Multilineage Dysplasia as Assessed by Immunophenotype in Acute Myeloid Leukemia: A Prognostic Tool in a Genetically Undefined Category.

Acute myeloid leukemia (AML) "with myelodysplasia-related changes (MRC)" is considered a separate entity by the World Health Organization (WHO) classification of myeloid neoplasms. While anamnestic and cytogenetic criteria provide objective attribution to this subset, with clear unfavorable prognostic significance, the actual role of multi-lineage dysplasia (MLD) as assessed by morphology is debated. The aim of our work was to study MLD by a technique alternative to morphology, which is multiparameter flow cytometry (MFC), in a large series of 302 AML patients intensively treated at our Center. The correlation with morphology we observed in the unselected analysis reiterated the capability of the MFC-based approach at highlighting dysplasia. MLD data, estimated through an immune-phenotypic score (IPS), provided no insight into prognosis when considered overall nor within well-defined genetic categories. Of interest, IPS-related dysplasia conveyed significant prognostic information when we focused on genetically undefined patients, triple-negative for NPM1, FLT3 and CEBPA (TN-AML). In this context, the lack of dysplastic features (IPS_0) correlated with a significantly higher CR rate and longer survival compared to patients showing dysplasia in one or both (neutrophil and erythroid) cell lineages. The impact of IPS category maintained its validity after censoring at allogeneic HSCT and in a multivariate analysis including baseline and treatment-related covariates. In a subgroup featured by the lack of genetic determinants, our data could help address the relative unmet needs in terms of risk assessment and treatment strategy, and provide insight into prediction of response in the rapidly evolving therapeutic scenario of AML.

Early peripheral blast cell clearance predicts minimal residual disease status and refines disease prognosis in acute myeloid leukemia.

Minimal residual disease (MRD) assessment in acute myeloid leukemia (AML) is increasingly used in risk stratification. However, several issues around this use are unresolved, including, among others, the most suitable time-point(s) for its application. Overall, late assessments appear more effective at distinguishing outcome but, in some studies, the early evaluations were already highly informative, anticipating the value of later ones. Our work integrated MRD with peripheral blast clearance (PBC), a treatment-related biomarker previously demonstrated to be a powerful predictor of response. From 2007 to 2014, we have studied 120 patients treated according to the NILG 02-06 trial and who achieved CR after induction. Patients in PBC-defined categories (separated by a 1.5-log threshold) showed significantly different probabilities of attaining MRD negativity, after either induction (MRD1) or consolidation (MRD2). Peripheral blast clearance combined with MRD1 largely anticipated MRD2-related information: when both biomarkers predicted chemosensitive disease (PBChigh /MRD1neg ), the rate of MRD2-negativity was 90%, and DFS and OS estimates were 68% and 76% at 3 years, respectively. When both markers were unfavorable (PBClow /MRD1pos ), rates of MRD2 negativity, DFS, and OS were 20%, 34%, and 24%, respectively, at 3 years. In fact, MRD2 added prognostic value only in cases with discordant PBC/MRD1 data. Our data support a reasoned timing for MRD-based therapeutic decisions, modulated on individual chemosensitivity, an approach we have implemented in a forthcoming prospective multi-center trial by Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA).

CEBPA-double-mutated acute myeloid leukemia displays a unique phenotypic profile: a reliable screening method and insight into biological features.

Mutations in CCAAT/enhancer binding protein α (CEBPA) occur in 5-10% of cases of acute myeloid leukemia. CEBPA-double-mutated cases usually bear biallelic N- and C-terminal mutations and are associated with a favorable clinical outcome. Identification of CEBPA mutants is challenging because of the variety of mutations, intrinsic characteristics of the gene and technical issues. Several screening methods (fragment-length analysis, gene expression array) have been proposed especially for large-scale clinical use; although efficient, they are limited by specific concerns. We investigated the phenotypic profile of blast and maturing bone marrow cell compartments at diagnosis in 251 cases of acute myeloid leukemia. In this cohort, 16 (6.4%) patients had two CEBPA mutations, whereas ten (4.0%) had a single mutation. First, we highlighted that the CEBPA-double-mutated subset displays recurrent phenotypic abnormalities in all cell compartments. By mutational analysis after cell sorting, we demonstrated that this common phenotypic signature depends on CEBPA-double-mutated multi-lineage involvement. From a multidimensional study of phenotypic data, we developed a classifier including ten core and widely available parameters. The selected markers on blasts (CD34, CD117, CD7, CD15, CD65), neutrophil (SSC, CD64), monocytic (CD14, CD64) and erythroid (CD117) compartments were able to cluster CEBPA-double-mutated cases. In a validation set of 259 AML cases from three independent centers, our classifier showed excellent performance with 100% specificity and 100% sensitivity. We have, therefore, established a reliable screening method, based upon multidimensional analysis of widely available phenotypic parameters. This method provides early results and is suitable for large-scale detection of CEBPA-double-mutated status, allowing gene sequencing to be focused in selected cases.

Multilineage dysplasia as assessed by immunophenotype has no impact on clinical-biological features and outcome of NPM1-mutated acute myeloid leukemia.

The presence of multilineage dysplasia (MLD) by morphology at diagnosis in acute myeloid leukemia (AML) defines a separate subset in the World Health Organization classification with still-debated prognostic value. A major controversy concerns MLD's role in NPM1-mutated (NPM1⁺) AML, which correlates with good prognosis. We used flow cytometry (FC), an emerging technique for assessing dysplasia, to investigate MLD in NPM1⁺ AML by an immunophenotypic score (IPS), a technique previously adopted in myelodysplastic syndrome. Eighty-five intensively treated NPM1⁺ AML cases were studied. Patients were grouped according to the combination of data in maturing cell compartments. FC-assessed dysplasia showed a significant correlation with morphology-assessed dysplasia, showing the efficacy of this method in highlighting dysplasia in AML. Except for MLD, IPS did not influence any patient- or disease-related characteristics at diagnosis. Furthermore, IPS did not influence complete remission rate, disease-free survival, or overall survival. By investigating NPM1 status on separated cell compartments, we established a correlation between FC-assessed MLD and belonging to AML clone. This study shows that dysplasia evaluated by immunophenotype has no impact on clinical-biological characteristics or on outcome of NPM1⁺ AML. Dysplasia is part of the spectrum of NPM1⁺ AML, and the prognostic stratification of this category of patients should not be based upon it.

CXCR4 expression accounts for clinical phenotype and outcome in acute myeloid leukemia.

BACKGROUND: In acute myeloid leukemia (AML), CXCR4 expression has been correlated with leukocytosis and prognosis. METHODS: We quantified CXCR4 expression by flow cytometry on leukemic cells in 142 AML patients. RESULTS: We confirm a correlation between high CXCR4 expression and leukemic burden. Furthermore, we documented a correlation with platelet count, dysplastic megakaryopoiesis, hepato-splenomegaly and extra-hematological disease. NPM1-mutated AML displayed a significantly higher intensity of CXCR4 compared to NPM1-wt cases: it is conceivable its clinical phenotype to be driven by high CXCR4 expression. CONCLUSIONS: CXCR4 expression resulted in an independent prognostic factor. Our data support CXCR4 targeting as a potential therapeutic strategy.

CXCR4 expression accounts for clinical phenotype and outcome in acute myeloid leukemia.

Background. In acute myeloid leukemia (AML), CXCR4 expression has been correlated with leukocytosis and prognosis. Methods. We quantified CXCR4 expression by flow cytometry on leukemic cells in 142 AML patients. Results. We confirm a correlation between high CXCR4 expression and leukemic burden. Furthermore, we documented a correlation with platelet count, dysplastic megakaryopoiesis, hepato-splenomegaly and extra-hematological disease. NPM1-mutated AML displayed a significantly higher intensity of CXCR4 compared to NPM1-wt cases: it is conceivable its clinical phenotype to be driven by high CXCR4 expression. Conclusions. CXCR4 expression resulted an independent prognostic factor. Our data support CXCR4 targeting as a potential therapeutic strategy. © 2014 Clinical Cytometry Society.

Overexpression of microRNA-16-2 contributes to the abnormal erythropoiesis in polycythemia vera.

Deregulated expression of microRNAs is associated with neoplasia. Here, we show that mature miR-16 levels are abnormally increased in CD34(+) cells of patients with polycythemia vera as a consequence of preferential expression of miR-16-2 on chromosome 3 rather than of miR-16-1 on chromosome 13. Forced expression of miRNA-16 in normal CD34(+) cells stimulated erythroid cell proliferation and maturation. Conversely, exposure of polycythemia vera CD34(+) cells to small interfering RNA against pre-miR-16-2 reduced erythroid colonies and largely prevented formation of erythropoietin-independent colonies; myeloid progenitors remained unaffected. Experiments with knock down of JAK2 indicated that overexpression of miR-16 was independent of JAK/STAT pathway activation. Mice injected with an miR-16 antagomir showed a blunted erythroid response to exogenous erythropoietin, which indicates a role of miR-16 in normal erythropoiesis. These data suggest that deregulation of miR-16-2 contributes to abnormal expansion of erythroid lineage in polycythemia vera. However, the mechanisms for miR-16-2 overexpression remain to be elucidated, because no genetic abnormalities at the miR-16-2 locus were discovered.

Early reduction of WT1 transcripts during induction chemotherapy predicts for longer disease free and overall survival in acute myeloid leukemia.

We investigated the prognostic significance of early peripheral blast clearance as assessed by WT1 transcript reduction during the first days of standard induction therapy in 57 adult patients with acute myeloid leukemia (AML). Quantification of WT1 transcript by real-time quantitative PCR in peripheral blood on days 1 and 5 of treatment was performed. WT1 ratio was defined as the ratio of copy number measured on day 1 and on day 5. The median WT1 ratio was greater in patients attaining CR as compared to non-responders (11.68 vs. 2.14, respectively; P=0.0006). Furthermore, DFS and OS were significantly longer in patients displaying a WT1 ratio greater than 5.82 (i.e. the median value of whole cohort) than in patients with WT1 ratio of 5.82 or under (P=0.024 and P<0.001, respectively). These data suggest that early decrease of WT1 copy number in peripheral blood predicts for better outcome and should be considered in the management of AML patients.

Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele.

A total of 186 patients with primary myelofibrosis (PMF) were genotyped for JAK2V617F at diagnosis aimed at analyzing the correlation of mutational status and mutated allele burden with outcome variables, including time to anemia, leukocytosis, leukopenia, thrombocytopenia, massive splenomegaly, leukemia, and with overall survival. A total of 127 JAK2V617F-mutated patients (68% of whole series) were divided in quartiles of V617F allele burden. After a median follow-up of 17.2 months, 23 patients died, 15 because of leukemia. A JAK2V617F mutated status did not impact on the rate of leukemia transformation or overall survival. Patients in the lower quartile had shorter time to anemia and leukopenia and did not progress to large splenomegaly. Furthermore, survival was significantly reduced in the lower quartile compared with upper quartiles and JAK2 wild-type patients. In multivariate analysis, factors associated with reduced survival were age, a blast count more than 1%, and a JAK2V617F burden within first quartile. Causes of death in the lower quartile were represented mainly by systemic infections. We conclude that a low JAK2V617F allele burden at diagnosis is preferentially associated with a myelodepletive rather than myeloproliferative phenotype and represents an independent factor associated with shortened survival in patients with PMF.

Increased risk of lymphoid neoplasms in patients with Philadelphia chromosome-negative myeloproliferative neoplasms.

Association of myeloproliferative neoplasm (MPN) with lymphoproliferative neoplasm (LPN) has been occasionally reported. The aim of this study, which included 353 patients with polycythemia vera and 467 with essential thrombocythemia, was to assess whether the risk of developing LPN is increased in MPN patients. Expected numbers of LPN incident cases were calculated based on 5-year age group, gender, and calendar time-specific cancer incidence rates in the general population of the same area. Standardized incidence ratios were computed to estimate the relative risk of developing LPN. Analyses were carried out for the whole series and then separately for essential thrombocythemia and polycythemia vera, gender, and JAK2V617F genotype. With 4,421 person-years, we found 11 patients developing LPN, including four chronic lymphocytic leukemias, five non-Hodgkin's lymphomas, and two plasma cell disorders, after a median interval time of 68 months from MPN diagnosis. Cumulative risk to develop LPN at 5 and 10 years was 0.93% (95% confidence interval, 0.39-2.22) and 2.96% (95% confidence interval, 1.52-5.72), respectively. There was a 3.44-fold increased risk of LPN compared with the general population, ranging from 2.86 for plasma cell disorder to 12.42 for chronic lymphocytic leukemia; the risk was significantly increased in JAK2V617F mutated patients (5.46-fold) and in males (4.52-fold). The JAK2V617F mutation was found in lymphoid tumor cells in two of three cases evaluated, indicating that, in some patients, LPN originated in a JAK2V617F mutated common lymphoid-myeloid hematopoietic progenitor cell. We conclude that the risk of developing LPN is significantly increased in MPN patients compared with the general population.

Anti-oxidant enzymes and related elements in term and preterm newborns.

BACKGROUND: Although oxidative stress-related diseases mostly affect neonates with extremely low birthweight, healthy preterm newborns might also be at risk of oxidative damages. The aim of the present study was to verify this possibility. METHODS: Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), erythrocyte glutathione peroxidase (GSHPx) and superoxide dismutase (SOD), plasma and erythrocyte concentrations of selenium, zinc and copper were measured until 100 days of life in 30 preterm infants with mean +/- SD birthweight and gestational age of 1605 +/- 122 g and 34.5 +/- 0.5 weeks. The control group included 30 term infants with birthweight 3123 158 g and gestational age 39.6 0.7 weeks. RESULTS: Throughout the study period urinary 8-OHdG, taken as a marker of oxidative stress, was significantly higher in the preterm than in the term group. Up until 20 days of life, GSHPx activity was significantly lower in the preterm than in the term infants but this was not associated with any apparent selenium deficiency. Conversely, up until 100 days, preterm infants had significantly reduced SOD levels that appeared to reflect a shortage of the elements needed for this enzyme's activity, notably copper, the plasma concentrations of which were constantly and significantly below the control values. CONCLUSION: The nutritional status of the elements related to the anti-oxidant enzymes, especially zinc and copper, should be carefully assessed in preterm infants, even if their birthweight is not extremely low.

A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction.

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.

Hypermethylation of CXCR4 promoter in CD34+ cells from patients with primary myelofibrosis.

Constitutive mobilization of CD34(+) cells in patients with primary myelofibrosis (PMF) has been attributed to proteolytic disruption of the CXCR4/SDF-1 axis and reduced CXCR4 expression. We document here that the number of circulating CD34(+)/CXCR4(+) cells in PMF patients, as well as the cellular CXCR4 expression, was directly related to CXCR4 mRNA level and that reduced CXCR4 mRNA level was not due to SDF-1-induced downregulation. To address whether epigenetic regulation contributes to defective CXCR4 expression, we studied the methylation status of the CXCR4 promoter using methylation-specific polymerase chain reaction and methylation-specific sequencing in the JAK2V617F-positive HEL cell line and in CD34(+) cells. We found that CD34(+) cells from PMF patients, unlike those from normal subjects, presented hypermethylation of CXCR4 promoter CpG island 1. Following incubation with the demethylating agent 5-Aza-2'-deoxycytidine (5-AzaD), the percentage of PMF CD34(+) cells expressing CXCR4 increased 3-10 times, whereas CXCR4 mRNA level increased approximately 4 times. 5-AzaD-treated PMF CD34(+) cells displayed almost complete reversal of CpG1 island 1 hypermethylation and showed enhanced migration in vitro in response to SDF-1. These data point to abnormal methylation of the CXCR4 promoter as a mechanism contributing to constitutive migration of CD34(+) cells in PMF. Disclosure of potential conflicts of interest is found at the end of this article.

Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.

BACKGROUND: Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2(V617F) mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2(V617F) mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients. DESIGN AND METHODS: In this single-center retrospective study, JAK2(V617F) allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria. RESULTS: Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2(V617F) allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3-6.8; p=0.01) in patients having a greater than 25% mutant allele burden. CONCLUSIONS: The JAK2(V617F) mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.

MicroRNA expression profile in granulocytes from primary myelofibrosis patients.

OBJECTIVE: Expression profiling of microRNA (miRNA) was performed in granulocytes isolated from patients with primary myelofibrosis (PMF), with the aim of identifying abnormally expressed miRNAs in comparison with normal subjects or patients with polycythemia vera (PV) or essential thrombocythemia (ET). PATIENTS AND METHODS: Using stem loop-primed reverse transcription and TaqMan quantitative real-time polymerase chain reaction, the expression of 156 mature miRNAs was evaluated using pooled granulocytes from PMF patients, either wild-type or JAK2(617V>F) mutant with >51% allele burden, and control subjects. Differentially expressed miRNAs were then validated on additional control and PMF samples, and also on PV or ET granulocytes. RESULTS: There was a global downregulation of miRNA expression in PMF granulocytes; 60 miRNAs, of 128 called present, displayed differential expression compared to normal samples. Twelve miRNAs, which had been selected based on statistically different expression level, were finally validated. In PMF granulocytes, levels of miR-31, -150, and -95 were significantly lower, while those of miR-190 significantly greater, than control and PV or ET samples; on the other hand, miR-34a, -342, -326, -105, -149, and -147 were similarly reduced in patients with PMF, PV, or ET compared to controls. Increased expression of miR-182 and -183 correlated with JAK2(617V>F) allele burden. Three in silico-predicted putative target genes (DTR, HMGA2, and MYB), showed deregulated expression in PMF granulocytes that correlated with expression level of regulatory miRNA. CONCLUSIONS: A defined miRNA profile distinguishes PMF granulocytes from those of normal subjects and, partially, also from PV or ET patients.

NADPH oxidase-dependent redox signaling in human heart failure: relationship between the left and right ventricle.

Oxidative stress resulting from increased superoxide generation by NADPH oxidase is implicated in the pathophysiology of human heart failure. Downstream targets of NADPH oxidase remain undefined and available information is restricted to the left ventricle (LV). Thus, we aimed to assess the cascade of events triggered by increased NADPH oxidase activity (lipid peroxidation and activation of mitogen-activated protein kinases ERK1/2, JNK and p38) and their mutual relationship in right (RV) and (LV) of end-stage failing human hearts. When compared to control ventricles, diseased RV and LV showed a significant increase in NADPH oxidase superoxide production that positively correlated with p47(phox) membrane translocation (RV: r=0.76, P<0.001; LV: r=0.79, P<0.001). MDA content, a marker of lipid peroxidation, was also enhanced and ERK and p38, but not JNK, were activated. For all these relevant steps of the oxidative stress pathway, a significant correlation was observed between LV and RV from the same heart (NADPH-dependent superoxide production: r=0.678, P<0.0055; MDA: r=0.95, P<0.0001; p-p38/p38 ratio: r=0.926, P<0.0001; p-ERK/ERK ratio: r=0.913, P<0.0001). We concluded that in human heart failure, both ventricles are targets of NADPH oxidase superoxide generation which in turn may trigger the coordinated activation of downstream signaling components. This pathway may contribute to adverse remodeling of the LV and RV and subsequent progression toward end-stage heart failure, suggestive of new therapeutic targeting strategy.

Beneficial effects of poly (ADP-ribose) polymerase inhibition against the reperfusion injury in heart transplantation.

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD+ and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD+ and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.

Acylphosphatase interferes with SERCA2a-PLN association.

We previously reported that acylphosphatase, a cytosolic enzyme present in skeletal and heart muscle, actively hydrolyzes the phosphoenzyme (EP) of cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), inducing an increased activity of this pump. We hypothesized that acylphosphatase-induced stimulation of SERCA2a, in addition to enhanced EP hydrolysis, may be due to a displacement of phospholamban (PLN), removing its inhibitory effect. To verify this hypothesis co-immunoprecipitation experiments were performed by adding recombinant muscle acylphosphatase to solubilized heart SR vesicles, used as a source of SERCA2a and PLN. With anti-acylphosphatase antibodies only SERCA2a was co-immunoprecipitated in an amount which increased in parallel to the concentrations of our enzyme. Conversely, using anti-SERCA2a antibody, both PLN and acylphosphatase were co-immunoprecipitated with SERCA2a, and the PLN amount in the precipitate decreased with increasing acylphosphatase concentrations. SERCA2a and PLN were co-immunoprecipitated by anti-phospholamban antibodies, but while the amount of precipitated phospholamban increased in the presence of acylphosphatase, the level of SERCA2a decreased. These preliminary results strengthen the supposed displacement of phospholamban by acylphosphatase.