Barbara Bigerna

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.).

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

Born to Be Exported: COOH-Terminal Nuclear Export Signals of Different Strength Ensure Cytoplasmic Accumulation of Nucleophosmin Leukemic Mutants

Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.

IRTA1 is selectively expressed in nodal and extranodal marginal zone lymphomas

Falini B, Agostinelli C, Bigerna B, Pucciarini A, Pacini R, Tabarrini A, Falcinelli F, Piccioli M, Paulli M, Gambacorta M, Ponzoni M, Tiacci E, Ascani S, Martelli M P, Dalla Favera R, Stein H & Pileri S A 
(2012) Histopathology 61, 930–941

A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14ARF. Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14ARF in a ‘dose-dependent tug of war’ fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14ARF) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14ARF) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.

Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice.

The anti‐CD30 immunotoxin (IT) Ber‐H2/saporin is effective in patients with refractory Hodgkin’s disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti‐CD30 ITs by covalently linking the mouse monoclonal antibody Ber‐H2 to the type 1 ribosome‐inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP‐S), which do not cross‐react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin’s disease and anaplastic large‐cell lymphoma (ALCL)‐derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 × 10−13 m to 2.75 × 10−11m, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3 d treatment with non‐toxic doses of Ber‐H2/MOM (50% LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were a dose‐related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber‐H2/MOM and 0.45 mg/kg for Ber‐H2/PAP‐S. Sequential administration of two anti‐CD30 ITs (Ber‐H2/MOM and Ber‐H2/saporin) was well tolerated and did not result in formation of antibodies cross‐reacting with the two plant toxins. The results presented in this paper suggest that, in the future, sequential administration of anti‐CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP‐S) should be feasible.

IL-17-producing CD4−CD8− T cells are expanded in the peripheral blood, infiltrate salivary glands and are resistant to corticosteroids in patients with primary Sjögren's syndrome

Objectives It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögrens syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage. Methods Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining. Results CD3+CD4−CD8− DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αβ-TCR+ DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone. Conclusions These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): a comparison with NPMc+ AML.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): A comparison with NPMc+ AML

CD4(-)CD8(-) T-cells in primary Sjögren's syndrome: association with the extent of glandular involvement.

OBJECTIVES Growing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögrens syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters. METHODS Paired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed. RESULTS In early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4(+)Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells. CONCLUSIONS Our findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease.

Cytoplasmic mutated nucleophosmin is stable in primary leukemic cells and in a xenotransplant model of NPMc+ acute myeloid leukemia in SCID mice

Findings of this study indicate that NPM1 mutations are stable in patients with acute myeloid leukemia, providing a rationale for monitoring of minimal residual disease. We investigated the NPM1 mutation status or subcellular expression of NPM protein (nuclear vs. aberrant cytoplasmic) at diagnosis and relapse in 125 patients with acute myeloid leukemia from Italy and Germany. All 52 patients with acute myeloidleukemia carrying at diagnosis mutated or cytoplasmic NPM (NPMc+ acute myeloid leukemia) retained this feature at relapse. Notably, cytoplasmic mutated NPM has now been retained for eight years in a xenotransplant model of NPMc+ acute myeloid leukemia in immunodeficient mice. None of 73 acute myeloid leukemia patients carrying at diagnosis wild-type NPM1 gene or showing at immunohistochemistry nucleus-restricted expression of nucleophosmin (NPMc− acute myeloid leukemia), which is predictive of NPM1 gene in germline configuration, acquired cytoplasmic mutated NPM at relapse. This finding further confirms that NPMc+ acute myeloid leukemia represents a primary event rather than a transformation stage of NPMc− acute myeloid leukemia. The stability of cytoplasmic mutated NPM in patients with acute myeloid leukemia, even at relapse in extramedullary sites, and in a xenotransplant model, suggest this event is crucial for leukemogenesis and represents the rationale for monitoring minimal residual disease and molecular targeted therapy in NPMc+ acute myeloid leukemia.