Bone Marrow Failure States Research Articles

Erythropoiesis-stimulating agents in the treatment of anaemia in cancer patients: ESMO Clinical Practice Guidelines for use

Erythropoiesis-stimulating agents in the treatment of anaemia in cancer patients: ESMO Clinical Practice Guidelines for use

Pretransplant Conditioning with Campath-1H (Alemtuzumab) in Pediatric Matched Unrelated Bone Marrow Transplants - An Institutional Experience.

Abstract 4651 IntroductionGraft versus host disease remains a major cause of mortality and morbidity following matched unrelated hematopoietic stem cell transplantation. Campath-1H (Alemtuzumab) is a humanized monoclonal antibody to CD52, an antigen expressed on T and B lymphocytes, monocytes and natural killer cells and is thought to reduce GVHD incidence through in vivo T cell depletion. Through the same mechanism it can potentially increase the risk of relapse by reducing the graft vs. leukemia effect and also possibly increase the risk of infection due to delayed immune recovery. This study looks at our experience with Campath-1H substituted in place of the conventional anti-thymocyte globulin (ATG) in our transplant conditioning regimen. Patients and MethodsThis retrospective case study included 17 pediatric (9 male; 8 female) matched unrelated bone marrow transplants done in our institution between January 2003 and June 2009 with Campath-1H as part of the pretransplant conditioning regimen. The primary transplant indication was leukemia/lymphoma (n=9), MDS/ MDS evolving into AML (n= 3), severe aplastic anemia (n=4) and Fanconi anemia (n=1). The conditioning regimen included Campath-1H given with cytoxan/ total body irradiation (TBI) in 14 patients, fludarabine/ TBI in 2 patients, and melphalan/ fludarabine in 1 patient. Campath-1H dosing was body weight based: 3mg (if between 5-15kg), 5 mg (if between 16-30kg) and 10 mg (if >30 kg) and 3 doses were administered when underlying condition was a malignancy and 4 doses when it was a bone marrow failure state. The last dose was given at least 24 hours prior to the bone marrow/ peripheral stem cell infusion. GVHD prophylaxis was with tacrolimus/methotrexate (n=12), tacrolimus (n=4) and cyclosporine/methotrexate (n=1). Standard institutional infectious prophylaxis was followed. ResultsThe median age at transplant was 12.2 years (range; 0.7-19.7 years). All but one patient engrafted with a median of 21 days (range; 14-25 days). 5 out of 17 developed Grade I-II acute GVHD which resolved with steroids. No patient developed chronic GVHD. One patient had a CMV reactivation but no patient developed active CMV disease. 4 patients had varicella one of whom died of disseminated infection (day +376). The same patient also had adeno viral infection and BK viremia. 1 patient developed PCP pneumonia and retinal toxoplasmosis. 5 patients (6 transplants) relapsed (range; 40-641 days) with 3 relapsing within +100 days. 1 patient developed PTLD which was successfully treated with rituximab. Of the 4 patient deaths 3 were due to relapse and one due to disseminated varicella infection. The median follow-up time was 719 days (range; 147-2175 days). Overall survival as calculated using the Kaplan-Meir analysis was 100 % at 100 days and 94% at 1 year. Event free survival censoring for death, relapse and rejection was 76% at 100 days and 64% at 1 year. ConclusionsBased on our experience, Campath-1H used as part of pretransplant conditioning regimen in pediatric matched unrelated transplants seems to reduce the risk of serious GVHD. This is in concordance with other published literature. T cell depletion is considered to increase the risk of life threatening infections. Our study had one infection related death. There were no patients with active CMV disease. This may in part be attributed to strict prophylactic measures and increased surveillance. Longer duration of follow-up is required to adequately analyze the relapse rates. Also, given our small patient numbers the effect of primary disease state and stage on relapse could not be assessed. Larger studies in the pediatric population with longer duration of follow-up comparing Campath-1H with conventional regimens are required to further assess its role with regards to graft vs. leukemia effect and also to establish if the decreased incidence of GVHD is sustained in larger cohorts. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Histology in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Abstract 4215 IntroductionParoxysmal Nocturnal Hemoglobinuria (PNH) is a disease of the hematopoietic stem cell (HSC) resulting in a clone of hematopoietic cells deficient in glycosyl phosphatidyl inositol anchored proteins. The clinical spectrum of PNH is highly variable with classical hemolytic PNH at one end, and PNH in association with aplastic anemia (AA/PNH) or other bone marrow failure states at the other end. It is still largely unknown what is causing these highly variable clinical presentations. Immune-mediated marrow failure has been suggested to contribute to the development of a PNH clone by selective damage to normal HSC. However, in classic PNH patients with no or only mild cytopenias, a role for immune mediated marrow failure is less obvious. No series of trephine biopsies has been previously documented of patients with PNH and AA/PNH to investigate the similarities and differences in these patients. MethodsWe have reviewed a series of trephine biopsies of 41 PNH patients at the time the PNH clone was first detected. The histology was compared of 27 patients with aplastic anemia and a PNH clone was compared to that of 14 patients with classic PNH. Age related cellularity, the ratio between myeloid and erythroid cells (ME ratio), and the presence of inflammatory cells (mast cells, lymphoid nodules and plasma cells) were evaluated. The relation with clinical and other laboratory parameters of PNH was established. ResultsClassic PNH patients showed a normal or hypercellular marrow in 79% of patients, whereas all AA/PNH patients showed a hypocellular marrow. Interestingly, a decreased myelopoiesis was observed not only in AA/PNH patients but also in 93% of classic PNH patients, despite normal absolute neutrophil counts (ANC ≥ 1,5 × 109/l) in 79% of these patients. The number of megakaryocytes was decreased in 29% of classic PNH patients although thrombocytopenia (< 150 × 109/l) was only present in 14% of the patients.Median PNH granulocyte clone size was 70% (range 8-95%) in classic PNH patients, whereas in AA/PNH patients this was only 10% (range 0.5-90%). PNH clones below 5% were exclusively detected in the AA/PNH group. Clinical or laboratory evidence of hemolysis was present in all classical PNH patients and in 52% of AA/PNH patients and correlated with PNH granulocyte clone size. Bone marrow iron stores were decreased in 71% of classic PNH patients. In contrast, increased iron stores were present in 63% of AA/PNH patients, probably reflecting their transfusion history. AA/PNH patients showed increased plasma cells in 15% of patients and lymphoid nodules in 37%, versus 0% and 11% in classic PNH. Increased mast cells (>2/high power field) were three times more frequent in AA/PNH (67%) than in PNH (21%). ConclusionClassic PNH patients were characterized by a more cellular bone marrow, increased erythropoiesis, larger PNH clones and clinically by less pronounced or absent peripheral cytopenias and more overt hemolysis. Decreased myelopoiesis and/or megakaryopoiesis was observed in both AA/PNH and classic PNH patients, even in the presence of normal peripheral blood counts, suggesting a role for bone marrow failure in classic PNH as well. More prominent inflammatory infiltrates were observed in AA/PNH patients compared to classical PNH patients. Disclosures:No relevant conflicts of interest to declare.

Consensus and controversies in platelet transfusion

Platelet transfusion therapy is the standard of care for thrombocytopenic patients with hemato-oncological disorders and bone marrow failure states due to intensive chemoradiotherapy. Guidelines to lower triggers for prophylactic and therapeutic transfusions are being developed based on better levels of evidence. The optimum transfusion dose, the choice of platelet concentrate and transfusion interval pose a challenge to balance scientific advances with cost-effective strategies. Platelet refractoriness requires “matched” platelets and is a difficult to treat phenomenon. Pathogen inactivation is a crucial issue in view of susceptibility of platelet concentrates to bacterial contamination. This article reviews the current developments and challenges in optimizing platelet transfusion therapy.

High Resolution Karyotyping of Large Granular Lymphocyte Leukemia by SNP Arrays Reveals Clonal Defects Leading to LOH at Loci Integral to Lymphocyte Function.

Large granular lymphocyte leukemia (LGL leukemia) is a semiautonomous clonal lymphoproliferation of cytotoxic T cells associated with various immune cytopenias. Within the spectrum of acquired immune-mediated bone marrow failure states, LGL leukemia can serve as monoclonal model of usually polyclonal T cell-mediated pathology. Mechanisms of unopposed clonal expansion of LGL cells in many aspects resemble true lymphoma and are not well understood. In addition to its reactive character, intrinsic clonal defects may be present in some patient with LGL, in particular those with more pronounced lymphoproliferative features. However, unlike in B-cell lymphomas, recurrent chromosomal abnormalities have not been frequently identified in LGL leukemia using traditional metaphase karyotyping techniques. We have applied Affymetrix 250K single nucleotide polymorphism-arrays (SNP-A) and 6.0 SNP-A in 28 patients with LGL leukemia, to elicit a far higher resolution of chromosomal content through genomic mapping of individual SNP and respective copy number analysis in LGL leukemia. SNP-A based cytogenetics have been applied successfully to MDS patients and has increased prognostic reliability. Blood mononuclear cells containing high proportion of clonal cells as determined by TCR Vβ flow cytometry were used as a source of DNA. For comparison, a large number of control blood and marrow specimens (N=119 for 6.0 and 124 for 250K SNP arrays) were analyzed. Data were processed using Genotyping Console v2.1 software (Affymetrix, Santa Clara, CA). After exclusion of known copy number variants (CNV) referenced in public databases and our own set of 178 normal controls, we found distinct chromosomal changes in 16/28 (57%) of LGL leukemia patients. Consensus regions of deletion/gain or uniparental disomy (UPD) were identified. The most common abnormalities included either UPD or copy number loss of chromosome 3q21.2–q21.31, which was identified in 6 (21%) patients. This region harbors CD86, the gene encoding the B7.2 protein responsible for T cell activation and regulation through costimulatory mechanisms. Copy number loss/UPD was also identified at chromosome 1p31.1–p32.3 in 4 (18%) patients. Interestingly, copy number gain in this same region was identified in 2 (9%) patients suggesting that this region may correspond to either a new, infrequent germ line encoded CNV or represents a somatic microdeletion. Recent studies indicated a role for SIL/SCL, which resides at this locus, in V(D)J recombination, lymphocyte development, and maturation. Additional conserved chromosomal abnormalities included copy number gains in 14q (11%) and copy number loss at 11p15 (14%), all possibly representing germ line or somatic CNV physiologically acquired during lymphocyte ontogenesis. We compared chromosomal lesions identified in our LGL cohort with clinical features including age, presence of neutropenia, anemia, thrombocytopenia, splenomegaly, immunophenotype, and degree of clonal expansion. No difference in clinical course was found between patients with and without cytogenetic abnormalities with regard to type and severity of cytopenias or size of the LGL clone. However, patients with 1p31.1–p32.3 deletions were found to have lesser degree of neutropenia compared to patients without 1p31.1–p32.3 deletions (p<0.001). While these data may reflect chromosomal variants and random somatic abnormalities, the recurrent loss of heterozygosity and gains within several loci with known regulatory function for lymphocyte biology suggests the involvement of these defects in the mechanism of clonal evolution.

Fluorescence Activated Cell Sorting (FACS) Followed by Fluorescence In Situ Hybridization (FISH) To Determine Clonal Origins of Cells in Myelodysplastic Syndrome (MDS) with Paroxysmal Nocturnal Hemoglobinuria (PNH).

The combination of FACS and FISH has been used to identify clonal cytogenetic abnormalities in small populations of cells in diseases such as multiple myeloma. We describe our refinement and application of this technique in a patient with known MDS/PNH to dissect the clonal origins of the disorder. The subject is a 45-year old woman with MDS diagnosed five years ago when she presented with macrocytic anemia and thrombocytopenia. Bone marrow was hypocellular (20% cellularity), with 4% blasts, FAB-RA, WHO- RCMD, Intermediate-1 by IPSS, del 20q clone by FISH. Family history is significant for a familial cytopenia with two sisters developing bone marrow failure, but negative for carriage of the hTERC mutation. HLA DRB1 type was 0101;1301. There was no evidence for T-LGL by flow cytometry or by TCR gene rearrangement assay. Management has been observational only without the need for transfusions. A new PNH clone was detected for the first time by flow cytometry six months ago. Heparinized blood was processed by the laboratory on the same day as collected. WBCs were separated from RBCs by a combination of dextran centrifugation and lysis of RBCs with ammonium chloride. Normal mouse IgG (3mg/mL) was added to block Fc receptors. The cells were stained with mAb cocktails containing CD16 PE (Clone 3G8, Invitrogen), CD55 PC5 (clone IA10, BD Bioscience), and CD15 APC (clone HI98, BD Bioscience) for granulocytes; or CD14 PE (clone TUK4, Invitrogen), CD55 PC5, and CD64 (clone M22, Trillium) for monocytes. Concurrent with mAb staining proaerolysin (FLAER Ax488, Protox Biotech) was added to each tube. The cells were incubated with mAbs and FLAER reagent at saturating concentrations in the dark, on ice, for 30 minutes. They were then washed once with PBS, fixed in 0.5% methanol free formaldehyde (Polysciences, Warrington, PA) for 20 minutes and finally resuspended in PBS. Cytofluorometric analysis and sorting was performed using a FACSAria (BD BioSciences) flow cytometer. Granulocytes were defined using a Boolean combination of forward, side scatter and CD15; monocytes were defined with forward, side scatter and CD64. Granulocytes and monocytes were sorted into PNH positive and negative cells based on FLAER, CD55 and either CD16 (granulocytes) or CD14 (monocytes) expression. Data was analyzed using WinList (Verity Software House, Topsham, ME). FISH analysis for del(20q) was performed on these cells using the Vysis LSI D20S108 SpectrumOrange Probe (Abbott Molecular Inc.). Our results (table 1) show that the PNH clone (FLAER negative) is a distinct non-overlapping population from the del(20q) dysplastic population.Conclusions: Our technique of flow-sorting followed by FISH is feasible for enriching and characterizing PNH positive and negative cellular fractions in bone marrow failure states. The PNH clone in this patient, contrary to other reports in the literature, does not overlap with the MDS clone, rather arising from normal non-dysplastic cells consistent with a putative immune evasion mechanism. This technique is a powerful clinical or research tool to elucidate clonal origins in PNH-associated bone marrow failure states.Table 1Pre-sortPost-sort20q− by FISHUnsorted WBCs13/200MonocytesPNH−67%76%34/200PNH+24581%5/200 = negative controlGranulocytesPNH−72%94%34/200PNH+26%94%5/200 = negative control

Hematopoietic Growth Factors in the Treatment of Acquired Bone Marrow Failure States

In severe aplastic anemia (SAA), the use of hematopoietic growth factors (HGFs) to support blood counts is of limited value, as predicted by in vitro studies and measurement of endogenous serum levels of hematopoietic growth factors (HGF), which are markedly elevated. Benefit is usually only seen in those with less severe disease who are unlikely to require HGFs in practice. HGFs administered alone play no role in the treatment of SAA. The main indication for using HGFs, most often granulocyte colony-stimulating factor (G-CSF), in SAA has been to determine whether they increase the response rate to immunosuppressive therapy (IST) and improve survival. While earlier neutrophil recovery occurs when G-CSF is administered with IST, studies to date show no significant advantage in hematologic response or overall survival. Conflicting results have been reported concerning whether G-CSF increases the known risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) after IST; follow-up of at least 10 years is required, lacking in many clinical studies reported to date. In MDS, HGFs have been used to counteract the intramedullary apoptosis, which leads to ineffective hematopoiesis. In several uncontrolled and controlled studies, especially in low-risk MDS, high-dose erythropoietin (EPO) or its glycosylated derivative darbepoetin (DPO), alone or in combination with G-CSF, increased hemoglobin levels and diminished the need for red blood cell transfusions, in selected patients with prior transfusion frequency of less than 2 units per month and EPO levels below 500 IU/L. Quality-of-life measures were claimed to have improved, but the cost-effectiveness of this approach is debated, as is safety with regard to the risk of progression. G-CSF is used in supportive care of MDS to improve neutropenia during infectious complications, but to date there is no compelling evidence for a survival benefit or alteration of the course of the disease through the use of HGFs in MDS.

Management of Thrombocytopenia in Bone Marrow Failure: A Review

The clinical course of many neoplastic and primary bone marrow diseases will result in cytopenias secondary to bone marrow failure or infiltration. Acute and chronic leukemias, the myelodysplastic syndromes (MDS), aplastic anemia, breast and prostate cancer, as well as other hematologic and solid tumors, all may lead to chronic, severe cytopenias. Management of anemia and neutropenia are well described in the medical literature. Less well detailed are management approaches for patients with chronic thrombocytopenia, with or without active bleeding. Severe thrombocytopenia presents many difficult management choices for caregivers, patients and their families, especially near the end of life. The use of platelet transfusions in this patient population presents complex issues; platelets are logistically more difficult to transfuse than red cells and carry risks including acute febrile episodes, alloimmunization, and infection. In this review, we discuss the association of chronic thrombocytopenia to serious bleeding and the role of various prophylactic and therapeutic interventions available to palliative care and hospice providers. Specifically, this review examines the following issues: What is the morbidity and mortality from chronic thrombocytopenia in the setting of cancer or other bone marrow failure states? Is there a role for prophylactic platelet transfusions in the palliative care setting, and if so, with what frequency of monitoring, and at what transfusion threshold? What is the impact of alloimmunization and how can it be minimized? What treatments are available besides, or in addition to, platelet transfusions for acute bleeding episodes?

Allogeneic Related HLA Mismatch Mesenchymal Stem Cells for the Treatment of Engraftment Failure Following Autologous Hematopoietic Stem Cell Transplantation.

Primary graft failure is usually associated with a high mortality rate despite infusion of back up graft and haematopoietic growth factors. In animal models mesenchymal stem cells (MSC) stimulate haematopoiesis recovery after TBI and enhance engraftment of haematopoietic stem cells (Almeida-Porada et al, Exp hematol 1999). Co-infusion of autologous blood stem cells and MSC in cancer patients receiving high dose chemotherapy speed up haematopoietic recovery (Koç et al, JCO 2000). We have previously shown that MSC can engraft and improve the bone marrow microenvironment in a patient with end stage severe aplastic anaemia (Fouillard et al, Leukemia 2003).We report a patient treated with MSC for aplastic anemia secondary to engraftment failure. A 40 year old nulliparous woman with acute myeloid leukaemia received an autologous bone marrow transplantation; conditioning regimen combined a 12 Gray TBI and cyclophosphamide (120 mg/kg). Primary graft failure occurred and persited despite back up marrow infusion. Partial recovery on polymorphonuclear (PMN) and haemoglobin (Hb) was obtained with granulocyte colony stimulating factor (G-CSF) and EPO. Thrombocytopenia remained below 50x109/l. No residual leukaemic cells were detectedThree years after ABMT, allogeneic MSC were infused at a dose of 2,78x106/kg. MSC were isolated from a HLA mismatched brother bone marrow (Osiris Therapeutics Inc Baltimore, MD). At time of MSC infusion, the marrow aspirate was hypocellular with no leukaemic blast cells. Blood cell counts were: PMN: 0.8x109/l, platelets: 45x109/l and Hb: 10.5 g/dl. No conditioning regimen and no prophylaxis of GVHD was given. Growth factors were discontinued.After MSC infusion, a rapid haematopoietic recovery was observed on both PMN and platelet which reached a normal level. With a follow up of 18 months, the patient is alive and well.Recovery of haematopoiesis was corroborated by an improvement of in vitro haematopoietic and stromal clonogenic assays. CFU-GM and CFU-F studied the day before MSC infusion, one month and one year after MSC infusion, increased strikingly (p<0.05). LTC-IC increased significantly one year after MSC infusion (p<0.05). There was no change in BFUE.To further characterize the effects seen on haematopoiesis, we utilized a custom RayBiotech antibody array and compared proteins secreted by MSC of recipient before and one year after infusion. This array evidenced an increased secretion of proteins implied in haematopoiesis (Flt3l, GM-CSF, G-CSF, IL1, IL6, TPO, SDF1) one year after MSC infusion. Real time PCR confirmed an up-regulation of gene expression for GCSF, GM-CSF, IL1, IL6.We studied MSC engraftment. We analysed the bone marrow biopsy extracted DNA for mesenchymal chimerism before MSC infusion, one month and one year post MSC infusion by real time quantitative PCR of the Y specific SRY gene: male DNA was not detected before infusion; a level of male DNA of 1/105 was detected one month after MSC infusion.This observation shows that MSC can induce haematopoietic tissue repair. MSC should be considered in the treatment of engraftment failure and other bone marrow failure states including severe idiopathic aplastic anaemia and accidental irradiation.

New Molecular Strategies for Detection and Quantitation of Clonal Cytotoxic T Cell Responses in Myelodysplasia and Aplastic Anemia.

Clinical similarities suggest that a T cell-mediated immune attack is involved not only in the pathophysiology of AA but also certain forms of MDS. Such a mechanism may result from a CTL attack directed against normal hematopoiesis or may reflect an immune surveillance reaction. Unlike in clonal T-LGL, immune responses in bone marrow failure states are polyclonal. We hypothesized that if immunodominant CTL clones are involved in the mechanisms of cytopenia in MDS and AA, the clonal size should correspond to the activity of disease. New molecular technologies based on TCR B-chain CDR3 region analysis allow for identification and monitoring of immunodominant T cell clones. These techniques, initially established in T-LGL (Wlodarski et al, Blood 2005), serve here as a basis for the systematic analysis of polyclonal CTL responses in bone marrow failure. We analyzed blood and paraffin-embedded marrow specimens from 30 AA, 21 PNH, 36 MDS and 60 T-LGL patients. By crude flow cytometric VB utilization analysis, we observed a continuum of VB skewing patterns from extreme monoclonal VB over-representation in T-LGL to multiple smaller expansions identified in AA and MDS. To determine whether the VB expansions are due to oligoclonal CTL proliferations, we have amplified and cloned the VB CDR3 regions of expanded VB families and sequenced large numbers of clonotypes. In both AA and MDS, most of the expanded VB families showed oligoclonality consistent with the presence of immunodominant clones. Pathogenic immunodominant clonotypes were not present in HLA-matched sibling donors or healthy identical twins discordant for the disease. A novel TCR VB multiplex PCR was applied for direct identification of immunodominant clones in the corresponding paraffin-embedded biopsies. Using this technique we retrospectively demonstrated the presence of skewed pattern of CTL clones in marrow biopsies of AA and some MDS patients; we also identified immunodominant clones in most of the biopsies studied.Moreover, biopsy-derived clonotypes were detected in blood; in many instances these clonotypes were also immunodominant as precisely quantitated by a novel clonotypic Taqman PCR utilizing CB- and clonotype-specific primer and JB-specific Taqman probes. Using a modified clonotypic PCR, blood-derived clonotypes were also found in patients' bone marrow biopsies, but not in controls. The frequency of dominant VB CDR3 clonotypes and the overall TCR repertoire were measured before and after administration of immunosuppressive therapy. Notably, the size of immunodominant clones decreased parallel to hematopoietic improvement, while the overall variability of TCR repertoire increased. Conversely, nonpathogenic clones remained unchanged. Globally, our analysis showed that immunodominant CTL clonotypes can be detected at comparable rates in AA and MDS irrespective of the morphologic subtype. Immunodominant clones derived from blood or even historical marrow specimens can be used as markers of the activity of the disease. Beyond the previously reported molecular TCR analysis in AA and MDS, we show that the CDR3 sequences of immunodominant clones can be exploited to design individualized quantitative assays that would facilitate recognition of clonotypic patterns in patients. In the future, the analysis of CDR3 amino acid sequence patterns may be used for structural comparisons of CTL clones in MDS and AA.

Immunogenetic Factors Determining Evolution of T-Cell Large Granular Lymphocyte Leukemia and Associated Cytopenias.

T cell large granular lymphocyte leukemia (T-LGL) is a chronic clonal lymphoproliferation of cytotoxic T cells (CTL). T-LGL presents with cytopenias, implying a relationship to other immune-mediated bone marrow failure states. T-LGL is often accompanied by autoimmune diseases, suggesting a clonal transformation in the context of an initially polyclonal autoimmune response. Immunogenetic predisposition factors have been described for both immune-mediated bone marrow failure as well as other autoimmune conditions and it is possible that some of these factors can promote evolution of T-LGL and/or the development of cytopenias. Here, we analyzed the association of T-LGL (and clinical variants) with a number of immunogenetic factors, including HLA and KIR genotype, KIR-L/KIR mismatch, CTLA4 (−658T/C, −318C/T, +49A/G) and cytokine single nucleotide polymorphisms (SNP) including: TNF-a (−308G/A), TGF-b 1 (codons 10 C/T, 25 G/C), IL-10 (−1082 G/A, −819 T/C, −592 C/A), IL-6 (−174 C/G), and IFN-g (+874 T/A). We studied a large cohort of patients with T-LGL (N=79) systematically followed at our institution. Using molecular analysis of TCR utilization as previously described (Wlodarski et al, Blood 2005) we have determined the relative and absolute size of the clonal LGL population for each case. We performed HLA typing as certain alleles serve as KIR ligands and can determine quality of immune response. HLA-B37 and Cw-7 were over expressed in LGL patients (13 vs 2%, p<.001; 60 vs 45%, p=.039); no other alleles correlated with the disease. No HLA association was found in patients presenting with neutropenia or splenomegaly, however HLA-Cw7 was found in 100% of patients with an increased number of clonal CTL (>1900/uL; N=13 vs 45% in controls and 33% in patients with low LGL count; p<.001). HLA-Cw alleles can be divided into C1 and C2 groups based on recognition by KIR; in the above 13 patients, C1/C1 phenotype was significantly increased compared to normal controls while C2/C2 was absent (p<.001, p=.04, respectively). This finding suggests a protective function for the C2/C2 phenotype in T-LGL patients. Subsequently, we analyzed KIR genotype by SSP PCR; no association between a specific KIR genotype and disease was found. LGL patients did not vary from controls in the ratio of inhibitory to stimulatory KIR. As KIR genotype may exert pathogenic influence in the context of KIR/KIR-L interactions, we hypothesized that a KIR/KIR-L mismatch would result in less regulation of CTL response and thus more severe cytopenias. According to the subdivision of HLA-Cw alleles into group C1 (ligands KIR2DL2 and 2DL3) and C2 (ligands 2DL1), genetic mismatch between KIR-L and KIR was found in 50% of patients, but proved statistically insignificant compared to normal controls. The severity of cytopenias and other hematologic manifestations was greater in patients without KIR-L/KIR mismatch. This finding may be due to an overshooting CTL response resulting from silencing NK-cells. SNP in cytokines and their promoters may play a pathogenic role in autoimmune conditions, as such we also analyzed the frequency of multiple cytokine polymorphisms in T-LGL to determine a potential secretion/stimulation phenotype. Among cytokine SNP studied in T-LGL, overrepresentation of TNF-a -308G/A polymorphism (for A/A: 14 vs 1% in controls, p=.009), and CTLA4 +49 SNP A/A genotype was found (50 vs 27%, p=.013), suggesting that the presence of G allele is protective.

Osteoblastic Cells (OBs) and the Hematopoietic Microenvironment: Parathyroid Hormone (PTH) Increases the Notch Ligand Jagged1 (J1) in a Specific Subpopulation of OBs.

Selective manipulation of the hematopoietic stem cell (HSC) niche has numerous clinical applications, including the potential for HSC expansion in the setting bone marrow failure states or marrow transplantation. We have previously shown that activation of the PTH/PTHrP Receptor (PTH1R) in OBs results in expansion of HSC through activation of the Notch signaling pathway in the bone marrow (Nature 425: 841). In particular, stromal cell expression of the Notch ligand Jagged1 (J1) has been previously shown to play a fundamental role in HSC support through cell-cell interactions. We hypothesized that the PTH1R ligand PTH(1-34), a peptide currently used for the treatment of osteoporotic patients, could increase osteoblastic expression of J1, and preferentially target a subpopulation of OBs at a particular differentiation stage. We therefore treated adult mice daily with either vehicle or PTH(1-34). After 5 days of treatment, in sections from the long bones of vehicle-treated animals, immunohistochemical analysis using an antibody to the C-terminus of Jagged1 (H114, Santa Cruz Biotechnology) showed localization of the J1 protein to few trabecular osteoblasts, as well as endosteal osteoblasts, and in spindle shaped cells in the bone marrow cavity. In bones from PTH-treated mice, J1 levels were heterogeneously increased in a subpopulation of trabecular and endosteal OBs. To further characterize the J1-expressing OBs, we then studied whether expression of J1 in primary OBs changes during their differentiation. Primary OBs grown in mineralizing conditions stereotypically progressed through proliferation, differentiation and mineralization stages, as confirmed by alkaline phosphatase and Von Kossa staining as well as by expression of markers of osteoblastic differentiation such as osteocalcin. Real time PCR analysis of J1 expression in mRNA from primary calvarial osteoblasts at sequential differentiation stages showed a rapid increase in J1 expression during osteoblastic proliferation, particularly during days 5 to 10. J1 expression peaked during osteoblastic differentiation (day 15), and declined during mineralization (days 19–24). In order to study whether PTH(1-34) increases J1 expression in a specific subpopulation of OBs, we treated with PTH(1-34) primary calvarial OBs at different stages of maturation. Surprisingly, we found that PTH had a stimulatory effect only at day 22, in mineralizing osteoblasts, while in late proliferating cells and in maturing OBs there was no significant PTH-dependent J1 stimulation at the times tested. These studies show that PTH is capable of regulating J1 expression in a subpopulation of mature and mineralizing OBs. While it is possible that intermittent PTH treatment may also selectively expand a subpopulation of OBs with high J1 levels, we have demonstrated with these studies that there is a rapid PTH effect on J1 stimulation, which is dependent upon the differentiation stage of the target OBs. Further definition of the osteoblastic maturation stage necessary to mediate hematopoietic-osteoblastic interactions is critical to a more precise understanding of the HSC microenvironment, as well as a possible in vivo therapeutic target for HSC niche manipulation.

Role of the p38 Mitogen-Activated Protein Kinase Pathway in Cytokine-Mediated Hematopoietic Suppression in Myelodysplastic Syndromes

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by IFNs and other cytokines to mediate signals for important cellular functions, including transcriptional regulation and apoptosis. We examined the role of the p38 pathway in the generation of the effects of myelosuppressive cytokines on human hematopoiesis. Pharmacologic inhibition of p38 using BIX-01208 resulted in reversal of IFN-, tumor necrosis factor-alpha (TNF-alpha)-, and transforming growth factor-beta (TGF-beta)-mediated suppression of human erythroid (blast-forming unit-erythroid) and myeloid (granulocyte-macrophage colony-forming unit) colony formation, consistent with a key role for p38 in the generation of myelosuppressive signals by different cytokines. Similarly, the myelosuppressive effects of TNF-alpha and TGF-beta were reversed by small interfering RNAs targeting p38alpha expression, further establishing the requirement of this kinase in the induction of myelosuppressive responses. As TNF overproduction has been implicated in the pathophysiology of bone marrow failure states, we determined whether pharmacologic inhibition of p38 reverses the hematopoietic defects seen in bone marrows from patients with myelodysplastic syndromes (MDS) and the anemia of chronic disease. Addition of pharmacologic inhibitors of p38 on such bone marrows resulted in increased numbers of erythroid and myeloid progenitors. Similarly, inhibition of the activity of the downstream effectors of p38, MAPK activated protein kinase-2, and mitogen and stress activated kinase 1 partially restored the hematopoietic defect seen in these bone marrows. Taken altogether, our data implicate the p38 MAPK in the pathophysiology of myelodysplasias and suggest that p38 pharmacologic inhibitors may have therapeutic applications in the treatment of MDS.

What is quality in a transplant program?

Abstract Hematopoietic stem cell transplantation (HSCT) as a field of medicine has been subject to rapid development and evolution since its inception. Traditionally, HSCT has been used for therapy of a diverse group of malignancies, bone marrow failure states, and inherited disorders. The rapid evolution of transplantation technology coupled with the diverse outcomes associated with a heterogeneous group of patients has stymied the development of consensus over objective programmatic indicators of quality, especially as they pertain to outcomes. In some regard, the lack of consensus has caused transplant programs to respond to a more consumer-driven paradigm of evaluation. The community of providers of transplantation therapies has responded by establishing standards for accreditation of facilities and uniform presentation of programmatic data. Rapid acceptance of the need for meaningful quality programs to address all aspects of the transplant facility has moved HSCT to the forefront of implementing standards for medical practice. Because definition of optimal outcomes in HSCT is likely to remain elusive, it is imperative that providers involved with HSCT continue to take a leadership role in defining program quality through further research.

Thrombopoietic Factors in Chronic Bone Marrow Failure States: The Platelet Problem Revisited

Thrombocytopenia is a serious clinical problem in several different clinical settings. In chronic bone marrow failure states, which include aplastic anemia, myelodysplastic syndrome, and graft failure, the prolonged nature of thrombocytopenia often leads to alloimunization after repeated platelet transfusions, the consequence of which is a platelet-refractory state and enhanced risk of bleeding. Despite the introduction of several thrombopoietic factors into clinical trials, an effective way to alleviate thrombocytopenia has been elusive, and the problem in chronic bone marrow failure states has remained poorly addressed by clinical investigations. Even so, several studies by our group and others suggest that a subset of patients suffering from chronic bone marrow failure can respond to appropriate growth factor therapy. The temporal pace of response appears, however, to be much slower than that observed after administering growth factors which act on neutrophils. On the other hand, durable responses can be secured in some patients given thrombopoietic factors for long periods of time. Herein, we provide an overview of the clinical research investigations of thrombopoietic factors in chronic bone marrow failure, and the emerging insights these studies provide for understanding the process of thrombopoiesis and its therapy in this setting.

O-61 Molecular analysis of CTL responses in myelodysplastic syndrome and other bone marrow failure states

O-61 Molecular analysis of CTL responses in myelodysplastic syndrome and other bone marrow failure states

Intermittent Low Dose Interleukin 11 Can Lead to Transfusion Independence in Patients with Chronic Myelo-Monocytic Leukemia and Thrombocytopenia.

Recombinant Interleukin- 11 (IL-11) is a thrombopoietic cytokine that stimulates megakaryocytopoiesis in vitro and platelet production in vivo. It attenuates post chemotherapy thrombocytopenia at a dose of 50 mcg/ kg/ day subcutaneously (SC). Unfortunately, prolonged administration is associated with significant toxicity including peripheral and pulmonary edema at this dose. Administration of low dose IL-11 at 10 mcg/kg/day SC has shown efficacy in bone marrow failure states without significant toxicity. We report two cases of chronic myelo-monocytic leukemia (CMML) with transfusion dependent thrombocytopenia who received intermittent low dose IL-11 without significant toxicity. Case Reports- Patient 1 is a 79-year-old male with history of CMML with pancytopenia of three years duration. Recently he required transfusion of platelets with his platelet counts falling to less than 15 x 109/L. His cytogenetic study showed normal karyotype, 46 XY. He required platelet transfusion at every 14 days. He initially was started on IL-11 at 10mcg/kg/day, 5 days per week.. His platelet count increased to above 30 x 109/L and he became transfusion independent within two weeks. Unfortunately on this schedule he developed edema and mild CHF. IL-11 was stopped for two weeks and upon resolution of toxicity, restarted at 10 mcg/kg/day on Monday, Wednesday and Friday. He has remained transfusion independent without recurrence of edema at 5 months on this schedule. Patient 2 was a 63-year-old male with previous history of chronic lymphocytic leukemia and diffuse large B cell lymphoma who developed CMML with severe pancytopenia. His karyotype was 46, XY, −7, +21. His platelet count was consistently less than 10 x 109/L. He required platelet transfusion twice a week. He was started on IL-11 at 10mcg/kg/day for 5 days per week, two weeks on and two weeks off. His platelet count increased to as high as 64 x 109/L after 2nd cycle. His platelet transfusion requirement decreased from every 3rd day to every 10th-14th day. He experienced no peripheral or pulmonary edema. Conclusion: Administration of low dose IL-11 in other bone marrow failure states has been reported but its use has not been described in CMML. Our observation in these 2 patients suggests that IL-11 has efficacy in CMML and is very well tolerated at low doses on an intermittent administration schedule. IL-11 may decrease the transfusion requirement in transfusion dependent patients. Further studies are needed to evaluate overall impact on larger number of patients who require regular platelet transfusion.

A Pilot Application of SELDI Serum Proteomics in Bone Marrow Failure Syndromes.

Clinically, bone marrow failure syndromes show significant overlap. Newer analytic approaches may lead to improved diagnosis and better understanding of underlying pathophysiology. In contrast to traditional biomarker diagnostic principles, proteomic technologies such as surface-enhanced laser desorption/ionization (SELDI) method enable screening of protein patterns without knowledge of the identity of targets. The recent refinement of SELDI mass spectrometry has allowed for the early diagnosis of occult solid tumors. We explored the potential application of SELDI proteomics to the study of bone marrow failure states. An extensive sample library included 28 Aplastic anemia, 129 Myelodysplastic syndrome (MDS), 28 Paroxysmal Nocturnal Hemoglobinuria (PNH) patients and 36 control specimens. Within the MDS cohort, subgroups with RA (N=77), RARS (N=15) and RAEB/t (N=37) were studied. Analyses were also performed for patients with specific cytogenetic abnormalities, including those with 5q-syndrome (N=21), trisomy-8 (N=15), aberrations of chromosome 7 (N=15) and compared to MDS patients with normal cytogenetics (N=57). In preliminary experiments, H4 chips were selected for further study; spectra with >80 peaks were considered as informative. Initially, our analysis included learning sets in which 20 randomly selected samples from individual categories were compared to each other in order to identify specific peptide peaks. Subsequently, the results obtained in the learning set were confirmed using separate validation sample sets. A scoring system was applied for analysis in which signal/noise ratio was a sole criterion for peak recognition and assignment of non-parametric scores. Reproducibility was assured by repeating measurements of standard plasma sample and multiple determinations.When AA spectra were compared to controls, AA could be distinguished with 87% sensitivity and 85% specificity based on the presence of a single peak at M/Z value of 3.095kDa. When compared to RA, AA was discriminated with 95% specificity and 80% sensitivity (1.02kDa). Globally, we devised a decision tree (using 3 different peaks in 6 steps of separation) that allows for the plasma-based discrimination of MDS and controls. Within MDS group, RA could be further differentiated from a more advanced MDS with 95% specificity and 90% sensitivity. A single peak at M/Z value of 3.88kDa distinguished RA from RAEB/t. Similar learning data sets performed for RARS patient can be obtained. Not overlapping sample sets are used for validation of obtained results. Finally, samples with atypical spectra can be investigated for features that may prompt diagnostic reassignment; e.g., in patients with hypocellular MDS to AA, and PNH to AA/PNH syndrome. Most intriguing is the possibility that specific cytogenetic defects may result in proteomic patterns that may provide important clinical clues. A large sample collection allows for subanalyses based on karyotype: for example, data and result sets exist for patients with 5q-, trisomy-8 and other more common chromosomal changes.Our ongoing study demonstrates the feasibility of SELDI technology in detecting diagnostic protein signatures for individual bone marrow failure states. In the future, proteomic biomarker pattern-based analysis may allow for more precise definition and supplement the traditional diagnostic approaches.

Sequential interleukin 3 and granulocyte-macrophage–colony stimulating factor therapy in patients with bone marrow failure with long-term follow-up of responses

BACKGROUND Interleukin-3 (IL-3) and granulocyte-macrophage–colony stimulating factor (GM-CSF) have synergistic, hematopoietic growth-promoting activity in preclinical studies. Because of the paucity of effective therapies for patients with chronic bone marrow failure states, the authors studied the biologic activity of sequential IL-3/GM-CSF in such patients. METHODS IL-3 was given subcutaneously for 5 days (at escalating doses of 0.15 μg/kg, 0.3 μg/kg, 0.6 μg/kg, 1.2 μg/kg, 2.5 μg/kg, 5.0 μg/kg, 10.0 μg/kg, or 15.0 μg/kg per day), and GM-CSF for was given subcutaneously for 9 days (at a dose of 5 μg/kg per day; Phase I 3 + 3 design) followed by 14 days of rest (total, 2 courses), then maintenance therapy. RESULTS The majority of 38 evaluable patients had aplastic anemia or myelodysplastic syndrome. Most patients (79%) had neutrophil responses. Ten patients (26%), all of whom were treated with IL-3 doses ≥ 1.2 μg/kg per day, had platelet responses, with a median increase of 132 × 109/L (range, 41–180 × 109/L) over baseline in responders. Six patients (16%) had trilineage recovery, which could be durable (the longest ongoing at 6.5 years after therapy completion). The most common toxicities were low-grade fever, headache, and fatigue. The maximum tolerated doses were IL-3 at 10 μg/kg per day and GM-CSF at 5 μg/kg per day. CONCLUSIONS Sequential IL-3/GM-CSF effectively raised blood counts in some patients with bone marrow failure at doses that were tolerated well. These results indicate that early-acting growth factors can induce durable, multilineage responses in a subset of individuals with bone marrow failure. Cancer 2003. © 2003 American Cancer Society.

We Welcome a New Associate Editor

As we begin publishing the 30th volume of Experimental Hematology, the editorial board and I welcome Andrea Bacigalupo, MD, as associate editor for the journal. Andrea is the head of the Department of Hematology and Oncology at the Ospedale San Martino in Genoa, Italy. Andreas'research focuses on the pathobiology and treatment of bone marrow failure states and on clinical stem cell transplantation. We believe he is a strong addition to an already excellent group of editors.