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Background: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB., Methods: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available., Results: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB., Conclusions: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples., (© 2021 International Clinical Cytometry Society.)

Background: Multidimensional flow cytometry (MFC) is routinely used for the diagnosis and follow-up of hematolymphoid neoplasms but its contribution to the identification of non-hematolymphoid malignant tumors is limited., Methods: The presence of non-hematolymphoid cells in clinical samples obtained via minimally invasive methods was ascertained by using a panel of monoclonal antibodies previously developed in our laboratory comprising a mixture of antibodies: CD9-PacB/CD45-OC515/CD57-FITC/CD56-PE/CD3-PerCP-Cy5.5/CD117-PE-Cy7/CD326-APC/CD81-APC-C750. Histopathological studies were performed using standard techniques., Results: 164 specimens of different origins were included. Malignancy was finally confirmed in 142 (86.5%), while 22 non neoplastic samples were identified. The most frequent diagnosis was small cell lung carcinoma (SCLC) (50%). High sensitivity (S = 98.6%) was reached combining MFC and conventional pathology. Individual markers differed according to the cellular origin of the neoplasm, with neuroendocrine tumors showing a unique immunophenotypic profile (CD56+ CD326+ CD117-/+ and variable tetraspanins expression). Principal component analysis efficiently distinguished SCLC from other tumor samples. In immune cell populations, differences between reactive and malignant biopsies were found in different cell compartments, especially in B cells and Plasma cells. Differences also emerged in the percentage of CD4+ CD8- T cells, CD4-CD8+ T cells and NK cells and these were dependent on the origin of the tumor cells., Conclusions: These results support the use of MFC as a rapid and valuable technique to detect non-hematolymphoid tumoral cells in clinical specimens, providing an initial orientation to complement hystopathological studies and allow a more precise diagnosis, especially in neuroendocrine neoplasms. The impact of different immune cell patterns warrants further research., (© 2022 International Clinical Cytometry Society.)

The exquisite coupling between herpesvirus and human beings is the result of millions of years of relationship, coexistence, adaptation, and divergence. It is probably based on the ability to generate a latency that keeps viral activity at a very low level, thereby apparently minimising harm to its host. However, this evolutionary success disappears in immunosuppressed patients, especially in haematological patients. The relevance of infection and reactivation in haematological patients has been a matter of interest, although one fundamentally focused on reactivation in the post-allogeneic stem cell transplant (SCT) patient cohort. Newer transplant modalities have been progressively introduced in clinical settings, with successively more drugs being used to manipulate graft composition and functionality. In addition, new antiviral drugs are available to treat CMV infection. We review the immunological architecture that is key to a favourable outcome in this subset of patients. Less is known about the effects of herpesvirus in terms of mortality or disease progression in patients with other malignant haematological diseases who are treated with immuno-chemotherapy or new molecules, or in patients who receive autologous SCT. The absence of serious consequences in these groups has probably limited the motivation to deepen our knowledge of this aspect. However, the introduction of new therapeutic agents for haematological malignancies has led to a better understanding of how natural killer (NK) cells, CD4+ and CD8+ T lymphocytes, and B lymphocytes interact, and of the role of CMV infection in the context of recently introduced drugs such as Bruton tyrosine kinase (BTK) inhibitors, phosphoinosytol-3-kinase inhibitors, anti-BCL2 drugs, and even CAR-T cells. We analyse the immunological basis and recommendations regarding these scenarios., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alonso-Álvarez, Colado, Moro-García and Alonso-Arias.)

The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P < 0.001) and overall survival (HR: 0.73, P = 0.001), but uncovered the limited prognostic value of MFC based MRD in multivariate and recursive partitioning models including other clinical, genetic and treatment related factors. Virtually all aspects related with methodological, interpretation, and reporting of MFC based MRD testing impacted in its ability to discriminate patients with different CIR. Thus, this study demonstrated that "real-world" assessment of MRD using MFC is prognostic in patients at first remission, and urges greater standardization for improved risk-stratification toward clinical decisions in AML., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P -TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Several risk stratification models have been proposed in recent years for systemic mastocytosis but have not been directly compared. Here we designed and validated a risk stratification model for progression-free survival (PFS) and overall survival (OS) in systemic mastocytosis on the basis of all currently available prognostic factors, and compared its predictive capacity for patient outcome with that of other risk scores., Methods: We did a retrospective prognostic modelling study based on patients diagnosed with systemic mastocytosis between March 1, 1983, and Oct 11, 2019. In a discovery cohort of 422 patients from centres of the Spanish Network on Mastocytosis (REMA), we evaluated previously identified, independent prognostic features for prognostic effect on PFS and OS by multivariable analysis, and designed a global prognostic score for mastocytosis (GPSM) aimed at predicting PFS (GPSM-PFS) and OS (GPSM-OS) by including only those variables that showed independent prognostic value (p<0·05). The GPSM scores were validated in an independent cohort of 853 patients from centres in Europe and the USA, and compared with pre-existing risk models in the total patient series (n=1275), with use of Harrells' concordance index (C-index) as a readout of the ability of each model to risk-stratify patients according to survival outcomes., Findings: Our GPSM-PFS and GPSM-OS models were based on unique combinations of independent prognostic factors for PFS (platelet count ≤100 × 10 9 cells per L, serum β2-microglobulin ≥2·5 μg/mL, and serum baseline tryptase ≥125 μg/L) and OS (haemoglobin ≤110 g/L, serum alkaline phosphatase ≥140 IU/L, and at least one mutation in SRSF2, ASXL1, RUNX1, or DNMT3A). The models showed clear discrimination between low-risk and high-risk patients in terms of worse PFS and OS prognoses in the discovery and validation cohorts, and further discrimination of intermediate-risk patients. The GPSM-PFS score was an accurate predictor of PFS in systemic mastocytosis (C-index 0·90 [95% CI 0·87-0·93], vs values ranging from 0·85 to 0·88 for pre-existing models), particularly in non-advanced systemic mastocytosis (C-index 0·85 [0·76-0·92], within the range for pre-existing models of 0·80 to 0·93). Additionally, the GPSM-OS score was able to accurately predict OS in the entire cohort (C-index 0·92 [0·89-0·94], vs 0·67 to 0·90 for pre-existing models), and showed some capacity to predict OS in advanced systemic mastocytosis (C-index 0·72 [0·66-0·78], vs 0·64 to 0·73 for pre-existing models)., Interpretation: All evaluated risk classifications predicted survival outcomes in systemic mastocytosis. The REMA-PFS and GPSM-PFS models for PFS, and the International Prognostic Scoring System for advanced systemic mastocytosis and GPSM-OS model for OS emerged as the most accurate models, indicating that robust prognostication might be prospectively achieved on the basis of biomarkers that are accessible in diagnostic laboratories worldwide., Funding: Carlos III Health Institute, European Regional Development Fund, Spanish Association of Mastocytosis and Related Diseases, Rare Diseases Strategy of the Spanish National Health System, Junta of Castile and León, Charles and Ann Johnson Foundation, Stanford Cancer Institute Innovation Fund, Austrian Science Fund., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: CD47 over expression has been reported in several tumor subtypes. CD47 interacts with SIRPalpha on macrophages inhibiting phagocytic signal, providing a survival advantage to tumor. CD47, therefore, represents a valuable target for immunotherapy and is currently under clinical investigation. We aimed to study CD47 expression in Hodgkin Reed Sternberg cells (HRS)., Methods: We tested a polyclonal CD47 antibody (LifeSpan Biosciences, Seattle, WA) expression along with classical HRS cell markers on a tissue array of 16 classical Hodgkin Lymphoma (CHL) tumor biopsies obtained from newly diagnosed, non-selected patients (8 Female, 8 Male patients) in our institution from October 2016 to January 2018. Histologic subtypes were nodular sclerosis in 11 cases, mixed Cellularity in 3 cases and lymphocyte rich in 2 additional cases. Median age was 53 years (Range: 8, 74). Early stage disease was found in three patients without unfavorable prognostic factors according to EORTC and GHSG criteria, one patient with unfavorable prognostic factors and nine patients had advanced disease. Bulk disease was present in one patient. Normal lymphoid tissue and normal prostate epithelium were used as normal controls as recommended by manufacturer. Approval from the Local Ethical committee was obtained before any analysis., Results: CD47 was overexpressed on all HRS cells with a characteristic dot-like pattern in 13/13 cases of CHL. HRS clearly expressed CD47 more intensely than infiltrating T and stromal cells., Discussion: We propose that HRS cells, by up-regulating CD47, might avoid innate immunity check on tumor growth, which could be circumvented using blocking monoclonal antibodies.

Background: Diagnosis of hematolymphoid neoplasm (HLN) requires different technologies which are performed on a patient basis instead of per protocol. We hypothesize that integration of hematimetric and cytological analysis along with multiparametric flow cytometry (MFC) provides a framework to evaluate peripheral blood (PB) samples from Primary Care., Methods: Samples from patients with persistent (>3 months) lymphocytosis (>5 × 10 9 /L) and/or monocytosis (>10 9 /L) or the presence of atypical and/or blast cells upon the smear review were analyzed by MFC concurrent to cytological analysis. MFC studies were carried out following standardized procedures., Results: In a 3-year period, smear review and MFC were performed simultaneously in 350 samples, demonstrating HLN in 194 cases (55.4%). In 156 cases, reactive cell populations were found. The combination of age, absolute lymphocyte count (ALC), hemoglobin and platelets provided the best correlation with MFC for the presence of a chronic lymphoproliferative disorder (CLPD) in lymphocytosis [area under the curve (AUC) 0.891, p < 0.05]. A model evaluating the probability of CLPD has been proposed and validated in an independent cohort., Conclusions: A strategy to perform MFC studies following standardized procedures has proven to be useful to evaluate samples from patients in Primary Care centers for HLN diagnosis or reactive conditions, providing a sensitive and rapid clinical orientation and avoiding unnecessary consultations in routine clinical practice. The probability for the presence of CLPD in PB can be calculated and help guide decision-making regarding further testing., (©2020 Walter de Gruyter GmbH, Berlin/Boston.)

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Flow cytometry (FC) is a valuable tool for the diagnosis of myelodysplastic syndromes (MDS). We present results of a survey carried out to evaluate FC current practice for MDS diagnosis in Latin America (LA), focusing on markers used and characteristics of the clinical diagnostic report. Compliance to IMDSflow recommendations was also evaluated. These practices were then compared with those used in other countries., Methods: An online survey was sent through the Grupo Latino-Americano de Mielodisplasia to LA cytometrists and other international scientific societies., Results: 91 responses from 15 LA countries were received. The median of the number of markers used was 20 ± 4.5, but only 8.1% of participants adopted the complete panel proposed by the International/European LeukemiaNet Working Group (IMDSflow). We received 140 eligible answers from regions other than LA (66 Europe, 59 USA-Canada, 8 Oceania, 6 Asia and 1 Africa). LA utilized more markers for MDS diagnosis than USA/Canada (p = 0.006), but similar to Europe. The use of MDS scoring systems differed among regions: 10.3% in LA, 0% USA/Canada and 25.7% Europe reported the "Ogata score". Finally, 52.0% of all participants included a general interpretation statement in the final report about the consistency of the FC results with MDS diagnosis, with no statistical differences between regions., Conclusions: This survey shows a low compliance with the IMDSflow recommendations and a scarce use of the scoring systems proposed in the literature. However, the number of surface markers used is high. We will work to develop a FC consensus for MDS diagnosis adapted to the clinical practice requirements in LA., (Copyright © 2019. Published by Elsevier Ltd.)

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF ( KITLG , KIT , PTPN11 , GNB1 , KRAS and NRAS ) were mutated in 12 patients (2.6%), and genes downstream of BRAF ( MAPK2K1 , MAPK2K2 , and MAPK1 ) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P =0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited., (Copyright© 2019 Ferrata Storti Foundation.)

Small lymphocytic lymphoma (SLL) is considered as the non-leukemic form of presentation of chronic lymphocytic leukemia (CLL). We have compared the features, genomic alterations, and outcome of 890 patients with CLL and SLL. One hundred and thirteen patients presented as SLL and more frequently had unmutated-IGHV, CD38 high , ZAP-70 high , CD49d high , +12, alterations in genes of NOTCH1, cell cycle, RNA metabolism, and NFkB pathways than CLL. During the follow-up, 46% of SLL patients developed CLL. Time to first treatment (TTFT) was shorter in SLL (10-year: 75% vs 62%; p = .006). Binet stage, SLL, and IGHV were independent predictive factors for TTFT. Transformation to diffuse large B-cell lymphoma was higher (10-year: 12% vs 6%; p = .003), and overall survival was shorter in SLL (10-year: 55% vs 66%; p = .004). When A0 CLL patients were excluded, only CD38 and CD49d expression, +12, and 10-year TTFT remained different between the SLL and CLL patients. In summary, SLL showed only minor clinicobiological differences when compared with CLL in similar clinical stages.

[This corrects the article DOI: 10.18632/oncotarget.16657.].

Severe hemorrhagic events occur in a significant fraction of acute promyelocytic leukemia patients, either at presentation and/or early after starting therapy, leading to treatment failure and early deaths. However, identification of independent predictors for high-risk of severe bleeding at diagnosis, remains a challenge. Here, we investigated the immunophenotype of bone marrow leukemic cells from 109 newly diagnosed acute promyelocytic leukemia patients, particularly focusing on the identification of basophil-related features, and their potential association with severe bleeding episodes and patient overall survival.From all phenotypes investigated on leukemic cells, expression of the CD203c and/or CD22 basophil-associated markers showed the strongest association with the occurrence and severity of bleeding (p ≤ 0.007); moreover, aberrant expression of CD7, coexpression of CD34 + /CD7 + and lack of CD71 was also more frequently found among patients with (mild and severe) bleeding at baseline and/or after starting treatment (p ≤ 0.009). Multivariate analysis showed that CD203c expression (hazard ratio: 26.4; p = 0.003) and older age (hazard ratio: 5.4; p = 0.03) were the best independent predictors for cumulative incidence of severe bleeding after starting therapy. In addition, CD203c expression on leukemic cells (hazard ratio: 4.4; p = 0.01), low fibrinogen levels (hazard ratio: 8.8; p = 0.001), older age (hazard ratio: 9.0; p = 0.002), and high leukocyte count (hazard ratio: 5.6; p = 0.02) were the most informative independent predictors for overall survival.In summary, our results show that the presence of basophil-associated phenotypic characteristics on leukemic cells from acute promyelocytic leukemia patients at diagnosis is a powerful independent predictor for severe bleeding and overall survival, which might contribute in the future to (early) risk-adapted therapy decisions.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

The clinical consequences of the infectious events in patients receiving azacitidine are poorly documented. Likewise, the role of primary antimicrobial prophylaxis is unknown. In this retrospective, single-center study, we compare the impact of prophylaxis on the incidence of infection and morbidity in all consecutive higher-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, during the first 4 azacitidine cycles. Seventy-six patients, corresponding to 283 azacitidine cycles, were studied. There were infectious events in 43% of the patients. Development of infections led to more hospital admissions, increased red blood cells and platelet requirements, and a delay in subsequent cycles. Median overall survival was comparable between patients with or without infections. In the multivariate analysis, a neutrophil count below 0.5 × 10 9 /L (OR 12.5 [2.6-50]) and antimicrobial prophylaxis (OR 0.1 [0.02-04]) were independent factors for the development of infection. We conclude that infectious events have a significant impact in the early clinical course of azacitidine-treated patients by increasing hospital admissions and transfusion requirements. Antimicrobial prophylaxis may prevent infections, leading to a decreased need for supportive care in these patients with poor outcome.

Background: Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings., Methods: Here we evaluate the efficiency of consensus medical indications for PNH testing in 3,938 peripheral blood samples submitted to FCM testing in 24 laboratories in Spain and one reference center in Brazil., Results: Overall, diagnostic screening based on consensus medical indications was highly efficient (14% of PNH + samples) both in the multicenter setting in Spain (10%) and the reference laboratory in Brazil (16%). The highest frequency of PNH + cases was observed among patients screened because of bone marrow (BM) failure syndrome (33%), particularly among those with aplastic anemia (AA; 45%) and to a less extent also a myelodysplastic syndrome (MDS; 10%). Among the other individuals studied, the most efficient medical indications for PNH screening included: hemolytic anemia (19%), hemoglobinuria (48%) and unexplained cytopenias (9%). In contrast, only a minor fraction of the patients who had been submitted for PNH testing because of unexplained thrombosis in the absence of cytopenia, were positive (0.4%)., Conclusions: In summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza®, AZA) and decitabine (Dacogen®, DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.

An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio between the lipidated and cytosolic forms of microtubule-associated protein 1A/1B-light chain 3 (LC3-II/LC3-I). We found a positive effect of estradiol and genistein treatment by partially preserving the impaired cell viability after the recurrent ischemic injury; however, this positive effect does not seem to be mediated neither by blocking apoptosis processes nor by decreasing ROS production. This work contribute to the better understanding of the molecular mechanisms triggered by recurrent ischemic damage in neuronal cells and, therefore, could help with the development of an effective treatment to minimize the consequences of this pathology.

Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune-related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-κB activation observed in Mmp25-deficient leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients., (© 2016 by The American Society of Hematology.)

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haematopoietic progenitor cells due to a somatic mutation in theX-linked phosphatidylinositol glycan class A gene. The disease is characterized by intravascular haemolytic anaemia, propensity to thromboembolic events and bone marrow failure. Other direct complications of haemolysis include dysphagia, erectile dysfunction, abdominal pain, asthenia and chronic renal failure (65% of patients). The disease appears more often in the third decade of life and there is no sex or age preference. Detection of markers associated with glucosyl phosphatidyl inositol deficit by flow cytometry is currently used in the diagnosis of PNH. For years, transfusions have been the mainstay of therapy for PNH. A breakthrough in treatment has been the approval of the humanized monoclonal antibody eculizumab, which works by blocking the C5 complement protein, preventing its activation and therefore haemolysis. Several studies have confirmed that treatment with eculizumab avoids or decreases the need for transfusions, decreases the probability of thrombosis, improves the associated symptomatology and the quality of life in patients with PNH, showing an increase in survival. Because of rapid advances in the knowledge of the disease and its treatment, it may become necessary to adapt and standardize clinical guidelines for the management of patients with PNH., (Copyright © 2016 Elsevier España, S.L.U. All rights reserved.)

AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2(V617F) mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

Solitary plasmacytoma represents a heterogeneous group of patients; approximately half develop multiple myeloma (MM) in 2 or 3 years, whereas others remain disease-free at 10 years. By definition, these patients do not have morphologic bone marrow (BM) plasma cell (PC) infiltration. Here, we investigated whether sensitive BM evaluation of patients with solitary bone plasmacytoma (SBP; n = 35) and extramedullary plasmacytoma (EMP; n = 29) through multiparameter flow cytometry (MFC) would unravel the presence of clonal PCs in otherwise disease-free BM, and whether BM clonality predicted higher risk of progression. BM clonal PCs were detected in 17 of 35 SBP (49%) and 11 of 29 EMP (38%) patients. Seventy-one percent of flow-positive vs only 8% of flow-negative SBP patients evolved to MM (median time to progression of 26 months vs not reached; hazard ratio, 17.4; P < .001). No significant differences were observed among EMP cases. Our results highlight the importance of MFC for sensitive BM evaluation of SBP patients, to predict risk of developing treatment-requiring MM and to plan disease monitoring., (© 2014 by The American Society of Hematology.)

Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor κB pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ≤50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ≤50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome., (© 2014 by The American Society of Hematology.)

Abstract Purine analogs are highly effective in hairy cell leukemia (HCL) with response rates of 85%, but with many late relapses. We have retrospectively reviewed the clinical data from 107 patients treated with pentostatin (n = 27) or cladribine (n = 80), to investigate the long-term efficacy and to identify factors associated with the treatment-free interval (TFI). Complete remission and minimal residual disease (MRD) rates were similar in both groups. Median TFI was shorter (95 vs. 144 months) in the pentostatin group, although the difference was not significant (p = 0.476). MRD+ patients had shorter TFI than MRD- patients (97 months vs. not reached, p < 0.049). A hemoglobin level < 10 g/dL predicted for a shorter TFI only in the pentostatin group. Quality of response and number of hairy cells in the bone marrow are independent risk factors of treatment failure. The relationship between MRD+ and shorter TFI makes it of special interest to explore consolidation therapy with monoclonal antibodies to achieve durable responses.

We evaluated the impact of detection of minimal residual disease by flow cytometry (FCMRD) and CD3 chimerism in relapse in a cohort of 87 patients with acute myeloid leukemia or myelodysplastic syndrome undergoing stem cell transplantation. Patients with a positive FCMRD at day +100 after transplantation showed higher relapse rates and worse overall survival. In multivariate analysis, a positive FCMRD after transplantation was a significant predictor of relapse. Mixed chimerism showed a trend to statistical signification. We conclude that FCMRD at day 100 after SCT is the best predictor of relapse after SCT in patients with aggressive myeloid malignancies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. Here, we investigated the clonal vs reactive nature of residual maturing bone marrow cells in 59 newly diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

Epigenetic deregulation is considered a common hallmark of cancer. Nevertheless, recent publications have demonstrated its association with a large array of human diseases. Here, we explore the DNA methylation dynamics in blood samples during hematopoietic cell transplant and how they are affected by pathophysiological events during transplant evolution. We analyzed global DNA methylation in a cohort of 47 patients with allogenic transplant up to 12 months post-transplant. Recipients stably maintained the donor's global methylation levels after transplant. Nonetheless, global methylation is affected by chimerism status. Methylation analysis of promoters revealed that methylation in more than 200 genes is altered 1 month post-transplant when compared with non-pathological methylation levels in the donor. This number decreased by 6 months post-transplant. Finally, we analyzed methylation in IFN-γ, FASL, IL-10, and PRF1 and found association with the severity of the acute graft-versus-host disease. Our results provide strong evidence that methylation changes in blood are linked to underlying physiological events and demonstrate that DNA methylation analysis is a viable strategy for the study of transplantation and for development of biomarkers.

ADAMTSs (a disintegrin and metalloprotease with thrombospondin domains) are a family of enzymes with both proteolytic and protein interaction functions, which have been implicated in distinct pathologies. In this work, we have investigated the putative role of ADAMTS-12 in inflammation by using a mouse model deficient in this metalloprotease. Control and mutant mice were subjected to different experimental conditions to induce colitis, endotoxic sepsis, and pancreatitis. We have observed that Adamts12-deficient mice exhibit more severe inflammation and a delayed recovery from these challenges compared with their wild-type littermates. These changes are accompanied by an increase in inflammatory markers including several cytokines, as assessed by microarray expression analysis and proteomic-based approaches. Interestingly, the clinical symptoms observed in Adamts12-deficient mice are also concomitant with an elevation in the number of neutrophils in affected tissues. Finally, isolation and in vitro culture of human neutrophils demonstrate that the presence of ADAMTS-12 induces neutrophil apoptosis. On the basis of these results, we propose that ADAMTS-12 is implicated in the inflammatory response by modulating normal neutrophil apoptosis.

Despite the advantage observed with novel drugs such as bortezomib, thalidomide, or lenalidomide, multiple myeloma (MM) remains incurable and there is a clear need for new drugs or combinations based on the pathogenetic mechanism of MM. One of the proposed mechanisms in MM pathogenesis is the involvement of kinase molecules in the growth and survival of myelomatous cells. In this study, we have explored the optimal combination for dasatinib, a tyrosine kinase inhibitor, in MM cells. A clear synergistic effect was observed with the triple combination of dasatinib with bortezomib and dexamethasone which was evident even in the presence of bone marrow microenvironment. Experiments performed on freshly isolated patients' cells also demonstrated potentiation of response in the triple as compared with the agents alone or in double combinations. Gene expression profiling experiments provided some clues on the transcriptional rationale underlying this potentiation, as the triple combination led to significant deregulation of genes involved in cell death, cell growth, proliferation, DNA replication, repair and recombination, and cell-cell signaling. Some of these results were further confirmed by apoptosis and cell cycle experiments and also by Western blot and PCR. These data provide the rationale for the use of this novel combination in MM patients.

Matrix metalloproteinase-8, released mainly from neutrophils, is a critical regulator of the inflammatory response by its ability to cleave multiple mediators. Herein, we report the results of a model of endotoxemia after intraperitoneal LPS injection in mice lacking MMP-8 and their wildtype counterparts. Control, saline-treated animals showed no differences between genotypes. However, there was an increased lung inflammatory response, with a prominent neutrophilic infiltration in mutant animals after LPS treatment. Using a proteomic approach, we identify alarmins S100A8 and S100A9 as two of the main differences between genotypes. Mice lacking MMP-8 showed a significant increase in these two molecules in lung homogenates, but not in spleen and serum. Mice lacking MMP-8 also showed an increase in MIP-1α levels and a marked activation of the non-canonical NF-κB pathway, with no differences in CXC-chemokines such as MIP-2 or LIX. These results show that MMP-8 can modulate the levels of S100A8 and S100A9 and its absence promotes the lung inflammatory response during endotoxemia.

Background: Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin., Design and Methods: The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid., Results: Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34(+) CD38(-) Lin(-)) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX)., Conclusions: The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients' samples provides the rationale for the investigation of this compound in clinical trials.

Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of myeloid blasts in the bone marrow. Here, we report the effects of the novel histone deacetylase inhibitor panobinostat (LBH589) in combination with doxorubicin on AML cells. Panobinostat exhibited potent anti-AML activity in all AML cell lines tested and in primary AML cells from patients (IC(50)<20 nM). In addition, panobinostat potentiated the action of several standard-of-care anti-AML compounds, particularly, doxorubicin. The molecular effects induced by panobinostat and doxorubicin treatment were investigated by analyzing gene expression, cell cycle, apoptosis and signaling pathways. Analyses of gene expression profiles identified 588 genes whose expression was exclusively affected by the combination of panobinostat and doxorubicin. The combination induced AML cell death by an increase in the mitochondrial outer membrane permeability and release of cytochrome c from the mitochondria, resulting in caspase-dependent apoptosis and accompanied by the upregulation of Bax, Bak and, particularly, Bad. The drug combination provoked a strong activation of a DNA damage response, indicating that this combination may trigger cell death by a mechanism that induced DNA double-strand breaks. These data indicate that the combination of panobinostat and doxorubicin may be an effective therapy for the treatment of AML.

We have prospectively evaluated the efficacy of an individualized pre-operative blood saving protocol in elective total hip arthroplasty (THA) or total knee arthroplasty (TKA). The primary aim was to obtain a pre-operative haemoglobin (Hb) level of > or =14 g dL(-1). A reduction in requirements for allogeneic transfusion was considered the second aim. Several strategies are available for increasing pre-operative Hb levels and reducing red blood cell (RBC) transfusions following THA or TKA, but the success of these programmes depends on selecting the most appropriate treatment for each patient. Three hundred and five patients with an indication of elective THA or TKA were individually assigned to the following strategies according to Hb and ferritin levels and medical conditions: (a) no pre-operative intervention, (b) oral iron therapy, (c) intravenous (i.v.) iron therapy, (d) recombinant human erythropoietin alpha with i.v. iron and (e) pre-operative autologous donation (PAD) plus oral iron. Eighty-two percent of the patients reached a pre-operative Hb level of > or =14 g dL(-1) compared with 62% of patients with Hb levels of > or =14 g dL(-1) at the baseline visit. Treatment with PAD showed a significant reduction in the pre-operative Hb levels. The rate of RBC transfusion was 18.8% compared with 31.5% of matched historic group (P < 0.001). In conclusion, all patients scheduled to undergo THA or TKA should be candidates for an individualized pre-operative blood salvage programme.

Several grading systems have been developed in the bone marrow transplantation setting in attempts to predict survival in patients with chronic graft-versus-host disease (cGVHD). In this study, we evaluated the prognostic value of the National Institutes of Health (NIH) scoring system and investigated for any additional prognostic factors in a series of 171 patients undergoing peripheral blood stem cell transplantation (PBSCT) from matched related donors. The cumulative incidence of cGVHD was 70%; cumulative incidences of mild, moderate, and severe cGVHD were 29%, 42% and 28%, respectively. Overall, 68% of patients were free from immunosuppression 5 years after transplantation. Absence of previous acute GVHD (aGVHD; hazard ratio [HR] = 2; P = .004) and mild cGVHD (HR = 4.2; P = .007) increased the probability of being off immunosuppressive treatment by the last follow-up. Overall survival (OS) at 5 years was 52%. Severe cGVHD, according to the NIH scoring system (HR = 13.27; P = .001) adversely influenced outcome, whereas de novo onset (HR = 0.094; P = .003) had a more favorable impact on survival. The combination of both variables allowed us to identify 4 different subgroups of patients with OS of 82%, 70%, 50%, and 25%. Our findings indicate that the NIH scoring system has some prognostic value in patients undergoing PBSCT and, together with the type of onset, must be considered to predict the possible outcome of patients who develop cGVHD.

Lenalidomide combined with dexamethasone has significant clinical activity in the treatment of multiple myeloma (MM). In previous clinical trials lenalidomide-induced neutropenia was a frequent side-effect, often leading to treatment delays and dose reductions. We describe three MM patients treated with lenalidomide plus dexamethasone, which developed grade 3/4 neutropenia during the initial cycles, but without serious infection. Administration of granulocyte-colony stimulating factor (G-CSF) for 3 d prevented further neutropenia, treatment delays, dose reductions, or infectious complications during the following cycles. Consequently, G-CSF could be effective in preventing further neutropenia-related complications without compromising treatment efficacy in MM patients with lenalidomide-induced neutropenia.

Background: Proteasome inhibition represents a promising novel anticancer therapy, and bortezomib is a highly selective reversible inhibitor of the proteasome complex. Acute myeloid leukemia (AML) is an immnunophenotypically heterogeneous group of diseases, with CD34(+) cases being associated with drug resistance and poor outcome. We investigated the effects of bortezomib on the growth and survival of AML cells., Design and Methods: We studied the in vitro activity and mechanism of action of bortezomib on both cell lines and fresh cells from 28 AML patients including CD34(+) and CD34(-) cases., Results: Bortezomib showed potent anti-AML activity (IC(50) < 50 nM), which was greater than that of conventional agents (doxorubicin, cytarabine and fludarabine). Moreover, synergistic effects were observed when bortezomib was administered in combination with doxorubicin and cytarabine. Mechanistically, bortezomib induced accumulation of cells in the G(2)/M phase, with up-regulation of p27, together with cell death through an increase in the mitochondrial outer membrane permeability involving caspase-dependent and -independent pathways. The apoptotic activity of bortezomib on fresh CD34(+) blast cells from patients was similar to that observed on CD34(-)blast cells. Importantly, bortezomib was significantly more active than doxorubicin in the immature CD34(+) cells, while there were no differences in its action on CD34(-) cells., Conclusions: Bortezomib induces apoptosis in acute myeloid leukemia cells in vitro. Whether this drug might be useful in the treatment of patients with acute myeloid leukemia can be established only in ad hoc clinical trials.