IntroductionA viable option for high-risk, acute leukemia patients without matched donors is hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA)-haploidentical 3-locimismatched family members who were readily available for almost all patients. 1,2 Until the 1990s, full-haplotype-mismatched, T cell-replete transplantations were unsuccessful because donoralloreactive T cells triggered a high incidence of severe graft-versushost disease (GVHD) despite posttransplantation immune suppression. 3,4 The breakthrough came with the use of a megadoses of extensively ex vivo T cell-depleted peripheral blood hematopoietic progenitor cells and a highly myeloablative conditioning regimen containing antithymocyte globulin (ATG), which exerts additional T-cell depletion in vivo. This approach ensures a high rate of primary engraftment in the absence of GVHD, 5 with more than 40% long-term event-free survival and excellent quality of life. 1,2 However, extensive ex vivo and in vivo T-cell depletion delays the recovery of immune responses against pathogens, leading to a high incidence of life-threatening infections. 1,2 Strategies to hasten posttransplantation immune reconstitution without triggering GVHD have included infusion of donor T cells after engineering with a suicide gene, 6 photodynamic purging, 7 and the use of an anti-CD25 monoclonal antibody (mAb) 8 to remove alloreactive cells. An alternative strategy might be based on donor CD4 ϩ CD25 ϩ T-regulatory cells (Tregs). In murine models of HSCT across major histocompatibility complex barriers, CD4 ϩ CD25 ϩ Tregs suppressed lethal GVHD 9 and favored posttransplantation immune reconstitution when coinfused with conventional T cells (Tcons). 10 The main obstacle to clinical application of human Tregs is their paucity in the peripheral blood. Although ex vivo-expanded polyclonal 11 or recipient-specific Tregs 12 were proposed to circumvent this potential barrier, we opted for closed, automated immunoselection 13 of naturally occurring Tregs. In the present study, for the first time in humans, we show that the early infusion of freshly isolated donor Tregs followed by Tcons at the time of full-haplotypemismatched HSCT prevented GVHD while favoring Tconmediated posttransplantation immune reconstitution. MethodsStudy design, conditioning regimen, stem cell mobilization, and supportive careIn 2008, the Umbria Regional Hospital Ethics Committee (CEAS Umbria) approved the protocol entitled "Adoptive Immunotherapy with Natural Regulatory T cells (Treg) and Effector T Cells in Allogeneic Hematopoietic Stem Cell Transplantation from 2-3 Loci Mismatched HLA-Haploidentical Family Donors for Patients with High Risk Haematologic Malignancies" (Protocol No 01/08). Written informed consent was obtained for all patients and donors in accordance with the Declaration of Helsinki. Inclusion criteria were: acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) in remission at high risk of relapse; acute leukemia with primary induction failure, in chemoresist...

Background Hairy cell leukemia (HCL) is a well defined clinico-pathological entity whose underlying genetic lesion is still obscure. Methods We searched for HCL-associated mutations by massively parallel sequencing of the whole exome of leukemic and matched normal mononuclear cells purified from the peripheral blood of one patient with HCL. Results Whole exome sequencing identified 5 missense somatic clonal mutations that were confirmed at Sanger sequencing, including a heterozygous V600E mutation involving the BRAF gene. Since the BRAF V600E mutation is oncogenic in other tumors, further analyses were focused on this genetic lesion. Sanger sequencing detected mutated BRAF in 46/46 additional HCL patients (47/47 including the index case; 100%). None of the 193 peripheral B-cell lymphomas/leukemias other than HCL that were investigated carried the BRAF V600E mutation, including 36 cases of splenic marginal zone lymphomas and unclassifiable splenic lymphomas/leukemias. Immunohistological and Western blot studies showed that HCL cells express phospho-MEK and phospho-ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic cells from 5 HCL patients with PLX-4720, a specific inhibitor of active BRAF, led to marked decrease of phosphorylated ERK and MEK. Conclusions The BRAF V600E mutation was present in all HCL patients investigated. This finding may have relevant implications for the pathogenesis, diagnosis and targeted therapy of HCL (Funded by the Associazione Italiana Ricerca Cancro and others).

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol-3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b∼25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b∼25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a protooncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, characterized by immunodeficiency. Hence, patients with CLL might be considered more susceptible to severe complications from COVID-19. We undertook this retrospective international multicenter study to characterize the course of COVID-19 in patients with CLL and identify potential predictors of outcome. Of 190 patients with CLL and confirmed COVID-19 diagnosed between 28/03/2020 and 22/05/2020, 151 (79%) presented with severe COVID-19 (need of oxygen and/or intensive care admission). Severe COVID-19 was associated with more advanced age (≥65 years) (odds ratio 3.72 [95% CI 1.79-7.71]). Only 60 patients (39.7%) with severe COVID-19 were receiving or had recent (≤12 months) treatment for CLL at the time of COVID-19 versus 30/39 (76.9%) patients with mild disease. Hospitalization rate for severe COVID-19 was lower (p < 0.05) for patients on ibrutinib versus those on other regimens or off treatment. Of 151 patients with severe disease, 55 (36.4%) succumbed versus only 1/38 (2.6%) with mild disease; age and comorbidities did not impact on mortality. In CLL, (1) COVID-19 severity increases with age; (2) antileukemic treatment (particularly BTK inhibitors) appears to exert a protective effect; (3) age and comorbidities did not impact on mortality, alluding to a relevant role of CLL and immunodeficiency.

The nucleophosmin (NPM1) gene encodes for a multifunctional protein with prominent nucleolar localization that shuttles between nucleus and cytoplasm. NPM1 mutations represent the most common genetic lesion in adult AML (about one-third of cases) and act deterministically to cause the aberrant cytoplasmic delocalization of NPM1 mutants. Because of its unique features, NPM1-mutated AML is recognized as a distinct entity in the 2016 World Health Organization classification of hematopoietic neoplasms. Here, we focus on recently identified functions of wild-type NPM1 in the nucleolus and address new biological and clinical issues on NPM1-mutated AML. The relevance of cooperation between NPM1 and other mutations in driving AML with different outcomes is presented. We also discuss the importance of eradicating NPM1-mutated clones to achieve AML cure and the impact of pre-leukemic clonal hematopoiesis persistence in predisposing to second AML. The contribution of HOX genes expression to the development of NPM1-mutated AML is also highlighted. Clinically, yet unsolved diagnostic issues in the 2016-WHO classification of myeloid neoplasms and the importance of NPM1 mutations in defining the framework of European LeukemiaNet genetic-based risk-stratification are discussed. Finally, we address the value and limits of NPM1-based minimal residual disease assessment for treatment guidance and present the results of promising preclinical studies with XPO1 and Menin-MLL inhibitors.

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After the discovery of NPM1-mutated acute myeloid leukemia (AML) in 2005 and its subsequent inclusion as a provisional entity in the 2008 World Health Organization classification of myeloid neoplasms, several controversial issues remained to be clarified. It was unclear whether the NPM1 mutation was a primary genetic lesion and whether additional chromosomal aberrations and multilineage dysplasia had any impact on the biologic and prognostic features of NPM1-mutated AML. Moreover, it was uncertain how to classify AML patients who were double-mutated for NPM1 and CEBPA. Recent studies have shown that: (1) the NPM1 mutant perturbs hemopoiesis in experimental models; (2) leukemic stem cells from NPM1-mutated AML patients carry the mutation; and (3) the NPM1 mutation is usually mutually exclusive of biallelic CEPBA mutations. Moreover, the biologic and clinical features of NPM1-mutated AML do not seem to be significantly influenced by concomitant chromosomal aberrations or multilineage dysplasia. Altogether, these pieces of evidence point to NPM1-mutated AML as a founder genetic event that defines a distinct leukemia entity accounting for approximately one-third of all AML. (Blood. 2011;117(4):1109-1120) IntroductionThe remarkable molecular heterogeneity of acute myeloid leukemia (AML) 1 has made a genetic-based classification essential for accurate diagnosis, prognostic stratification, monitoring minimal residual disease, and developing targeted therapies. The category of "AML with recurrent genetic abnormalities," which includes the genetically best defined myeloid neoplasms, underwent major changes in the 2008 World Health Organization (WHO) classification. 2 The 4 molecularly distinct entities that had been described in the 2001 WHO classification were expanded to include AML with t(6;9), AML with inv(3) or t(3;3), and AML (megakaryoblastic) with; t(1;22) and 2 provisional entities: AML with mutated CEBPA and AML with mutated nucleophosmin (NPM1) ( Table 1). The latter accounts for approximately one-third of all AMLs 3 and has distinct genetic, pathologic, immunophenotypic, and clinical characteristics. 4,5 The WHO synonym for AML with mutated NPM1, NPMc ϩ AML (c ϩ indicates "cytoplasmic positive"), 3 focuses on its most distinguishing functional feature, that is, aberrant expression of nucleophosmin in the cytoplasm of leukemic cells. 6 This unique immunohistochemical pattern, which led in 2005 to the discovery of NPM1 mutations in AML, 3 is an excellent surrogate marker for molecular studies because it is fully predictive of NPM1 mutations. 7,8 The present review is an update of the distinct genetic and clinical features of AML with mutated NPM1. AML with mutated NPM1 shows distinct genetic featuresSeveral pieces of evidence suggest the NPM1 mutation is a founder genetic alteration (Table 2) in AML.With the exception of rare cases of myelodysplastic syndrome (MDS)/myeloproliferative neoplasms 9 that require further confirmation, the NPM1 mutation or its immunohistochemical surrogate (cytoplasmic nucleophosmin)...