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Background .There has been a decrease in the use of unrelated cord blood transplants (UCBT) in the past years: this is probably due to slow hematologic and immune recovery, resulting in a relatively high non relapse mortality (NRM). The addition of anti-thymocyte globulin (ATG) in the conditioning prevents graft versus host disease (GvHD) but makes immune recovery very slow. In addition there is a growing competition of unmanipulated haploidentical transplants. Aim of the study. We have opened a pilot study to test whether high dose post-transplant cyclophosphamide (PT-CY) would prevent GvHD but still allow for robust immune and hematologic recovery . Methods. We have grafted 10 patients with an unrelated CB unit and PT-CY. The conditioning regimen was thiotepa (10 mg/kg), busulfan 9.6 mg/kg and fludarabine 150 mg/m^2 (TBF). GvHD prophylaxis was cyclosporin (CSA) starting day 0 (3 mg/kg/day(i.v.), mycophenolate (MMF) 30 mg/kg starting day +1 (p.o) , and PT-CY 30 mg/kg days +3 and +5. The median patients' age was 58 (43-66), and the median weight was 75 kg (54-85) the diagnosis was AML in 8 patients, Ph'+ALL in one and RAEB in one patient; 6 patients were in remission and 4 had active disease. CB units. The HLA matching of the CB unit was 5/8 antigens/alleles (A,B,C,DRB1) in six patients, 4/8 in two and 2/8 in one. The median nucleated cell dose was 3.1x10^7/kg (range 1.8- 4.5). The ABO was mismatched in all 10 patients. Hematologic recovery: median time to neutrophils 0.5x10^9/l was day 23 days (range 17-27) and the median time to a platelet count of 20x10^9/L was 38 days (range 34-40). The median counts on day +50 were as follows: Hb 9,1 gr/dL (range 8.7-11.1), Neutrophils 2,3 x10e9/L (range 1-5), PLTs 56 x10e9/L (10-90). One patient failed to engraft and received a second transplant from an unrelated donor, which was successful. No patient developed pure red cell aplasia despite 9/10 being ABO major mismatched. CD4 recovery : the median CD4 count on day +50 was 74 /cmm (range 67-116) and on day +100 it was 111/cmm(range 100-136). CMV pre-emptive therapy occurred in 3/6 evaluable patients Outcome: two patients with advanced disease, died early of infections, within day +20. GvHD was seen in 1 patient as a transient rash. No patient was treated for GvHD. No patient developed chronic GvHD. No patient relapsed. Eight patients survive in remission, with a median follow up of 6 months, and a projected one year actuarial survival of 80%. Readmissions were extremely rare. Conclusions. These first 10 patients suggest that UCBT followed by PT-CY, CSA, MMF, as GvHD prophylaxis is feasible and leads to encouraging hematologic and immunologic recovery. We were particularly impressed with the lack of GvHD, the absence of relapses and the good quality of life. Figure Disclosures No relevant conflicts of interest to declare.

Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates.

Two forms of NADH-cytochrome b5 reductase (b5R), an erythrocyte-restricted soluble form, active in methemoglobin reduction, and a ubiquitous membrane-associated form involved in lipid metabolism, are produced from one gene. In the rat, the two forms are generated from alternative transcripts differing in the first exon, however, biogenesis of human b5R was less understood. Recently, two different transcripts (M and S), differing in the first exon were also described in humans. Here, we have investigated the tissue-specificity and the role of the S-transcript in the generation of soluble b5R. By RNase protection assays designed to simultaneously detect alternative b5R transcripts in the same sample, the S transcript was undetectable in nonerythroid and in erythroleukemic K562 cells induced to differentiate, but was present in terminal erythroblast cultures, and represented a major b5R transcript in reticulocytes. Analysis of the translation products of the M- and S-transcripts in HeLa cells transfected with the corresponding cDNAs demonstrated that the S-transcript generates soluble b5R, presumably from an internal initiation codon. Our results indicate that the S-transcript is expressed at late stages of erythroid maturation to generate soluble b5R.

Human erythrocyte pyruvate kinase plays an important role in erythrocyte metabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutations have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and purified. The tetrameric protein exhibited properties characteristic of authentic human erythrocyte pyruvate kinase, including response to substrate, phosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleaved and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, which is the most frequently occurring mutation among Northern European population, was also generated and purified. The mutant protein retained its binding capacity to and could be activated by fructose 1,6-bisphosphate and showed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wild-type enzyme. Conversely, the mutant protein has a dramatically decreased stability toward heat and is more susceptible to ATP inhibition. The enzyme instability decreases the enzyme level in the cell, accounting for the clinically observed “pyruvate kinase deficiency” of patients who are homozygous for this mutation. This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase. These findings will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.

We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.

Background: Continuing growth in the incidence of leukemia suggests a possible environmental etiology correlated to the increase of environmental pollution. Recently, environmental particulate pollution (EPP) has been declared by IARC a Class I carcinogenic agent; it looks reasonable to presume that not only chemicals like benzene and its derivatives, but also other components like EPP could be worth of study. No specific researches have up to now focused the role of EPP on acute myeloid leukemia; we thus have identified a suitable instrumentation and protocol to show the presence and composition of particulate matter in blood samples of patients affected by acute myeloid leukemia patients and in healthy controls. Methods: 38 peripheral blood samples (19 acute myeloid leukemia, 19 healthy controls) were analyzed by means of an Environmental Scanning Electron Microscopy (ESEM) coupled with an Energy Dispersive Spectroscopy (EDS) a sensor capable of identifying the composition of micro- and nano-particles of exogenous nature in pathological tissues (applied for the first time in the current study on blood samples). The results were statistically treated with unpaired two-tailed Student's t-test, MANOVA and Principal Component Analysis. Results: A consistent quantity of micron-, submicron- and nano-sized foreign bodies (from 20 micron down to 100nm) was documented in 18/19 AML cases, whereas they were absent or rare in the controls. The particles appeared as singlet and aggregates (ranging from 5 to 20micron), either in close contact with blood elements or interacting with plasma. Some reacted with blood proteins thus forming composite clusters. A total of 141 aggregates (median 8, range 0-18) in AML, compared to a total of 12 aggregates in controls (median 1, range 0-3) were counted. The aggregate analysis showed variable sizes and number of particles, with a total of 5394 particles in leukemia cases compared to a total of 207 in controls. The total numbers of aggregates and particles were statistically different between cases and controls (MANOVA, P Conclusion: In conclusion, we demonstrated the exposure of a subset of AML patients to environmental contaminants, with invasive character in the human body, not biocompatible and biopersistent. AML, as well as myelodysplastic syndromes, are derived from precursor cells critical in innate immunity, that submicronic particles could have triggered. New etiopathogenic hypotheses involving an interaction among sub-micron and nanosized particles with blood components are under evaluation. Disclosures No relevant conflicts of interest to declare.

Introduction. The granulocyte transfusions (GTXs) are used to booster antimicrobial drugs in severely neutropenic hematological patients. However, the optimal GTX dose and the actual efficacy of this practice are debated. Methods. We retrospectively evaluated the infection-attributable mortality (IAM, i.e. the mortality at 30 days after the last GTX) in 84 consecutive patients with hematological malignancies receiving GTXs (January 2009- December 2014). The indications for GTXs were i) presence of absolute neutrophil count (ANC) Results. Among 84 patients, 101 infectious episodes requiring GTXs were recorded (422 transfusions in total). Patients characteristics are summarized in Table I. Bacterial infections were documented in 94 episodes (Klebsiella pneumonia in 35 cases, Escherichia coli in 16 and Pseudomonas aeruginosa in 13), invasive fungal infections (IFI) in 34 cases (including 18 pulmonary aspergillosis and 14 candidemia); 8 cases were considered as FUO. The infection was mono-microbial in 60 cases and poly-microbial in 33. Sepsis occurred in 67 cases. The overall IAM was 26.7 % (27 deaths among 101 infective episodes). At univariate analysis we failed to detect statistical association between IAM and several evaluated variables, either patient-related (age, sex, diagnosis, status of disease, allo-HSCT, aplasia duration) or infection-related (bacterial infection or IFI, sepsis, XDR, G-SCF concurrent administration) or GTX-related (number of GTXs received, PMN /Kg/course, PMN/Kg/day of neutropenia). However, when we grouped patients according to the value of the median dose of PMN per transfusion, we found that patients receiving 1.5 - 3 x 10^8/kg (GTXs A) had a lower IAM than patients receiving less than 1.5 (GTXs B) or more than 3 x 10^8 /kg (GTXs C) (15,7%, 35,3% and 44,4%, for GTXs A, B and C, respectively, p=0,014 at chi-square test). The dose's cut off were derived from the Guide to the preparation, Use and Quality assurance of Blood Components of the European Committee on Blood Transfusion (16th Edition). If the analysis was carried out by pooling together GTXs B and C, the association between PMN dose and IAM was even more pronounced (p value =0.006 at Fisher test for GTXs A versus GTXs B+C). At Kaplan-Meier analysis, the median survival was 59 days for GTXs A-patients and 30 days for GTXs B+C-patients (p =0,010). When patients with bacterial of fungal infections were separately evaluated, the effect of PMN dose on IAM was confirmed in bacterial (n=54, p=0,008) but not in fungal (n=23, p=0,588) infections. We then introduced the PMN dose (GTXs A or GTXs B+C) in a Cox proportional-hazards regression model together with variables with p Conclusions. These findings suggest that appropriate GTX doses can improve the post-infection survival of severely neutropenic hematological patients. Transfusion-related immunomodulation, leukostasis or transfusion-associated GVHD may underlie the detrimental effect of high PMN doses and deserve to be better explored. Table 1. Clinical characteristics of 84 patients treated with GTXs. A total of 101 courses were recorded. Characteristics Age (years, median value range) 46 (20-74) Male/Female 54/30 Underlying disease (n, %)Acute myeloid leukemiaLymphomaAcute lymphoblastic leukemiaMyelodysplastic syndromeMultiple myelomaChronic lymphocytic leukemia 63 (75%)12 (14%)5 (6%)2 (3%)1 (1%)1 (1%) Disease status at PMN transfusion (n, %)OnsetRelapse/resistanceComplete remission 49 (48,5%)41 (40.5%)11 (10.8%) Duration of neutropenia (days, median value, range) 18 (3-79) Site of infection (n, %)Sepsis LungBowelOthersMultiples (≥3 involved sites) 67 (66.3%)22 (21.9%)4 (3.9%)8 (7.9%)4 (4%) Allo-HSCTYes No 21(20.7)80(79.2) Transfusions per course (median value, range) 4 (1-14) PMN x 108/kg/course (median value, range) 8.78 (0.53-53.23) PMN x 108/kg/transfusion (median value, range) 2.11 (0.46-7.34) Disclosures No relevant conflicts of interest to declare.

Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics.

Inherited pyrimidine 5′-nucleotidase type I (P5′N-1) deficiency is the third most common erythrocyte enzymopathy that causes hemolysis. Fourteen different mutations have been identified to date. We have investigated the molecular bases of the disease by studying the biochemical properties of the recombinant wild-type human enzyme and 4 variant proteins (D87V, L131P, N179S, and G230R) bearing missense mutations found in patients affected by nonspherocytic hemolytic anemia. P5′N-1 is a relatively stable protein and has essentially identical catalytic efficiency toward cytidine monophosphate (CMP) and uridine monophosphate (UMP). All investigated mutant proteins display impaired catalytic properties and/or reduced thermostability, providing a rationale for the pathological effects of the mutations. Despite the substantial changes in the kinetic and thermostability parameters, the enzyme activity detected in the red blood cells of patients homozygous for mutations L131P and G230R exhibits moderate alterations. This suggests that P5′N-1 deficiency is compensated, possibly by other nucleotidases or alternative pathways in nucleotide metabolism. Therefore, nucleotidase activity may not be considered a prognostic indicator in patients affected by the enzymopathy. (Blood. 2005;105:3340-3345)

We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.

Abstract 3913 The possibility of measuring the circulating free light chains (FLC) improved our ability to detect the amyloidogenic clone, allowed refined risk stratification in combination with cardiac biomarkers, and provided a formidable tool for assessing response to treatment in AL amyloidosis. Recently, a novel method for FLC quantitation based on monoclonal antibodies has been developed. We evaluated its performance in the diagnosis, prognostication of survival, and response assessment in 353 consecutive newly diagnosed patients with AL amyloidosis enrolled between 2007 and 2011. Serum and urine immunofixation electrophoresis (IFE) was performed with a Hydragel 2IF/BJ(HR) kit on a Hydrasis apparatus (Sebia). Serum FLC concentration was measured in duplicate on frozen sera by a polyclonal (Binding Site) and a monoclonal (Siemens) immunoassay on a Behring BNII nephelometer. Reference ranges are k-FLC 3.3–19.4 mg/L, l-FLC 5.7–26.3 mg/L, k/l ratio 0.26–1.65 for the Binding Site (BS) assay, and k-FLC 6.7–22.4 mg/L, l-FLC 8.3–27.0 mg/L, k/l ratio 0.31–1.56 for the Siemens (S) assay. Response was evaluated 3 months after treatment initiation. The deposited amyloidogenic light chain identified by immuno electron microscopy was k in 69 patients (19%) and l in 284 (81%). Fifteen patients (4%) were excluded from the calculation of the diagnostic sensitivity because a biclonal gammapathy was detected by IFE. The two FLC assays had similar diagnostic sensitivity (Table 1). We calculated the concordance correlation coefficient to evaluate the agreement between the two assays, which was better for k (0.92, 95%CI 0.87–0.91) than for l (0.78, 95%CI 0.73–0.82) FLC. A total of 161 patients (46%) died. Median survival was 31 months. We evaluated the prognostic relevance of the difference between involved (amyloidogenic) and uninvolved FLC concentration (dFLC). Median values of dFLC measured with the two methods were 180 mg/L by BS and 165 mg/L by S. Patients with dFLC greater than the median value had a worse outcome (2 year survival 43% vs. 65%, P=0.001, by BS; 42% vs. 66%, P=0.001, by S). These thresholds were incorporated into a staging system modeled on the revised Mayo Clinic system (Figure 1). The S assay provided better discrimination between stages III and IV. We evaluated the use of the S assay in the response criteria of the International Society of Amyloidosis (ISA). A serum sample collected 3 months after treatment initiation was available in 226 patients. Among them, baseline dFLC was ≥50 mg/L (evaluable for response) in 171 subjects with the BS, in 170 with the S assay, and in 146 patients by both methods. Table 2 reports the response categorization according to the two methods. The greatest discrepancy was observed in the partial response group, although also the very good partial response group presented notable deviations. The novel monoclonal FLC assay has a diagnostic sensitivity comparable to that of the standard polyclonal assay and can be used for prognostic stratification. However, response assessment presents significant discrepancies between the two methods, indicating that further studies are needed to assess the performance of the S assay in the evaluation of response. Table 1. Diagnostic sensitivity of IFE and FLC k/l ratio by Binding Site (BS) and Siemens (S) in 338 patients with systemic AL amyloidosis Patients with k clones (N=67) Patients with l clones (N=271) Overall population (N=338) N positive % (95% CI) N positive % (95% CI) N positive % (95% CI) Serum IFE 55 82 (71–90) 259 96 (93–98) 314 93 (90–95) Urine IFE 54 81 (69–89) 239 88 (84–92) 293 87 (83–90) Serum and urine IFE 56 84 (72–91) 262 97 (94–98) 318 94 (91–96) FLC k/l ratio BS 65 97 (90–100) 214 80 (74–83) 279 82 (78–86) FLC k/l ratio S 60 89 (80–96) 225 83 (78–87) 285 84 (80–88) Serum and urine IFE + FLC k/l ratio BS 67 100 (96–100) 264 97 (95–99) 331 98 (96–99) Serum and Urine IFE + FLC k/l ratio S 64 95 (87–99) 268 99 (97–100) 332 98 (96–99) Table 2. Concordance between dFLC response with the two assays Number responding by the Siemens assay Response by the Binding Site assay CR VGPR PR NR CR (22 patients) 21 1 0 0 VGPR (29 patients) 1 16 5 7 PR (37 patients) 0 3 18 16 NR (58 patients) 0 3 3 52 Complete response (CR), negative serum and urine immunofixation and normal FLC k/l ratio; very good partial response (VGPR), dFLC concentration after chemotherapy 50%; no response (NR), all other patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3016 Poster Board II-992 Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. It is a relatively small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-BPG or 3-PG, whereas the C-terminal domain binds Mg-ADP or Mg-ATP. During the catalytic cycle, the enzyme undergoes large conformational rearrangements, proceeding from an open form to a closed form. Two isozymes, PGK1 and PGK2, are present in humans, encoded by two distinct genes. Whereas PGK2 is a testis-specific enzyme, PGK1 is expressed in all the somatic cells. The PGK1 gene is located on the X-chromosome q-13.1, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency, that is characterized by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. To date, 20 different mutations with worldwide distribution have been described. To investigate the genotype-phenotype relationship of PGK deficiency, recently we have undertaken a characterization of the all PGK mutant enzymes so far reported. In this study we describe the molecular abnormalities of the G158V, R206P, V266M and D285V variants obtained from E.coli as recombinant proteins. All patients were affected by moderate to severe hemolytic anemia. Moreover, patients bearing GI58V, R206P, and D285V variants displayed muscular disorders. Neurological dysfunctions were present in patients with R206P and V266M. The desired mutations were introduced into the PGK cDNA by site directed mutagenesis. All mutant enzymes were expressed and purified to homogeneity as previously indicated (Morera et al., Blood, ASH, Annual Meeting Abstracts, 2008;112:2875). Each variant was subjected to kinetic analysis and to different heat treatments in the absence and in the presence of specific ligands. The enzyme activity was determined following the backward reaction. Variants G158V and D285V turned out to be affected in their catalytic activities, displaying kcat values towards ATP and 3-PG 7-fold and 19-fold, respectively, lower than that of the wild type enzyme previously characterized. Variant R206P displayed reduced affinity vs 3-PG, the Km value being 8-fold higher than that of the wild type. Variant V266M showed kinetic properties similar to those of the wild type. The mutant enzymes subjected to heat treatments exhibited different protein stability. Whereas the wild type enzyme preserved 70% of its activity after one hour-incubation at 45°C, mutants G158V and D285V at the same temperature halved their activities after only 5 min and 2 min, respectively. Mutants R206P and V266M turned to be quite heat stable, their T50 (the temperature to which an enzyme halves its activity in 10 min) being 2°C lower than that of the wild type enzyme (47°C vs 49°C). Moreover, at a temperature 3-4 °C higher than its own T50, no one mutant was properly protected by the presence of Mg-ATP. In addition, variants G158V and D285V were not even protected by 3-PG. Therefore, these studies suggest that G158V and D285V substitutions affect amino acid residues located in key positions for allowing the enzyme to preserve its protein stability, especially during the red cell life span, and to adopt its proper conformations in fulfilling the catalytic cycle. The reduced RBC concentration of PGK and the energy pathway deficiency would account for the dysfunctions displayed by patients with G158V and D285V. With regard to R206P variant, the mutation affects an amino acid residue located in the hinge of the enzyme, far away from the 3-PG binding site. Owing to the fact that the variant displayed a reduced affinity versus 3-PG, it is likely that Arg206 plays an important role in the structuring of the 3-PG binding site, via long-distance interactions. Thus mutation R206P would lead to a distortion of the 3-PG binding site, impairing the PGK activity under physiological 3-PG concentrations. Consequently, the reduced energy supply would be the cause of the hemolysis displayed by the PGK deficient patient. Finally, with regard to V266M mutant, no acceptable explanation of the enzyme deficiency can be drawn by the present biochemical studies, the mutant behaving as the wild type enzyme. Disclosures: No relevant conflicts of interest to declare.

Abstract 4699 Objectives We have evaluated the possibility to use the new BD Cytometric Bead Array to detect the presence of the BCR-ABL fusion protein2 in peripheral blood samples with a routine blood hematology analyser, the Abbott Cell-Dyn Sapphire, for a quick identification of those positive samples, both at diagnosis and at follow up to detect the MRD. This pilot study was carried out on 5 samples with different level of BCR-ABL fusion protein2 plus 2 control samples positive and negative respectively: results were compared with those obtained with the BD FACScanto and the standard FISH analysis reference method. Methods Flow cytometry allows for the discrimination of particles on the basis of attributes such as size and fluorescence. BD Cytometric Bead Array (CBA) systems provide a way of coupling a soluble analyte or set of analytes with beads of known size and fluorescence, making it possible to detect analytes through flow cytometry.1 The BD” BCR-ABL Protein Kit uses this technology to detect BCR-ABL fusion proteins2 in human blood research samples. Cell-Dyn Sapphire is a routine automated hematology analyzer for full blood count. Moreover the system employs immuno-fluorescence analysis technology, similar to that used on a dedicated fluorescence flow cytometer, for analysis of MAb applications. BD FacScant is a routine dedicated flow cytometer using 6 fluorescence channels. FISH (Fluorescence In Situ Hybridation) is a cytogenetic technique using fluorescent probes that bind to only those part of the chromosome with which they show a high degree of sequence similarity in interphase nuclei and on metaphase chromosome. FISH detects and localizes the presence, the absence or the translocation of specific DNA sequences on chromosomes. In this patients' category, LSI bcr/abl dual fusion DNA probe hybridises to chromosome 22q11.2 (breakpoint cluster region SpectrumGreen) and to chromosome 9q34 (abl oncogene SpectrumOrange). In interphase nuclei of normal cells, the probe signals generally appear as two distinct signals of each colour. In the pathological cells with bcr/abl translocation, individual orange and green signals from the normal 9 and 22 chromosome and two orange/green fusion signals (one each from the derivative 9 and 22 chromosomes) is observed. We have analysed 200 nuclei in fluorescence microscopy for each sample. Statistical analyses were performed using the MedCalc program comparing the median fluorescence values obtained by the 2 cytometers and with the standard diagnostic procedure. Results Correlation of median fluorescence data between the BD FACScanto and the CD Sapphire is excellent with a Correlation coefficient r = 1,0000 and a Significance level P The Box –and –whisker Multiple comparison graph of the CD method versus the FISH reference one is represented in Figure 2. The results of our pilot study demonstrate the robustness of this new diagnostic tool in detecting the presence of the BCR-ABL fusion protein2 in the routine Laboratory of Hematology. The main limit to use this tool is now represented by the high cost of the BD Cytometric Bead Array. Disclosures: No relevant conflicts of interest to declare.

Objective: To evaluate the efficacy of Caspofungin in patients with hematological malignancies (HM) and candidemia Design: The study was prospectively conducted in 11 Hematology Divisions in a tertiary care or university hospital setting.; neutropenic hematological patients with clinical evidence of infection and a positive blood culture for Candida were enrolled. Patients and results: Between January 2005 and November 2007, 38 episodes of candidemia among patients with HM were registered. Among these, 24 eligible neutropenic patients, collected in 8/11 participating centers, were evaluated. All these patients had received chemotherapy for their underlying hematological diseases: acute myeloid leukemia (AML) in 11 patients (46%); non Hodgkin’s lymphoma (NHL) in 6 patients (25%); acute lymphoblastic leukemia (ALL) in 4 patients (16%); multiple myeloma (MM) in 2 patients (8%); chronic lymphoblastic leukemia (CLL) in 1 patient. In 11 patients (46%) the infection onset after a HSCT procedure (7 allogeneic and 4 autologous). Before the infection onset, all patients were neutropenic (ANC Conclusions: Our data confirm the efficacy of Caspofungin in the treatment of neutropenic pts with HM and concomitant candidemia as well as it was reported for non hematological subgroups. Caspofungin was well tolerated also in very compromised pts.

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phoshoryl-group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP forming 3-phosphoglycerate (3-PG) and ATP. PGK is a typical two-domain hinge-bending enzyme, with a highly conserved structure. The N-terminal domain binds 1,3-BPG/3-PG, whereas the C-terminal domain binds Mg-ADP/Mg-ATP.Humans have two PGK isozymes, PGK1 and PGK2, where PGK1 is an ubiquitous enzyme that is expressed in all somatic cells and PGK2 is a testis-specific enzyme. The PGK1 gene is located on the X-chromosome q-13.1, contains 11 exons and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency that is for the most clinically characterized by mild-to severe hemolytic anemia and various defects in the central nervous system. To date, 19 different mutations with worldwide distribution have been reported. No correlation between the residual PGK activity and the severity of the clinical manifestations have been documented so far. To analyze the mutations at protein level and possibly to correlate the genotype to clinical phenotype, we started with the molecular characterization of the wild-type PGK1 enzyme and three mutants (I47N, D164 and S320N) obtained from E.coli as recombinant proteins. The corresponding mutations, i.e., c.140T>A, c.491A>T and c.959G>A, have been identified in patients with PGK deficiency and affected by severe hemolytic anemia and progressive mental retardation. The cDNA encoding the PGK1 was prepared starting from a blood sample of a healthy donor, with normal PGK1 activity. Site-directed mutagenesis was used to introduce the desired mutations into the PGK1 cDNA. The wild type enzyme was expressed to its maximum level (about 80–100 mg of enzyme per liter of culture) after 5 hours of induction with 0.5 mM IPTG at 37 °C. For mutant enzymes the induction temperature was lowered to 25°C. All recombinant enzymes were purified to homogeneity after a single chromatographic step on DEAE Sepharose column. The wild-type enzyme was crystallized in both free form or complexed with 3-PG. The corresponding structures were solved to high resolution (1.8 and 1.6 A, respectively) and compared. Essentially, binding 3-PG caused a 6° rotation of the N-domain in respect to the C-domain. The recombinant enzyme exhibited kinetic properties similar to those of the authentic enzyme, displaying vs 3-PG and ATP alike specific activities (about 1000 U/mg) and alike Km values (about 1mM). I47N and S320N mutant enzymes showed kcat values 3-fold lower than the wild-type enzyme. The D164V was characterized by a Km value vs 3-PG 15 times higher than that of the other enzymes studied and a catalytic efficiency 70 times lower. Finally, all mutant enzymes turned out to be highly heat unstable with respect to the wildtype enzyme, losing half of their activity after approximately 10 minutes of incubation at 37 °C. At higher temperatures, the wild-type enzyme was protected from heat inactivation by Mg-ATP or 3-PG. On the contrary, no one mutant was protect by Mg-ATP and the D164V and S320N mutants were not even protected by 3-PG. Therefore, these preliminary studies indicate that all mutations target amino acid residues located in positions primarily important for preserving the protein stability during the red cell life span.

Backround: Gentuzumab Ozogamicin (GO) is effective as single agent in the treatment of poor risk acute myeloid leukemia (AML) patients (pts). The aim of this study was to evaluate the efficacy and safety of a chemotherapy including growth factors, cytabine and GO in the treatment of secondary AML (sAML) elderly pts. Patients and Treatments: From September 2003 to September 2006, a total of 23 pts, median age 69 years (range 58–77) with sAML were enrolled in G-AraMy protocol which was divided in two phases. In the first phase from September 2003 to December 2004 11/23 pts received G-AraMy-1 treatment: rhG-CSF (5 μg/kg, on days 1–8), Aracytin as continuous perfusion (100 mg/m2 on days 4–8), GO (6 mg/m2 iv on day 9). In the second phase from January 2005 to September 2006 12 pts was treated according G-AraMy-2 protocol: G-CSF (5 μg/kg, on days 1–8), Ara-C as continuous perfusion (100 mg/m2 on days 2–8), GO (6 mg/m2 iv on day 9). In pts reaching complete (CR) or partial remission (PR), consolidation therapy was performed. In G-AraMy-1 this consisted of: G-CSF(5 μg/kg, on days 1–6), Ara-C as continuous perfusion (100 mg/m2 on days 2–6), GO (6 mg/m2 iv on day 7). G-Ara-My-2 group was consolidated with: G-CSF(5 μg/kg, on days 1–5), Ara-C (1 g/m2 every 12 hours on days 2–5), GO (6 mg/m2 iv on day 6). Results: Among the 23 treated pts 11 (48%) presented a post-MDS AML while 12 pts (52%) had received chemotherapy for a prior malignancy (3 Hodgkin’s lymphoma, 5 breast, 2 thyroid, 1 gut, 1 bladder). Ten out 23 pts (43.5%) had previously received chemotherapy for AML being relapsed (4) or primary resistant pts (6) while 13 (56.5%) were untreated pts. Cytogenetic study was performed in all pts; karyotype was at “intermediate prognosis” in 11 cases, at “worse prognosis” in 7 cases, at “good prognosis” in 2 cases. In 3 pts no metaphases were observed. After induction and consolidation therapy 14 pts (6 group 1; 8 group 2) (61%) achieved a CR and 2 pts obtained PR. Five pts (22%) resulted refractory to treatment and 2 died during the aplasia period post induction treatment (1 due to sepsis, 1 due to cerebral haemorrhage). The most common adverse event was myelosuppression, as expected. No VOD was recorded. Seven pts (30%) developed documented infection (including pulmonary aspergillosis in 2 cases). Two pts died while in CR, 1 due to bladder cancer relapse and 1 to ischemic stroke. Nine of CR pts (39%) relapsed; at March 2007 5 pts (22%) are alive, of whom 1 are still in CR (4%). Median time to treatment failure (TTF) and median overall survival (OS) of whole population were 7.1 months (range 1–40.6+) and 8.6 months (range 1.7–40.6+) respectively. Stratifying pts according the two treatment groups median TTF was 4.4 months (range 3–10.5) in the first group and 7.2 months (range 1–40.6+) in the second; median OS was 6 months (range 1–13.6) in the first group and 9.1 months (range 1.7–40.6+) in the second. Conclusions: G-AraMy protocol could be considered an useful approach for elderly sAML pts considering the low reported side effects with a CR rate similar to that reported in literature. Unfortunately CR duration is brief. The modification of protocol schedule in the G-AraMy 2 group with the addition of more aggressive consolidation therapy seems to improve the duration of CR and OS.

Pyrimidine 5′-nucleotidase (P5′N-1) is a dephosphorylating enzyme that catalyzes the hydrolysis of various pyrimidine nucleoside 5′-monophosphates, particularly UMP and CMP, to produce the corresponding nucleosides. In RBC the reaction is essential for the removal of the nucleotides mainly arising from ribosomal RNA degradation during final erythroid maturation. Hereditary P5′N-1 deficiency is the third most common enzymopathy causing hereditary non-spherocytic hemolytic anemia. The disorder is transmitted as an autosomal recessive trait and is usually characterized by mild-to-moderate hemolytic anemia and accumulation of pyrimidine nucleotides within the erythrocyte. The enzyme is strongly inactivated by heavy metals; thus P5′N-1 deficiency can be acquired as a result of lead poisoning. The P5′N-1 gene is localized on 7p15-p14 and the cDNA has been cloned and sequenced. 24 different mutations have been identified so far, most of them at the homozygous level. Recently, five pathological variants of P5′N-1 have been in-depth characterized, and the molecular bases of the P5′N-1 deficiency has been elucidated. To unravel the cause of the P5′N-1 deficiency found in patients with hemolytic anemia and homozygous for 3 newly identified missense mutations (c.187T>C, c.469G>C, c.740T>C; Balta et al, Blood ASH2006, 108:3743; Manco et al, Haematologica2006, 91:266–267), we have undertaken a functional analysis of the 3 mutant enzymatic forms. The C63R, G157R and I247T proteins were produced as recombinant forms, purified and biochemically characterized. All enzymes were altered, although to a different extent, either in their catalytic efficiency or in thermal stability, the G157R being the most impaired enzyme. Catalytic efficiency of all mutants turned expecially towards UMP (about 50 to 200 times), owing to the increased Km values (about 10–25 times higher). The kinetic behaviour vs CMP was partly affected, the catalytic activity being moderately reduced (Kcat lowered to 5–20%). The G157R protein was highly heat unstable, halving the activity in about 23 min at 37°C, whereas C63R and I247T mutants at the same temperature maintained fully activity for more than 2 hours. However, at higher temperature also C63R and I247T mutants resulted less stable than the wild-type enzyme losing the activity in few minutes (t1/2 at 46°C, about 5 min vs 2 hours of the wild-type enzyme). Therefore, although mutations targeted different regions of the P5′N-1 structure, unexpectedly they produced similar aberrant effects on the molecular properties of the enzyme. Gly157 is a conserved amino acid, located close to the substrate binding site. Very likely, position 157 cannot tolerate the large and charged arginine side-chain introduced by c.469G>C mutation. Thus, it is conceivable that the drastic G157R substitution not only indirectly affects the binding of the substrate(s), but also weakens the protein stability. Cys63 and Ile247 are located far away from the catalytic site. Nevertheless, our biochemical data indicate that they are functionally and structurally important for preserving the enzyme activity. Thus, as in other cases, the decreased catalytic efficiency of C63R and I247T enzymes seems to result from secondary effects related to propagating conformational changes.

Background: To evaluate the incidence and the outcome of fungal infections in patients (pts) affected by hematological malignancies (HM). Methods: A retrospective study, conducted over 1999–2003, in pts with HM, admitted in 18 Italian Hematology divisions in tertiary cares or university hospitals, who developed proven or probable fungal infections. Results: The population included 11,802 pts: 3,012 AML (25.5%), 1,173 ALL (9.9%), 596 CML (5%), 1,104 CLL (9.4%), 1,616 MM (13.7%), 3,457 NHL (29.3%), 844 HL (7.2%). Pts who underwent HSCT were included in a different analysis. A fungal infection occurred in 538 pts, with an incidence of 4.6%; in particular we registered 346 episodes sustained by moulds (incidence 2.9%) and 193 by yeasts (1.6%). The incidence rate depends upon underlying malignancy (12.3% in AML, 6.5% in ALL, 2.7% in CML, 0.6% in CLL, 0.5% in MM, 1.6% in NHL, 0.9% in HL). Among moulds, the etiological agents were Aspergillus (310 episodes, incidence 2.8%), Mucorales (13 pts, 0.1%), Fusarium (15 pts, 0.1%), and other rare fungi (7 pts, 0.1%). Among yeasts we registered septicemia due to Candida (175 pts, 1.6%). Other yeast infections were caused by Cryptococcus (8 pts, 0.1%), Tricosporon (7 pts, 0.1%) and other rare agents (2 pts). We did not observed an increase of A.terreus infections while the number of episodes sustained by A.flavus increased from 1999 to 2003 (RR 2.10; IC95% 0.8–5.49; p-value 0.117). The overall mortality rate was 1.8%. Among 538 pts with fungal infection 39% died for this complication, with differences between aspergillosis (42%), zygomycosis (63%), fusariosis (53%) and candidemia (33%). There was not variation in mortality rate during the study period; comparing these pts with those observed in our previous studies during the period 1987–1988 we observed a significant reduction of death due to aspergillosis (RR 1.90; IC 95% 1.17–3.09), but no differences in mortality rate due to Candida. Conclusions: Our study confirms the general trends already described: infections due to moulds are more frequent than those caused by yeast. Aspergillus remains the main etiologic agent, followed by Candida. The other agents (Mucorales, Fusarium, Trichosporon) remain rare. AML represents the most frequently involved category. The mortality rate due to aspergillosis is actually about 40%, with a remarkable decrease when compared to past years; as for candidemia, we observed a reduction in the incidence, but not in the mortality rate.

Hereditary pyrimidine 5′ nucleotidase deficiency (P5′N) is the most frequent abnormality of the red cell nucleotide metabolism causing hereditary non-spherocytic hemolytic anemia. The disorder is characterized by mild-to-moderate hemolytic anemia associated with reticulocytosis and hyperbilirubinemia and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. P5′N-1 gene is localized on 7p15-p14; eighteen mutations have been so far identified in 27 unrelated families, 6 of them of Italian origin. The aim of this study is to describe the hematological, biochemical and molecular characteristics of two new Italian patients affected by P5′N deficiency. Case1: The propositus was a 37 yrs old woman of Northern Italian origin affected by chronic hemolytic anemia with Hb levels ranging from 8.2 to 10.5 g/dL. At the time of the study Hb was 8.4 g/dL, reticulocytes 300x109/L, unconjugated bilirubin 3.2 mg/dL. Peripheral blood smear examination showed basophilic stippling and purines/pyrimidines ratio (OD260/280) was decreased (1, ref. values 1.4 – 2.98). P5′N activity, measured by capillary electrophoresis, was undetectable. Molecular analysis of P5′N-1 gene showed the presence of a new homozygous deletion of two bp (ag) at the splice junction between intron 7 and exon 8, which probably results in a splicing alteration and in the absence of a functional protein. Case2: The propositus, a 37 yrs old woman of Northern Italian origin carrying the hemoglobin variant HbD Punjab, had an history of chronic hemolytic anemia since childhood; at the age of 14 yrs splenectomy and colecystectomy were performed. The patient needed blood transfusions because of exacerbation of anemia (Hb 4.7g/dL) during parvovirus B19 infection. Iron status parameters were increased requiring desferrioxamine treatment. At the time of the study Hb was 9.3 g/dL, reticulocytes 752x109/L, unconjugated bilirubin 13.3 mg/dL. Serum ferritin was 1980 mg/mL and transferrin saturation 115%. The propositus was found to be homozygous for Gilbert’s syndrome and heterozygous for mutation H63D of HFE gene. Basophilic stippling (6%) was observed in peripheral blood smear. Pur/pyr ratio was 0.8 and residual P5′N activity was 40% of normal. Complete sequencing of P5′N-1 gene showed the presence of the frameshift mutation ins GG710-711, already described in Italian and Turkish patients, and the new in-frame aminoacidic deletion of Gln 143.

Background: The acute myeloid leukemia (AML)-M4 subtype is frequently associated to eosinophilia and/or to the cytogenetic alteration inv(16)/t(16;16). The presence of these features is generally associated with good prognosis, but the studies concerning their exact role are hampered by the low number of cases. We retrospectively analyzed patients with AML-M4 enrolled in two consecutive GIMEMA studies to assess the influence of eosinophilia and of the inv(16) cytogenetic abnormality on the prognosis of acute myelomonocytic leukemia (M4) and acute myelomonocytic leukemia with abnormal eosinophils (M4eos). Setting: A retrospective study, conducted over 9 years in patients affected by AML, admitted to 35 Italian hematological divisions. Patients and methods: Between December 1993 and December 2002, 1686 patients aged over 15 years with a diagnosis of AML were admitted to the EORTC-GIMEMA AML10 and AML 99p trials; of these, 400 patients (355 M4 and 45 M4Eo) were studied. The diagnosis of M4 and M4eos was first established at each institution and subsequently centrally reviewed at the time of study entry. The following parameters were evaluated: morphology, immunophenotype, cytogenetics performed at the onset of the disease, complete remission achievement and duration, overall survival (OS) and event-free survival (EFS) from AML diagnosis. Patients with M4eo were younger and more frequently associated with inv(16) compared to M4. Cytogenetic analisis failed or was not carried out in 40% of cases, while it was successfully analyzed in 240 cases; inv(16) was found in 17% of them. Results: Concerning the probability of obtaining a CR after standard treatment, at univariate analysis M4Eo had a non significant advantage compared to M4, while presence of inv(16) was significantly correlated to a higher CR probability; multivariate analysis showed a significant advantage only of M4Eo+ inv(16) compared to M4-without eosinophilia and without inv(16). DFS was not different in univariate analysis between patients carrying or not inv(16), while a borderline advantage of M4Eo was observed with respect of M4, not confirmed at multivariate analysis. OS curves showed at univariate analysis a significant advantage both of the presence of eosinophilia (P=0.004) and of inv(16) (P=0.01); at multivariate analysis, patients with M4Eo+ inv(16) had a highly significant advantage compared to M4 without eosinophilia and without inv(16) (P=0.004), but also compared to M4+ inv(16) (P=0.045), and M4Eo-without inv(16) (P=0.076). Conclusions: AML-M4 with or without eosinophilia represent 23.7% of AML. The presence of eosinophilia and of inv(16)/t(16;16) can be both considered favorable prognostic factors; however, only the association of both features allows a highly significant advantage in terms of CR and OS.

Pyrimidine 5′-nucleotidase type-I (P5′N-1) catalyzes the dephosphorylation of UMP and CMP to their respective nucleosides. In red blood cells, the enzyme has a major role in the catabolism of nucleotides formed from RNA degradation. P5′N-1 possesses also phospho-transferase activity suggesting an additional role of the enzyme in nucleotide metabolism. P5′N-1 deficiency is an autosomal recessive disorder characterized by hemolytic nonspherocytic anemia, heavy basophilic stippling in the peripheral blood smear, and accumulation of pyrimidine nucleotides within the erythrocytes. P5′N-1 deficiency is the third most common RBC enzymopathy causing hemolysis after G6PD and PK deficiency. Fourteen different mutations have been identified at the DNA level to date including four missense mutations. To increase our understanding on molecular basis of the P5′N-1 deficiency, after mutants N190S and G241R (Chiarelli et al, Blood 2003, abstract; Chiarelli et al, The Hematology Journal 2004, abstract), we have undertaken the biochemical characterization of D98V and L142P enzymes, identified respectively in a Norwegian family and in Japanese patients. The proteins were produced in E. coli cells as recombinant forms, and purified to homogeneity. The L142P protein showed a drastic reduction in the thermal stability (t1/2 at 37°C about 6 min compared to a fully stable wild-type), and kinetic properties slightly altered (kcat values nearly halved and Km 3–5 times higher). D98V exhibited reduced heat stability (t1/2 at 37° about 25 min) and catalytic efficiency turned especially versus UMP (about 25 times) owing the increased Km values. Thus, the decreased activity observed in Japanese patients homozygous for the L142P mutation is essentially due to lowered enzyme levels caused by protein instability, whereas the D98V mutation of Norwegian patients alters both stability and catalytic efficiency. We suggest that substitution D98V affects an amino acid residue involved in substrates binding site.

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6- contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.