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Hematopoietic stem and progenitor cells (HSPCs) emerge from artery-derived hemogenic endothelium (HE) in the vertebrate embryo during development. This process entails a cellular reprogramming from endothelial to hematopoietic gene signatures driven by the Runx1 transcription factor (TF), which is referred to as the endothelial-to-hematopoietic transition (EHT). A major goal of cellular therapeutics is to derive patient-specific HSPCs from iPSCs for clinical use, yet current differentiation protocols largely fail to recapitulate EHT to produce or expand long-lived multi-potent HSPCs in vitrosuggesting an incomplete understanding of the in vivoregulatory cues. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in embryos are one such cue required during EHT to generate HSPCs from HE, however the mechanisms by which these forces are sensed and converted into a “stemness” regulatory module remain undefined.

To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse.

To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse.

Baseline platelet production is dependent on thrombopoietin (TPO). TPO is constitutively produced and primarily regulated by receptor-mediated uptake by platelets. Inflammatory thrombocytosis is thought to be related to increased interleukin-6 (IL-6) levels. To address whether IL-6 might act through TPO to increase platelet counts, TPO was neutralized in vivo in C57BL/10 mice treated with IL-6, and hepatic TPO mRNA expression and TPO plasma levels were studied. Transcriptional regulation of TPO mRNA was studied in the hepatoblastoma cell line HepG2. Furthermore, TPO plasma levels were determined in IL-6–treated cancer patients. It is shown that IL-6–induced thrombocytosis in C57BL/10 mice is accompanied by enhanced hepatic TPO mRNA expression and elevated TPO plasma levels. Administration of IL-6 to cancer patients results in a corresponding increase in TPO plasma levels. IL-6 enhances TPO mRNA transcription in HepG2 cells. IL-6–induced thrombocytosis can be abrogated by neutralization of TPO, suggesting that IL-6 induces thrombocytosis through TPO. A novel pathway of TPO regulation by the inflammatory mediator IL-6 is proposed, indicating that the number of platelets by themselves might not be the sole determinant of circulating TPO levels and thus of thrombopoiesis. This regulatory pathway might be of relevance for the understanding of reactive thrombocytosis.

Baseline platelet production is dependent on thrombopoietin (TPO). TPO is constitutively produced and primarily regulated by receptor-mediated uptake by platelets. Inflammatory thrombocytosis is thought to be related to increased interleukin-6 (IL-6) levels. To address whether IL-6 might act through TPO to increase platelet counts, TPO was neutralized in vivo in C57BL/10 mice treated with IL-6, and hepatic TPO mRNA expression and TPO plasma levels were studied. Transcriptional regulation of TPO mRNA was studied in the hepatoblastoma cell line HepG2. Furthermore, TPO plasma levels were determined in IL-6–treated cancer patients. It is shown that IL-6–induced thrombocytosis in C57BL/10 mice is accompanied by enhanced hepatic TPO mRNA expression and elevated TPO plasma levels. Administration of IL-6 to cancer patients results in a corresponding increase in TPO plasma levels. IL-6 enhances TPO mRNA transcription in HepG2 cells. IL-6–induced thrombocytosis can be abrogated by neutralization of TPO, suggesting that IL-6 induces thrombocytosis through TPO. A novel pathway of TPO regulation by the inflammatory mediator IL-6 is proposed, indicating that the number of platelets by themselves might not be the sole determinant of circulating TPO levels and thus of thrombopoiesis. This regulatory pathway might be of relevance for the understanding of reactive thrombocytosis.

The spleens from two children and one adult with hereditary spherocytosis were studied in the electron microscope. Stagnation of the erythrocytes within the splenic cords is attributable to their lack of plasticity as evidenced by the absence of bilobed, tailed, or squeezed forms in transit through the walls of the sinuses. In contrast to the sections studied by conventional light microscopy, the splenic sinuses in hereditary spherocytosis were not "empty," but contained red blood cells, the majority of which had lost their hemoglobin content. Cordal macrophages were increased in all three cases and were abundant in the splenic cords of the adult patient, causing a further impediment to the rapid passage of erythrocytes. Macrophages, and, to a lesser degree, sinus endothelial cells contained the products of hemoglobin breakdown. The macrophages showed active erythrophagocytosis. Sinus endothelial cells rarely contained intact red blood cells, but showed pronounced pinocytotic activity, a probable mechanism of hemoglobin incorporation. Platelets within the endothelial cells of the sinuses were much more frequently seen in the three cases of hereditary spherocytosis than in control spleens. The presence of ferritin in platelets suggests that they too may play a role in clearing the end products of hemolysis from the spleen.

The spleens from two children and one adult with hereditary spherocytosis were studied in the electron microscope. Stagnation of the erythrocytes within the splenic cords is attributable to their lack of plasticity as evidenced by the absence of bilobed, tailed, or squeezed forms in transit through the walls of the sinuses. In contrast to the sections studied by conventional light microscopy, the splenic sinuses in hereditary spherocytosis were not “empty,” but contained red blood cells, the majority of which had lost their hemoglobin content. Cordal macrophages were increased in all three cases and were abundant in the splenic cords of the adult patient, causing a further impediment to the rapid passage of erythrocytes. Macrophages, and, to a lesser degree, sinus endothelial cells contained the products of hemoglobin breakdown. The macrophages showed active erythrophagocytosis. Sinus endothelial cells rarely contained intact red blood cells, but showed pronounced pinocytotic activity, a probable mechanism of hemoglobin incorporation. Platelets within the endothelial cells of the sinuses were much more frequently seen in the three cases of hereditary spherocytosis than in control spleens. The presence of ferritin in platelets suggests that they too may play a role in clearing the end products of hemolysis from the spleen.

Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Dyskeratosis congenita (DC) belongs to the group of inherited bone marrow failure syndromes (IBMFS) and is characterized by premature telomere shortening caused by mutations in components of the telomerase or the shelterin complexes. The main cause of death in affected patients is hematopoietic failure, but there is also a 10-15% risk of malignant transformation into secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Critically short telomeres activate a DNA damage response with p53-mediated cell cycle inhibition, senescence and/or apoptosis, the latter mediated primarily by PUMA, a BCL-2 family member belonging to the group of pro-apoptotic BH3-only proteins and transcriptionally regulated by p53. Activation of p53 and expression of its target genes are critical for the exhaustion of hematopoietic stem cells (HSCs) in DC patients. Inactivation of the DNA damage checkpoint could possibly mitigate hematopoietic failure but poses a significant risk of genomic instability and leukemia. Based on our earlier mouse model of secondary leukemia (Genes Dev, 24(15):1602-7), we hypothesized that selective inhibition of p53-mediated apoptosis - while all other p53-checkpoint-induced pathways remain active - could both delay hematopoietic failure and prevent malignant transformation.

Platelet-driven blood clot contraction reduces and compacts clot volume, promoting hemostasis while restoring blood flow past otherwise obstructive thrombi. Clot contraction is driven by traction forces in the range of tens of pN per platelet that are generated by platelet non-muscle myosin IIa and actin on αIIbβ3 bound to extracellular fibrin fibers. In an analysis of the kinetics of thrombin-induced clot formation and contraction in platelet-rich plasma, we found that clot contraction has two discernable phases: an exponential initiation phase and a second exponential contraction phase. It is noteworthy that Azam et al (Mol Cell Biol21:2213-20, 2001) observed that deleting the µ-calpain gene in mice impairs platelet-mediated clot contraction. In this context, we found that inhibiting calpain activity with ALLM (N-Acetyl-Leu-Leu-Methional), a cell-permeable calpain inhibitor, significantly prolonged the duration of the initiation phase and decreased the kinetic rate constants for both the initiation and contraction phases of thrombin-stimulated platelet-mediated clot contraction, but without affecting the overall extent of contraction. The calpain inhibitor ALLN (N-acetyl-Leu-Leu-Norleu) had the same effect. Thus, these studies imply that activation of platelet calpain facilitates the transmission of traction forces from the platelet cytoskeleton to the αIIbβ3 bound to fibrin fibers. μ-Calpain is calcium-dependent cytosolic neutral cysteine protease that is activated 30-60 seconds after the onset of platelet aggregation stimulated by platelet agonists such as thrombin. To identify the calpain-cleaved protein or proteins involved in clot contraction, we incubated washed human platelets with the thrombin activation peptide TRAP in the presence or absence of calpain inhibitors and identified calpain-cleaved proteins using subtiligase-mediated biotin-labeling of nascent N-termini followed by mass spectrometry. We identified 32 proteins in the platelet cytosol that undergo calpain-mediated cleavage after TRAP stimulation. Many of these are cytoskeletal proteins, most prominently talin which connects integrins to the cytoskeleton and vinculin which is required for myosin-contractility-dependent effects on traction force and adhesion strength. Accordingly, we focused our attention on these two proteins. Talin, a 250 kD protein, is composed of a 45 kD N-terminal head domain attached via a flexible linker to a 200 kD C-terminal rod domain. The rod domain consists of 62 amphipathic α-helices organized into a series of four- and five-helix bundles. Vinculin is a 116 kD protein composed of 5 domains and has an autoinhibited structure in which domains 1-3 bind to domain 5. We detected 8 calpain-mediated cleavages in talin, 2 previously identified cleavage sites in unstructured regions and 6 others in α-helical regions known to interact with other proteins. Four of the latter are located within the first 12 talin helices, a region that contains four vinculin binding sites (VBSs). Likewise, the remaining two cleavage sites are in proximity to a VBS. A VBS is a vinculin-binding α-helix that is buried in a helical bundle due to extensive hydrophobic interactions with other amphipathic helices. Because VBS are packed into the interior of a helical bundle, a structural rearrangement is required to initiate vinculin binding. Using magnetic tweezers and atomic force microscopy, del Rio et al demonstrated that force-induced stretching of single talin rod molecules activates VBSs (Science323:638-41, 2009)), implying that stretching causes the conformational change required to expose buried VBSs. Thus, based on the location of the calpain-mediated cleavages in talin, we hypothesize that by cleaving talin in proximity to a VBS, calpain facilitates vinculin to binding talin, thereby strengthening the coupling of fibrin-bound αIIbβ3 to the platelet cytoskeleton to promote fibrin clot contraction.

INTRODUCTION

Ashby: University of Arkansas for Medical Sciences: Employment. Barlogie:Signal Genetics: Patents & Royalties. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Purified poloxamer188 (P188) (Mast Therapeutics) is a non-ionic, linear block copolymer composed of a central chain of hydrophobic polyoxypropylene and two flanking chains of hydrophylic polyoxyethylene (MW 8.5 kDa). This agent has hemorheologic properties which result in improved microvascular blood flow. P188 has been investigated in a number of indications and is currently under study in an international phase 3 clinical trial in sickle cell patients with vaso-occlusive crisis. Dextrans represent branched polysaccharides of 10-70 kDa that have been used as antithrombotic agents and plasma expanders. Sickle cell disease (SCD) represents a complex hemorheologic condition due to RBC aggregation and cell-fibrin/fibrinogen interactions. The erythrocyte sedimentation rate (ESR) is reflective of RBC and plasma interactions. This study was designed to compare the effect of P188 and dextrans on ESR’s in blood obtained from healthy subjects and patients with sickle cell disease who were seen at Loyola University Medical Center clinics.Whole EDTA blood collected from normal individuals (n=8) and sickle cell patients confirmed by electrophoresis (n=11) were supplemented with P188 or dextran 10K, 18K , 40K and 70K at various concentrations (or saline control). ESR was measured using standard laboratory technique.The ESR’s for sickle cell patients (26.4 ± 7.1 mm/hr) were significantly higher in comparison to the ESR’s for healthy subjects (14.6 ± 2.1 mm/hr). Supplementation of P188 decreased ESR’s in both populations. Normal blood ESR’s decreased to 9.1 ± 1.3 mm/hr (38%), whereas the sickle cell patient values decreased to 14.1 ± 4.6 mm/hr (47%).  At comparable concentrations, none of the dextrans changed ESR’s in healthy subjects or patients with sickle cell disease.These results demonstrate that ESR in SCD patients are elevated compared to healthy subjects. P188 supplementation decreased (up to 50%) ESR’s in both the healthy subjects and sickle cell patients. This may be due to the inhibition of rouleaux formation resulting from P188 effects on RBC membranes or cell-protein interactions. None of the dextrans produced a similar decrease, suggesting that the observed lowering of ESR by P188 is unlikely to be due to a non-specific effect related to polymer molecular weight.P188 is a potential therapeutic agent which may facilitate blood flow and reduce cell-fibrin/fibrinogen interactions in a variety of hemorrheologic disorders. The observed decrease in ESR both in normal and sickle cell blood samples by P188 may primarily be due to increased membrane hydration, fibrinogen dispersion and anti-adhesive effects of this agent.Emanuele: Mast Therapeutics: Employment. Fareed:Mast Therapeutics: Research Funding.

Purified poloxamer188 (P188) (Mast Therapeutics) is a non-ionic, linear block copolymer composed of a central chain of hydrophobic polyoxypropylene and two flanking chains of hydrophylic polyoxyethylene (MW 8.5 kDa). This agent has hemorheologic properties which result in improved microvascular blood flow. P188 has been investigated in a number of indications and is currently under study in an international phase 3 clinical trial in sickle cell patients with vaso-occlusive crisis. Dextrans represent branched polysaccharides of 10-70 kDa that have been used as antithrombotic agents and plasma expanders. Sickle cell disease (SCD) represents a complex hemorheologic condition due to RBC aggregation and cell-fibrin/fibrinogen interactions. The erythrocyte sedimentation rate (ESR) is reflective of RBC and plasma interactions. This study was designed to compare the effect of P188 and dextrans on ESR's in blood obtained from healthy subjects and patients with sickle cell disease who were seen at Loyola University Medical Center clinics.

Abstract 2031

The role of High-Dose Therapy (HDT) and Autologous Stem Cell Transplantation (ASCT) as salvage therapy in relapsed Follicular Lymphoma (FL) has been questioned since the introduction of Rituximab in the treatment of FL. Our objectives were to evaluate the long-term event-free survival (EFS) and overall survival (OS) rates of patients with relapsed or refractory FL who received HDT and ASCT as salvage therapy. Our secondary objective was to compare EFS of patients who received ASCT before and after 2002 (the year Rituximab become fully available for use in our country).We conducted a retrospective analysis of 123 consecutive patients with relapsed or refractory FL who had received HDT and ASCT as salvage therapy in five Argentinean transplant centers from 1992 to 2012.One hundred twenty tree patients (median age: 48 years, range: 23–72) were transplanted, 71 (58%) male, 37/106 (35%) had FL Grade 1, 47/106 (44%) FL Grade 2 and 22/106 (21%) FL Grade 3; 75/102 (73%) of patients have a stage III-IV disease at the moment of diagnosis and 27/102 (26%) have a disease stage I-II. Before transplantation 78/121 (64%) patients were in Complete Response (CR), 42/121 (35%) were in Partial Response (PR) and one patient had a Progressive Disease. Fifty four patients (44%) were transplanted before 2002 (before Rituximab era in our country). The conditioning regimens used were CBV 94 (76%), BEAM/BEAC 27 (22%), others regimens two patients; only one patient received TBI containing conditioning regimen and was excluded for conditioning regimen PFS curve calculation. The median time from FL diagnosis to ASCT was 29 (range 1–300) months. The transplant related mortality (until day 90 after ASCT) was 5%. With a median follow-up of 52 (range 0–247)months, the median EFS was reached at 44 months and a plateau on the EFS curve was evident starting at 6 years after ASCT. The 10 years-projected EFS was 36% (Figure 1). The median OS was not reached during follow-up. Median EFS for patients who were transplanted before 2002 (before Rituximab era) and for those who were transplanted after 2002 was 41 months and 47 months respectively [HR 1.01; IC (0.61–1.65); p=0.97]. EFS rates difference was not statically significant between patients who previously transplantation have achieved a CR or a PR, median EFS 47 and 68 months respectively [HR 0,94; IC (0,56–1,58); p= 0,82]. There was no difference in EFS rates between patients who received CBV as conditioning regimen and patients who received BEAM/BEAC, median EFS 57.6 months and 16.3 months respectively [HR 0.67; IC (0,34–1.35); p=0.2]. The median EFS rate for patients with FL Grade 1, FL Grade 2 and FL grade 3 were 58, 44 and 16 months respectively, P for trend = 0.78.Seven cases (5,7%) of secondary malignancies were detected, six cases (6/94; 6,4%) in patients who received CBV as conditioning regimen (urotelial carcinoma, pancreas carcinoma, lynfoprolipherative disorders, non-melanoma skin malignancies and prostate carcinoma) and one case (1/27; 3,7%) in patients who received BEAM (urotelial carcinoma), p=0,85; there were no reports of secondary myelodysplastic syndromes/acute myeloid leukemia.In this study, 50% of the patients analyzed were free from progression after 44 months of ASCT and approximately a 30% of patients achieve long term remission. According to this, patients with relapsed or refractory FL can achieve long-term EFS with HDT followed by ASCT.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 1255

No relevant conflicts of interest to declare.

Abstract 4915

No relevant conflicts of interest to declare.

Abstract 152

BACKGROUND: Autologous graft versus host disease (GVHD) and engraftment syndrome (ES) probably result from host immune dysfunction during the recovery from high dose chemotherapy and radiation. Since impaired immunity has been associated with myelodysplastic syndrome, we explore the risk factors of post-transplant myelodysplastic syndrome/acute myeloid leukemia (MDS), specifically, in relation to the GVHD and ES. PATIENTS AND METHODS: Consecutive patients with lymphoma undergoing autologous transplantation in our institution from 1991 to 2006. RESULTS: There is total of 452 lymphoma patients undergoing autologous transplants in this period; 274 males and 178 females, median age of 50 years (range 16–76). There are 85 patients with Hodgkin’s lymphoma (HL) and 367 non-Hodgkin’s lymphoma (NHL), of which, 291 are B-cell, 47 T-cell and 29 unknown. Total of 277 received TBI-based and 175 chemotherapy-alone conditioning regimens; 98 patients received transplantation of the bone marrow (BM), 343 peripheral blood stem cells (PBSC) and 11 both. Eleven patients had second autologous transplantation for progressive lymphoma and another four patients have second allogeneic transplant for MDS. Thirty-two patients (7%) died of regimen-related toxicity within 100 days of transplant. Eleven patients developed severe engraftment syndrome (high fever, skin rash ± pulmonary infiltrate requiring systemic steroid); 27 patients had skin and 2 patients had gastrointestinal biopsies consistent with GVHD. The median follow-up of the patients was 6.2 years and median overall survival 5.3 years. Univariate analysis using Kaplan-Meier plots and logrank tests, younger age, HL, B-phenotype, source of stem cells (BM vs PBSC), chemo-sensitivity, less prior chemotherapy are better prognostic indicators. Conditioning regimens (TBI-based vs non-TBI) do not affect the overall survival. Twenty-four patients (5.3%) developed MDS with median time of onset of 4.2 years (range 8 months-7.5 years). Additional 5 patients developed clonal karyotypic abnormalities in the bone marrow without clinical MDS. Actuarial probabilities of developing MDS 5 and 8 years after transplant are 5% and 15% respectively. The incidences of MDS are similar in HL and NHL. Significant risk factors of developing MDS include older age, advanced stage, onset of ES or GVHD, and longer intervals between the initial diagnoses to transplant. CONCLUSION: Although overall incidence of MDS is only 5.3%, the actuarial risk at 8 years is up to 15% and may be higher in selected patients such as older age, and prolonged interval from initial diagnosis to transplant (a surrogate for prior chemotherapy). The association of engraftment syndrome and GVHD to MDS is intriguing. It is conceivable that perturbation to the host immunity caused by either prior chemotherapy, conditioning regimens in the elderly may play a role in the development of MDS after autologous transplant.

Table 1.Samples to p53 deletions

BACKGROUND: Autologous graft versus host disease (GVHD) and engraftment syndrome (ES) probably result from host immune dysfunction during the recovery from high dose chemotherapy and radiation. Since impaired immunity has been associated with myelodysplastic syndrome, we explore the risk factors of post-transplant myelodysplastic syndrome/acute myeloid leukemia (MDS), specifically, in relation to the GVHD and ES.

Table 1. Samples to p53 deletions p 53 deletion FISH aCGH FISH and aCGH MGUS 142 - - SMM 108 - - Newly diagnosed MM - 182 - Relapsed/refractory MM 62 156 19 HMCLs - 48 - Total 312 386 19 Background: Inactivation of p53 by mutation or allelic loss is a rare event in multiple myeloma (MM) at the time of disease diagnosis and believed to be more common in the late stages of the disease. Here we defined the prevalence of p53 deletions in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), newly diagnosed MM, relapsed MM and human myeloma cell lines (HMCLs). Indirect mechanisms of p53 inactivation such as amplification of MDM2, or deletion of CDKN2A (p14ARF) were also investigated. Patients and Methods: A total of 631 MM samples and 48 HMCLs were analyzed for p53 abnormalities (Table 1) and compared with a cohort of plasma cell leukemia (PCL) previously published by us. Interphase fluorescence in situ hybridization (FISH) and highresolution array-based comparative genomic hybridization (aCGH) were used to detect p53 deletions. MDM2 amplification and p14ARF loss were evaluated by aCGH. Overall survival (OS) was estimated through Kaplan Meier method. Results: The prevalence of p53 deletions was 1.4%, 3.7%, 8.9%, 20% and 87.5% in MGUS, SMM, newly diagnosed MM, relapsed MM and HMCLs respectively (Figure 1). Of interest patients in first relapse showed a p53 deletion prevalence of 18% in comparison with 33% for patients in second relapse or more. We obtained p53 deletion status in 8 patients noted to have deletion at the time of relapse and in whom we had previous samples stored. In 7 of 8 cases deletions had been acquired (absent in the previous samples). The median OS for relapsed MM patients with or without p53 deletion were 4.2 months and 37.8 months respectively (p <0.001). MDM 2 amplification and p14ARF loss was detected in 0.4% and 2% of new diagnosed MM, 0.6% and 5.1% of relapsed MM and 2% and 29% of HMCLs respectively. Discussion: Emergence of p53 deletion/mutations denotes progression genetic events in MM. Importantly we show for the first time the overriding importance of p53 deletions as prognostic markers in relapsed MM. A molecular staging system that is based on the presence of p53 inactivation may be more powerful than classifying patients based on the loss of p53. Other abnormalities involving p53 pathway regulators like MDM2 gain and p14ARF loss are infrequent events in MM, even in advanced disease. Figure 1. Prevalence of p53 deletions Figure 1. Prevalence of p53 deletions

Iron overload at the time of bone marrow transplantation has recently been associated with increased mortality. The mechanism by which iron overload negatively impacts survival is unclear. In an effort to better define iron overload and its potential role in the transplant setting, we examined 78 consecutive patients at risk for transfusion-related iron overload (AML, ALL, MDS, and aplastic anemia) undergoing either autologous or allogeneic stem cell transplant at our institution between 1999 and 2004. To estimate transfusion-related iron overload, we devised a novel scoring system based on routinely available laboratory variables obtained prior to transplant. In our scoring system, a single point was awarded for each of the following: number of red cell units transfused prior to transplantation > 25; serum ferritin > 1000 ng/ml; and a semi-quantitative bone marrow iron stain of 6+. The Transfusion Iron Overload Score (range of 0 to 3) predicted for overall survival in our patient sample (Fig. 1, p = 0.0003 by Kaplan-Meier log rank trend) and was more powerful than using serum ferritin alone (p = 0.02). The calculated score retained its significant association with overall survival while controlling for traditional risk factors (age, sex, diagnosis, transplant type, donor relation, and remission status), inflammation (c-reactive protein), and measures of organ damage (transaminases, total bilirubin, INR, and cardiac ejection fraction) (p = 0.04 by Cox proportional-hazards regression). The 27 patients (35%) with a high score (≥ 2) had a dramatically lower median survival of 5.0 months compared to 29.3 months in those with an iron score of less than two (unadjusted hazard ratio of 2.5; 95% CI of 1.6 to 6.3). The observed mortality seen in those with a high score was primarily attributable to treatment-related mortality (p = 0.018) rather than disease-related mortality (p = 0.48). This finding likely accounts for the significant survival impact of iron overload seen in the allogeneic (p = 0.006) but not the autologous (p = 0.48) subgroup. In conclusion, the Transfusion Iron Overload Score served as a strong independent predictor of survival in our stem cell transplant population, identifying patients at significant risk for early treatment-related mortality. Our results lend further support to the notion that iron overload is detrimental in the transplant setting, and for the first time suggest that transfusion-related iron overload increases mortality via a mechanism independent of pre-existing inflammation and end-organ dysfunction. Figure 1. Overall post-transplant survival by the Transfusion Iron Overload Score. Figure 1. Overall post-transplant survival by the Transfusion Iron Overload Score.

Background: The main mechanism of action of rituximab is through antibody-dependent cellular cytotoxicity via Fc receptors for immunoglobulin G. Recently a polymorphism of Fc was reported, which consists in the substitution of phenylalanine for valine in position 158. Patients with homozygous 158 valine/valine (V/V) alleles of Fc showed a higher response rate to rituximab treatment in contrast to patients with phenylalanine/valine (F/V) or homozygous phenilalanine (F/F). It would have clinical value to know which patients could have a greater benefit from the treatment with rituximab.

Background: The main mechanism of action of rituximab is through antibody-dependent cellular cytotoxicity via Fc receptors for immunoglobulin G. Recently a polymorphism of Fc was reported, which consists in the substitution of phenylalanine for valine in position 158. Patients with homozygous 158 valine/valine (V/V) alleles of Fc showed a higher response rate to rituximab treatment in contrast to patients with phenylalanine/valine (F/V) or homozygous phenilalanine (F/F). It would have clinical value to know which patients could have a greater benefit from the treatment with rituximab. Aims: 1-to determine the Fc genotype in patients with Non-Hodgkin lymphoma (NHL) in our population. 2- to analyze the response rate to rituximab of these patients according to the Fc polymorphism. Methods: DNA was isolated from peripheral blood. Genotyping of FCyIIIa polymorphism was performed using a polymerase chain reaction and were confirmed by direct sequencing of the region of interest. To analyse the results the patients were divided into two groups: with valine expression: V/V or V/F, and without valine expression F/F. The response was evaluated after 3 months of treatment and at the end of it. Chi-square and Fisher’s exact tests were used for statistical analysis. Results: 34 patients with NHL: 19 follicular lymphoma, 12 diffuse large B-cell lymphoma, 3 mantle lymphoma. The FcyIIIa polymorphism expression was 11.8% V/V, 52.9% V/F and 35.3% F/F. The complete response (CR) after 3 months of treatment was 50% and 41.7% for V/V-V/F and F/F respectively (not statistically significant (ns)). After end of treatment CR was 86.7% in V/V-V/F and 72.7% in F/F(ns). In patients with follicular lymphoma the CR after end of treatment was 87.5% in V/V-V/F and 79% in F/F (ns). In patients with diffuse large B-cell lymphoma the CR was reached in all patients regardless of polymorphism. Conclusions: FcyIIIa gene polymorphism did not help in predicting response after treatment with rituximab in our group of patients with NHL. A large number of patients would be necessary to draw conclusions, especially in the group with follicular lymphoma.

Background: Two procoagulant factors are associated with the tumor cell: tissue factor (TF) and cancer procoagulant (CP). The activation of the coagulation system can cause thrombosis and it has also been involved in the development of metastasis. Aims: 1- to establish the relationship between the presence and the levels of both procoagulants and the development of thrombosis or disease progression. 2- to analyse if the treatment with chemotherapy increases the levels of TF or CP. Methods: Patients with recent diagnosis of cancer were included in this prospective study. TF and CP determinations were done at diagnosis, at three months after chemotherapy, and at either event of progression or thrombosis. TF evaluation was done through ELISA test (normal value 159 pg/ml) and CP evaluation by a coagulometric method (Gordon et al, Thromb Res1989;56:431–40). In the last test negative values indicate high CP activity. Statistical analysis of the data was performed by two-tailed Fisher exact test and Mann-Withney/Wilcoxon test. Results: 31 patients, 61% female sex, age median 54 years. Digestive cancer 49%, breast 32%, genitourinary 13%, lung 2%. Stages of the disease were: early (I and II) 48% and advanced (III and IV) 52%. Median follow-up was 11.6 months. High levels of TF and CP at diagnosis were seen in 48% and 84% respectively. No patients developed thrombosis. Nine patients had disease progression (29%) and 4 (13%) died. Eigthy eight percent patients of this last group had advanced stages of the disease. The type of tumor more frequently associated with the event was digestive cancer (45%) and genitourinary cancer (33%). TF and CP values were increased by 40% and 50% respectively during chemotherapy treatment. Level of CP at diagnosis predicted progression (-26 sec versus -8 sec in patients with and without progression p 0.006) and also mortality (-34 sec versus -10 sec p0.003). TF at diagnosis predicted neither progression nor mortality. All patients who had progressive disease showed increased levels of TF (388 pg/ml vs 217 pg/ml p0.002) and CP (-63 sec vs -23 sec p.006) with respect to the values at diagnosis. Conclusions: Higher levels of CP at diagnosis are associated with a greater frequency of progression and mortality. No patients developed thrombosis. TF and CP levels were increased at the time of progression with respect to the values obtained at diagnosis. Chemotherapy increases the values of both procoagulants. A larger follow-up and a large number of patients could clarify the role of these procoagulants in the development of disease progression (metastasis).

Iron overload at the time of bone marrow transplantation has recently been associated with increased mortality. The mechanism by which iron overload negatively impacts survival is unclear. In an effort to better define iron overload and its potential role in the transplant setting, we examined 78 consecutive patients at risk for transfusion-related iron overload (AML, ALL, MDS, and aplastic anemia) undergoing either autologous or allogeneic stem cell transplant at our institution between 1999 and 2004. To estimate transfusion-related iron overload, we devised a novel scoring system based on routinely available laboratory variables obtained prior to transplant. In our scoring system, a single point was awarded for each of the following:

Background:Two procoagulant factors are associated with the tumor cell: tissue factor (TF) and cancer procoagulant (CP). The activation of the coagulation system can cause thrombosis and it has also been involved in the development of metastasis.

High dose cytarabine (HiDAC) is the most effective single agent for the treatment of acute myeloid leukemia (AML); clofarabine (CLOF) is also an active agent in AML. Preclinical data suggest synergy between cytarabine and clofarabine. We conducted a two step limited phase I trial of sequential HiDAC (2g/m2 over 3 hours) followed by CLOF (30 or 40 mg/m2 infused over 2 hours), each given daily for 5 days, in adults with AML in first or second relapse or refractory to initial induction chemotherapy. Patients with persistent leukemia on day 12–14 received a second course of HiDAC→CLOF; phase I toxicity evaluation was based on cycle 1 data only. Nine patients (6 men and 3 women) were treated. The median age was 55.5 years (range 29.2 – 68.1). All had relapsed AML; two had prior autologous stem cell transplant. The initial cohort of 3 patients received clofarabine 30 mg/m2 with one dose limiting toxicity (DLT); an additional 3 patients were treated in cohort 1. The second cohort was treated with CLOF 40 mg/m2, the target dose for a planned phase II trial of HiDAC→CLOF. Hematologic toxicities and infections were not considered DLT. In the first cohort (30 mg/m2; n = 6) there was 1 DLT - grade 4 skin rash in a patient who subsequently died on day 17 with sepsis-related multi-organ failure; 3 patients had reversible grade 3 elevations in AST/ALT, 1 had grade 3 skin toxicity. In cohort 2 (40 mg/m2 ; n = 3) there was no DLT; 1 patient had grade 3 AST/ALT; 2 had grade 3 skin. Three of nine patients received a second course of induction HiDACCLOF. Two of six patients in cohort 1 achieved complete remission (CR), 1/3 patients in cohort 2 achieved CRi(CRp). Two of three CR/CRi patients received one course and one received two courses of HiDAC→CLOF induction. Conclusion: HiDAC→CLOF was associated with transient elevation in AST/ALT (4/9) and skin rash (3/9; primarily extensive palmar/plantar); skin toxicity appeared especially prominent in patients with palmar/plantar toxicity during prior therapy with HiDAC. Toxicities (other than skin) were comparable to other salvage regimens for relapsed and refractory AML. This combination is active in relapsed AML with 3/9 CR/CRp. A phase II trial of HiDAC→CLOF is underway; prophylactic intravenous hydrocortisone has been incorporated in an attempt to decrease skin toxicity.

High dose cytarabine (HiDAC) is the most effective single agent for the treatment of acute myeloid leukemia (AML); clofarabine (CLOF) is also an active agent in AML. Preclinical data suggest synergy between cytarabine and clofarabine. We conducted a two step limited phase I trial of sequential HiDAC (2g/m2over 3 hours) followed by CLOF (30 or 40 mg/m2infused over 2 hours), each given daily for 5 days, in adults with AML in first or second relapse or refractory to initial induction chemotherapy. Patients with persistent leukemia on day 12–14 received a second course of HiDAC→CLOF; phase I toxicity evaluation was based on cycle 1 data only. Nine patients (6 men and 3 women) were treated. The median age was 55.5 years (range 29.2 – 68.1). All had relapsed AML; two had prior autologous stem cell transplant. The initial cohort of 3 patients received clofarabine 30 mg/m2with one dose limiting toxicity (DLT); an additional 3 patients were treated in cohort 1. The second cohort was treated with CLOF 40 mg/m2, the target dose for a planned phase II trial of HiDAC→CLOF. Hematologic toxicities and infections were not considered DLT. In the first cohort (30 mg/m2; n = 6) there was 1 DLT - grade 4 skin rash in a patient who subsequently died on day 17 with sepsis-related multi-organ failure; 3 patients had reversible grade 3 elevations in AST/ALT, 1 had grade 3 skin toxicity. In cohort 2 (40 mg/m2; n = 3) there was no DLT; 1 patient had grade 3 AST/ALT; 2 had grade 3 skin. Three of nine patients received a second course of induction HiDACCLOF. Two of six patients in cohort 1 achieved complete remission (CR), 1/3 patients in cohort 2 achieved CRi(CRp). Two of three CR/CRi patients received one course and one received two courses of HiDAC→CLOF induction. Conclusion: HiDAC→CLOF was associated with transient elevation in AST/ALT (4/9) and skin rash (3/9; primarily extensive palmar/plantar); skin toxicity appeared especially prominent in patients with palmar/plantar toxicity during prior therapy with HiDAC. Toxicities (other than skin) were comparable to other salvage regimens for relapsed and refractory AML. This combination is active in relapsed AML with 3/9 CR/CRp. A phase II trial of HiDAC→CLOF is underway; prophylactic intravenous hydrocortisone has been incorporated in an attempt to decrease skin toxicity.

Anemia is prevalent in older adults. Although the majority of cases are due to nutritional deficiencies, chronic inflammation, or renal disease, anemia remains unexplained in about one third of cases (Guralnik et al., Blood, 2004, p2263). Copper deficiency has been reported as a rare cause of anemia and neutropenia. We hypothesized that copper deficiency could account for some cases of unexplained anemia in adults. To test this hypothesis, we examined the relationship between serum copper and unexplained anemia in 11,240 adults (15 years and older) who participated in the Second National Health and Nutrition Examination Survey (NHANES II). Anemia was defined as hemoglobin level below 13.5 g/dL in males and below 11.5 g/dL in females. In this study population, 638 or 5.7% of adults were anemic. Of these, anemia was unexplained by vitamin B12, folate, or iron deficiencies, chronic inflammation, or renal disease in 421 or 3.7% of the total subjects studied. Thus, unexplained anemia constituted approximately 65% of all anemia cases in this sample of US adults. Median serum copper level was significantly higher in all anemia cases (126 µg/dL, p <0.0001), in explained anemia (133 µg/dL, p<0.0001), and in unexplained anemia (122 µg/dL, p<0.02) as compared to non-anemic adults (119 µg/dL, ref). Spline regression showed a U-shaped relationship between serum copper levels and unexplained anemia. The prevalence of unexplained anemia increased significantly at both lower and higher copper levels as compared to the median (119 µg/dL): odds ratios for unexplained anemia were 1.2 (95% CI: 1.1–1.3) in the association with a decrease of serum copper from median to 10th percentile (92 µg/dL) and 1.8 (95% CI: 1.6–2.2) in the association with an increase of serum copper to 90th percentile (161 µg/dL). This study likely underestimates the prevalence of anemia of chronic illness, as only a single marker of inflammation (serum iron) was available to capture these subjects. Furthermore, copper levels are known to be elevated in inflammation. Thus, unrecognized cases of anemia of chronic illness might explain the observed association between high serum copper levels and unexplained anemia. On the other hand, subjects with low serum copper levels and unexplained anemia might have nutritional deficiency. In conclusion, this analysis of the NHANES II data confirms (1) that unexplained anemia is common among the US adults, and (2) that both lower and higher than median serum copper levels are positively associated with unexplained anemia. These findings support the need for further study of copper status in adults with unexplained anemia.

Copper deficiency is a rare cause of anemia. Copper deficiency anemia has been described in patients on total parenteral or forced enteral nutrition deficient in copper, post gastric resection, short bowel syndrome (from prior intestinal resections or gastric by-pass) or malabsorptive disease states such as Crohn’s or celiac disease, and in patients taking excessive amounts of zinc. We have shown that in the NHANES II adult population, low copper levels are associated with increased risk of unexplained anemia (Molnar et al., abstract submitted at this meeting). We recently started to routinely check serum copper levels in patients referred to our hematology clinic for evaluation of unexplained anemia. Between December 2003 and March 2005, we found seven patients who had lower than normal copper levels and unexplained anemia. Six out of the seven patients were treated with copper supplementation and had improvement in their hematological abnormalities. Most patients had undergone an expensive, unproductive diagnostic evaluation before copper deficiency was diagnosed. In five of the seven patients, the anemia was severe. Two patients were red cell transfusion-dependent. Six out of seven patients had associated granulocytopenia. Even in the presence of severe granulocytopenia, recurrent, clinically significant infections were rare. Thrombocytopenia was present in two patients. Peripheral blood findings were non-specific. Bone marrow findings varied with two patients demonstrating normal marrow histology, two patients with hypercellular marrow, one exhibited myeloid maturation arrest, and the remaining two patients demonstrating frank dysplastic changes with ring sideroblasts. Five out of seven patients had cytogenetic examination of the bone marrow revealing a normal karyotype. The response to copper treatment was frequently rapid, and improvement in anemia and neutropenia was evident within a few weeks. The characteristics of our patient population are outlined in the table below. In conclusion, copper deficiency anemia is not uncommon in a hematology practice. Copper deficiency should be considered in patients presenting with unexplained anemia associated with neutropenia, a history of malabsorption, gastrointestinal resection or gastric bypass, nephrotic syndrome, or dysplastic bone marrow changes with a normal karyotype analysis. The details of clinical and pathological features of the patients will be presented in detail at the meeting. Copper Deficiency Anemia Patient Characteristics Case Age /Sex Anemia Neutropenia Bone Marrow Findings Co-morbid conditions Copper Level μg/dL Hematological Response to Copper Treatment 1 71 /Female Transfustion dependent Yes Hypercellular Chronic diarrhea, nephrotic syndrome, diabetes mellitus < 10 Partial 2 60 /Male Yes Yes Normal Chronic diarrhea, renal failure status-post renal transplant, celiac sprue 41 Not treated Complete 3 33 /Female Mild Yes Ringed sideroblasts, myeloid and erythroid dysplasia None 37 Complete response 4 32 /Female Yes Yes Myeloid maturation arrest Gastric bypass surgery < 10 Complete response 5 45 /Female Transfusion dependent Yes Ringed sideroblasts, Dyserythropoiesis Billroth II operation < 10 Complete response 6 42 /Female Mild Yes Hypercellular, myeloid maturation arrest None < 10 Complete response 7 51 /Male Yes No Normal Acute myeoid leukemia, short bowel syndrome 45 Partial

Anemia is prevalent in older adults. Although the majority of cases are due to nutritional deficiencies, chronic inflammation, or renal disease, anemia remains unexplained in about one third of cases (Guralnik et al., Blood, 2004, p2263). Copper deficiency has been reported as a rare cause of anemia and neutropenia. We hypothesized that copper deficiency could account for some cases of unexplained anemia in adults. To test this hypothesis, we examined the relationship between serum copper and unexplained anemia in 11,240 adults (15 years and older) who participated in the Second National Health and Nutrition Examination Survey (NHANES II). Anemia was defined as hemoglobin level below 13.5 g/dL in males and below 11.5 g/dL in females. In this study population, 638 or 5.7% of adults were anemic. Of these, anemia was unexplained by vitamin B12, folate, or iron deficiencies, chronic inflammation, or renal disease in 421 or 3.7% of the total subjects studied. Thus, unexplained anemia constituted approximately 65% of all anemia cases in this sample of US adults.

Imatinib mesylate, an inhibitor of BCR/ABL tyrosine kinase, has remarkable activity in CML resulting in up to 87% major cytogenetic response, but further treatment is needed for the remaining 13% of patients and others who progress on therapy. Studies suggest that hematopoietic growth factors (HGF) induce the selective terminal differentiation of CML progenitors at concentrations that allow optimal growth of normal progenitors. Treatment with HGF may be able to stop the expansion of BCR/ABL positive cells while promoting the expansion of non-leukemic myeloid cells, thereby encouraging a cytogenetic response. Initially, these HGF were used to improve the safety of chemotherapies but had not been studied specifically to improve cytogenetic response. Our institution has an open phase II study of GM-CSF in patients with chronic phase CML who have not achieved a complete cytogenetic remission after at least 9 months of therapy. Our first patient on study was a woman originally diagnosed in 4/1996 with a variant Philadelphia chromosome [t(9;9;22)] positive CML. She was initially started on hydroxyurea and was then switched to interferon-α for approximately 5 ½ years. When imatinib became available her interferon was discontinued and she was treated with imatinib at 400 mg daily. Bone marrow biopsy 5/2003 was morphologically normal but on cytogenetic analysis all cells contained the same aberrant karyotype. Her imatinib therapy was complicated by pancytopenia requiring frequent transfusions. She had received approximately 22 units of packed red blood cells over 11 months. Her white count had been running in the 1,500 to 2,500/μL range and her platelet count had dropped to 64,000/μL. She presented to us 1/2004 at the age of 56 and was evaluated for a reduced-intensity allogeneic transplant but did not have any donors available in her family. She was not felt to be a candidate for an unrelated donor transplant because of co-morbid risks. Given her continued cytogenetic abnormalities and lack of transplant candidacy she was enrolled in our GM-CSF trial March 2004. At the time of entry onto study she had been on imatinib for 2 1/2 years with no cytogenetic improvement. She began treatment with daily GM-CSF 100 μg/m2 in addition to her imatinib. Cytogenetic studies revealed a gradual increase in percentage of normal cells from start, 4 months, 9 months, and 15 months at 0%, 10%, 55%, and 85% respectively. She also had reversal of her pancytopenia and has not required transfusion of blood or platelets since enrollment. She developed an eosinophilia which we attribute to GM-CSF. Clinical studies have examined the use of GM-CSF in addition to interferon alpha. However, with imatinib becoming the new standard of care it is more relevant to examine the combined effect of imatinib and GM-CSF. The use of GM-CSF with imatinib in CML still needs more evaluation, but it appears to be a promising approach to improve cytogenetic response.

Transfusional iron overload is common in survivors of acute leukemia and hematopoietic stem cell transplantation and might cause long-term liver dysfunction. Routine evaluation for iron overload in such patients is recommended because excess iron can be readily removed from the body via phlebotomy or chelation. Iron overload might be associated with worse survival after stem cell transplantation in these diseases. We were interested in determining whether levels of the iron binding protein ferritin or the serum transferrin receptor (TfR) were predictive for survival. In a prospective study, we examined the correlation between iron parameters at the time of transplantation and overall survival. Serum ferritin, transferrin saturation, and TfR were measured before preparative regimen on patients who underwent hematopoietic stem cell transplantation between 1999 and 2004 for the diagnosis of aplastic anemia, MDS or acute leukemia (n=79). We used the number of transfusions before transplantation as a measure of iron load. Among these iron markers, serum ferritin correlated the most with the number of transfusions, regardless of remission status. High ferritin (>1,500 ng/ml), low TfR (≤4 μg/ml) and low TfR/log ferritin ratio (≤2) were associated with shorter survival (p=0.005, 0.04, and 0.001 respectively)(Figure 1). Among acute leukemia patients in remission, there was no difference in overall survival between patients with high or low iron markers. Markers of iron excess (serum ferritin >1,500 ng/ml, TfR/log ferritin ratio ≤2) at the time of stem cell transplantation is associated with shorter survival in MDS, aplastic anemia and acute leukemia with active disease. These results demonstrate that knowledge of patient ferritin and TfR levels can aid in risk stratification. The results also suggest that patients with high levels of ferritin may benefit from iron chelation before treatment.

Transfusional iron overload is common in survivors of acute leukemia and hematopoietic stem cell transplantation and might cause long-term liver dysfunction. Routine evaluation for iron overload in such patients is recommended because excess iron can be readily removed from the body via phlebotomy or chelation. Iron overload might be associated with worse survival after stem cell transplantation in these diseases. We were interested in determining whether levels of the iron binding protein ferritin or the serum transferrin receptor (TfR) were predictive for survival. In a prospective study, we examined the correlation between iron parameters at the time of transplantation and overall survival. Serum ferritin, transferrin saturation, and TfR were measured before preparative regimen on patients who underwent hematopoietic stem cell transplantation between 1999 and 2004 for the diagnosis of aplastic anemia, MDS or acute leukemia (n=79). We used the number of transfusions before transplantation as a measure of iron load. Among these iron markers, serum ferritin correlated the most with the number of transfusions, regardless of remission status. High ferritin (>1,500 ng/ml), low TfR (≤4 μg/ml) and low TfR/log ferritin ratio (≤2) were associated with shorter survival (p=0.005, 0.04, and 0.001 respectively)(Figure 1). Among acute leukemia patients in remission, there was no difference in overall survival between patients with high or low iron markers. Markers of iron excess (serum ferritin >1,500 ng/ml, TfR/log ferritin ratio ≤2) at the time of stem cell transplantation is associated with shorter survival in MDS, aplastic anemia and acute leukemia with active disease. These results demonstrate that knowledge of patient ferritin and TfR levels can aid in risk stratification. The results also suggest that patients with high levels of ferritin may benefit from iron chelation before treatment. Figure 1: Overall Survival based on iron parameters (A) Serum Ferritin (B) Serum Transferrin Receptor (C) TfR (mcg/ml) divided by log ferritin (ng/ml) Figure 1:. Overall Survival based on iron parameters (A) Serum Ferritin (B) Serum Transferrin Receptor (C) TfR (mcg/ml) divided by log ferritin (ng/ml)

Copper deficiency is a rare cause of anemia. Copper deficiency anemia has been described in patients on total parenteral or forced enteral nutrition deficient in copper, post gastric resection, short bowel syndrome (from prior intestinal resections or gastric by-pass) or malabsorptive disease states such as Crohn's or celiac disease, and in patients taking excessive amounts of zinc. We have shown that in the NHANES II adult population, low copper levels are associated with increased risk of unexplained anemia (Molnar et al., abstract submitted at this meeting). We recently started to routinely check serum copper levels in patients referred to our hematology clinic for evaluation of unexplained anemia. Between December 2003 and March 2005, we found seven patients who had lower than normal copper levels and unexplained anemia. Six out of the seven patients were treated with copper supplementation and had improvement in their hematological abnormalities. Most patients had undergone an expensive, unproductive diagnostic evaluation before copper deficiency was diagnosed. In five of the seven patients, the anemia was severe. Two patients were red cell transfusion-dependent. Six out of seven patients had associated granulocytopenia. Even in the presence of severe granulocytopenia, recurrent, clinically significant infections were rare. Thrombocytopenia was present in two patients. Peripheral blood findings were non-specific. Bone marrow findings varied with two patients demonstrating normal marrow histology, two patients with hypercellular marrow, one exhibited myeloid maturation arrest, and the remaining two patients demonstrating frank dysplastic changes with ring sideroblasts. Five out of seven patients had cytogenetic examination of the bone marrow revealing a normal karyotype. The response to copper treatment was frequently rapid, and improvement in anemia and neutropenia was evident within a few weeks. The characteristics of our patient population are outlined in the table below. In conclusion, copper deficiency anemia is not uncommon in a hematology practice. Copper deficiency should be considered in patients presenting with unexplained anemia associated with neutropenia, a history of malabsorption, gastrointestinal resection or gastric bypass, nephrotic syndrome, or dysplastic bone marrow changes with a normal karyotype analysis. The details of clinical and pathological features of the patients will be presented in detail at the meeting.

Imatinib mesylate, an inhibitor of BCR/ABLtyrosine kinase, has remarkable activity in CML resulting in up to 87% major cytogenetic response, but further treatment is needed for the remaining 13% of patients and others who progress on therapy. Studies suggest that hematopoietic growth factors (HGF) induce the selective terminal differentiation of CML progenitors at concentrations that allow optimal growth of normal progenitors. Treatment with HGF may be able to stop the expansion of BCR/ABLpositive cells while promoting the expansion of non-leukemic myeloid cells, thereby encouraging a cytogenetic response. Initially, these HGF were used to improve the safety of chemotherapies but had not been studied specifically to improve cytogenetic response. Our institution has an open phase II study of GM-CSF in patients with chronic phase CML who have not achieved a complete cytogenetic remission after at least 9 months of therapy. Our first patient on study was a woman originally diagnosed in 4/1996 with a variant Philadelphia chromosome [t(9;9;22)] positive CML. She was initially started on hydroxyurea and was then switched to interferon-α for approximately 5 ½ years. When imatinib became available her interferon was discontinued and she was treated with imatinib at 400 mg daily. Bone marrow biopsy 5/2003 was morphologically normal but on cytogenetic analysis all cells contained the same aberrant karyotype. Her imatinib therapy was complicated by pancytopenia requiring frequent transfusions. She had received approximately 22 units of packed red blood cells over 11 months. Her white count had been running in the 1,500 to 2,500/μL range and her platelet count had dropped to 64,000/μL. She presented to us 1/2004 at the age of 56 and was evaluated for a reduced-intensity allogeneic transplant but did not have any donors available in her family. She was not felt to be a candidate for an unrelated donor transplant because of co-morbid risks. Given her continued cytogenetic abnormalities and lack of transplant candidacy she was enrolled in our GM-CSF trial March 2004. At the time of entry onto study she had been on imatinib for 2 1/2 years with no cytogenetic improvement. She began treatment with daily GM-CSF 100 μg/m2in addition to her imatinib. Cytogenetic studies revealed a gradual increase in percentage of normal cells from start, 4 months, 9 months, and 15 months at 0%, 10%, 55%, and 85% respectively. She also had reversal of her pancytopenia and has not required transfusion of blood or platelets since enrollment. She developed an eosinophilia which we attribute to GM-CSF. Clinical studies have examined the use of GM-CSF in addition to interferon alpha. However, with imatinib becoming the new standard of care it is more relevant to examine the combined effect of imatinib and GM-CSF. The use of GM-CSF with imatinib in CML still needs more evaluation, but it appears to be a promising approach to improve cytogenetic response.

Background: The benefits and harms of platelet transfusions in critically ill patients are unclear. Objectives: To describe the frequency of, indications for, and effects of platelet transfusions in patients admitted to a medical- surgical intensive care unit (ICU). Design: Single center cohort study (January 2001 to January 2002). Methods: We identified all patients who developed thrombocytopenia (platelet count <150 x 109/l) during their ICU admission from a prospective study which enrolled consecutive adults admitted to ICU with an expected length of stay ≥72 hours, and which excluded patients with trauma, orthopedic surgery, cardiac surgery, pregnancy, or receiving palliative care. Retrospectively, using a priori criteria, bleeding severity and the indications for platelet transfusions were assessed; 23.7% of bleeding events and 89.5% of transfusion indications were reviewed in duplicate independently. Initial agreement was calculated using Cohen’s unweighted kappa and all assessments of bleeding severity and transfusion indications were adjudicated by third person. Results: Of 261 ICU patients, 118 (45.2%) had thrombocytopenia. Initial agreement between primary reviewers for assessments of bleeding severity was good (k= 0.69), and for indications for platelet transfusions was poor (k=0.35); consensus was achieved in all cases. One third of thrombocytopenic patients had major bleeding (37/118, 31.4%), and one fifth had minor bleeding (24/118, 20.3%). Among thrombocytopenic patients, 27/118 (22.9%) received a total of 76 platelet transfusions, 24 (31.6%) of which were administered to treat bleeding, and 52 (68.4%) of which were to prevent bleeding. Of the prophylactic platelet transfusions, 18/52 (34.6%) preceded invasive procedures. The mean ± SD platelet count prior to therapeutic platelet transfusions was 54 ± 40 x109/l; for peri-procedural transfusions, 55 ± 38 x109/l; and for other prophylactic platelet transfusions, 37 ± 21 x109/l. Most transfusions (69/76, 91%) were administered as pools of 5.2 ± 1.2 random donor platelets, and few (7/76, 9.2%) were apheresis platelets. A single platelet transfusion (pool of 5 random donor units or 1 apheresis unit) resulted in a platelet increment of 14 ± 29 x109/l at 6.6 ± 5.9 hours following transfusion. No rise in platelet count was observed following 17 transfusions given to 13 patients. Conclusions: Platelet transfusions are frequently administered to thrombocytopenic critically ill patients, and, although the indication is not always clear, the most common reason is to prevent bleeding. Nearly half of transfused ICU patients were refractory to one or more platelet transfusion. Further prospective studies are needed on the indications for, and effects of, platelet transfusions in the ICU setting.

Background:The benefits and harms of platelet transfusions in critically ill patients are unclear.

Being self-renewable, the hematopoietic stem cells are seemingly unlimited. Scarcity of donor tissues, unlike the solid organ transplantation, is not a limiting criterion for stem cell transplantation (SCT). We are interested in the public opinion of the upper age limit for transplantation and a pilot questionnaire survey was designed. The survey was approved by IRB and conducted among ASCO participants who visited one of our posters in 6/2004. Forty-seven questionnaires were returned on site. Among those who completed the respective questions, 63% were males, median age was 39 (range 30–60) years, 81% were medical doctors, 51% currently worked in the US, 50% graduated their highest academic degrees in the US, and median time interval from graduation was 8.5 (range 1–37) years. When asked to comment on "There should be an upper age limit to transplantation", 11% strongly and 39% somewhat disagreed, 11% were neutral to the statement, and 35% somewhat and 4% strongly agreed. Among those who completed the respective questions, 52% and 62% felt that 70 years should be the upper age limit for kidney and autologous SCT respectively; whereas 61%, 71% and 68% felt that 60 years should be the upper age limit for liver, heart/lung and allogeneic SCT respectively. "Should transplantation be offered to people older than 65?" For kidney and autologous SCT, the majority (64% and 94% respectively) answered yes. For liver, heart/lung and allogeneic SCT, the majority (60%, 56% and 59% respectively) answered no. "Should transplantation be offered to people older than 70?" For autologous SCT, 52% still answered yes. For kidney, liver, heart/lung and allogeneic SCT, the majority (60%, 76%, 77% and 83% respectively) answered no. To investigate specifically SCT, we asked, "Should autologous SCT be offered in the following scenario - 5-year overall survival with transplantation is 50% compared to <20% without transplantation and early transplant mortality is 5%?" Over 86% felt that autologous SCT should be offered in people 70 years of age or younger. When we asked, "Should allogeneic SCT be offered in the following scenario - 5-year overall survival with transplantation is 50% compared to <20% without transplantation and early transplant mortality is 20%?" Over 86% felt that allogeneic SCT should only be offered to people 60 years of age or younger. We analyzed the possible correlation of various factors, age, gender, occupation, current residence, country of graduation, and time interval of graduation, to their comments to the statement, "There should be an upper age limit to transplantation", by chi-square test. Interestingly, the country of graduation, but not the current residence, correlated with their answers. Participants who graduated from outside US are more likely to agree (52%) to the statement than those who graduated in the US (25%) (chi sq p= 0.02). CONCLUSION: In this pilot study, about half of the participants strongly and somewhat disagreed that there should be upper age limit to transplantation. Among those who completed the questions, 70 years should be the upper age limit for kidney and autologous SCT, and 60 years for liver, heart/lung and allogeneic SCT. In addition, there is a significant difference in the opinion of U.S. versus non-U.S. graduates whether there should be an upper age limit for transplantation. Further study is required to confirm these findings.

Being self-renewable, the hematopoietic stem cells are seemingly unlimited. Scarcity of donor tissues, unlike the solid organ transplantation, is not a limiting criterion for stem cell transplantation (SCT). We are interested in the public opinion of the upper age limit for transplantation and a pilot questionnaire survey was designed. The survey was approved by IRB and conducted among ASCO participants who visited one of our posters in 6/2004. Forty-seven questionnaires were returned on site. Among those who completed the respective questions, 63% were males, median age was 39 (range 30–60) years, 81% were medical doctors, 51% currently worked in the US, 50% graduated their highest academic degrees in the US, and median time interval from graduation was 8.5 (range 1–37) years. When asked to comment on "There should be an upper age limit to transplantation", 11% strongly and 39% somewhat disagreed, 11% were neutral to the statement, and 35% somewhat and 4% strongly agreed. Among those who completed the respective questions, 52% and 62% felt that 70 years should be the upper age limit for kidney and autologous SCT respectively; whereas 61%, 71% and 68% felt that 60 years should be the upper age limit for liver, heart/lung and allogeneic SCT respectively. "Should transplantation be offered to people older than 65?" For kidney and autologous SCT, the majority (64% and 94% respectively) answered yes. For liver, heart/lung and allogeneic SCT, the majority (60%, 56% and 59% respectively) answered no. "Should transplantation be offered to people older than 70?" For autologous SCT, 52% still answered yes. For kidney, liver, heart/lung and allogeneic SCT, the majority (60%, 76%, 77% and 83% respectively) answered no. To investigate specifically SCT, we asked, "Should autologous SCT be offered in the following scenario - 5-year overall survival with transplantation is 50% compared to <20% without transplantation and early transplant mortality is 5%?" Over 86% felt that autologous SCT should be offered in people 70 years of age or younger. When we asked, "Should allogeneic SCT be offered in the following scenario - 5-year overall survival with transplantation is 50% compared to <20% without transplantation and early transplant mortality is 20%?" Over 86% felt that allogeneic SCT should only be offered to people 60 years of age or younger. We analyzed the possible correlation of various factors, age, gender, occupation, current residence, country of graduation, and time interval of graduation, to their comments to the statement, "There should be an upper age limit to transplantation", by chi-square test. Interestingly, the country of graduation, but not the current residence, correlated with their answers. Participants who graduated from outside US are more likely to agree (52%) to the statement than those who graduated in the US (25%) (chi sq p= 0.02).