Alessia Tabarrini

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

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.

Absence of BRAF-V600E in the human cell lines BONNA-12, ESKOL, HAIR-M, and HC-1 questions their origin from hairy cell leukemia

To the editor: Hairy cell leukemia (HCL) shows distinct clinicopathologic, immunophenotypic, and gene expression features.[1][1][⇓][2]–[3][3] We previously identified the BRAF -V600E mutation as the disease-defining genetic event in HCL.[4][4] This mutation is present in virtually all cases of

FRI0001 IL-17 producing-DN T cells are expanded in the blood, infiltrate salivary glands and are resistant to corticosteroids in sjÖgren’s syndrome

Background The discovery of IL-17-producing CD4+ T helper (Th17) cells challenged the long-standing paradigm of Th1/Th2 cell immune response and shed some light on the pathogenic mechanisms of systemic autoimmune disorders. IL-17 in a pro-inflammatory cytokine which mediates target organs damage during the disease. To note, it has been recently observed that asmall CD3+ T-cell population, that lacks of both CD4 and CD8 molecules, defined as double negative (DN), is expanded in the peripheral blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during lupus nephritis. Objectives Since IL-17 production is enhanced in salivary gland infiltrates of patients with primary Sjögren’s syndrome (SS), we aimed to investigate whether DN T cells may be involved in the pathogenesis of salivary gland damage Methods Thirty patients with SS and 16 normal controls (NC) were enrolled. CFSE-stained PBMCs were cultured in anti-CD3-coated plates in presence or absence of dexamethasone (Dex) at different concentrations. Phenotypic characterization was performed by flow cytometry in freshly isolated cells and after culture using anti-CD3, CD4, αβ-TCR, γδ-TCR, RORγt, IL-17 antibodies and respective isotypes. SS minor salivary glands (MSG) were processed for immunofluorescence staining. Results Total circulating DN T cells were increased in SS compared to NC. This was due to an increase of both αβ-TCR+ and γδ-TCR+ cells. NC and SS freshly isolated DN T cells express RORγt and produce consistent amounts of IL-17. Notably, DN T cells were found in the SS-MSG infiltrate. αβ-TCR+, but not γδ-TCR+, DN T-cell expansion in SS was dependent on disease activity according to the EULAR Sjögren’s syndrome disease activity index (ESSDAI). Dex was able to down-regulate IL-17 in vitro production in NC and SS CD4+ cells and in NC DN T cells, but not in DN T cells from SS samples. Conclusions DN T cells are expanded in SS PB, produce IL-17 and infiltrate SS MSG. The expansion is dependent on disease activity. In SS, conventional Th17 cells are inhibited by Dex, but DN T cells appear to be resistant to this effect. The recognition of a pathogenic T-cell subset, that appears to be in vivo activated and not sensitive to CS, in an autoimmune disorder such as SS may represent an intriguing finding with potential therapeutic implications of great interest. References Crispin C et al. J Immunol 2008;181:8761 Nguyen CQ et al. Arthritis Rheum 2008;58:734 Disclosure of Interest None Declared