Roberta Pacini

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.).

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.

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.

CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice

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 patients 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.

Constant activation of the RAF-MEK-ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia

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.

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

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.

Absence of nucleophosmin leukaemic mutants in B and T cells from AML with NPM1 mutations: implications for the cell of origin of NPMc+ AML.

Absence of nucleophosmin leukaemic mutants in B and T cells from AML with NPM1 mutations: implications for the cell of origin of NPMc+ AML

Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma

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.