and in 1 patient treated with 60 mg/day. There were no deaths caused by toxicity; 12 of the 15 patients received all four weekly doses and the remaining 3 received three doses. Of 10 patients with fully evaluable data, all achieved a complete response at the primary site and 9 had a complete response in the neck. One patient had persistent neck disease after chemoradiation, and this tumor was removed with neck dissection. CONCLUSIONS: 13-Cis-retinoic acid and cis-platinum are strongly synergistic against head and neck squamous cell cancer in vitro. Pretreatment with retinoic acid results in stronger synergy than concurrent drug exposure alone. Preliminary clinical experience with combined retinoic acid and cis-platinum in a design that parallels the in vitro study indicates that toxicity is acceptable with 13-cis-retinoic acid dosages of 20 mg/day in a high-dose-intensity intraarterial chemoradiation regimen.     

Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome

Pancytopenia is a frequent manifestation of myelodysplastic syndromes (MDS). In the presence of an empty bone marrow, clinical distinction from aplastic anemia may be difficult. The hypoplastic marrow morphology seen in some cases of MDS raises questions about etiologic and pathophysiologic relationships between aplastic anemia and MDS. The goal of our study was to compare the degree of the hematopoietic failure in these diseases at the level of the most immature progenitor and stem cells that can be measured in vitro. In a systemic, prospective fashion, we have studied bone marrow (n = 45) and peripheral blood (n = 33) of patients with MDS for the number of long-term culture initiating cells (LTC-IC) in comparison to 17 normal controls and patients with new, untreated aplastic anemia (46 marrow; 62 blood samples). Due to the low numbers of cells available for the analysis, formal limiting dilution analysis could not be performed, instead secondary colony-forming cells (CFC) after 5 weeks of LTBMC were measured. As the number of these cells is proportional to the input number of LTC-IC, the number of secondary CFC per 10(6) mononuclear cells (MNC) initiating the LTBMC can be used as a measure of the content of immature stem cells in bone marrow and peripheral blood. The MDS group consisted of 34 RA, three RARS, eight RAEB and two RAEB-T patients with mean absolute neutrophil values of 1992, 1413, 1441, and 380 per mm3, respectively. The diagnosis was established based on bone marrow morphology and results of cytogenetic studies. In comparison to controls (147 +/- 38/10(6) MNC), significantly decreased numbers of bone marrow secondary CFC were found in MDS: in patients with RA and RARS, 21 +/- 7 secondary CFC per 10(6) bone marrow MNC (P < 0.001); patients with RAEB and RAEB-T: 39 +/- 12 CFC per 10(6) marrow MNC (P < 0.001). In all groups tested, the decrease in peripheral blood secondary CFC numbers was consistently less pronounced. In MDS patients with hypocellular bone marrow, secondary CFC were lower but not significantly different in comparison to MDS with hypercellular marrow (18 +/- 6 vs 35 +/- 11; NS; hypoplastic bone marrow also was not associated with significantly lower neutrophil counts). However, in 24% of patients with MDS, bone marrow secondary CFC were within the normal range, while in the aplastic anemia group only one of the patients showed secondary CFC number within normal range. Bone marrow and blood secondary CFC numbers in hypoplastic RA were significantly higher than those in severe aplastic anemia 919 +/- 5 in bone marrow, P < 0.01; 7 +/- 2 in blood, P < 0.05). This trend was even more pronounced in hypoplastic RA with chromosomal abnormalities. However, no significant differences were found between the secondary CFC numbers in hypoplastic RA and moderate aplastic anemia. We concluded that, although the deficiency in the stem cell compartment is less severe in MDS than in aplastic anemia, depletion of early hematopoietic cells is an essential part of the pathophysiology in both diseases.     

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.     

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations 'RA with severe dysplasia (RASD)' and 'RA with minimal dysplasia (RAminiD)'. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger-Huet anomalies of mature neutrophils > or =3% and/or micromegakaryocytes (mMgk) of megakaryocytes > or =10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P=0.06; RASD vs RAEB, P=0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.     

HPLC-fluorescence determination of unconjugated estrogens in pharmaceuticals

The demonstration of synergistic interaction between differentiation inducing agents and DNA synthesis inhibitors suggests that these two groups act by two different mechanisms. We prospectively studied the response rate, response duration, survival, and toxicity in 10 patients with myelodysplastic syndrome (MDS) treated with all trans retinoic acid (ATRA) and low dose cytosine arabinoside (ara-C). These patients diagnosed between October 1993 and May 1995 were treated with ATRA (45 mg/M2/day) for 90 days followed by 90 mg/M2 on alternate day till Day 275; together with Ara-C (10 mg/m2) subcutaneously twice daily for 21 days for a total of 6 cycles. These patients were analyzed for response after 3 cycles of LD Ara-C and at the time of completion of therapy. Toxicity was recorded at the end of each cycle of Ara-C. There were 6 male and 4 female patients in the age range of 24 to 76 years. The morphological diagnosis was chronic myelomonocytic leukemia in 2, refractory anemia with excess blasts in 4 and refractory anemia with excess blasts in transformation in 4. Only 1 patient achieved a complete remission and 1 patient achieved a partial response. Four patients had progressive disease on treatment. One patient died of neutropenic sepsis and 1 of resistant thrombocytopenia and intracranial hemorrhage while on treatment. One patient refused further treatment after a minor clinical response and in 1 patient treatment was stopped due to toxicity. This data in a pilot study with a limited number of patient suggests that ATRA in combination with Ara-C has little effect in MDS.     

Relationship between autoantibody and dermatosis in myelodysplastic syndrome

We analyzed the relationship between autoantibody and dermatosis in 22 patients with myelodysplastic syndrome (MDS). These MDS patients consisted of five cases with refractory anemia (RA), three RA with ringed sideroblasts (RARS), eight RA with excess of blasts (RAEB), four RAEB in transformation (RAEB-t), and two chronic myelomonocytic leukemia (CMMoL) according to the FAB classification of MDS. The autoantibody was detected in seven patients, of whom four had rheumatoid factor (RF) and three had antinuclear antibody (ANA). Neither RF-positive nor ANA-positive MDS patients had other autoantibodies. Dermatosis was observed in nine cases of these 22 MDS patients. Five of 7 MDS patients (71%) with autoantibody developed dermatosis in their clinical course, as did four of 15 MDS patients (27%) without autoantibody. All four MDS patients with RF had dermatosis such as anaphylactoid purpura, xerotic dermatitis, thrombophlebitis, ephelides, and genital herpes. One of three MDS patients with ANA had pruritus senilis. The four MDS patients without autoantibody had dermatosis such as erythema nodosum, ichthyosis vulgaris, Sweet syndrome, and thrombophlebitis. Three of four MDS patients with RF had normal liver function tests, while three MDS patients with ANA showed liver dysfunction. Our studies presented here suggested that the dermatosis could develop frequently in MDS patients with autoantibody and that RF was closely related to development of dermatosis in MDS patients, although the dermatosis is not specially fixed.     

Myelodysplastic syndrome with bone marrow eosinophilia: clinical and cytogenetic features

We investigated the hematological and clinical status of 145 patients with de novo myelodysplastic syndrome (MDS), 14 of whom (10%) had eosinophilia in the bone marrow (MDS-Eo). Most of these 14 patients had severe anemia. Their bone marrow cells exhibited trilineage dysplasia and some morphological abnormalities in the eosinophils, including disproportion of eosinophilic granules, basophilic granules, a ring-shaped nucleus, and vacuolation in the cytoplasm. However, these abnormalities were less prominent than those of acute myelomonocytic leukemia with eosinophilia (FAB: M4Eo). Three of the 14 MDS-Eo patients had refractory anemia (RA), seven had RA with excess of blasts (RAEB), and four had RAEB in transformation. Cytogenetic analysis revealed chromosomal abnormalities in 12 of 13 MDS-Eo patients (92%), in particular, there were major karyotypic abnormalities (MAKA) in eight patients (62%). Cytotoxic agents were not effective in the treatment of four patients after leukemic transformation occurred. These four patients died of the leukemic transformation while seven died of bone marrow failure. The other three MDS-Eo patients are still alive; two of them have already transformed to a leukemic phase. The duration of survival of these patients was significantly shorter than that of the other MDS patients. These findings suggest that bone marrow eosinophilia in MDS may be a poor prognostic factor that is strongly related to the existence of MAKA.     

Purification of a tumoral marker recognized by monoclonal antibody Po66 and associated with human lung squamous cell carcinoma

This paper reports on the production of tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor (GM-CSF) by cultured mononuclear adherent cells derived from bone marrow of 25 patients affected by myelodysplastic syndrome (MDS) of different FAB subtypes. Mean production of GM-CSF was much lower than in controls, without significant differences among different subtypes. Mean production of TNF was similar in MDS patients and in controls, but noteworthy differences were observed between patients with RA, RAEB and RAEB-t and patients with RARS and CMML. Growth of bone marrow granulocyte macrophage and erythroid progenitors did not correlate with TNF and GM-CSF production, although in MDS subtypes with higher GM-CSF levels, colony growth was slightly higher than in subtypes with lower GM-CSF production.     

Antithymocyte globulin for patients with myelodysplastic syndrome

Twenty-five transfusion-dependent myelodysplastic syndrome (MDS) patients (with < 20% blasts) were treated in a phase II study with antithymocyte globulin (ATG) at 40 mg/kg/d for four doses and then followed with blood counts every 2 weeks and clinic visits every 3 months, for a median of 14 months (range 1-38 months). 11 (44%) patients responded and became transfusion-independent after ATG, including three complete responses, six partial responses, and two minimal responses. Responses were observed in 9/14 patients (64%) with refractory anaemia (RA) and 2/6 patients (33%) with refractory anaemia with excess blasts (RAEB). Median response duration was 10 months (range 3-38 months). The Kaplan-Meier estimate of overall survival was 84% at 38 months, with one early death due to pneumonia and two deaths from disease progression to leukaemia. Side-effects consisted mainly of mild serum sickness in all patients. A single course of ATG restored haemopoiesis in some patients with MDS and was well tolerated.     

Hematologic, clinical, and cytogenetic analysis in 109 patients with primary myelodysplastic syndrome. Prognostic significance of morphology and chromosome findings

One hundred and nine patients with primary myelodysplastic syndrome (MDS) were classified according to the French-American-British (FAB) criteria: 27 refractory anemia (RA, 25%), 26 RA with ringed sideroblasts (RARS, 24%), 16 RA with excess of blasts (RAEB, 15%), 10 RAEB in transformation (RAEB-t, 9%), 25 chronic myelomonocytic leukemia (CMMoL, 23%), and five unclassifiable MDS (4%). Forty-three were women and 66 were men (sex ratio 2:3). Age ranged from 30-92 years (mean 69 years) with nine patients aged less than 50 years (8%). A cytogenetic result was obtained in all cases. At initial study, a chromosome defect was observed in 56% of patients. Rates of abnormality depended on FAB subtype: 52% in RA, 100% in RA 5q-, 50% in RARS, 56% in RAEB, 70% in RAEB-t and 44% in CMMoL. The most frequent single defects were del(5q), -7/del(7q), del(20q), Y loss, and +8. Except for the 5q- syndrome entity, specific chromosome defects were not associated with particular FAB subtypes. Bone marrow (BM) insufficiency (22%) and leukemic transformation (21%) were the most important causes of death. The rate of leukemic transformation increased with the number of dysplastic BM cell lineages and was also associated with karyotype complexity and the proportion of abnormal/normal metaphases. The longest median survivals were observed in RARS (142 months) and RA/RA5q- (91 months) types. Median survivals decreased with increasing Bournemouth score values. Patients with three abnormal cell lineages had a median survival shorter than those with one or two abnormal lineages. Similarly, patients with complex defects had shorter survival than those with single or double defects or a normal karyotype. There was no statistically significant difference between survival of NN (normal), AN (abnormal/normal), and AA patients or between survival of patients with del(5q), -7/del(7q), +8 or del(20q).     

Prevention of infections in a case with myelodysplastic syndrome by an intermittent subcutaneous administration of G-CSF

An 83-year-old male was admitted to our hospital because of pancytopenia and low grade fever on April 19, 1993. On admission, hematological data were as follows: WBC 1,000/microliters with 19% neutrophils, RBC 367 x 10(4)/microliters, Hb 9.5 g/dl and platelets 6.7 x 10(4)/microliters. Bone marrow examination revealed 6.6% myeloblasts and 33.5% erythroblasts. Morphological abnormalities included hypersegmentation, degranulation and pseudo-Pelger's nuclear anomaly in neutrophils. Based on these findings the diagnosis of refractory anemia with excess of blasts (RAEB) of the myelodysplastic syndrome (MDS) was made and therapy with low dose Cytarabine (Ara-C) was initiated in April 1993. The patient had two episodes of severe pneumonia in June and July. Therefore, 75 micrograms/day of G-CSF was given in addition to antibiotic therapy for the second episode of infection in July. Thereafter the severe infection subsided, and G-CSF administration was switched to an intermittent schedule (75 micrograms twice a week) since September. Cytarabine ocfosfate (100 mg/day) was added for 10-14 days at interval 1-2 months from October,1993. He has been well with no episode of infection for more than two year. One major concern regarding the clinical application of G-CSF in MDS patients is related to the possible stimulation of leukemic cell proliferation. Frequent hematological monitoring is necessary in patients with RAEB who are prone to develop acute myeloid leukemia. However, we administered G-CSF at a relatively low dose twice a week for over two year and could successfully prevent infections without inducing the leukemic changes.     

Clinical implications of chromosomal abnormalities in 401 patients with myelodysplastic syndromes: a multicentric study in Japan

It is well known that cytogenetic analysis in patients with myelodysplastic syndrome (MDS) provides information useful in determining their prognosis. Based on the chromosomal results obtained from 401 MDS patients by a multicentric study in Japan, we studied correlations between chromosomal findings and prognosis or leukemic transformation in MDS patients. Patients with complex aberrations (cytogenetic abnormalities at more than three chromosomes), of any subtype, had a poor prognosis; for example, > 60% of patients with refractory anemia (RA) showing complex aberrations died within one year, but only 11% of them developed leukemia. In patients with RA with ringed sideroblasts (RARS), > 70% of those with complex aberrations evolved into the leukemic phase and survived for less than one year, suggesting a biologic heterogeneity in RARS patients. By contrast, about 5% of patients with RA or RARS exhibiting chromosomal findings other than -7/7q-, +8, two aberrations, and complex aberrations, developed leukemia and had a favorable prognosis. Therefore, the presence of chromosome abnormalities alone in patients with RA or RARS is not a factor in predicting leukemic transformation or poor prognosis. In patients with refractory anemia with an excess of blasts (RAEB), the presence of chromosome aberrations at MDS diagnosis affected the occurrence of leukemic transformation (24% versus 43%), however, no particular difference was noted in patients with RAEB in transformation with regard to whether they had chromosome changes or not, and about 60% of them evolved into leukemia. The poor prognosis related to complex aberrations was consistently noted in all MDS subtypes or age-matched groups, indicating that this cytogenetic anomaly is an independent risk factor for a poor prognosis in MDS patients. The duration between MDS diagnosis and development of the leukemic phase and that between the latter and death were significantly shorter in patients with complex aberrations than those without this change. Although the clinical significance of certain chromosomal abnormalities differs among subtypes of MDS, a new scoring system for predicting prognosis by cytogenetic changes, in combination with hematologic parameters, was proposed.     

Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes, and cell lines

Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All-trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage.     

Ethnic disparities in patient recall of physician recommendations of diagnostic and treatment procedures for coronary disease

A pilot study was performed to determine the efficacy of 13-cis-retinoic acid (CRA) and alpha-tocopherol (AT) with increasing doses of recombinant human erythropoietin (rHuEPO) in anemic patients with primary myelodysplastic syndrome (MDS), to determine response rate and to determine the dose requirement and long-term effects of rHuEPO therapy on the transition to acute non-lymphocytic anemia and survival of these patients. Twenty-four consecutive MDS patients were entered into the study. Patients were stratified according to their FAB classification at study entry. Therapy consisted of a 6 month trial of CRA (100 mg m(-2) day(-1) and AT (800 mg day(-1)) with rHuEPO (150 units kg(-1) body weight subcutaneously three times a week). The rHuEPO dose was escalated to daily doses at 2 months, and 300 U kg(-1) body weight given three times a week for another 2 months and continuing therapy after 6 months in responsive patients. Response was measured by elimination of transfusions requirement (partial response, PR) and normal hemoglobin level and complete blood counts (complete response, CR). Observed responses for the 23 evaluable patients were 2 CR and 6 PR (34.8%). Odds ratio analysis showed that patients with anemia alone were 14 times more likely to respond than patients with pancytopenia (p = 0.06). In our study, two patients (8%) transformed to acute leukemia in CRA + AT + rHuEPO-treated patients. Median survival of 34 months with a median follow-up of 17 months (range 3-70 months) was observed. The response rates with the addition of rHuEPO to CRA and AT was similar but occurs earlier at 2 months compared to 6-10 months with CRA and AT alone and did not alter survival. There was no increase in the risk for leukemia in the group treated with rHuEPO. Response to either therapy appeared to be limited to patients in the early stages of MDS.     

Genetic localization of Cd63, a member of the transmembrane 4 superfamily, reveals two distinct loci in the mouse genome

We describe a patient with Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukaemia (ALL) who developed it 2.5 years after being diagnosed with myelodysplastic syndrome (MDS). The patient initially had refractory anaemia (RA), but progressed to refractory anaemia with excess blasts (RAEB) 2 years later, that terminated in ALL. An immunophenotypic analysis of the lymphoblasts revealed CD10 and CD19 positive cells. The karyotype was normal 46,XY in RA phase, 46,XY,20q-during the RAEB phase, and 46,XY,t(9;22)(q34;q11),20q-during the ALL phase. Furthermore, p190 BCR-ABL mRNA was detected in the ALL blasts. These findings indicate that this ALL arose from the MDS clone through multiple cytogenetic evolutions, the final event of which was the acquisition of p190 BCR-ABL type Ph1.     

Carcinoid of the appendix. Observations on 4 cases

Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders characterized by peripheral cytopenias in the presence of normo- or hypercellular dysplastic marrow. It has been suggested that premature intramedullary apoptosis may contribute to this phenomenon. We used terminal dUTP nick-end labeling (TUNEL) of bone marrow biopsy specimens and cytocentrifuge preparations from patients with MDS and a variety of other hematopoietic disorders to determine whether there is increased intramedullary apoptosis in MDS and whether any such effect is specific to MDS. TUNEL labeling of bone marrow from 24 patients with MDS revealed significant positivity in 10 of 11 patients with refractory anemia (RA), five of seven with RA and excess of blasts (RAEB), all three patients with RAEB in transformation (RAEB-t), and all three patients with RA with ring sideroblasts (RARS). The percent of positive cells ranged from 5 to 50% but showed no apparent correlation with morphological subtype. In a series of 29 patients with acute leukemia, 17 showed significant positivity (13 of 13 with myeloid disease: three M1, seven M2, one M3, two M4; four of 16 patients with lymphoid disease: one Burkitt-type lymphoma, two null acute leukemia, and one common acute lymphoid leukemia). Intramedullary apoptosis was associated with myeloid or early committed progenitor cells and was highest in secondary acute myeloid leukemia (AML). Normal bone marrow samples from 12 individuals showed no evidence of apoptosis. Our results suggest that an increased level of intramedullary apoptosis is apparent in both patients with MDS and those with AML; those with secondary AML have the highest levels. The relative absence of such findings in lymphoid malignancy suggests that the apoptotic pathways are different in this lineage.     

beta Interferon inhibits HIV-1 Tat-induced angiogenesis: synergism with 13-cis retinoic acid

Kaposi's sarcoma (KS) is a highly angiogenic lesion which frequently presents as an aggressive form in HIV-infected male patients. We have previously shown that the HIV-1 Tat protein induces endothelial cell migration and invasion in vitro and a rapid angiogenic response in vivo, suggesting that it acts as a cofactor in epidemic KS. In this study we tested beta interferon (IFN beta) and retinoic acid (RA) for the inhibition of Tat-induced angiogenesis using in vivo and in vitro models. IFN beta, at a concentration above 2500 U/ml, was an effective inhibitor of Tat-stimulated growth, migration and morphogenesis of an endothelial cell line in vitro and of angiogenesis in vivo. A strong reduction of properties associated with neovascularisation was induced by 10,000 U/ml. In vivo, RA alone was on ineffective inhibitor of angiogenesis, and in vitro gave only a limited inhibition of endothelial cell growth. However, 13-cis RA used in combination with IFN beta impressively potentiated its effects. A combination of lower doses of IFN beta (2500 U/ml) and 13-cis RA induced a virtually complete inhibition of the Tat-related angiogenic phenotype both in vivo and in vitro. The potentiation of the anti-angiogenic activity of IFN beta by 13-cis RA suggests that this combination could be a useful approach for the therapy of epidemic KS.     

Factors associated with the therapeutic efficacy of retinoic acids on malignant lymphomas

We recently reported the successful use of retinoic acids in the treatment of refractory lymphoma. The biologic determinants predicting response of lymphomas to retinoic acid remain unknown. This study was conducted to explore this question using in vitro models. Sensitivity of representative lymphoma cells to 13-cis-retinoic acid was determined. Sensitive and resistant cell lines were then compared for their baseline and/or retinoic-acid-regulated expression of total cellular retinoic acid binding protein, retinoic acid receptor (RAR)-alpha, RAR-beta, RAR-gamma mRNA, retinoid X receptor (RXR)-alpha, RXR-beta, RXR-gamma mRNA, transforming growth factor (TGF)-beta 1 and TGF-beta 1 receptors, and Fas (Apo-I) mRNA. The results showed that four of five T, two of three Hodgkin's, and none of six B cell lymphoma cell lines were sensitive (IC30 < 1.5 mmol/L) to 13-cis-retinoic acid. Further analyses revealed several of the above-mentioned parameters may be relevant to retinoic acid sensitivity. Baseline expression of TGF-beta 1 receptors was present in all of the five sensitive cell lines examined, but in only one of the four resistant cell lines. The correlation of Fas expression and retinoic acid sensitivity was good for B cell lines, but not apparent for T cell or Hodgkin's cell lines. On exposure to retinoic acid, an immediate and prolonged upregulation of RAR-alpha mRNA expression, lasting for more than 12 hours, occurred in all sensitive cell lines, but only minimal or transient induction was seen in resistant cells. Together, these data suggested that; 1) retinoic acid has a preferential effect on T cell and Hodgkin's lymphoma cell lines; 2) autoregulation of RAR-alpha by retinoic acids, and the presence of TGF-beta 1 receptors may be relevant to the response of lymphomas to treatment with retinoic acids.     

Hypermethylation of the p15INK4B gene in myelodysplastic syndromes

Previous studies have shown that the cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and that they are candidate tumor-suppressor genes. Although genetic alterations in these genes may be limited to lymphoid malignancies, it has been reported that their inactivation by aberrant methylation of 5' CpG islands may be involved in various hematologic malignancies. In this study, we investigated the p15INK4B and p16INK4A genes to clarify their roles in the pathogenesis of myelodysplastic syndrome (MDS). Southern blotting analysis showed no gross genetic alterations in either of these genes. However, hypermethylation of the 5' CpG island of the p15INK4B gene occurred frequently in patients with MDS (16/32 [50%]). Interestingly, the p15INK4B gene was frequently methylated in patients with high-risk MDS (refractory anemia with excess blasts [RAEB], RAEB in transformation [RAEB-t], and overt leukemia evolved from MDS; 14/18 [78%]) compared with patients with low-risk MDS (refractory anemia [RA] and refractory anemia with ring sideroblast [RARS]; 1/12 [8%]). Furthermore, methylation status of the p15INK4B gene was progressed with the development of MDS in most patients examined. In contrast, none of the MDS patients showed apparent hypermethylation of the p16INK4A gene. These results suggest that hypermethylation of the p15INK4B gene is involved in the pathogenesis of MDS and is one of the important late events during the development of MDS.