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Reduced levels of human myeloid nuclear differentiation antigen (MNDA) gene transcripts have been detected in both familial and sporadic cases of myelodysplastic syndromes (MDS). Numerous reports implicate elevated apoptosis/programmed cell death and death ligands and their receptors in the pathogenesis of MDS. MNDA and related proteins contain the pyrin domain that functions in signaling associated with programmed cell death and inflammation. We tested the hypothesis that MNDA is involved in the regulation of programmed cell death in human myeloid hematopoietic cells. Clones of K562 cells (MNDA-null) that expressed ectopic MNDA protein were established using retroviral transduction. MNDA-expressing K562 clones were resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)–induced apoptosis, but were not protected from programmed cell death induced with genotoxic agents or H2O2. MNDA protein expression assessed in control and intermediate and high-grade MDS marrows showed several patterns of aberrant reduced MNDA. These variable patterns of dysregulated MNDA expression may relate to the variable pathophysiology of MDS. We propose that MNDA has a role regulating programmed cell death in myeloid progenitor cells, and that its down-regulation in MDS is related to granulocyte-macrophage progenitor cell sensitivity to TRAIL-induced programmed cell death. (Cancer Res 2006; 66(9): 4645-51)

Supplementary Figure 2 from Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis

Supplementary Figure 1 from Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis

Diagnosis, classification, and requisite ancillary studies (flow cytometry, cytogenetics, and molecular genetics) of acute myeloid leukemia, critical to optimal patient care, have changed dramatically in the last 15 years and continue to evolve at a rapid pace. We present in this chapter background information for understanding these changes to date, review current diagnosis and classification of these diseases, and provide insights into future possible evolution of our understanding of the biology of this set of diseases.

BACKGROUND Autoimmune hemolytic anemia (AIHA) due to anti-Ena has been previously reported in association with massive intravascular hemolysis, disseminated intravascular coagulation, and fatal outcomes. Here we report a case of successfully treated AIHA due to anti-Ena. CASE REPORT A 69-year-old male with a past medical history of cirrhosis due to nonalcoholic steatohepatitis status post–orthotopic liver transplant presented with 1-month history of progressive anemia. At presentation, his hemoglobin (Hb) was 5.6 g/dL, hematocrit (Hct) 16%, reticulocytes 0.3%, direct bilirubin (bili) 4 g/dL, lactate dehydrogenase 533 units/L (reference, 125-220 units/L), and haptoglobin 254 mg/dL (reference, 40-273 mg/dL). Blood bank testing revealed an autoantibody present in his plasma and a direct antiglobulin test positive for immunoglobulin G (IgC) but negative for complement. He received 1 unit of an incompatible blood group O phenotypically matched red blood cell unit. RESULTS Over the course of the next 5 days, the Hb and Hct decreased to 4.1 g/dL and 12%, respectively, direct bili increased to 12.3 mg/day, reticulocytes slightly increased to 0.9%, and haptoglobin decreased to less than 8 mg/dL. Marrow study showed a hypercellular marrow with erythroid hyperplasia. Additional workup performed at a reference laboratory identified an anti-Ena autoantibody. He received prednisone and weekly rituximab infusions and was monitored weekly. At the 2-month visit, Hb and Hct were 10 g/dL and 32%, respectively. CONCLUSION Unlike two of the previously reported fatal cases of AIHA with anti-Ena specificity, this 69-year-old male treated with weekly rituximab infusion underwent clinical recovery and significant anemia improvement.

The myelodysplastic syndromes (MDS) comprise a heterogenous group of myeloid disorders with a highly variable disease course. Diagnostic criteria to better stratify patients with MDS continue to evolve, based on morphology, cytogenetics, and the presence of cytopenias. More accurate classification of patients will allow for better treatment guidance. Treatment encompasses supportive care, treatment of anemia, low-intensity therapy, and high-intensity therapy. This portion of the guidelines focuses on diagnostic classification, molecular abnormalities, therapeutic options, and recommended treatment approaches.

Context.— Detailed diagnostic features of acute myeloid leukemia in Down syndrome are lacking, leading to potential misdiagnoses as standard acute myeloid leukemia occurring in patients with Down syndrome. Objective.— To evaluate diagnostic features of acute myeloid leukemia and myelodysplastic syndrome in patients with Down syndrome. Design.— Diagnostic bone marrow samples from 163 patients enrolled in the Children's Oncology Group study AAML0431 were evaluated by using central morphologic review and institutional immunophenotyping. Results were compared to overall survival, event-free survival, GATA1 mutation status, cytogenetics, and minimal residual disease results. Results.— Sixty myelodysplastic syndrome and 103 acute myeloid leukemia samples were reviewed. Both had distinctive features compared to those of patients without Down syndrome. They showed megakaryocytic and erythroid but little myeloid dysplasia, and marked megakaryocytic hyperplasia with unusual megakaryocyte morphology. In acute myeloid leukemia cases, megakaryoblastic differentiation of blasts was most common (54 of 103, 52%); other cases showed erythroblastic (11 of 103, 11%), mixed erythroid/megakaryoblastic (20 of 103, 19%), or no differentiation (10 of 103, 10%). Myelodysplastic syndrome and acute myeloid leukemia cases had similar event-free survival and overall survival. Leukemic subgroups showed interesting, but not statistically significant, trends for survival and minimal residual disease. Cases with institutional diagnoses of French American British M1-5 morphology showed typical features of Down syndrome disease, with survival approaching that of other cases. Conclusions.— Myelodysplastic syndrome and acute myeloid leukemia in Down syndrome display features that allow discrimination from standard cases of disease. These distinctions are important for treatment decisions, and for understanding disease pathogenesis. We propose specific diagnostic criteria for Down syndrome–related subtypes of acute myeloid leukemia and myelodysplastic syndrome.

Objectives: To determine the effect of iterative refinement of standard ordering protocols on test utilization and results for bone marrow biopsy specimens. Methods: Eighteen months of test utilization and result data were used to revise the protocols that determine cytogenetic and molecular test selection on bone marrow specimens and then compared with data obtained following protocol revision. Results: Revision of protocols resulted in reduction in total tests and associated charges, due to a decrease in tests both concordant and discordant with the protocols. These reductions only occurred in diseases for which revisions were made and were limited to cases in which reflex testing was performed. There was an increase in the fraction of positive tests, which was also limited to reflex testing. Conclusions: Data-driven iterative revision of protocols further improves test utilization and performance, while reducing cost. Analysis of testing data can be used to continuously improve test ordering decisions.

The National Comprehensive Cancer Network (NCCN) defines the following types of acute myeloid leukemia (AML) as favorable-risk: acute promyelocytic leukemia with t(15;17) (APL); AML with core-binding factor (CBF) rearrangements, including t(8;21) and inv(16) or t(16;16) without mutations in KIT (CBF-KITwt); and AML with normal cytogenetics and mutations in NPM1 (NPM1mut); or biallelic mutations in CEBPA (CEBPAmut/mut), without FLT3-ITD. Although these AMLs are categorized as favorable risk by NCCN, clinical experience suggests that there are differences in clinical outcome amongst these cytogenetically and molecularly distinct leukemias. This study compared clinical and genotypic characteristics of 60 patients with favorable-risk AML, excluding APL, and demonstrated significant differences between them. Patients with NPM1mut AML were significantly older than those in the other groups. Targeted next-generation sequencing on DNA from peripheral blood or bone marrow revealed significantly more mutations in NPM1mut AML than the other favorable-risk diseases, especially in genes related to DNA splicing and methylation. CEBPAmut/mut AMLs exhibited more mutations in transcription-related genes. Patients with NPM1mut AML and CEBPAmut/mut AML show significantly reduced overall survival in comparison with CBF-KITwt AML. These findings emphasize that favorable-risk AML patients have divergent outcomes and that differences in clinical and genotypic characteristics should be considered in their evaluation and management.

The NCCN Guidelines for Myelodysplastic Syndromes (MDS) comprise a heterogeneous group of myeloid disorders with a highly variable disease course that depends largely on risk factors. Risk evaluation is therefore a critical component of decision-making in the treatment of MDS. The development of newer treatments and the refinement of current treatment modalities are designed to improve patient outcomes and reduce side effects. These NCCN Guidelines Insights focus on the recent updates to the guidelines, which include the incorporation of a revised prognostic scoring system, addition of molecular abnormalities associated with MDS, and refinement of treatment options involving a discussion of cost of care.

Patients with myeloid leukemia of Down syndrome (ML-DS) have favorable event-free survival (EFS), but experience significant treatment-related morbidity and mortality. ML-DS blast cells ex vivo have increased sensitivity to cytarabine (araC) and daunorubicin, suggesting that optimizing drug dosing may improve outcomes while reducing toxicity. The Children’s Oncology Group (COG) AAML0431 trial consisted of 4 cycles of induction and 2 cycles of intensification therapy based on the treatment schema of the previous COG A2971 trial with several modifications. High-dose araC (HD-araC) was used in the second induction cycle instead of the intensification cycle, and 1 of 4 daunorubicin-containing induction cycles was eliminated. For 204 eligible patients, 5-year EFS was 89.9% and overall survival (OS) was 93.0%. The 5-year OS for 17 patients with refractory/relapsed leukemia was 34.3%. We determined the clinical significance of minimal residual disease (MRD) levels as measured by flow cytometry on day 28 of induction I. MRD measurements, available for 146 of the 204 patients, were highly predictive of treatment outcome; 5-year disease-free survival for MRD-negative patients (n = 125) was 92.7% vs 76.2% for MRD-positive patients (n = 21) (log-rank P = .011). Our results indicated that earlier use of HD-araC led to better EFS and OS in AAML0431 than in past COG studies. A 25% reduction in the cumulative daunorubicin dose did not impact outcome. MRD, identified as a new prognostic factor for ML-DS patients, can be used for risk stratification in future clinical trials. This trial was registered at www.clinicaltrials.gov as #NCT00369317.

Fanconi anemia is a rare autosomal recessive disease which is associated with an increased risk of malignancy. Acute myeloid leukemia and myelodysplastic syndrome are the most common malignancies. This case report is on an infant who was diagnosed with Fanconi anemia and developed myelodysplastic syndrome in his first year of life which later likely transformed into T acute lymphoblastic leukemia. These findings were demonstrated by FISH. The case highlights a rare lymphoid neoplasm in an FA patient and also the increased incidence of T lineage neoplasms when MDS transforms into a lymphoid leukemia.

Background: Between January 1999 and December 2008, 469 patients treated for acute myeloid leukemia (AML) were included in this single-institution study. Methods: We performed a case–control analysis to study the rate of obesity among patients with acute promyelocytic leukemia (APL) and non-APL AML. Results: A total of 81% of APL patients analyzed were obese compared with 41.7% in the non-APL group ( p 30 was seen in 57% of APL patients compared with 31% for the non-APL group ( p = 0.01). Neither obesity nor the chemotherapy dosing based on ideal body weight affected survival. Conclusions: Our findings generate the hypothesis that APL and metabolic syndromes may share a common pathogenic pathway via retinoic acid receptors (RARs), the ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth and survival. If this link is confirmed in larger studies, our data will instigate further studies using RXR and RAR modulators as a preventive strategy among obese individuals.

These suggested practice guidelines are based on extensive evaluation of the reviewed risk-based data and indicate useful current approaches for managing patients with MDS. Four drugs have recently been approved by the FDA for treating specific subtypes of MDS: lenalidomide for MDS patients with del(5q) cytogenetic abnormalities; azacytidine and decitabine for treating patients with higher-risk or nonresponsive MDS; and deferasirox for iron chelation of iron overloaded patients with MDS. However, because a substantial proportion of patient subsets with MDS lack effective treatment for their cytopenias or for altering disease natural history, clinical trials with these and other novel therapeutic agents along with supportive care remain the hallmark of management for this disease. The role of thrombopoietic cytokines for management of thrombocytopenia in MDS needs further evaluation. In addition, further determination of the effects of these therapeutic interventions on the patient's quality of life is important.(116,119,120,128,129) Progress toward improving management of MDS has occurred over the past few years, and more advances are anticipated using these guidelines as a framework for coordination of comparative clinical trials.

The purpose of the current study was to evaluate the activity and toxicity of dexamethasone, high-dose cytarabine, and carboplatin (DAC) combination therapy in children with newly diagnosed large-cell non-Hodgkin lymphoma (NHL) and to estimate the event-free and overall survival rates achieved when DAC is incorporated into a conventional regimen.From 1991 to 1997, 20 boys and 5 girls aged 4.2 to 17.7 years who had stage III (according to the St. Jude staging system) (n = 21) or stage IV (n = 4) large-cell NHL were treated in this study. DAC therapy was administered at the beginning of the induction phase in 2 sequential cycles and incorporated throughout a continuation phase (modified from the ACOP+ regimen, which features doxorubicin, cyclophosphamide, vincristine, and prednisone) with doxorubicin, cyclophosphamide, vincristine, and dexamethasone. The total duration of treatment was approximately 10 months.DAC therapy yielded a response in 22 of 25 patients (88%; 95% confidence interval [95% CI], 68%-97%): complete remission in 13 cases (52%), and partial response in 9 (36%). After additional treatment with doxorubicin, cyclophosphamide, vincristine, and dexamethasone, complete remission was attained in 18 patients (72%) and partial remission in 3 (12%). The event-free survival rate (+/- the standard error [SE]) was 64% +/- 9% and the overall survival rate was 80% +/- 8% at 5 years.The results of the current study indicate that the DAC regimen is well tolerated and effective for pediatric patients with large-cell NHL.

Induction therapy for acute myeloid leukemia (AML) usually consists of 7 days of cytarabine at 100–200 mg/m2/day and an anthracycline. Such combinations produce complete response (CR) rates of 60–80% in patients with de novo AML. On the basis of a previous report, suggesting a higher CR rate using a regimen of standard daunomycin and cytarabine followed by 3 days of high-dose cytarabine (HDAC), 101 eligible patients received this regimen in a phase II trial. Sixty patients [59%, 95% confidence interval (CI) 49–69%] achieved a CR, and 10 patients died of infection during induction. Although cytogenetic risk group affected overall survival (P = 0.0016) and relapse-free survival (P = 0.0043), it had no impact on CR rate (P = 0.63). Patients received postremission therapy with repetitive courses of alternate day high-dose cytarabine; this was associated with considerable toxicity and the majority of patients could not receive all of the scheduled postremission therapy. The estimated median survival was 23 months (95% CI 15–34 months), and the estimated probability of surviving 5 years was 34% (95% CI 24–43%). The results of this intensive induction regimen were similar to that seen in previous trials and were not as promising as reported in the previous pilot study. Am. J. Hematol., 2007. © 2007 Wiley-Liss, Inc.

The effectiveness of bifunctional alkylating nitrogen mustard compounds in chemotherapy is related to their ability to form DNA inter-strand crosslinks. Patients exposed to DNA inter-strand crosslinking (ICL) agents subsequently experience an elevated incidence of myelodysplastic syndromes (MDS) and MDS related acute myeloid leukemia. Fanconi's anemia (FA) patients are deficient in the repair of crosslink DNA damage and they experience a high incidence of MDS. These observations indicate that hematopoietic cells are specific target for the transforming effects of DNA crosslinking damage. Changes in transcript levels were characterized in human hematopoietic cells occurring in response to the nitrogen mustard, mechlorethamine (HN2), but not in response to monofunctional analogs. Only modest changes in a few gene transcripts were detected in HL60 cells exposed to levels of HN2 tittered to maximal dose that caused growth suppression with minimal cell death and allowed eventual resumption of normal cell growth. Under conditions of transient growth suppression, a subset of glutathione-S-transferase (GST) isoenzyme genes was consistently upregulated three to fourfold by HN2, but not by monofunctional analogs. Subsequent efforts to confirm the changes detected by microarray analyses revealed an unexpected dependence on treatment conditions. The GST alpha class A2 subfamily member transcripts were upregulated 24 h after a 1 h exposure to HN2 that caused an extensive, but transient block in late S/G2 cell cycle phase, but were minimally altered with continuous exposure. The 1-h exposure to HN2 caused a transient late S/G2 cell cycle arrest in both the HL-60 cell line and the Colo 320HSR human colon cancer cell line. Overexpression of GSTA2 by transient transfection protected Colo 320HSR cells against both cycle arrest and apoptosis following exposure to HN2. Overexpression of GSTA2 in Colo 320HSR cells induced after exposure to HN2 did not alter cycle arrest or apoptosis. The results indicate that human GSTA2 facilitates the protection of cells from HN2 damage and not repair. Our results are consistent with the possibility that GSTA2 polymorphisms, variable isoenzyme expression, and variable induced expression may be factors in the pathogenesis of MDS.

Idiopathic myelofibrosis is a rare hematologic disorder that is occasionally associated with pulmonary hypertension and has been cured with bone marrow transplantation (BMT). Most cases occur in older adults, but children with similar clinical and pathologic findings have been described. The authors describe a critically ill male infant with idiopathic myelofibrosis and subtle findings suggestive of pulmonary hypertension who was treated with BMT after failing to respond to chemotherapy. After BMT, the patient's clinical course improved in all respects, but he ultimately died of progressive pulmonary hypertension.

Myelodysplastic and myeloproliferative disorders are rare in childhood and there is no widely accepted system for their diagnosis and classification. We propose minimal diagnostic criteria and a simple classification scheme which, while based on accepted morphological features and conforming with the recent suggestions of the WHO, allows for the special problems of myelodysplastic diseases in children. The classification recognizes three major diagnostic groups: (1) juvenile myelomonocytic leukemia (JMML), previously named chronic myelomonocytic leukemia (CMML) or juvenile chronic myeloid leukemia (JCML); (2) myeloid leukemia of Down syndrome, a disease with distinct clinical and biological features, encompassing both MDS and AML occurring in Down syndrome; and (3) MDS occurring both de novo and as a complication of previous therapy or pre-existing bone marrow disorder (secondary MDS). The main subtypes of MDS are refractory cytopenia (RC) and refractory anemia with excess of blasts (RAEB). It is suggested retaining the subtype of RAEB-T with 20-30% blasts in the marrow until more data are available. Cytogenetics and serial assessments of the patients are essential adjuncts to morphology both in diagnosis and classification.

Purpose/objectives To compare the accuracy of three coagulation tests (prothrombin time [PT], activated partial thromboplastin time [aPTT], and fibrinogen [FBG]) performed on blood samples collected through heparinized tunneled venous access devices (TVADs) with those from venipuncture. Design Descriptive comparative with patients serving as their own controls. Setting Pediatric comprehensive care setting for children and adolescents experiencing catastrophic diseases. Sample 53 patients who had TVADs, had not received asparaginase during the previous 14 days, and had coagulation studies ordered. Patients ranged in age from 2-20 years (mean = 9.2 years, SD = 5). The most common diagnoses were neuroblastoma and acute myelocytic leukemia. Methods Blood was collected through TVADs within seconds of collection of the venipuncture sample. The first 3 ml of blood from a TVAD was discarded; the research nurse then drew three sequential samples of 3 ml each. Laboratory personnel were blinded to the source of all four samples until all analyses had been completed. Main research variables PT, aPTT, and FBG. Findings For all patients, results of PT, aPTT, and FBG tests on each of the three blood samples obtained through the TVAD differed significantly from results of the same tests on blood obtained by venipuncture. Conclusions These findings indicate that neither a 6 ml, 9 ml, nor 12 ml discard from a heparinized TVAD is sufficient to yield clinically trustworthy PT, aPTT, or FBG values. Implications for nursing Nurses who have been persuaded by patients or parents to withdraw blood samples for coagulation indicators from a TVAD rather than from a venipuncture should have access to this research-based information that the three indicators, particularly aPTT, differ significantly from each other as to make it unreliable and potentially unsafe to sample blood from a TVAD to assess coagulation.

The human myeloid nuclear differentiation antigen (MNDA) is a hematopoietic cell specific nuclear protein. MNDA and other related gene products interact with and alter the activity of a large number of proteins involved in regulating specific gene transcription. MNDA and related genes exhibit expression characteristics, which suggest functions unique to specific lineages of cells, in addition to mediating the effects of interferons. Cells of the human K562 myeloid line do not express MNDA and are relatively immature compared to lines that express MNDA (HL-60, U937, and THP1). The hypothesis that MNDA influences the expression of specific genes was tested by creating MNDA expressing K562 cells using stable retroviral mediated gene transfer followed by evaluation of transcription profiles. Two macroarrays containing a total of 2,350 cDNAs of known genes showed a specific up-regulation of Dlk1 expression in MNDA expressing K562 cell clones. Real time quantitative RT-PCR analysis confirmed an average of over 3- and 7-fold upregulation of Dlk1 in two clones of MNDA expressing K562 cells. The effects on Dlk1 were also confirmed by Northern blotting. Dlk1 is essential for normal hematopoiesis and abnormal expression is a proposed marker of myelodysplastic syndrome. Additional screening of transcription profiles after induced erythroid and megakaryoblastic differentiation showed no additional gene transcripts altered by the presence of MNDA. These results indicate that MNDA alters expression of a gene essential for normal hematopoiesis.

The 2008 WHO classification scheme of hematolymphoid neoplasms recognizes a category of myeloid and lymphoid neoplasms (MLNs) with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1.1 The postulated cell of origin for PDGFRA or FGFR1-rearranged diseases is a pluripotent progenitor capable of giving rise to myeloid neoplasms (myeloproliferative neoplasms and acute leukemias) and to lymphoblastic leukemia/lymphoma. Historically, PDGFRB translocations have not been associated with malignancies of the lymphoid lineage. Myeloid neoplasms with abnormalities of PDGFRB have been described to have hematologic features of chronic myelomonocytic leukemia (CMML), sometimes with eosinophilia, or as various other myeloproliferative and/or myelodysplastic neoplasms.2 In recognition of this distinction, the classification scheme designates specific categories of “MLNs” with PDGFRA and FGFR1 rearrangement, but omits the word “lymphoid” from the PDGFRB-associated entity.1 To call attention to the rare occurrence of lymphoid and mixed MLNs with abnormalities of PDGFRB, we present the clinical and pathological details of 2 cases.

Among 511 patients with therapy-related myelodysplastic syndrome or acute leukemia (t-MDS/t-AL) and balanced chromosome aberrations, 162 (32%) had translocations involving 11q23. The recurring translocation partners were 9p22 (48%), 19p13.3 (11%), 19p13.1 (10%), 4q21 (9%), 6q27 (6%), 1p32 (2%), 16p13.1 (2%), 10p13 (1%), and 17q25 (1%); in 9%, the translocations were seen only once. The remaining 349 patients were divided into five subgroups based on the balanced aberration: 21q22, inv(16), t(15;17), Rare, and Unique aberrations. Patients in the 11q23 subgroup had a sole cytogenetic abnormality more often than those in the 21q22, inv(16), Rare, and Unique subgroups, and a complex karyotype or -5/del(5q) and/or -7/del(7q) less often than patients in the 21q22, Rare, and Unique subgroups. Clinically, 11q23 patients had acute lymphoblastic leukemia (ALL) more often as their primary disease and a shorter latency from start of treatment for the primary disease to their t-MDS/t-AL diagnosis, except when compared with the inv(16) subgroup. The 11q23 subgroup demonstrated a younger age at t-MDS/t-AL diagnosis, but this finding was not significant when patients with AL as their primary diagnosis were excluded. Survival from the time of diagnosis of t-MDS/t-AL was significantly shorter for the 11q23 subgroup compared with that of the 21q22, inv(16), and t(15;17) subgroups (median 8 vs. 14, 28, and 29 months, respectively). Inferior survival occurred even though 11q23 patients were younger and more often received blood or marrow transplantation (BMT). Even among patients receiving BMT, 11q23 patients had a shorter median survival (9 vs. 12-31 months for the other subgroups). However, among 11q23 patients, those receiving BMT survived longer, with 1- and 5-year survivals of 43% and 18% compared with 23% and 7% for patients not transplanted. With regard to prior therapy, 11q23 patients, compared with other patients, received radiotherapy less often as their sole therapy and chemotherapy more often. They had received VP16, methotrexate, 6MP/6TG, L-asparaginase, daunorubicin, cytarabine, and VM26 more often, likely attributed to the high frequency of AL as their primary disease. More patients in the 11q23 subgroup had received doxorubicin, except in comparison with the 21q22 subgroup; more vincristine, except in comparison with the Rare and Unique subgroups; and more prednisone, except in comparison with the Unique subgroup. Patients in the 11q23 subgroup more often received alkylating agents (AAs) (86% vs. 59-82% for the other subgroups), and topoisomerase II inhibitors (TIs) (84% vs. 49-75%), and they more often reported exposure to AAs plus TIs without radiotherapy (33% vs. 12-21%), except in comparison with the 21q22 subgroup (36%). We performed a multivariate analysis to determine whether the adverse survival of 11q23 patients compared to other Workshop patients was explained by factors other than the presence of the 11q23 abnormality. Covariates in the final model were the five cytogenetic subgroup indicators, where the 11q23 subgroup was the referent (P < 0.0001); age at t-MDS/t-AL (P = 0.0036); previous exposure to lomustine (P < 0.0001) and mitoxantrone (P = 0.0225); BMT for t-MDS/t-AL (P = 0.0006); and karyotype complexity (P = 0.0114). The risk of death for 11q23 patients relative to patients in the 21q22, inv(16), t(15;17), and Unique subgroups was significant, even after adjustment for other risk factors (relative risks 2.3, 3.6, 3.1, and 1.5, respectively; P < 0.0001 for the first three comparisons and P = 0.0125 for the last). When a multivariable model was constructed, excluding patients with AL or MDS as their primary diagnosis, the relative risk of death for 11q23 patients was significantly higher than that of all five other cytogenetic subgroups. We conclude that among t-MDS/t-AL patients with balanced aberrations, 11q23 translocations are an independent adverse risk factor. Although BMT is the current therapy of choice, new treatment is required.

Cyclosporine A (CsA) inhibits P-glycoprotein (Pgp)–mediated cellular export of anthracyclines at clinically achievable concentrations. This randomized controlled trial was performed to test the benefit of CsA addition to treatment with cytarabine and daunorubicin (DNR) in patients with poor-risk acute myeloid leukemia (AML). A total of 226 patients were randomly assigned to sequential treatment with cytarabine and infusional DNR with or without intravenous CsA. Remitting patients received one course of consolidation chemotherapy that included DNR with or without CsA as assigned during induction. Addition of CsA significantly reduced the frequency of resistance to induction chemotherapy (31% versus 47%,P = .0077). Whereas the rate of complete remission was not significantly improved (39% versus 33%, P = .14), relapse-free survival (34% versus 9% at 2 years,P = .031) and overall survival (22% versus 12%,P = .046) were significantly increased with CsA. The effect of CsA on survival was greatest in patients with moderate or bright Pgp expression (median 12 months with CsA versus 4 months for controls) compared to patients with absent or low Pgp expression (median 6 months in both arms). The frequency of induction deaths was 15% with CsA and 18% in controls. Steady-state serum concentrations of DNR (P = .0089) and daunorubicinol (P

To describe the clinical and biologic features of pediatric acute megakaryoblastic leukemia (AMKL) and to identify prognostic factors, experience at St Jude Children's Research Hospital was reviewed. Of 281 patients with acute myeloid leukemia treated over a 14-year period, 41 (14.6%) had a diagnosis of AMKL. Six patients had Down syndrome and AMKL, 6 had secondary AMKL, and 29 had de novo AMKL. The median age of the 22 boys and 19 girls was 23.9 months (range, 6.7-208.9 months). The rate of remission induction was 60.5%, with a 48% rate of subsequent relapse. Patients with Down syndrome had a significantly higher 2-year event-free survival (EFS) estimate (83%) than did other patients with de novo AMKL (14%) or with secondary AMKL (20%; P < or =.038). Among patients who had de novo AMKL without Down syndrome, 2-year EFS was significantly higher after allogeneic bone marrow transplantation (26%) than after chemotherapy alone (0%; P =.019) and significantly higher when performed during remission (46%) than when performed during persistent disease (0%; P =.019). The 5-year survival estimates were significantly lower for de novo AMKL (10%) than for other forms of de novo AML (42%; P

PURPOSE: To develop more effective chemotherapy regimens for childhood acute myelogenous leukemia (AML). PATIENTS AND METHODS: Between June 1991 and December 1996, we administered the nucleoside analog 2-chlorodeoxyadenosine (2-CDA) to 73 children with primary AML and 20 children with secondary AML or myelodysplastic syndrome (MDS). Patients received one or two 5-day courses of 2-CDA (8.9 mg/m2/d) given by continuous infusion. All patients then received one to three courses of daunomycin, cytarabine, and etoposide (DAV) remission induction therapy. RESULTS: Seventy-two patients with primary AML were assessable for response. Their rate of complete remission (CR) was 24% after one course of 2-CDA, 40% after two courses of 2-CDA, and 78% after DAV therapy. Of the 57 patients who entered CR, 11 subsequently underwent allogeneic bone marrow transplantation (BMT), and 40 underwent autologous BMT. Twenty-nine patients remain in continuous CR after BMT. Two patients remain in CR after chemotherapy only. The 5-year event-free survival (EFS) estimate was 40% (SE = 0.080%). Patients with French-American-British (FAB) M5 AML had a higher rate of CR after treatment with 2-CDA (45% after one course and 70.6% after two courses) than did others (P = .002). In contrast, no patient with FAB M7 AML (n = 10) entered CR after treatment with 2-CDA. Similarly, no patient with primary MDS (n = 6) responded to 2-CDA. Seven patients with secondary AML or MDS (n = 14) had a partial response to one course of 2-CDA. CONCLUSION: This agent was well tolerated, and its toxicity was acceptable. Future trials should examine the effectiveness of 2-CDA given in combination with other agents effective against AML.

The associations of cytogenetics with complete remission (CR) rates, overall survival (OS), and outcomes after CR were studied in 609 previously untreated AML patients younger than 56 years old in a clinical trial comparing 3 intensive postremission therapies: intensive chemotherapy, autologous transplantation (ABMT), or allogeneic bone marrow transplantation (alloBMT) from matched related donors. Patients were categorized into favorable, intermediate, unfavorable, and unknown cytogenetic risk groups based on pretreatment karyotypes. CR rates varied significantly (P

Older patients with acute myelogenous leukemia (AML) have overexpression of P-glycoprotein (Pgp+), and this has been shown to correlate quantitatively with therapeutic outcome. Since Pgp-mediated efflux of cytotoxic drugs can be inhibited by the cyclosporine analogue, PSC 833, we investigated the use of this agent with a 5-day mitoxantrone/etoposide regimen in patients over age 55 with newly diagnosed AML. Previous studies suggested a 33% incidence of grade IV/V non-hematologic toxicity with the use of mitoxantrone 10 mg/M(2) and etoposide 100 mg/M(2), each for 5 days, in this patient population. Since PSC 833 alters the pharmacokinetic excretion of MDR-related cytotoxins, this phase I dose-finding study was performed to identify doses of mitoxantrone/etoposide associated with a similar 33% incidence of grade IV/V non-hematologic toxicity, when given with PSC 833. Mitoxantrone/etoposide (M/E) doses were escalated in fixed ratio from a starting dose of M: 4 mg/M(2) and E: 40 mg/M(2), to M: 7 mg/M(2) and E: 70 mg/M(2), in successive cohorts of eight patients each. PSC 833 was well tolerated and the MTD of this M/E regimen with PSC 833 in this population was M: 6 mg/M(2) and E: 60 mg/M(2). The complete response (CR) rate for all patients was 50% (15/30) and was considerably higher for de novo than for secondary AML. These data suggest that the addition of PSC 833 to an M/E regimen for older patients with untreated AML is well tolerated but requires a reduction in M/E dosing to avoid increased toxicity.

The MLL gene at 11q23 is frequently disrupted by chromosomal translocations in de novo acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), and in secondary leukemia induced by treatment with inhibitors of topoisomerase II, including the epipodophylotoxins. The CBFA2 gene at 21q22 is also frequently disrupted in de novo ALL and AML and less commonly in secondary AML. Rearrangements of MLL and CBFA2 have been described in de novo and secondary myelodysplastic syndrome (MDS). There have been no previous descriptions of coexisting abnormalities of MLL and CBFA2 in cases of MDS or acute leukemia. We describe a patient who developed secondary MDS after chemotherapy for hyperdiploid ALL. At the time of conversion to MDS, the patient had 46 chromosomes, with an 11q23/MLL translocation involving a new partner breakpoint at 2p23 and a 21q22/CBFA2 translocation involving a new partner breakpoint at 6p22. This report is the first to describe new partner breakpoints at 2p23 and 6p22 for MLL and CBFA2 genes, respectively, and concurrent rearrangements of these genes in a patient with secondary MDS. Genes Chromosomes Cancer 28:227–232, 2000. © 2000 Wiley-Liss, Inc.

Idiopathic myelofibrosis can develop in children as well as adults. However, the disease appears to be much more aggressive in adults, being characterized by poor survival rates and a high frequency of malignant transformation. Here, we describe three cases of idiopathic myelofibrosis in infants, two of whom were followed for 16 and 22 years after diagnosis. Neither of these patients required more than minimal supportive care, and both have had spontaneous erythropoietic recovery as early as 2–3 years after diagnosis. There have been no indications of malignant transformation or clinical deterioration. Thus, idiopathic myelofibrosis may have a different pathogenesis and clinical course in infants from adults, requiring a more conservative approach to management.

Three pediatric patients with refractory anemia with ringed sideroblasts (RARS) are presented. Bone marrow aspirates were examined using Romanowsky and Prussian blue iron stains in all three patients, and electron microscopic analysis was performed in one patient. All three patients had cytogenetic analysis of the bone marrow. Other studies included analysis of serum iron, total iron-binding capacity, ferritin, copper, vitamins B6 and B12, and folate levels. Antibody titers to Parvovirus, HIV, and other viruses were measured. The patients had contrasting clinical courses. Patients 1 and 2 had dysplastic hematopoietic features and cytogenetic findings (with either partial or one allele loss of chromosome 7), suggestive of myelodysplastic syndrome. Patient 1 experienced acute myeloid leukemia (AML) and had a good response to AML-directed therapy. Patient 2 had prolonged cytopenias and underwent bone marrow transplantation (BMT). Patient 3 had features suggestive of refractory anemia associated with mitochondrial cytopathy, including normal cytogenetics with pronounced vacuolization of marrow precursors. His anemia regressed spontaneously a few months after diagnosis. These patients represent two subgroups of pediatric RARS. Patients with the myelodysplastic syndrome (MDS) type may progress to cytopenias or leukemia and may require aggressive therapy; the type is characterized by clonal cytogenetic findings. The non-MDS type, which may relate to mitochondrial cytopathy, often shows spontaneous regression and requires only supportive treatment; it has normal cytogenetic findings.

The aim of this study is to determine whether the addition of mitoxantrone to high dose cytarabine improves the outcome of treatment in patients with relapsed or refractory acute myeloid leukemia (AML). One hundred and sixty-two eligible patients, 14-76 years of age, with AML either in first relapse or that failed to respond to initial remission induction therapy, with no CNS involvement were randomized to receive therapy with cytarabine 3 gm/M2 i.v. over 2 h every 12 h for 12 doses on days 1-6 (Arm I) (HIDAC); or HIDAC plus mitoxantrone 10 mg/M2 i.v. daily on days 7 9 (Arm II) (HIDAC + M). Patients achieving complete remission were treated with three courses of consolidation including HIDAC (Ara-C 3 gm/M2 i.v. 12 h days 1 3; 2 gm/M2 over age 50) alone (ARM I) or with mitoxantrone (10 mg/M2 i.v. day 1) (ARM II). Among 162 patients (81 HIDAC, 81 HIDAC + M) evaluated for induction toxicity, there were 10 (12%) induction deaths with HIDAC and 13 (17%) with HIDAC + M (2-tailed P = 0.65). Most early deaths were due to infection and/or hemorrhage. Among 162 patients evaluated for responses to induction therapy, 26/81 (32%) HIDAC and 36/81 (44%) HIDAC + M patients achieved complete remission (two-tailed P = 0.15). Although this difference was not statistically significant in univariate analysis, it was after adjusting for the effects of WBC and PMN percentage in multivariate analysis (P=0.013). Median survivals from study entry were 8 months (HIDAC) and 6 months (HIDAC + M); 2-tailed logrank P = 0.58. Among 48 patients registered for consolidation, the median disease-free survivals from that registration were 8 months with HIDAC and 11 months with HIDAC + M (P = 0.60). There were three treatment-related deaths during consolidation (1 HIDAC, 2 HIDAC + M), all due to infections. In this randomized trial, the addition of mitoxantrone to high-dose cytarabine was associated with a trend toward a higher CR rate. There was less evidence for an advantage in disease-free or overall survival, although any such conclusion is limited by the size of the study.

The association of human herpesvirus-8 (HHV-8) with a small non-cleaved cell lymphoma is described in a child with the acquired immunodeficiency syndrome (AIDS) who developed a malignant pleural effusion and radiologic evidence of multiple solid tumors. HHV-8 DNA and Epstein-Barr virus DNA were identified in pleural fluid cells by polymerase chain reaction (PCR) amplification. The serum antibody titer against lytic HHV-8 proteins was 1:640; antibodies to latent HHV-8 proteins were not detected. Cytogenetic analysis of malignant cells revealed three abnormal karyotypes sharing the common finding of a t(8;14) translocation. Rearrangement of c-myc was demonstrated by PCR analysis. Oligoclonal JH immunoglobulin bands were found. Insufficient pleural fluid cells were available to permit localization of HHV-8 to malignant cells by in situ hybridization. This malignancy contrasts with HHV-8-associated lymphomas reported in adult patients with AIDS with respect to cell morphology, c-myc translocation, and oligoclonal immunoglobulin gene rearrangement. HHV-8 is associated with a wider spectrum of malignancies than recognized previously.

PURPOSE To reassess the clinical and biologic significance of myeloid-associated antigen expression in childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS We prospectively studied 334 newly diagnosed cases of this disease, using a comprehensive panel of antibodies that represented five myeloid cluster groups (CD13, CD14, CD15, CD33, and CD65). Blast cells were tested for ETV6 and MLL rearrangement using Southern blot analysis. RESULTS CD13 was expressed in 13.7% of cases, CD14 in 1%, CD15 in 6.6%, CD33 in 16%, and CD65 in 9.7%. Approximately one third of cases (31.4%) expressed one or more of these antigens (B-cell precursor, 31.9%; T-cell, 28.8%), while 10.5% expressed two or more (B-cell precursor, 11.3%; T-cell, 6.1%). Among the B-cell precursor leukemias, myeloid-associated antigen expression was significantly associated with a lack of hyperdiploidy and rearrangements of ETV6 or MLL gene. Most of the cases with MLL rearrangements (82%) expressed CD65, CD15, and CD33, either alone or in combination, whereas 48% of those with a rearranged ETV6 gene expressed CD13, CD33, or both. Myeloid-associated antigen expression did not correlate with event-free survival, whether the analysis was based on any of the five antigens in our panel or on the three more commonly tested antigens (CD13, CD33, and CD65). Importantly, this finding was not affected by exclusion of patients with ETV6 or MLL gene rearrangements. CONCLUSION Even though blast cell expression of myeloid-associated antigen expression shows significant associations with specific genetic abnormalities, it lacks prognostic value in childhood ALL.

A 19-month-old boy was referred to our institution because of chronic macrocytic anemia and severe thrombocytopenia. At age 17 months, he had developed petechiae. He had a leukocyte count of 4.4 x 10(9)/L, hemoglobin concentration of 7.9 g/dL, packed cell volume of 21%, mean corpuscular volume of 101 fL, and platelet count of 19 x 10(9)/L. At the time of referral, a bone marrow aspirate and biopsy revealed myelodysplastic changes that included megakaryocytic hyperplasia with hypolobated megakaryocytes, megaloblastoid erythropoiesis, 12% blast cells, and bone marrow fibrosis; the diagnosis was refractory anemia with excess blasts (RAEB). Cytogenetic analysis showed the following abnormalities: 47, XY, inv(3)(p21q25), del(5)(q22q31), +21/46, XY. By dinucleotide polymorphism analysis, the 5q22-q31 loci were normal in peripheral blood granulocytes. Because of severe thrombocytopenia that became refractory to platelet transfusions and because of possible progression to leukemia, the patient received an unrelated-donor bone marrow transplant. Recovery was complicated by a visceral fungal infection, but the patient now has normal, fully reconstituted bone marrow function. This patient is the youngest to be reported with RAEB and a 5q- anomaly accompanied by thrombocytopenia, megakaryocytic hyperplasia with hypolobated megakaryocytes, and macrocytic anemia with megaloblastoid erythropoiesis, similar to "5q- syndrome" in adults.

Central nervous system (CNS) relapse has been an obstacle to uniformly successful treatment of childhood acute lymphoblastic leukemia (ALL) for many years. We therefore intensified intrathecal chemotherapy (simultaneously administered methotrexate, hydrocortisone, and cytarabine) for 165 consecutive children with newly diagnosed ALL enrolled in Total Therapy Study XIIIA from December 1991 to August 1994. The 64 patients (39%) who had 1 or more blast cells in cytocentrifuged preparations of cerebrospinal fluid at diagnosis, with or without associated higher-risk features, received additional doses of intrathecal chemotherapy during remission induction and the first year of continuation treatment. Patients with higher-risk leukemia, regardless of cerebrospinal fluid findings, also received additional doses of intrathecal chemotherapy during the first year of continuation treatment. Cranial irradiation was reserved for patients with higher-risk leukemia (22% of the total). The 5-year cumulative risk of an isolated CNS relapse among all 165 patients was 1.2% (95% confidence interval, 0% to 2.9%), whereas that of any CNS relapse was 3.2% (0.4% to 6.0%). The probability of surviving for 5 years without an adverse event of any type was 80.2% ± 9.2% (SE). Our results suggest that early intensification of intrathecal chemotherapy will reduce the risk of CNS relapse to a very low level in children with ALL, securing a higher event-free survival rate overall.

Older age is a poor prognosis factor in acute myeloid leukemia (AML). This double-blind trial was designed to test the hypothesis that granulocyte colony-stimulating factor (G-CSF) used as supportive care could improve the treatment of elderly AML patients. Two hundred thirty-four patients 55 or more years of age with a morphologic diagnosis of de novo or secondary AML, French-American-British (FAB) M0-M7, excluding M3, were randomly assigned to a standard induction regimen (daunorubicin at 45 mg/m2 intravenously [IV] on days 1 through 3 and Ara-C at 200 mg/m2 IV continuous infusion on days 1 through 7) plus either placebo or G-CSF (400 μg/m2 IV over 30 minutes once daily). Results are reported here for 211 centrally confirmed cases of non-M3 AML. The two groups were well balanced in demographic, clinical, and hematological parameters, with median ages of 68 years in the G-CSF and 67 years in the placebo groups. The complete response (CR) rate was not significantly better in the G-CSF group: 50% in the placebo and 41% in the G-CSF group (one-tailedP = .89). Median overall survival was also similar, 9 months (95% confidence interval [CI], 7 to 10 months) in the placebo and 6 months (95% CI, 3 to 8 months) in the G-CSF arms (P = .71). We found a significant 15% reduction in the time to neutrophil recovery in the G-CSF group (P = .014). G-CSF had no impact on recovery from thrombocytopenia (P = .80) or duration of first hospitalization (P = .27). When infection complications were evaluated, G-CSF had a beneficial effect on the duration but not on incidence of infection. G-CSF patients had fewer days with fever and shorter duration of antibiotic use. However, there was no difference in the frequency of total documented infections or in the number of fatal infections (19% placebo v 20% G-CSF). In this study of elderly AML patients, G-CSF improved clinical parameters of duration of neutropenia and antibiotic use, but did not change CR rate or survival or shorten hospitalization.

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.

Compared with younger patients, elderly patients with acute myeloid leukemia (AML) respond poorly to conventional chemotherapy. To determine if this poor response is due to differences in the biologic characteristics of AML in the elderly, we studied 211 patients (161 de novo, 50 secondary AML) over 55 years of age (median, 68 years) registered to a single clinical trial for previously untreated AML (SWOG 9031, Phase III randomized trial of standard dose cytosine arabinoside and daunomycin ± rhG-CSF ). Pretreatment leukemic blasts were karyotyped and were also analyzed for intrinsic drug resistance by quantitating expression of the multidrug resistance glycoprotein MDR1 and functional drug efflux using sensitive flow cytometric techniques. Results were correlated with clinical variables and outcome. These elderly AML patients had a high frequency of unfavorable cytogenetics (32%), MDR1 protein expression (71%), and functional drug efflux (58%); each of these factors occurred at high frequencies in both de novo and secondary AML patients and was associated with a significantly poorer complete remission (CR) rate. In multivariate analysis, secondary AML (P = .0035), unfavorable cytogenetics (P = .0031), and MDR1 (P = .0041) were each significantly and independently associated with lower CR rates. Resistant disease was associated with unfavorable cytogenetics (P = .017) and MDR1 expression (P = .0007). Strikingly, elderly MDR1(−) de novo AML patients with favorable/intermediate cytogenetics had a CR rate of 81%; with increasing MDR1 expression, CR rate decreased in this cytogenetic group. MDR1(+) secondary AML patients with unfavorable cytogenetics had a CR rate of only 12%. Thus, AML in the elderly is associated with an increased frequency of unfavorable cytogenetics and MDR1 expression, both of which independently contribute to poor outcomes. The high frequencies of these features in both de novo and secondary elderly AML patients suggest a common biologic mechanism for these leukemias distinct from that in younger patients. Investigation of biologic parameters at diagnosis in AML in the elderly may help identify patients with a high likelihood of achieving CR with conventional regimens, as well as those who may require alternate regimens designed to overcome therapy resistance.

We conducted a clinical trial to determine the feasibility of growth factor mobilization of CD34+ progenitor cells in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Eight asymptomatic, HIV-1-infected adults (median CD4+ T-cell count, 415 cells/microL), received 480 micrograms/d of granulocyte colony-stimulating factor (G-CSF) for 6 days without evidence of viral activation. Despite concerns that HIV-1 might inhibit hematopoiesis, CD34+ cells were successfully mobilized to the periphery of all donors, independent of the baseline CD4+ T-cell count, and the status of antiretroviral therapy. Leukapheresis was performed on day 6, and yielded a median of 194 x 10(6) CD34+ cells per leukapheresis (n = 7). CD34-enriched cells from the leukapheresis were predominantly myeloid-committed, but between 0.2% and 1.7% were primitive CD34+/CD38- progenitors. A median of 21.7% of the mobilized CD34+ cells were dimly positive for CD4. Consequently, CD34(+)-enriched cells were purified on the cell sorter (mean purity, 97.7% +/- 2.4%; n = 7), and examined for HIV-1 DNA. Purified CD34+ cells from two of seven donors were polymerase chain reaction (PCR)-positive for HIV-1, but only from one of three samples from each donor. We conclude that G-CSF can safely mobilize CD34+ progenitor cells in HIV-1-infected subjects, and that these cells are suitable for consideration in gene-transfer strategies.

Interest in high-dose cytarabine (HDAC) for both induction and postremission therapy for acute myeloid leukemia (AML) prompted the Southwest Oncology Group (SWOG) to initiate a randomized trial comparing HDAC with standard-dose cytarabine (SDAC) for remission induction of previously untreated AML and to compare high-dose treatment versus conventional doses for consolidation therapy. Patients less than 65 years of age with de novo or secondary AML were randomized for induction between SDAC 200 mg/ m2/d for 7 days by continuous infusion or HDAC at 2 g/ m2 intravenously every 12 hours for 12 doses; both groups received daunorubicin (DNR) at 45 mg/m2/d intravenously for 3 days. Complete responders to SDAC were randomized to receive either two additional courses of SDAC plus DNR or one course of HDAC plus DNR. Complete responders to HDAC were nonrandomly assigned to receive one additional course of HDAC plus DNR. Of patients randomized between SDAC (n = 493) and HDAC (n = 172) induction, 361 achieved complete remission (CR). The CR rate was slightly poorer with HDAC: 55% versus 58% with SDAC for patients aged less than 50, and 45% (HDAC) versus 53% (SDAC) for patients aged 50 to 64 (age-adjusted one-tailed P = .96). With a median follow-up time of 51 months, survival was not significantly better with HDAC (P = .41); the estimated survival rate at 4 years was 32% (HDAC) versus 22% (SDAC) for those aged less than 50, and 13% (HDAC) versus 11% (SDAC) for those aged 50 to 64. However, relapse-free survival was somewhat better following HDAC Induction (P = .049): 33% (HDAC) versus 21% (SDAC) at 4 years for those aged less than 50, and 21% (HDAC) versus 9% (SDAC) for those aged 50 to 64. Induction with HDAC was associated with a significantly increased risk of fatal (P = .0033) and neurologic (P < .0001) toxicity. Among patients who achieved CR with SDAC, survival and disease-free survival (DFS) following consolidation randomization were not significantly better with HDAC compared with SDAC (P = .77 and .46, respectively). Patients who received both HDAC induction and consolidation had the best postremission outcomes; however, the proportion of CR patients who did not go on to protocol consolidation therapy was more than twice as high after HDAC induction compared with SDAC. Induction therapy with HDAC plus DNR was associated with greater toxicity than SDAC plus DNR, but with no improvement in CR rate or survival. Following CR induction with SDAC, consolidation with HDAC increased toxicity but not survival or DFS. In a nonrandomized comparison, patients who received both HDAC induction and consolidation had superior survival and DFS compared with those who received SDAC induction with either SDAC or HDAC consolidation.

We prospectively evaluated the ability of magnetic resonance imaging (MRI) of the thoraco-lumbar vertebrae to determine the relative amount of red and fatty marrow in children with aplastic anemia.Twenty pediatric patients (ages 1-19 years) with aplastic anemia underwent T1-weighted (T1W, n = 31) and short T1 inversion recovery (STIR, n = 30) MRI of the midline sagittal thoraco-lumbar spine. Bone marrow (BM) biopsies from the posterior iliac crest (n = 29) were also performed. All studies were evaluated by blinded observers; MR grading was based on visual inspection of signal intensity. Biopsy-estimated cellularity was compared with T1W and STIR grading when these were performed within 14 days of each other (n = 16). All studies were compared to a simultaneous absolute neutrophil count (ANC), absolute reticulocyte count (ARC), and platelet count.BM cellularity estimated by BM biopsy was significantly associated with STIR grading (p = 0.032, Jonckheere-Terpstra test), as were peripheral ANC (p = 0.044), ARC (p = 0.007), and platelet count (p = 0.003). T1W grade was significantly associated with ANC (P = 0.011) but not ARC (p = 0.053) or platelet count (p = 0.377). Biopsy-estimated cellularity was associated with ANC (p = 0.032) and ARC (p = 0.036), but not platelet count (p = 0.282).In childhood aplastic anemia patients, STIR (and, to a lesser extent, T1W) MRI of the thoraco-lumbar spine reflects BM activity, as measured by peripheral blood ANC, ARC, and platelet count, and BM cellularity, as measured by BM biopsy. MRI may thus provide a noninvasive measure of hematopoietic status.

Resistance to chemotherapy is a major factor limiting successful treatment of acute myeloid leukemia (AML); one of the best characterized drug resistance mechanisms is extrusion of drugs by the energy-dependent multidrug resistance (MDR1) transport protein. Expression of MDR1 is common in AML and has been linked to lower remission induction rates and decreased remission durations. Because MDR1 efflux function may be modified by drugs such as cyclosporin A, accurate identification of MDR1+/efflux+ AML cases will be critical to identify patients who may benefit from therapies that contain such MDR1 modulators. We have optimized single and multiparameter flow cytometric assays to detect efflux of drugs or fluorescent dyes by previously cryopreserved AML blasts. These assays allowed precise identification of efflux by leukemic blasts, and correlation with CD34 and MDR1 expression. We subsequently studied a series of 60 previously untreated AML cases. Functional efflux was identified in 39 cases and was significantly correlated with MDR1 expression (P = .0002). However, discrepant cases were identified; 10 cases were efflux+ without significant MDR1 expression, whereas 6 MDR1+ cases were efflux-. There was also a highly significant correlation of efflux with CD34; 31 (79%) of the 39 efflux+ cases were CD34+ in comparison with only 5 (24%) of the 21 efflux- cases (P < .0001). Multivariate analysis showed that efflux was significantly associated with independent effects of both CD34 (P = .0011) and MDR1 expression (P = .034); the majority of efflux+ cases were CD34+, whereas 5 of the 6 MDR1+ efflux- cases lacked CD34 expression. Cyclosporin A blocked efflux in all but 2 cases regardless of MDR1 expression. Functional efflux in AML is frequently detected without the classic MDR1+ phenotype indicating that alternate non-MDR1-mediated efflux mechanisms may be important. Efflux assays may better identify patients who would benefit from therapies that include efflux modulators.

Balanced translocations affecting the 11q23 region are among the most frequent chromosomal abnormalities in childhood acute lymphoblastic leukemia (ALL), comprising 5% to 6%. These cases consistently have a rearranged MLL gene and are associated with high-risk presenting features, hyperleukocytosis and younger age, and a poor treatment outcome. To assess the clinical and biologic significance of 11q23- associated structural chromosomal abnormalities other than translocations, we studied 17 cases of childhood ALL [14 with del(11)(q23) and 3 with inv(11)(p12q23)] that were identified among 785 cases with successful chromosome analysis. In contrast to reported cases with 11q23 and MLL gene rearrangement, our series was characterized by relatively low leukocyte counts (median, 15.1 x 10(9)/L), expression of CD10 antigen but not myeloid-associated CD15 and CDw65 antigens, a relatively high frequency of T-cell immunophenotypes, and a generally favorable prognosis. All 13 cases with interpretable molecular analysis lacked MLL gene rearrangements. We suggest that most cases with deletions or inversions affecting the 11q23 region represent clinically and biologically different entities as compared with those defined by 11q23 translocation.

The Southwest Oncology Group analyzed outcome with cytotoxic chemotherapy for previously untreated acute myeloblastic leukemia (AML) from 1982 through 1986. Results with acute promyelocytic leukemia (APL) prompted comparison with patients from 1986 through 1991 and analysis of factors contributing to APL results. Patient and disease characteristics and treatment outcome were compared for all evaluable patients, with more detailed analysis of factors affecting APL treatment outcome. From 1982 through 1986, median survival and disease- free survival in 45 APL patients were 106 months and greater than 105 months, respectively, versus 6 and 14 months for 417 other AML patients. Such differences were not seen from 1986 through 1991. In the 141 APL patients from 1982 through 1991, after adjusting for significant patient and disease characteristics, higher daunomycin (DNR) doses during induction were significantly associated with higher complete remission rates (P < .0001), longer survival (P < .0001), and longer DFS (P < .0001). Cytosine arabinoside (Ara-C) induction dose, the inclusion of other chemotherapy agents in induction, postremission therapy (consolidation, maintenance, or bone marrow transplantation) other than DNR, APL subtype, and patient age did not appear to significantly affect outcome of APL, except for a significant detrimental effect of high-dose Ara-C in consolidation (P = .0042). Morphologic AML subtypes other than APL did not affect outcome. We conclude that high-dose DNR selectively increases survival in APL. This good survival is important for evaluation of combined all-trans retinoic acid (ATRA)/chemotherapy protocols and for planning future combinations of chemotherapy and ATRA. These results illustrate the need to individualize chemotherapy for subtypes of AML. Therapeutic response of APL is independent of age. Except for APL, morphologic subclassification of AML contributed little prognostic information.

Inv(16)(p13q22) is one of the most frequent chromosomal rearrangements found in acute myelogenous leukemia (AML), representing approximately 16% of documented karyotypic abnormalities. The inv(16) breakpoints have been cloned and shown to involve the non-DNA binding component of the AML-1 transcription factor complex termed core binding factor beta gene (CBF beta) on 16q and the smooth muscle myosin heavy chain gene (MYH11) on 16p. In this study, we analyzed 37 cases of inv(16)-containing AML and 4 cases with t(16;16)(p13;q22) for expression of the CBF beta/MYH11 chimeric message by reverse transcription-polymerase chain reaction (PCR) analysis. CBF beta/MYH11 chimeric messages were detected in 33 of 37 cases with the inv(16) and in the 4 t(16;16)-containing cases. Sequence analysis of PCR products showed extensive breakpoint heterogeneity in both CBF beta and MYH11. In addition to the previously described breakpoint in CBF beta at nucleotide (nt) 495 (amino acid 165), we have identified a second novel fusion point at nt 399 (amino acid 133) in 7% of the cases. Similarly, a unique breakpoint site was identified in MYH11 at nt 1098, as well as at three previously characterized sites at nts 994, 1201, and 1921. Of the 4 PCR-negative cases, 2 of 3 tested lacked CBF beta rearrangements by Southern blot analysis, suggesting the possible involvement of a different genomic locus in some cases with cytogenetic evidence of inv(16). To assess whether the portions of CBF-beta contained within the CBF beta/MYH11 chimeric products retain the ability to interact with their heterodimeric DNA-binding partner AML-1, we performed in vitro DNA- binding analysis. Recombinant CBF-beta polypeptides consisting of the N-terminal 165 amino acids retained their ability to interact with AML-1, whereas mutants containing only the N-terminal 133 amino acids interacted with AML-1 less efficiently. These data suggest that different CBF beta/MYH11 products may vary subtly in their biologic activities.

The t(2;5)(p23;q35) translocation was initially identified in cases of anaplastic large-cell lymphoma (ALCL) that expressed the Ki-1 (CD30) antigen. We have recently cloned this translocation and shown it to encode a chimeric product consisting of the N-terminal portion of a nonribosomal nucleolar phosphoprotein, nucleophosmin (NPM), from chromosome 5, fused to the kinase domain of a novel transmembrane tyrosine-specific protein kinase, anaplastic lymphoma kinase (ALK), from chromosome 2. To better define the spectrum of lymphomas that contain this translocation, we have analyzed 70 cases of non-Hodgkin's lymphoma (NHL) for expression of the t(2;5)-derived NPM/ALK chimeric message by reverse transcriptase-polymerase chain reaction (RT-PCR). Using a previously described set of oligonucleotide primers, NPM/ALK chimeric transcripts were detected in 21 of 22 cases that contained the t(2;5) by cytogenetic analysis and in 10 of 48 cases that either lacked evidence of the t(2;5) or had unsuccessful cytogenetics. In all but 1 case, the NPM/ALK PCR products were of identical size and sequence, suggesting that the genomic chromosome breaks are clustered in a single intron in both NPM and ALK. The NPM/ALK-expressing cases were not confined to NHLs with anaplastic morphology and included 15 ALCLs, 6 immunoblastic lymphomas, and 10 diffuse large-cell lymphomas. Moreover, only slightly greater than half of the cases with anaplastic morphology and 59% of CD30-expressing cases were NPM/ALK positive. Thus, neither anaplastic morphology nor the expression of CD30 accurately predicted the presence of this molecular genetic subtype of lymphoma.

Summary. The clinicopathological features and the prognostic significance of acute myeloid leukaemia (AML) with trisomy 11 are currently unknown. In this study we describe 15 adult AML cases with trisomy 11. Trisomy 11 was the sole chromosomal anomaly in eight cases; the remaining seven cases were characterized by +11 in association with other karyotypic aberrations. Patient ages ranged from 34 to 79 years. 12 patients were male; three were female. Although there was no correlation of trisomy 11 with any specific FAB subgroup [M2 (n=7). Ml (n=5), M4/5 (n=2), M3 (n=l)] less mature forms predominated. Immunologically, the leukaemic blasts showed a strikingly consistent stem cell phenotype with expression of HLA-DR, CD34 and the myeloid antigens (CD15, CD33 and/or CD13). In addition, two cases expressed the B-cell associated antigen CD19. The presence of trilineage dysplasia, suggesting the presence of an underlying myelodysplasia (MDS), was observed at presentation in five cases; in another case MDS was evident at relapse only. Unexpectedly, MLL gene rearrangements were observed in two of four cases characterized by trisomy 11 as the sole karyotypic abnormality; however, MLL aberrations were not identified in three cases with trisomy 11 accompanied by other karyotypic anomalies. The majority of patients in each subgroup (i.e. those with and without additional cytogenetic abnormalities) achieved a short first complete remission (CR) (mean 8 months) and failed to obtain a second CR. Only one patient in each trisomy 11 subgroup is in a continuous CR for >34 months. These findings suggest that trisomy 11 leukaemia is characterized by a stem/progenitor cell immunophenotype with poor response to standard chemo-therapeutic regimens and an unfavourable prognosis.

The t(1;19)(q23;p13) or its derivative encodes an E2A-PBX1 fusion transcript and protein that has been shown to have important prognostic and therapeutic implications in patients with acute lymphoblastic leukemia (ALL). We describe two childhood cases in which a der(22)t( 1;22)(q21-23;p13) cytogenetically mimicked a der( 19)t( 1; 19)(q23;p 13). In one case, which was phenotyped as early pre-B ALL with hyperdiploidy but lacked evidence of an E2A-PBX1 gene fusion by molecular study, the poor banding quality of chromosomes led to misinterpretation of the cytogenetic findings; a correct diagnosis was established only after analysis by the fluorescence in situ hybridization (FISH) method. The second case, which was classified as pseudodiploid pre-B ALL, had both a derivative 19 and a derivative 22 but lacked sufficient cells for evaluation of E2A-PBX1 gene fusion. This case was included in order to compare the der(19)t(1;19) and the der(22)t(1;22) and to pinpoint the difficulty in distinguishing these markers. FISH analysis can resolve diagnostic uncertainty in cases of ALL with equivocal chromosome 19 markers. © 1995 Wiley-Liss, Inc.