Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c(+)), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non-exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c(+) cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n 5 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

Nucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (∼30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)-retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1 mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics.

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 Sjogren's 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, BIT 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 ON T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating ON 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. (C) 2014 Elsevier Ltd. All rights reserved.

The BRAF-V600E mutation defines genetically hairy cell leukemia among B-cell leukemias and lymphomas. In solid tumors, BRAF-V600E is known to aberrantly activate the oncogenic MEK-ERK pathway, and targeted BRAF and/or MEK inhibitors have shown remarkable efficacy in clinical trials in melanoma patients. However, the MEK-ERK pathway status in hairy cell leukemia has not been thoroughly investigated. We assessed phospho-ERK expression in 37 patients with hairy cell leukemia and 44 patients with neoplasms mimicking hairy cell leukemia (40 splenic marginal zone lymphoma, 2 hairy cell leukemia-variant and 2 splenic lymphoma/leukemia unclassifiable) using immunohistochemistry on routine biopsies and/or Western blotting on purified leukemic cells, and correlated the phospho-ERK status with the BRAF-V600E mutation status. Besides confirming the constant presence of BRAF-V600E in all patients with hairy cell leukemia, we observed ubiquitous phospho-ERK expression in this malignancy. Conversely, all 44 cases with neoplasms mimicking hairy cell leukemia were devoid of BRAF-V600E and none expressed phospho-ERK. Furthermore, the two exceptionally rare cases of non-hairy cell leukemia unclassifiable chronic B-cell neoplasms previously reported to be BRAF-V600E+ on allele-specific polymerase chain reaction lacked phospho-ERK expression as well, suggesting the presence of the mutation in only a small part of the leukemic clone in these cases. In conclusion, our findings support the use of phospho-ERK immunohistochemistry in the differential diagnosis between hairy cell leukemia and its mimics, and establish the MEK-ERK pathway as a rational therapeutic target in this malignancy.

Aims: The aim of this study was to search for a molecule selectively expressed by marginal zone (MZ) lymphomas (MZLs), whose diagnosis is currently based on morphological criteria and negativity for markers detectable in other B-cell lymphomas.Methods and results: Two thousand one hundred and four peripheral lymphomas of various types were immunostained with a monoclonal antibody against immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), which recognizes the equivalents of MZ in human lymphoid tissues other than spleen. IRTA1 expression was restricted to extranodal (93%) and nodal MZLs (73%) and to lymphomas with MZ differentiation. Extranodal MZL cells with the strongest IRTA1 expression were usually located adjacent to epithelia, mimicking the IRTA1 expression pattern of normal and acquired mucosa-associated lymphoid tissue (MALT). The cytological features, growth pattern and IRTA1 positivity in nodal MZLs suggest they may derive from IRTA1(+) perifollicular B cells or monocytoid B cells detectable in reactive lymph nodes. Double immunostaining for IRTA1/bcl-6 tracked the colonization of B-cell follicles by MZL cells, and showed modulation of their phenotype (e.g. acquisition of bcl-6) during recirculation through germinal centres. MZL cells differentiating into plasma cells usually lost IRTA1.Conclusions: These results further expand our knowledge of the biology of MZLs, and highlight IRTA1 as the first positive marker for MZLs, enabling more accurate diagnosis of these neoplasms.

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.).

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Abstract Abstract 480 Acute myeloid leukemia with mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features [Falini B et al, Blood 2007;109:874-885] which include negativity for CD34 antigen expression at both immunohistochemistry and gene expression profiling. Flow cytometric analysis shows that, in most NPM1-mutated AML, percentages of CD34+ cells are in the low range (< 5-10%). Detection of NPM1 mutations by molecular techniques and/or immunohistochemistry and Western Blot analysis with specific antibodies provides an important tool for tracking the genetic lesion in leukemic cells at different hierarchical stage. We previously reported involvement by NPM1 gene mutation of the CD34+ cell fraction isolated from patients with NPM1-mutated AML, and, in one case, the involvement, in particular, of the early progenitor CD34+/CD38- [Martelli MP et al, Blood (ASH Annual Meeting Abstracts) 2008;112:307]. Here we expand and confirm our previous observation in 5 cases of CD34-negative NPM1-mutated AML. CD34+/CD38- cells were isolated by either FACS (3 cases, purity >98%) or MACS-sorting (2 cases, purity >92%) and analyzed by molecular analysis and Western Blot with a specific anti-NPM1 mutant antibody, respectively. The presence of either NPM1 gene mutation or mutant protein was demonstrated in all samples analyzed proving the CD34+/CD38- cells belong to the leukemic clone. This cell subpopulation displayed also immunophenotypic features classically associated to leukemic stem cells (LSCs) (CD123+/CD33+/CD90-) in all (16/16) samples analyzed, suggesting they might actually represent the LSCs in NPM1-mutated AML. Indeed, CD34+ cell fraction isolated from NPM1-mutated AML was able to generate leukemia in immunocompromised mice resembling the original patient's disease. However, there is experimental evidence that, at least in some CD34-negative AML, also the CD34- population may contain LSCs. Whether the CD34- cell compartment in NPM1-mutated AML is also able to engraft and outgrow into leukemia in mice remains to be clarified. For this purpose, we assessed the engraftment ability of CD34- cells from 5 NPM1-mutated AML patients. No engraftment was observed in one case. Interestingly, in three patients with myelomonocytic (M4, 2 cases) and myelocytic (M2, 1 case) AML, the CD34- fraction resulted into marrow engraftment by human CD45+/CD33+ myeloid cells that, at morphological and immunohistological grounds, consisted of a mixed population of macrophage cells expressing the CD68 (PG-M1) antigen and mature looking myeloperoxidase (MPO)-positive cells. This pattern possibly reflects short-term engraftment by leukemic cells devoid of self-renewal potential that differentiated into mature elements. However, the neoplastic nature of engrafted cells could be established with certainty only in one case by western blotting detection of NPM1 mutant protein. Immunohistochemistry could not help in these cases to establish the leukemic nature of human cells since terminally differentiated leukemic cells in NPM1-mutated AML show nucleus-restricted NPM1 positivity. In contrast, the pure CD34+ fraction (availabel for comparison in one of these three cases) engrafted as AML with clear blastic morphology and cytoplasmic dislocation of nucleophosmin. In a fourth patient, the highly purified CD34- fraction from relapsed NPM1-mutated AML engrafted in mice with a typical AML picture. These preliminary findings suggest that in general the CD34- fraction from NPM1-mutated AML may have more limited engraftment potential than the CD34+ fraction. Further studies are ongoing to address this issue. Disclosures: Falini: Xenomics: Patents & Royalties.

Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005; 352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood 2007; 109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood 2006; 108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity >90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient's, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1.

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.

Pileri, Cristina Mecucci, Franco Mandelli, Massimo Fabrizio Martelli and Brunangelo Falini Enrico Tiacci, Giovanna Meloni, Giorgina Specchia, Nicola Cantore, Francesco Di Raimondo, Stefano Tabarrini, Manola Carini, Barbara Bigerna, Alessandra Pucciarini, Roberta Mannucci, Ildo Nicoletti, Laura Pasqualucci, Arcangelo Liso, Maria Paola Martelli, Niccolò Bolli, Roberta Pacini, Alessia acute myeloid leukemia: impact on WHO classification Mutated nucleophosmin detects clonal multilineage involvement in

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.