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The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.

Clonal hematopoiesis (CH) is an age-related condition predisposing to blood cancer and cardiovascular disease (CVD). Murine models demonstrate CH-mediated altered immune function and proinflammation. Low-grade inflammation has been implicated in the pathogenesis of osteoarthritis (OA), the main indication for total hip arthroplasty (THA). THA-derived hip bones serve as a major source of healthy hematopoietic cells in experimental hematology. We prospectively investigated frequency and clinical associations of CH in 200 patients without known hematologic disease who were undergoing THA. Prevalence of CH was 50%, including 77 patients with CH of indeterminate potential (CHIP, defined as somatic variant allele frequencies [VAFs] ≥2%), and 23 patients harboring CH with lower mutation burden (VAF, 1% to 2%). Most commonly mutated genes were DNMT3A (29.5%), TET2 (15.0%), and ASXL1 (3.5%). CHIP is significantly associated with lower hemoglobin, higher mean corpuscular volume, previous or present malignant disease, and CVD. Strikingly, we observed a previously unreported association of CHIP with autoimmune diseases (AIDs; multivariable adjusted odds ratio, 6.6; 95% confidence interval, 1.7-30; P = .0081). These findings underscore the association between CH and inflammatory diseases. Our results have considerable relevance for managing patients with OA and AIDs or mild anemia and question the use of hip bone–derived cells as healthy experimental controls.

Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1− B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5-year OS of 0% to 27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); 20.3% had intermediate-risk genotypes with 5-year OS of 33% to 45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like, and TP53 mutations in patients who were low-hypodiploid (54%) and BCL2/MYC-rearranged (33%) but were not independently associated with outcome. Of patients considered high risk based on presenting age and white blood cell count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like, and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non–risk-adapted approach on a single trial show the prognostic importance of genomic analyses, which may translate into future therapeutic benefits.

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+ B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid–restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid–restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid–restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid–restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.

Background: Randomised trials examining continuous lenalidomide maintenance after autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM) have demonstrated progression free survival (PFS) and overall survival benefit compared to placebo. In 2021 the UK National Institute of Clinical Excellence (NICE) approved lenalidomide maintenance for all newly diagnosed National Health Service (NHS) patients receiving ASCT after induction chemotherapy. Patient-reported outcomes from observational studies suggest lenalidomide causes tolerable adverse effects. However, clinical trial data demonstrate that neutropenia, fatigue, neuropathy, and gastrointestinal issues are common, particularly in the first six months of treatment, and that lenalidomide increases the risk of secondary malignancies.

Introduction:

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion and variable risk of progression to myeloid malignancy. DNMT3Ais the most commonly mutated gene and while single mutations do not raise the likelihood of progression, high variant allele frequencies (VAF) and presence of co-mutations do. In myeloid diseases, the bone marrow (BM) microenvironment exhibits alterations promoting malignant transformation. However, it is unknown whether similar alterations exist in the pre-malignant niche. We set out to analyze whether human mesenchymal stromal cells (MSC) from CHIP individuals with a DNMT3Amutation ( DNMT3A mut) are altered and whether they are able to remodel the HSPC compartment in healthy hematopoiesis.

Background

A natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4. Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.

Pathogenic gain-of-function variants in the genes encoding phosphoinositide 3-kinase δ (PI3Kδ) lead to accumulation of transitional B cells and senescent T cells, lymphadenopathy, and immune deficiency (activated PI3Kδ syndrome [APDS]). Knowing the genetic etiology of APDS afforded us the opportunity to explore PI3Kδ inhibition as a precision-medicine therapy. Here, we report in vitro and in vivo effects of inhibiting PI3Kδ in APDS. Treatment with leniolisib (CDZ173), a selective PI3Kδ inhibitor, caused dose-dependent suppression of PI3Kδ pathway hyperactivation (measured as phosphorylation of AKT/S6) in cell lines ectopically expressing APDS-causative p110δ variants and in T-cell blasts derived from patients. A clinical trial with 6 APDS patients was conducted as a 12-week, open-label, multisite, within-subject, dose-escalation study of oral leniolisib to assess safety, pharmacokinetics, and effects on lymphoproliferation and immune dysregulation. Oral leniolisib led to a dose-dependent reduction in PI3K/AKT pathway activity assessed ex vivo and improved immune dysregulation. We observed normalization of circulating transitional and naive B cells, reduction in PD-1+CD4+and senescent CD57+CD4−T cells, and decreases in elevated serum immunoglobulin M and inflammatory markers including interferon γ, tumor necrosis factor, CXCL13, and CXCL10 with leniolisib therapy. After 12 weeks of treatment, all patients showed amelioration of lymphoproliferation with lymph node sizes and spleen volumes reduced by 39% (mean; range, 26%-57%) and 40% (mean; range, 13%-65%), respectively. Thus, leniolisib was well tolerated and improved laboratory and clinical parameters in APDS, supporting the specific inhibition of PI3Kδ as a promising new targeted therapy in APDS and other diseases characterized by overactivation of the PI3Kδ pathway. This trial was registered at www.clinicaltrials.govas #NCT02435173.

The Philadelphia chromosome positive arm of the UKALLXII/ECOG2993 study for adult acute lymphoblastic leukemia (ALL) enrolled 266 patients between 1993 and 2003 (pre-imatinib cohort). In 2003 imatinib was introduced as a single-agent course following induction (N = 86, late imatinib). In 2005 imatinib was added to the second phase of induction (N = 89, early imatinib). The complete remission (CR) rate was 92% in the imatinib cohort vs 82% in the preimatinib cohort (P = .004). At 4 years, the overall survival (OS) of all patients in the imatinib cohort was 38% vs 22% in the preimatinib cohort (P = .003). The magnitude of the difference between the preimatinib and imatinib cohorts in event-free survival (EFS), OS, and relapse-free survival (RFS) seen in univariate analysis was even greater in the multivariate analysis. In the preimatinib cohort, 31% of those starting treatment achieved hematopoietic stem cell transplant (alloHSCT) compared with 46% in the imatinib cohort. A Cox multivariate analysis taking alloHSCT into account showed a modest additional benefit to imatinib (hazard ratio for EFS = 0.64, 95% confidence interval 0.44-0.93, P = .02), but no significant benefit for OS and RFS. Adding imatinib to standard therapy improves CR rate and long-term OS for adults with ALL. A proportion of the OS benefit derives from the fact that imatinib facilitates alloHSCT. This trial was registered at clinicaltrials.gov as NCT00002514.

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4–receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4–driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα–induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4–driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurkaand Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurkaby Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkbis regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

The biology and outcome of adult T-cell acute lymphoblastic leukemia are poorly understood. We present here the clinical and biologic features of 356 patients treated uniformly on the prospective trial (UKALL XII/ECOG 2993) with the aim of describing the outcome and identifying prognostic factors. Complete remission was obtained in 94% of patients, and 48% survived 5 years. Positivity of blasts for CD1a and lack of expression of CD13 were associated with better survival (P = .01 and < .001, respectively). NOTCH1 and CDKN2A mutations were seen in 61% and 42% of those tested. Complex cytogenetic abnormalities were associated with poorer survival (19% vs 51% at 5 years, P = .006). Central nervous system involvement at diagnosis did not affect survival (47% vs 48%, P = not significant). For 99 patients randomized between autograft and chemotherapy, 5-year survival was 51% in each arm. Patients with a matched sibling donor had superior 5-year survival to those without donors (61% vs 46%, χ2, P = .02); this was the result of less relapse (25% vs 51% at 5 years, P < .001). Only 8 of 123 relapsed patients survive. This study provides a baseline for trials of new drugs, such as nelarabine, and may allow risk-adapted therapy in patients with poor-prognosis T-cell ALL.

RUNX1 (AML1) encodes the core binding factor α subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the δ catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)–kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

Prospective data on the value of allogeneic hematopoietic stem cell transplantation (alloHSCT) in Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) are limited. The UKALLXII/ECOG 2993 study evaluated the outcome of assigning alloHSCT with a sibling (sib) or matched unrelated donor (MUD) to patients younger than 55 years of age achieving complete remission (CR). The CR rate of 267 patients, median age 40, was 82%. Twenty-eight percent of patients proceeded to alloHSCT in first CR. Age older than 55 years or a pre-HSCT event were the most common reasons for failure to progress to alloHSCT. At 5 years, overall survival (OS) was 44% after sib alloHSCT, 36% after MUD alloHSCT, and 19% after chemotherapy. After adjustment for sex, age, and white blood count and excluding chemotherapy-treated patients who relapsed or died before the median time to alloHSCT, only relapse-free survival remained significantly superior in the alloHSCT group (odds ratio 0.31, 95% confidence interval 0.16-0.61). An intention-to-treat analysis, using the availability or not of a matched sibling donor, showed 5-year OS to be nonsignificantly better at 34% with a donor versus 25% with no donor. This prospective trial in adult Ph+ALL indicates a modest but significant benefit to alloHSCT. This trial has been registered with clinicaltrials.gov under identifier NCT00002514 and as ISRCTN77346223.

Activating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F− patients and a single V617F+ patient. Patients carrying the W515K allele had a significantly higher allele burden than did those with the W515L allele, suggesting a functional difference between the 2 variants. Compared with V617F+ ET patients, those with MPL mutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth, and reduced bone marrow erythroid and overall cellularity. Compared with V617F− patients, those with MPL mutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinicopathologic subgroup. MPL mutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

Activating mutations of MPLexon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPLmutations outside exon 10 are uncommon in platelet cDNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPLmutations were identified in 8.5% of JAK2 V617F−patients and a single V617F+patient. Patients carrying the W515Kallele had a significantly higher allele burden than did those with the W515Lallele, suggesting a functional difference between the 2 variants. Compared with V617F+ET patients, those with MPLmutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth, and reduced bone marrow erythroid and overall cellularity. Compared with V617F−patients, those with MPLmutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinicopathologic subgroup. MPLmutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

An international collaboration was set up to prospectively evaluate the role of allogeneic transplantation for adults with acute lymphoblastic leukemia (ALL) and compare autologous transplantation with standard chemotherapy. Patients received 2 phases of induction and, if in remission, were assigned to allogeneic transplantation if they had a compatible sibling donor. Other patients were randomized to chemotherapy for 2.5 years versus an autologous transplantation. A donor versus no-donor analysis showed that Philadelphia chromosome–negative patients with a donor had a 5-year improved overall survival (OS), 53% versus 45% (P = .01), and the relapse rate was significantly lower (P = .001). The survival difference was significant in standard-risk patients, but not in high-risk patients with a high nonrelapse mortality rate in the high-risk donor group. Patients randomized to chemotherapy had a higher 5-year OS (46%) than those randomized to autologous transplantation (37%; P = .03). Matched related allogeneic transplantations for ALL in first complete remission provide the most potent antileukemic therapy and considerable survival benefit for standard-risk patients. However, the transplantation-related mortality for high-risk older patients was unacceptably high and abrogated the reduction in relapse risk. There is no evidence that a single autologous transplantation can replace consolidation/maintenance in any risk group. This study is registered at http://clinicaltrials.gov as NCT00002514.

The role of histopathology in the diagnosis of essential thrombocythemia (ET) is controversial, and there has been little attempt to quantitate interobserver variability. Diagnostic bone marrow trephine biopsy specimens from 370 patients with ET by Polycythemia Vera Study Group (PVSG) criteria were assessed by 3 experienced hematopathologists for 16 different morphologic features and overall diagnosis according to the World Health Organization (WHO) classification. Our results show substantial interobserver variability, particularly for overall diagnosis and individual cellular characteristics such as megakaryocyte morphology. Reticulin grade was the dominant independent predictor of WHO diagnostic category for all 3 hematopathologists. Factor analysis identified 3 independent factors likely to reflect underlying biologic processes. One factor related to overall and lineage-specific cellularity and was significantly associated with JAK2 V617F status (P < .001), a second factor related to megakaryocyte clustering, and a third was associated with the fibrotic process. No differences could be discerned between patients labeled as having “prefibrotic myelofibrosis” or “true ET” in clinical and laboratory features at presentation, JAK2 status, survival, thrombosis, major hemorrhage, or myelofibrotic transformation. These results show that histologic criteria described in the WHO classification are difficult to apply reproducibly and question the validity of distinguishing true ET from prefibrotic myelofibrosis on the basis of subjective morphologic criteria. This study was registered at http://isrctn.org as #72251782 and at http://eudract.emea.europa.eu/ as #2004-000245-38.

Pretreatment cytogenetics is a known predictor of outcome in hematologic malignancies. However, its usefulness in adult acute lymphoblastic leukemia (ALL) is generally limited to the presence of the Philadelphia (Ph) chromosome because of the low incidence of other recurrent abnormalities. We present centrally reviewed cytogenetic data from 1522 adult patients enrolled on the Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 trial. The incidence and clinical associations for more than 20 specific chromosomal abnormalities are presented. Patients with a Ph chromosome, t(4;11)(q21;q23), t(8;14)(q24.1;q32), complex karyotype (5 or more chromosomal abnormalities), or low hypodiploidy/near triploidy (Ho-Tr) all had inferior rates of event-free and overall survival when compared with other patients. In contrast, patients with high hyperdiploidy or a del(9p) had a significantly improved outcome. Multivariate analysis demonstrated that the prognostic relevance of t(8;14), complex karyotype, and Ho-Tr was independent of sex, age, white cell count, and T-cell status among Ph-negative patients. The observation that Ho-Tr and, for the first time, karyotype complexity confer an increased risk of treatment failure demonstrates that cytogenetic subgroups other than the Ph chromosome can and should be used to risk stratify adults with ALL in future trials.

Most adults with acute lymphoblastic leukemia (ALL) who achieve complete remission (CR) will relapse. We examined the outcome of 609 adults with recurring ALL, all of whom were previously treated on the Medical Research Council (MRC) UKALL12/ECOG2993 study, where the overall survival (OS) of newly diagnosed patients is 38% (95% confidence interval [CI] = 36%-41%) at 5 years. By contrast, OS at 5 years after relapse was 7% (95% CI = 4%-9%). Factors predicting a good outcome after salvage therapy were young age (OS of 12% in patients younger than 20 years vs OS of 3% in patients older than 50 years; 2P < .001) and short duration of first remission (CR1) (OS of 11% in those with a CR1 of more than 2 years versus OS of 5% in those with a CR1 of less than 2 years; 2P < .001). Treatment received in CR1 did not influence outcome after relapse. In a very highly selected subgroup of patients who were able to receive HSCT after relapse, some were long-term survivors. We conclude from a large, unselected series with mature follow-up that most adults with recurring ALL, whatever their prior treatment, cannot be rescued using currently available therapies. Prevention of recurrence is the best strategy for long-term survival in this disease.

Background:

Outcome of acute lymphoblastic leukemia (ALL) in adults with central nervous system (CNS) disease at diagnosis is unclear. We treated 1508 de novo ALL patients with 2-phase induction and then high-dose methotrexate with l-asparaginase. Patients up to 50 years old in first remission (CR1) with a matched related donor (MRD) underwent an allogeneic stem cell transplantation (SCT); the remainder in CR1 were randomized to an autologous SCT or intensive consolidation followed by maintenance chemotherapy. Philadelphia chromosome (Ph)–positive patients were offered a matched unrelated donor (MUD) allogeneic SCT. Seventy-seven of 1508 (5%) patients a median age of 29 years had CNS leukemia at presentation; 13 of the 77 (17%) had Ph-positive ALL. Sixty-nine of 77 (90%) patients attained CR1. Thirty-six patients underwent transplantation in CR1 (25 MRD, 5 MUD, and 6 autografts). Eleven of 25 patients with MRD transplantation remain alive at 21 to 102 months, 2 of 5 with MUD at 42 and 71 months, and 1 of 6 with autologous SCT at 35 months. Seven of 27 treated with consolidation/maintenance remain in CR1 56 to 137 months after diagnosis. Overall survival at 5 years was 29% in those with CNS involvement at diagnosis versus 38% (P = .03) for those without. CNS leukemia in adult ALL is uncommon at diagnosis. Adult Ph-negative ALL patients, however, can attain long-term disease-free survival using SCT as well as conventional chemotherapy.

Children with Down syndrome (DS) with acute megakaryocytic leukemia (AMkL) have very high survival rates compared with non-DS AMkL patients. Somatic mutations identified in the X-linked transcription factor gene, GATA1, in essentially all DS AMkL cases result in the synthesis of a shorter (40 kDa) protein (GATA1s) with altered transactivation activity and may lead to altered expression of GATA1 target genes. Using the Affymetrix U133A microarray chip, we identified 551 differentially expressed genes between DS and non-DS AMkL samples. Transcripts for the bone marrow stromal-cell antigen 2 (BST2) gene, encoding a transmembrane glycoprotein potentially involved in interactions between leukemia cells and bone marrow stromal cells, were 7.3-fold higher (validated by real-time polymerase chain reaction) in the non-DS compared with the DS group. Additional studies confirmed GATA1 protein binding and transactivation of the BST2 promoter; however, stimulation of BST2 promoter activity by GATA1s was substantially reduced compared with the full-length GATA1. CMK sublines, transfected with the BST2cDNA and incubated with HS-5 bone marrow stromal cells, exhibited up to 1.7-fold reduced cytosine arabinoside (ara-C)-induced apoptosis, compared with mock-transfected cells. Our results demonstrate that genes that account for differences in survival between DS and non-DS AMkL cases may be identified by microarray analysis and that differential gene expression may reflect relative transactivation capacities of the GATA1s and full-length GATA1 proteins.

The international acute lymphoblastic leukemia (ALL) study was designed to prospectively define the optimal therapy for adults 60 years of age or younger with newly diagnosed ALL. All patients received identical induction therapy, and 91% achieved complete remission (CR). Patients 50 years of age or younger with a compatible sibling were assigned to undergo allogeneic transplantation; the others were randomly assigned to autologous transplantation or to consolidation/maintenance therapy for 2.5 years. Patients who did not achieve CR after induction had an overall survival rate of 5% compared with 45% for patients who achieved CR. Factors at diagnosis predictive of overall survival and disease-free survival were age (P= .001), white blood cell count less than 30 × 109/L for B lineage or less than 100 × 109/L for T lineage (P= .001) and immunophenotype, T lineage versus B lineage (P= .001). The data demonstrate that achieving CR with induction therapy is indispensable for long-term survival in adult patients with ALL. Furthermore, with a response rate greater than 90%, the induction regimen was highly efficacious as remission-inducing therapy. This large database has validated several previously identified independent prognostic factors in ALL, such as age, white blood cell count at presentation, cytogenetics, and immunophenotype. However, the achievement of CR within 4 weeks does not appear to be an independent prognostic factor.

Immunotherapeutic approaches to limit cytomegalovirus (CMV) morbidity and mortality after hematopoietic stem cell transplants (HSCTs) are currently under investigation as alternatives to antiviral drugs. In this context, we have inserted full-length and ubiquitin-modified CMV phosphoprotein 65 (pp65), phosphoprotein 150 (pp150), and immediate early protein 1 (IE1) immunodominant antigens into the virulent Western Reserve strain of vaccinia virus (VV) and the highly attenuated strain, modified vaccinia Ankara (MVA). Recombinant (r) VV or rMVA stimulated vigorous expansion of CMV-specific CD8+ T cells in CMV-positive donor peripheral blood mononuclear cells (PBMCs), which showed minimal alloreactivity and high levels of HLA tetramer binding, cytokine production, and cytotoxicity. Ubiquitinated antigens had a profound effect when expressed in VV. Single antigen rMVA expressing pp65 or IE1, either ubiquitin-modified or native, stimulated both cytotoxic T lymphocyte (CTL) populations to be expanded up to 500-fold in a 60-mL blood draw from the same donor. This result demonstrates the clinical feasibility of simultaneously amplifying multiple CMV-CTL populations. Transgenic HLA A2.1 (HHD II) mice, immunized with the same rMVA as used with human PBMCs, produced a robust cytotoxic response to both CMV pp65 and IE1. The specificity of the vigorous immunologic response to rMVA, both in vitro and in vivo, makes them candidates for clinical evaluation in the context of adoptive immunotherapy for hematopoietic stem cell transplant (HSCT) recipients or donor vaccination.

The optimal dose of interferon-alfa (IFN) for chronic myeloid leukemia (CML) is unknown. Retrospective analyses suggest that low doses are as effective as high doses, with less toxicity and fewer patients abandoning the drug. The Dutch Hemato-Oncology Association (HOVON) and British Medical Research Council (MRC) cooperative groups jointly performed randomized trials in newly diagnosed CML patients, comparing high-dose IFN (5 MIU/m2 daily) with low-dose (3 MIU, 5 times a week). Both arms allowed additional hydroxyurea to keep the white blood cell count lower than 5 × 109/L. Quality of life data were collected in a subset of patients. Between 1993 and 2001, 407 patients were randomized. At a median follow-up of 53 months, there were no significant differences in overall survival (odds ratio = 1.09, 95% confidence interval, 0.81-1.46), progression-free survival, and complete hematologic or major cytogenetic responses. Fewer patients in the low-dose group abandoned IFN for reasons other than transplant or progressive disease (P = .002, 58% vs 72% at 5 years). Quality of life data showed comparable results in both arms for most factors. There is no evidence of benefit for high-dose IFN compared with low-dose for the treatment of CML. Therefore, when IFN is combined with other drugs, low-dose IFN is advised, to minimize toxicity and costs. (Blood. 2004;103:4408-4415)

The conditioning regimen prior to stem cell transplantation in 36 patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) was intensified by treating patients with a rhenium 188–labeled anti-CD66 monoclonal antibody. Dosimetry was performed prior to therapy, and a favorable dosimetry was observed in all cases. Radioimmunotherapy with the labeled antibody provided a mean of 15.3 Gy of additional radiation to the marrow; the kidney was the normal organ receiving the highest dose of supplemental radiation (mean 7.4 Gy). Radioimmunotherapy was followed by standard full-dose conditioning with total body irradiation (12 Gy) or busulfan and high-dose cyclophosphamide with or without thiotepa. Patients subsequently received a T-cell–depleted allogeneic graft from a HLA-identical family donor (n = 15) or an alternative donor (n = 17). In 4 patients without an allogeneic donor, an unmanipulated autologous graft was used. Infusion-related toxicity due to the labeled antibody was minimal, and no increase in treatment-related mortality due to the radioimmunoconjugate was observed. Day +30 and day +100 mortalities were 3% and 6%, respectively, and after a median follow-up of 18 months treatment-related mortality was 22%. Late renal toxicity was observed in 17% of patients. The relapse rate of 15 patients undergoing transplantation in first CR (complete remission) or second CR was 20%; 21 patients not in remission at the time of transplantation had a 30% relapse rate.

Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT390) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 µg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.

Three distinct classes of epitopes on human CD164 have been identified. Two of these, recognized by the monoclonal antibodies 105A5 and 103B2/9E10, are the CD164 class I and class II functionally defined epitopes, which cooperate to regulate adhesion and proliferation of CD34+ cell subsets. In this article, we demonstrate that these 2 CD164 epitopes are expressed on CD34+ cells throughout ontogeny, in particular on CD34+ cell clusters associated with the ventral floor of the dorsal aorta in the developing embryo and on CD34+ hematopoietic precursor cells in fetal liver, cord blood, and adult bone marrow. While higher levels of expression of these CD164 epitopes occur on the more primitive AC133hiCD34hiCD38lo/- cell population, they also occur on most cord blood Lin-CD34lo/-CD38lo/- cells, which are potential precursors for the AC133hiCD34hiCD38lo/- subset. In direct contrast to these common patterns of expression on hematopoietic precursor cells, notable differences in expression of the CD164 epitopes were observed in postnatal lymphoid and nonhematopoietic tissues, with the class I and class II CD164 epitopes generally exhibiting differential and often reciprocal cellular distribution patterns. This is particularly striking in the colon, where infiltrating lymphoid cells are CD164 class I–positive but class II–negative, while epithelia are weakly CD164 class II–positive. Similarly, in certain lymphoid tissues, high endothelial venules and basal and subcapsular epithelia are CD164 class II–positive, while lymphoid cells are CD164 class I–positive. It therefore seems highly likely that these CD164 class I and II epitopes will mediate reciprocal homing functions in these tissue types.

Background:

The high event-free survival rates of Down syndrome (DS) children with acute myeloid leukemia (AML) are due, in part, to increased in vitro sensitivity of DS myeloblasts to cytosine arabinoside (ara-C) and daunorubicin and the greater generation of ara-C triphosphate (ara-CTP) from ara-C compared with myeloblasts from non-DS patients (Taub et al,Blood 87:3395, 1996). This study further explores the molecular basis of chemotherapy sensitivity of DS AML patients by examining the expression of chromosome 21-localized genes in myeloblasts from newly diagnosed AML patients. Transcript levels of two chromosome 21-localized genes, cystathionine-ß-synthase (CBS) and superoxide dismutase (SOD), measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were 12.0- and 3.8-fold higher in DS compared with non-DS myeloblasts (P < .0001 and P < .0001, respectively). Conversely, there were no significant increases in transcripts for 2 other chromosome 21-localized genes, carbonyl reductase and the reduced folate carrier. CBS transcript levels correlated with both in vitro ara-C sensitivity measured by the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT) assay (P = .003) and the generation of 3H-ara-C triphosphate (ara-CTP) after in vitro incubations with 5 µmol/L3H-ara-C (P = .0003). Transcripts of deoxycytidine kinase were 2.6-fold higher in DS compared with non-DS cells and may be a factor in the enhanced metabolism of ara-C in DS cells. There was no significant correlation of SOD transcripts with in vitro ara-C and daunorubicin sensitivities. Increased CBS transcripts could result in elevated CBS activity, which modulates ara-C metabolism by altering reduced folate pools, deoxycytidine triphosphate pools, S-adenosylmethionine levels, and/or methylation of the deoxycytidine kinase gene. The further identification of the molecular mechanisms of chemotherapy sensitivity of DS AML patients may lead to significant improvements in the treatment and cure of AML.

Phenotypic analysis of hematopoietic stem and progenitor cells (HSCs) has been an invaluable tool in defining the biology of stem cell populations. We have recently described the production of AC133, a monoclonal antibody (MoAb) that binds to a novel cell surface antigen present on a CD34bright subset of human HSCs. This antigen is a glycosylated protein with a molecular weight of 120 kD. Here, we report the molecular cloning of a cDNA encoding this antigen and show that it does not share homology with any previously described hematopoietic or other cell surface antigen(s). The AC133 polypeptide has a predicted size of 97 kD and contains five-transmembrane (5-TM) domains with an extracellular N-terminus and a cytoplasmic C-terminus. Whereas the expression of tetraspan (4-TM) and 7-TM molecules is well documented on mature and immature hematopoietic cells and leukocytes, this 5-TM type of structure containing two large (255–amino acid [aa] and 290-aa) extracellular loops is unique and does not share sequence homology with any known multi-TM family members. Expression of this protein appears limited to bone marrow in normal tissue by immunohistochemical staining; however, Northern analysis suggests that the mRNA transcript is present in a variety of tissues such as the kidney, pancreas, placenta, and fetal liver. The AC133 antigen is also expressed on subsets of CD34+ leukemias, suggesting that it may be an important early marker for HSCs, as well as the first described member of a new class of TM receptors.

AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133- population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

By immunization with nuclear lysates of L428 cells, we raised a monoclonal mouse antibody, Ki-S2 (IgG1 ). In Western blots, this antibody recognizes a nuclear antigen with an apparent molecular mass of 100 kD, termed p100. Protein sequencing of p100 showed that this is a hitherto unknown protein. Immunohistochemical examination of cryostat and paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the nuclei of a fraction of proliferating cells. Cell sorting and fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition G1/S until the end of cytokinesis. During mitosis, this protein is strictly associated with the spindle pole and with the mitotic spindle, whereas during S and G2 , p100 is diffusely distributed throughout the cell nucleus. Immediately after completion of cytokinesis, p100 was rapidly degraded. In L428 cells, p100 is phosphorylated at least during mitosis. It has a turnover time of about 1 hour. Studies on routinely processed paraffin sections of specimens of malignant lymphoma, benign and malignant nevocellular tumors, and breast cancer showed that in all cases less than 40% of the Ki-67–positive growth fraction expressed p100. Thus, p100 might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer prognosis, beyond its general biologic relevance as a cell cycle protein.

The relationships between dihydrofolate reductase (DHFR) levels or methotrexate membrane transport and acute lymphoblastic leukemia (ALL) immunophenotype were evaluated on 51 T-cell and 44 B-precursor ALL specimens from 90 pediatric ALL patients at diagnosis and relapse, using a fluorescent methotrexate analog (PT430) and flow cytometry assay (Matherly et al, Blood 85:500, 1995). For T-cell ALL, 35 of 45 (78%) of newly diagnosed patients' specimens exhibited elevated DHFR relative to DHFR levels in ALL blasts from methotrexate-responsive patients. For 30 of 45 diagnostic T-ALL specimens, DHFR expression was heterogeneous, with up to 3 separate subpopulations covering a 44-fold range; the DHFR-overproducing fractions comprised 10% to 88% of the total blasts. Elevated DHFR was less common in B-precursor ALL at diagnosis, being detected in only 17 of 36 specimens (47%); 11 of these samples exhibited DHFR heterogeneity. Median maximal DHFR levels were fourfold higher in T-cell than B-precursor ALL at diagnosis. Within a particular phenotypic group, there were no correlations between DHFR levels and patient prognostic features, including age, sex, chromosomal abnormalities, white blood cell counts (WBCs), and percentage of S-phase. However, for B-precursor ALL, there was a notably higher fraction of African-American than white patients with elevated DHFR. For patients (both phenotypes) with low WBCs (<50,000/µL), event-free survival times were significantly shorter for those expressing DHFR above a threshold level than for patients expressing DHFR below this level (P < .016); this relationship was not seen for patients with high WBCs (<50,000/µL). Elevated DHFR was detected in 11 of 14 relapse specimens (5 of 6 T-cell and 6 of 8 B-precursor). Two of five paired relapse specimens (both T-cell) from patients treated with methotrexate exhibited increased DHFR levels over those at diagnosis (2.5- to 5-fold); the fraction of DHFR-overproducing blasts was also increased in 4 of 5 paired relapse specimens (2 B-precursor and 2 T-cell). In contrast to the variations in DHFR, highly impaired methotrexate transport was detected in only 6 of 95 ALL specimens, including both diagnosis and relapse. There was no correlation between phenotype and impaired transport. These data provide further rationale for the use of mechanistically based prognostic factors to complement known biologic or disease-based prognostic indicators in the design of ALL therapy including methotrexate, particularly with patients presenting with low WBCs. The finding of a markedly increased frequency of DHFR overexpression in T-cell over B-precursor ALL suggests that this difference is associated with the poorer prognosis of patients with T-cell ALL treated with standard-dose antimetabolite therapy and implies that higher-dose methotrexate (=1 g/m2) during consolidation therapy may be useful in the treatment of this disease.

Down syndrome (DS) children with acute myeloid leukemia (AML) have significantly higher event-free survival (EFS) rates compared with non- DS children when treated with protocols containing 1-beta-D- arabinofuranosylcytosine (ara-C). Sensitivity and metabolism of ara-C was examined in myeloblasts from DS and non-DS patients with AML, DS infants with the transient myeloproliferative disorder, and Epstein- Barr Virus (EBV) transformed lymphoblastoid cell lines with and without trisomy 21. DS myeloblasts were approximately 10-fold more sensitive to ara-C (measured by the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) colorimetric sensitivity assay), compared with non-DS myeloblasts, following exposure to ara-C for 72 hours. Mean levels of l-beta-D-arabinofuranosylcytosine 5′-triphosphate (ara-CTP) were significantly higher in DS myeloblasts compared with non-DS myeloblasts after incubation with 5 micromol/L ara-C (621.4 v 228.4 pmol/mg protein). DS cell lines also generated higher levels of ara-CTP compared with cell lines with diploid chromosome numbers (66.5 v 13.6 pmol/mg protein and 137.6 v 41.7 pmol/mg protein at 1 and 5 micromol/L ara-C, respectively). Elevated ara-CTP levels in the DS cells were accompanied by slightly lower levels of endogenous deoxycytidine triphosphate (dCTP) pools, slightly greater extent of ara-C incorporation into DNA, and increased relative numbers of double strand DNA strand breaks. There were no significant differences in the cell cycle distributions of DS and non-DS cells. These in vitro studies support our hypothesis that enhanced metabolism of ara-C in DS cells may be a contributing factor to the superior survival rate of DS children with AML and is possibly based on a gene dosage effect of genes localized to chromosome 21 including cystathionine-beta-synthase. Further study of the mechanisms (ie, alterations in dCTP pools and DNA methylation) involved may lead to improvements in the treatment of all AML patients.

Human monocyte/macrophage serine esterase (HMSE), commonly known as acid esterase or α-naphthylacetate esterase, comprises a group of five enzyme variants that can be distinguished by their isoelectric points from esterase variants of the other normal human blood cell populations. A cDNA for one of the monocytic enzyme variants (HMSE1) was cloned from a U-937 λgt11 cDNA library by screening with an oligonucleotide mixture designed according to amino acid sequence data of the purified enzyme. The cDNA contains 1,727 bp with an open reading frame of 1,512 bp coding for a protein of 503 amino acid residues. HMSE1 cDNA represents the first cloned monocyte/macrophage-specific serine esterase and its sequence shows up to 77% homology to other known serine esterases of different species. The amino acid composition of the putative active site of HMSE1 as deduced from the nucleotide sequence corresponds with the active sites of other serine esterases but not with the active sites of serine proteases. Hybridization of the cDNA with RNA of separated normal blood cell populations and hematopoietic cell lines shows restricted expression within the monocyte/macrophage lineage.

Recombinant tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces proliferation of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms involved in regulating TNF responsiveness, we have examined TNF receptor expression on neoplastic B-CLL cells. We have demonstrated that freshly isolated neoplastic B cells from patients with CLL did not express TNF receptors. After 1 day of incubation in culture medium, TNF receptors were detectable in the range of 540 to 1,500/cell. Kinetic experiments revealed that receptor expression was half-maximal after 3 hours of culturing and required de novo protein synthesis. The Scatchard plots of TNF-alpha binding indicated a single set of high- affinity TNF receptors with a dissociation constant of 70 pmol/L. TNF receptor expression in vitro was found in all examined cases. All cytokines tested, with the exception of IL-2, did not influence the expression of TNF receptors. The TNF receptor expression is enhanced in B-CLL cells cultured in the presence of interleukin-2 when compared with the receptor expression of cells cultured in medium alone. Our data suggest that neoplastic B-CLL cells in patients with stable disease do not express TNF receptors in vivo and that an unknown mechanism suppressing TNF receptor expression in vivo may play a role in growth regulation of neoplastic B cells.

The biologic effects of recombinant tumor necrosis factor-alpha (rTNF- alpha) and the expression of specific TNF membrane receptors on isolated neoplastic B cells from previously untreated patients with chronic lymphocytic leukemia (CLL) were investigated in vitro. Isolated B cells were incubated up to six days with various concentrations of rTNF-alpha (0.1 to 100 ng/mL). B cells from most patients proliferated ranged from two to 104 times that of unstimulated cells from the same patients. An optimal proliferative effect was achieved at 25 ng/mL rTNF- alpha and an incubation time between 96 and 120 hours, whereas a low concentration of rTNF-alpha (1 ng/mL) reduced [3H]TdR incorporation in four cases. Metaphase cells were detected in the rTNF-alpha-stimulated cultures that proliferated in response to rTNF-alpha. B cells from three of ten patients proliferated spontaneously and proliferation was further enhanced in two patients by rTNF-alpha. TNF binding assays gave a value of approximately 390 to 1,400 binding sites/cell for TNF and a dissociation constant (kd) of approximately 60 pmol/L. These data indicate that rTNF-alpha, in contrast to its cytotoxic/cytostatic effects, can also induce proliferation of tumor cells.

Down syndrome (DS) children with acute myeloid leukemia (AMD have significantly higher event-free survival (EFS) rates compared with non-DS children when treated with protocols containing 1-β-D-arabinofuranosylcytosine IsraC). Sensitivity and metabolism of ara-C was examined in myeloblasts from DS and non-DS patients with AML, DS infants with the transient myeloproliferative disorder, and Epstein-Barr Virus (EBV) transformed lymphoblastoid cell lines with and without trisomy 21. DS myeloblasts were approximately 10-fold more sensitive to ara-C (measured by the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) colorimetric sensitivity assay), compared with non-DS myeloblasts, following exposure to ara-C for 72 hours. Mean levels of 1-β-D-arabinofuranosylcytosine 5’-triphosphate (ara-CTP) were significantly higher in DS myeloblasts compared with non-DS myeloblasts after incubation with 5 μmol/L ara-C (621.4 v228.4 pmol/mg protein). DS cell lines also generated higher levels of ara-CTP compared with cell lines with diploid chromosome numbers (66.5 v 13.6 pmol/mg protein and 137.6 v41.7 pmol/mg protein at 1 and 5 μmol/L ara-C, respectively). Elevated ara-CTP levels in the DS cells were accompanied by slightly lower levels of endogenous deoxycytidine triphosphate (dCTP) pools, slightly greater extent of ara-C incorporation into DNA, and increased relative numbers of double strand DNA strand breaks. There were no significant differences in the cell cycle distributions of DS and non-DS cells. These in vitro studies support our hypothesis that enhanced metabolism of ara-C in DS cells may be a contributing factor to the superior survival rate of DS children with AML and is possibly based on a gene dosage effect of genes localized to chromosome 21 including cystathionine-β-syntnase. Further study of the mechanisms (ie, alterations in dCTP pools and DNA methylation) involved may lead to improvements in the treatment of all AML patients.

A retrospective study of clinical resistance to methotrexate (MTX) was performed on 29 archival specimens of frozen lymphoblasts obtained from children with acute lymphoblastic leukemia (ALL), including 19 at initial presentation and 10 at first relapse. Blasts were assayed for dihydrofolate reductase and MTX transport by flow cytometry using the fluorescent methotrexate analog, PT430 (Rosowsky et al, J Biol Chem 257:14162, 1982). In contrast to tissue culture cells, patient blasts were often heterogeneous for dihydrofolate reductase content. Of the 19 specimens at initial diagnosis, 7 exhibited dual blast populations, characterized by threefold to 10-fold differences in relative dihydrofolate reductase; the dihydrofolate reductase-overproducing populations comprised 12% to 68% of the total blasts for these specimens. Remission duration intervals for patients exhibiting dual blast populations were notably shorter than for patients expressing a single blast population with lower dihydrofolate reductase ( < or = 9 months v > or = 15 months, respectively), a difference that was statistically significant (P = .045). There was no apparent correlation between expression of increased dihydrofolate reductase at diagnosis and known patient and disease prognostic features (immunophenotype, age, sex, and white blood count). For the relapsed patients, 4 of 10 exhibited dual lymphoblast populations with elevated dihydrofolate reductase. The majority of the patient lymphoblast specimens were entirely competent for MTX transport and, likewise, expressed immunoreactive reduced folate carriers by indirect immunofluorescence staining with specific antiserum to the transporter. Three patients (2 at relapse and 1 at diagnosis) exhibited heterogeneous expression of imparied MTX transport (14% to 73% of blasts). In only 1 of these patients did the majority of the lymphoblasts (73%) show impaired MTX transport and for this specimen, immunoreactive carrier proteins were virtually undetectable. These results suggest that heterogeneous expression of elevated dihydrofolate reductase and impaired MTX transport are important modes of resistance in childhood ALL patients undergoing chemotherapy with MTX and that these parameters may serve as predictive indices of clinical response to MTX.