Arcangelo Liso

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.).

Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin

Acute myeloid leukemia (AML) carrying NPM1 mutations and cytoplasmic nucleophosmin (NPMc+ AML) accounts for about one-third of adult AML and shows distinct features, including a unique gene expression profile. MicroRNAs (miRNAs) are small noncoding RNAs of 19–25 nucleotides in length that have been linked to the development of cancer. Here, we investigated the role of miRNAs in the biology of NPMc+ AML. The miRNA expression was evaluated in 85 adult de novo AML patients characterized for subcellular localization/mutation status of NPM1 and FLT3 mutations using a custom microarray platform. Data were analyzed by using univariate t test within BRB tools. We identified a strong miRNA signature that distinguishes NPMc+ mutated (n = 55) from the cytoplasmic-negative (NPM1 unmutated) cases (n = 30) and includes the up-regulation of miR-10a, miR-10b, several let-7 and miR-29 family members. Many of the down-regulated miRNAs including miR-204 and miR-128a are predicted to target several HOX genes. Indeed, we confirmed that miR-204 targets HOXA10 and MEIS1, suggesting that the HOX up-regulation observed in NPMc+ AML may be due in part by loss of HOX regulators-miRNAs. FLT3-ITD+ samples were characterized by up-regulation of miR-155. Further experiments demonstrated that the up-regulation of miR-155 was independent from FLT3 signaling. Our results identify a unique miRNA signature associated with NPMc+ AML and provide evidence that support a role for miRNAs in the regulation of HOX genes in this leukemia subtype. Moreover, we found that miR-155 was strongly but independently associated with FLT3-ITD mutations.

Simple diagnostic assay for hairy cell leukaemia by immunocytochemical detection of annexin A1 (ANXA1)

A marker capable of distinguishing with certainty between hairy cell leukaemia and other B-cell malignant disease would be of great diagnostic value. Through gene expression profiling, annexin A1 (ANXA1) has been identified as a gene that is upregulated in hairy cell leukaemia. We did immunostaining of 500 B-cell tumours with a specific anti-ANXA1 monoclonal antibody and showed that ANXA1 protein expression is specific to hairy cell leukaemia. Immunocytochemical detection of ANXA1 represents a simple, inexpensive, highly sensitive and specific (100%) assay for diagnosis of hairy cell leukaemia. This assay will be especially useful in distinguishing hairy cell leukaemia from splenic lymphoma with villous lymphocytes and variant hairy cell leukaemia, both of which usually respond poorly to treatments that are effective in hairy cell leukaemia.

Gene expression profiling of hairy cell leukemia reveals a phenotype related to memory B cells with altered expression of chemokine and adhesion receptors

Hairy cell leukemia (HCL) is a chronic B cell malignancy characterized by the diffuse infiltration of bone marrow and spleen by cells displaying a typical “hairy” morphology. However, the nature of the HCL phenotype and its relationship to normal B cells and to other lymphoma subtypes remains unclear. Using gene expression profiling, we show here that HCL displays a homogeneous pattern of gene expression, which is clearly distinct from that of other B cell non-Hodgkin lymphomas. Comparison with the gene expression profiles of purified normal B cell subpopulations, including germinal center (GC), pre-GC (naive), and post-GC (memory) B cells, shows that HCL cells are more related to memory cells, suggesting a derivation from this B cell population. Notably, when compared with memory cells, HCL cells displayed a remarkable conservation in proliferation, apoptosis, and DNA metabolism programs, whereas they appeared significantly altered in the expression of genes controlling cell adhesion and response to chemokines. Finally, these analyses have identified several genes that are specifically expressed in HCL and whose expression was confirmed at the protein level by immunocytochemical analysis of primary HCL cases. These results have biological implications relevant to the pathogenesis of this malignancy as well as clinical implications for its diagnosis and therapy.

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.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

Nucleophosmin (NPM1) is a highly conserved nucleo-cytoplasmic shuttling protein that shows a restricted nucleolar localization. Mutations of NPM1 gene leading to aberrant cytoplasmic dislocation of nucleophosmin (NPMc+) occurs in about one third of acute myeloid leukaemia (AML) patients that exhibit distinctive biological and clinical features. We discuss the latest advances in the molecular basis of nucleophosmin traffic under physiological conditions, describe the molecular abnormalities underlying altered transport of nucleophosmin in NPM1-mutated AML and present evidences supporting the view that cytoplasmic nucleophosmin is a critical event for leukaemogenesis. We then outline how a highly specific immunohistochemical assay can be exploited to diagnose NPM1-mutated AML and myeloid sarcoma in paraffin-embedded samples by looking at aberrant nucleophosmin accumulation in cytoplasm of leukaemic cells. This procedure is also suitable for detection of haemopoietic multilineage involvement in bone marrow trephines. Moreover, use of immunohistochemistry as surrogate for molecular analysis can serve as first-line screening in AML and should facilitate implementation of the 2008 World Health Organization classification of myeloid neoplasms that now incorporates AML with mutated NPM1 (synonym: NPMc+ AML) as a new provisional entity. Finally, we discuss the future therapeutic perspectives aimed at reversing the altered nucleophosmin transport in AML with mutated NPM1.

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.

PAX5 Expression in Acute Leukemias Higher B-Lineage Specificity Than CD79a and Selective Association with t(8;21)-Acute Myelogenous Leukemia

The transcription factor PAX5 plays a key role in the commitment of hematopoietic precursors to the B-cell lineage, but its expression in acute leukemias has not been thoroughly investigated. Hereby, we analyzed routine biopsies from 360 acute leukemias of lymphoid (ALLs) and myeloid (AMLs) origin with a specific anti-PAX5 monoclonal antibody. Blasts from 150 B-cell ALLs showed strong PAX5 nuclear expression, paralleling that of CD79a in the cytoplasm. Conversely, PAX5 was not detected in 50 T-cell ALLs, including 20 cases aberrantly coexpressing CD79a. Among 160 cytogenetically/molecularly characterized AMLs, PAX5 was selectively detected in 15 of 42 cases bearing the t(8;21)/AML1-ETO rearrangement. Real-time reverse transcription-PCR studies in t(8;21)-AML showed a similar up-regulation of PAX5 transcript in all of the 8 tested samples (including 4 cases that were negative at anti-PAX5 immunostaining), suggesting that PAX5 is expressed in t(8;21)-AML more widely than shown by immunohistochemistry. Interestingly, PAX5+ t(8;21)-AML also expressed CD79a and/or CD19 (major transcriptional targets of PAX5 in B-cells) in 10 of 12 evaluable cases. Our results indicate that PAX5 is a more specific marker than CD79a for B-cell ALL diagnosis. Moreover, among AMLs, PAX5 expression selectively clusters with t(8;21), allowing its immunohistochemical recognition in a proportion of cases, and likely explaining a peculiar biological feature of this subset of myeloid leukemias, i.e. the aberrant expression of B-cell genes.

Poor mobilization is an independent prognostic factor in patients with malignant lymphomas treated by peripheral blood stem cell transplantation.

Haemopoietic stem cell therapy is an increasingly adopted procedure in the treatment of patients with malignant lymphoma. In this retrospective analysis, we evaluated 262 patients, 57 (22%) with Hodgkins and 205 (78%) with non-Hodgkins lymphomas (NHL), and 665 harvesting procedures in order to assess the impact of poor mobilization on survival and to determine the factors that may be predictive of CD34+ poor mobilization. The mobilization chemotherapy regimens consisted of high-dose cyclophosphamide in 92 patients (35.1%) and a high-dose cytarabine-containing regimen (DHAP in 87 patients –(33.2%), MAD in 83 (31.7%)). The incidence of poor mobilizers (<2 × 106 CD34+ cells/kg) was 17.9% overall, with a 10% of very poor mobilizers (⩽1 × 106/kg). Refractory disease status and chemotherapeutic load (>3 regimens) before mobilization played a negative role and were associated with poor mobilization. Survival analysis of all harvested patients showed an overall survival at 3 years of 71% in good mobilizers vs 33% in poor mobilizers (P=0.002). The event-free survival at 3 years was 23% in poor mobilizers and 58% in good mobilizers (P=0.04). We conclude that in NHL patients, poor mobilization status is predictive of survival.