Paolo Sportoletti

Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

BRAF Mutations in Hairy-Cell Leukemia

BACKGROUND Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and 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 hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Identification of the miR-106b∼25 microRNA cluster as a proto-oncogenic PTEN-targeting intron that cooperates with its host gene MCM7 in transformation

A microRNA network regulates the tumor suppressor PTEN in prostate cancer. A Malignant Combination The abundance of microRNAs (miRNAs), tiny non–protein-coding RNAs that act as posttranscriptional regulators of gene expression, is frequently altered in cancer; indeed, various miRNAs are thought to act as oncogenes or tumor suppressors. Poliseno et al. investigated the possible role of miRNA regulation of the tumor suppressor PTEN in prostate cancer. They identified miRNAs from several families that targeted the gene encoding PTEN, thereby decreasing PTEN abundance, and showed that the abundance of some of these miRNAs was increased in human prostate cancer. Intriguingly, three PTEN-targeting miRNAs located within an intron of the gene encoding the DNA helicase minichromosome maintenance protein 7 (MCM7), which shows increased abundance in various human cancers, cooperated with MCM7 to transform fibroblasts in vitro and to initiate tumors when overexpressed in the prostates of transgenic mice. Thus, the MCM7 gene locus appears to encode multiple oncogenic elements that cooperate to promote prostate cancer development. 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 proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

Mesenchymal cells recruit and regulate T regulatory cells

OBJECTIVE Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. MATERIALS AND METHODS Human MSCs and different T cell populations (CD3(+), CD3(+)/CD45RA(+), CD3(+)/CD45RO(+), CD4(+)/CD25(+), CD4(+)/CD25(+)/CD45RO(+), CD4(+)/CD25(+)/CD45RA(+)) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. RESULTS We demonstrate MSC recruit Tregs from a fraction of CD3(+) and from immunoselected CD3(+)/CD45RA(+) and CD3(+)/CD45RO(+) fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4(+)/CD25(bright)/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25(+), CD4/CD25(+)/CD45RA(+), and CD4/CD25(+)/CD45RO(+)) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. CONCLUSIONS In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time.

Acute myeloid leukemia with mutated nucleophosmin (NPM1): is it a distinct entity?

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.

Npm1 is a haploinsufficient suppressor of myeloid and lymphoid malignancies in the mouse

Nucleophosmin (NPM1) gene has been heavily implicated in cancer pathogenesis both as a putative proto-oncogene and tumor suppressor gene. NPM1 is the most frequently mutated gene in acute myeloid leukemia (AML), while deletion of 5q, where NPM1 maps, is frequent in patients with myelodysplastic syndromes (MDS). We have previously shown that mice heterozygous for Npm1 (Npm1+/-) develop a hematologic syndrome with features of human MDS. Here we analyzed Npm1+/- mutants to determine their susceptibility to cancer. Npm1+/- mice displayed a greater propensity to develop malignancies compared with Npm1+/+ mice. The Npm1+/- cohort frequently developed hematologic malignancies of both myeloid and lymphoid origin with myeloid malignancies displaying the highest incidence. Malignant cells retained the wild-type allele with normal localization and expression of Npm1 at the protein level, suggesting that complete Npm1 loss is not a prerequisite for tumorigenesis. Our results conclusively demonstrate that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment.

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

Prosper, J.Y., Campbell, K., Sutherland, D.R., Metcalfe, P., Horsfall, W. & Ouwehand, W.H. (2002) A tyrosine703serine polymorphism of CD109 defines the Gov platelet alloantigens. Blood, 99, 1692– 1698. Smith, J.W., Hayward, C.P., Horsewood, P., Warkentin, T.E., Denomme, G.A. & Kelton, J.G. (1995) Characterization and localization of the Gova/b alloantigens to the glycosylphosphatidylinositol-anchored protein CDw109 on human platelets. Blood, 86, 2807–2814.

Acute myeloid leukemia with mutated NPM1: diagnosis, prognosis and therapeutic perspectives.

Purpose of review Nucleophosmin (NPM1) gene mutations, which cause aberrant cytoplasmic expression of nucleophosmin (NPMc+), are the most frequent genetic alteration in acute myeloid leukemia (AML), being found in about 30% cases. The present review summarizes recent advances in the biology, diagnosis, prognosis and therapy of NPM1-mutated AML. Recent findings Diagnostic criteria of NPM1-mutated AML are discussed in the light of its recent inclusion in the 2008 WHO classification of myeloid neoplasms. We also outline the most recent findings on prognosis and monitoring of minimal residual disease in NPM1-mutated AML and their implications for therapeutic decisions. Moreover, new insights are presented into the molecular mechanisms underlying perturbed nucleophosmin traffic in NPM1-mutated AML, which provides the rationale for the development of targeted therapies. Summary AML with mutated NPM1 is a leukemia entity with distinct molecular, pathological, and prognostic features.

The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model

Although NPM1 gene mutations leading to aberrant cytoplasmic expression of nucleophosmin (NPMc(+)) are the most frequent genetic lesions in acute myeloid leukemia, there is yet no experimental model demonstrating their oncogenicity in vivo. We report the generation and characterization of a transgenic mouse model expressing the most frequent human NPMc(+) mutation driven by the myeloid-specific human MRP8 promoter (hMRP8-NPMc(+)). In parallel, we generated a similar wild-type NPM trans-genic model (hMRP8-NPM). Interestingly, hMRP8-NPMc(+) transgenic mice developed myeloproliferation in bone marrow and spleen, whereas nontransgenic littermates and hMRP8-NPM transgenic mice remained disease free. These findings provide the first in vivo evidence indicating that NPMc(+) confers a proliferative advantage in the myeloid lineage. No spontaneous acute myeloid leukemia was found in hMPR8-NPMc(+) or hMRP8-NPM mice. This model will also aid in the development of therapeutic regimens that specifically target NPMc(+).

Mutational landscape of AML with normal cytogenetics: Biological and clinical implications

Acute myeloid leukemia (AML) is a molecularly heterogeneous disease. Based on cytogenetics and FISH, AML patients are stratified into three major risk categories: favourable, intermediate and unfavourable. However, prognostic stratification and treatment decision for the intermediate risk category, that mostly comprises AML patients with normal cytogenetics (CN-AML), has been difficult due to the clinical heterogeneity and scarce knowledge of the molecular alterations underlying this large AML subgroup. During the past decade, the identification of several mutations associated with CN-AML has resulted into important advances in the AML field. In this review, we address the biological features of the main mutations associated with CN-AML and the impact of next generation sequencing studies in expanding our knowledge of the molecular landscape of CN-AML. In addition, we outline the prognostic value of mutations for risk stratification of CN-AML patients and discuss the potential of mutations discovery process for developing new molecular targeted therapies.