Mutation of the Bcr–Abl oncoprotein is one of most frequent mechanisms by which chronic myelogenous leukemia (CML) cells become resistant to imatinib. Here, we show that treatment of cell lines ...harbouring wild type or mutant BCR–ABL with carboxyamidotriazole (CAI), a calcium influx and signal transduction inhibitor, inhibits cell growth, the expression of Bcr–Abl and its downstream signalling, and induces apoptosis. Moreover, we show that CAI acts by increasing intracellular ROS. Clinically significant, CAI has also inhibitory effects on T315I Bcr–Abl mutant, a mutation that causes CML cells to become insensitive to imatinib and second generation abl kinase inhibitors.
1 Institute of Respiratory Pathophysiology, National
Research Council, and 3 Department of Hematology and
Laboratory of Clinical Pathology, Ospedale V. Cervello, 90146 Palermo; 2 Department of ...Experimental Medicine,
University of Palermo, 90129 Palermo; and 4 Istituto
Superiore di Sanità, 00161 Rome, Italy; and
5 University of British Columbia, Vancouver, British
Columbia, Canada V6T 1Z4
Because
endurance exercise causes release of mediators and growth factors
active on the bone marrow, we asked whether it might affect
circulating hematopoietic progenitor cells (HPCs) in amateur runners
n = 16, age: 41.8 ± 13.5 (SD) yr, training:
93.8 ± 31.8 km/wk compared with sedentary controls
( n = 9, age: 39.4 ± 10.2 yr). HPCs,
plasma cortisol, interleukin (IL)-6, granulocyte colony-stimulating factor (G-CSF), and the growth factor fms-like tyrosine kinase-3 (flt3)-ligand were measured at rest and after a marathon (M;
n = 8) or half-marathon (HM; n = 8).
Circulating HPC counts (i.e., CD34 + cells and their
subpopulations) were three- to fourfold higher in runners than in
controls at baseline. They were unaffected by HM or M acutely but
decreased the morning postrace. Baseline cortisol, flt3-ligand, IL-6,
and G-CSF levels were similar in runners and controls. IL-6 and G-CSF
increased to higher levels after M compared with HM, whereas cortisol
and flt3-ligand increased similarly postrace. Our data suggest that
increased HPCs reflect an adaptation response to recurrent,
exercise-associated release of neutrophils and stress and inflammatory
mediators, indicating modulation of bone marrow activity by habitual running.
cytokines; growth factors; marathon; endurance training
Abstract 2638
We recently demonstrated that over 30% of cases with splenic marginal-zone lymphoma (SMZL) express distinctive immunoglobulin (IG) receptors that utilize a single polymorphic variant of ...the IGHV1-2 gene (IGHV1-2*04) and also exhibit restricted antigen-binding site motifs and precise targeting of somatic hypermutation (SHM). On these grounds, we proposed the existence of molecular subtypes of SMZL defined by immunogenetic analysis of the IG receptors with implications for selection by specific (super) antigenic element(s) in the development of at least a major subset of SMZL. In order to gain insight as to whether antigen involvement is relevant only prior to the malignant transformation or if it continues to affect the SMZL clone itself leading to intraclonal diversification (ID) through ongoing SHM, we conducted a large-scale subcloning study of rearranged IG heavy variable genes, in a total of 471 subcloned sequences from 22 SMZL cases. The analysis was intentionally biased towards cases expressing IGHV1-2*04 receptors that exhibit a series of distinctive immunogenetic features, including biased usage of the IGHD3-3 and IGHD3-10 genes, unusually long heavy complementarity-determining region 3 (VH CDR3) and minimally/borderline mutated status (identity to the germline in the range of 97–99.6%). Hence, the study group included 16 IGHV1-2*04 cases and 6 cases utilizing other IGHV genes. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and a median of 21 (9–46) colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. Overall, 15/22 cases (68%) carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, of which 12 utilized the IGHV1-2*04 gene whereas the remaining 3 utilized other IGHV genes. The high frequency of ID within the IGHV1-2*04 group, ranging from limited to (often) pronounced, is noteworthy in view of the generally low level of SHM among IGHV1-2*04 receptors. Detailed analysis of the distribution and molecular features of CMs revealed: (i) restricted ID patterns, in the sense of identical mutations in certain VH positions among subclones of different cases; (ii) “hotspots” of ID, i.e. particular codons exhibiting intense ongoing mutational activity (especially notable in this respect was codon 39 in VH FR2); (iii) a predominance of conservative amino acid changes, characterized by somatically introduced amino acid belonging to the same biochemical category as the mutating amino acid; and (iv) limited diversification within VH CDR3. Additionally, in 7/12 IGHV1-2*04 cases with CMs, identified ID patterns delineate distinct “clusters” of subcloned sequences with unique as well as shared mutations and closely similar if not identical VH CDR3s (including identical VH CDR3 length), pointing to “branching” of the malignant clone into distinct subclones, perhaps able to evolve along related yet distinct pathways. In conclusion, our study indicates that the SHM mechanism may continuously operate in certain subsets of SMZL, especially those expressing IGHV1-2*04 receptors. Although the precise timing of interactions with antigen(s) and their functional implications for SMZL evolution will likely remain difficult to define accurately, the results reported here suggest a role for persistent antigenic stimulation, at least for certain immunogenetically defined subsets of SMZL.
Mollejo:Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.
BACKGROUND. Monoallelic and biallelic mutations of the PRF1 gene have been reported in some cases of childhood lymphoma. Anaplastic large cell lymphoma (ALCL) accounts for 10% to 15% of all childhood ...lymphomas. To assess the possible role of PRF1 mutations in ALCL, the authors screened a series of patients collected by the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP). METHODS. The authors investigated 44 patients with ALCL by direct sequence of the PRF1 gene. To address the issue of the prevalence of the most frequently observed PRF1 mutations in the control population, the authors examined a series of 400 healthy white control subjects for the 272C>T mutation (A91V). RESULTS. A total of 6 different mutations were identified in 12 patients (27.3%). Eleven patients had 1 mutation whereas 1 patient was found to have 2 mutations. Of the 6 PRF1 mutations identified, 2 were novel mutations: 529C>T (resulting in R177C) and 1471G>A (resulting in D491N). The remaining 4 mutations were previously described; in particular, the 272C>T mutation (resulting in the A91V amino acid change) was found in 8 patients, whereas the 368G>A (R123H), 695G>A (R232H), and 1262T>G (F421C) mutations were all found in 1 case each. Overall, the incidence of PRF1 mutations was found to be significantly higher in patients with ALCL compared with 400 control subjects, among whom only heterozygous A91V was observed in 41 subjects (10.2%) (chi-square test, 10.9; P <.01). CONCLUSIONS. Patients with childhood ALCL have a higher probability of being a carrier of a PRF1 mutation compared with healthy controls, suggesting a possible predisposing role. Cancer 2007.
Abstract 634
We systematically explored the immunoglobulin (IG) gene repertoire in 337 cases with splenic marginal-zone lymphoma (SMZL), by far the largest series yet. To resolve classification ...uncertainties, we included in the analysis only cases with a diagnosis of SMZL based on spleen histopathological findings or cases fulfilling the 2008 SBLG criteria (Matutes et al. Leukemia 2008). We here report that the IG heavy variable (IGHV) gene repertoire in SMZL is remarkably biased, with only three genes accounting for 45.8% of cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23: 8.1%, respectively), significantly extending previous similar observations. Particularly for the IGHV1-2 gene, strong biases became evident at the level of utilization of different alleles, since 79/86 rearrangements (92%) utilized allele *04 vs. only 7/86 rearrangements (8%) that utilized allele *02. This is noteworthy, taking into consideration that these two alleles differ in a single nucleotide, leading to a single amino acid change in framework region (FR)-3. The repertoire biases became more pronounced when the analysis was focused on 171 rearrangements from 163 cases classified as SMZL based on splenic histopathology, according to the 2008 WHO criteria. Within this subgroup, 56/171 cases (32.7%) utilized IGHV1-2*04. Noticeably, only 1/17 cases with a diagnosis of splenic diffuse red pulp lymphoma utilized IGHV1-2*04 (p<0.02 for comparison to SMZL). The IGHV1-2*04 rearrangements carried significantly longer heavy complementarity-determining region-3 (VH CDR3) than all other cases (median, 22 vs. 17 amino acids, respectively; p<0.001). In addition, 52/79 IGHV1-2*04 cases (65.8%) employed one of the IGHD3-3, IGHD3-9 or IGHD3-10 genes. In 28/32 IGHV1-2*04/IGHD3 rearrangements, the IGHD gene was utilized in the same reading frame, leading to VH CDR3s with common “IGHD-derived” amino acid (AA) motifs. Using bioinformatics tools previously applied to CLL, biased associations of IGHV, IGHD and IGHJ genes with stereotyped VH CDR3s were identified in 25/345 sequences (7.2%). Noticeably, only 10/28 IGHV1-2*04/IGHD3-3 rearrangements with “IGHD-derived” VH CDR3 amino acid motifs could be assigned to “stereotyped” clusters. Despite exhibiting restricted usage of the IGHV1-2*04 and IGHD3-3 genes leading to great overall VH domain similarity, the remaining cases did not fulfill the established criteria for VH CDR3 “stereotypy”, as defined in other lymphoid malignancies, namely CLL. Based on somatic hypermutation (SHM) analysis, the sequences were divided into three groups: (i) truly unmutated (100% germline identity, GI): 46/345 sequences (13.3%); (ii) minimally/borderline mutated (97-99.9% GI): 130/345 sequences (37.7%); and (iii) significantly mutated (<97% identity): 169/345 sequences (49%). At the individual gene level, the distribution of rearrangements of IGHV genes according to SHM status varied significantly. In particular, 56/79 IGHV1-2*04 rearrangements (71%) were predominantly “borderline mutated”, whereas the majority (>67%) of rearrangements utilizing the IGHV3-23, IGHV3-30 and IGHV3-7 genes were “significantly mutated”; finally, IGHV4-34 gene rearrangements were evenly distributed to the three mutational subgroups. Shared (“stereotyped”) AA changes were identified for IGHV1-2*04 rearrangements, with certain FR2 and FR3 codons emerging as “hotspots” for recurrent, conservative AA changes. In conclusion, we demonstrate that more than 30% of cases with a histopathological diagnosis of SMZL on the spleen express IGHV1-2*04 receptors with unusually long VH CDR3s, biased usage of the IGHD3-3 gene, leading to shared “IGHD-derived” VH CDR3 motifs, and very precise molecular features of SHM. The biased expression of a distinctive germline-encoded VH specificity might be considered as evidence for heavy chain dominance in the clonogenic IG receptors in SMZL. These findings allude to selection by specific (super)antigenic element(s) in the pathogenesis of at least a major subset of SMZL. In addition, they raise the intriguing possibility that certain subtypes of SMZL could derive from progenitor cell populations adapted to particular antigenic challenges through cellular selection of VH domain specificities.
No relevant conflicts of interest to declare.
Introduction.Pax-5 gene codifies for a transcription factor central to B-cell differentiation and function, expressed during the early stage of differentiation and alternatively spliced during B-cell ...development. In addition to the full-length isoform (Pax-5a), isoforms arising from the inclusion or exclusion of exons 2, 7, 8, and/or 9, and lacking the DNA-binding and the transactivating domains, have been described. These isoforms and their levels of expression increase during B-cell maturation. In particular, Pax-5b isoform (deleted of exon 2), resulting in protein with partial DNA-binding domain, is related with the differentiation stage. Recently, mutations of Pax-5 gene were reported in 31.7% of 242 children (Mullighan, Nature 2007) and 30% of 119 adults (Familiades, ASH 2007 abs. 2806) with B-cell precursor acute lymphoblastic leukemia (ALL). We investigated Pax-5 gene mutations and isoforms expression in patients with ALL at the diagnosis and in selected cell populations from control subjects.
Methods.Pax-5 mutations and mRNA isoforms were investigated by sequence analysis. Pax-5a and Pax-5b expression levels were evaluated by quantitative PCR in leukemic cells. Total RNA was obtained from leukemic blasts of 95 patients with B-cell precursor ALL at diagnosis, 45 adults and 50 children. These last had been selected according to the presence of adverse translocations: BCR/ABL p190 (n=10), E2A-PBX1 (n=10), TEL/AML (n=10), MLL/AF4 (n=10), or none (n=10). mRNA was obtained from purified peripheral blood CD19+ lymphocytes (6 samples), from bone marrow CD34+ hematopoietic progenitor cells (6 samples), and pro-B CD34+CD19+ cells (3 samples) from a total of 8 healthy bone marrow donors.
Results. Pax-5 gene point mutations were found in 8/45 adults (18%) and 14/50 children (28%) (p=n.s.), resulting in a total of 20 different mutations. Mutations were located as follows: exon 2 (53G>C, 76delG, 101C>G, 113G>A, 197G>A), exon 3 (223A>G, 247delA, 296T>C), exon 4 (446A>T, 526G>A), exon 5 (580A>C, 601G>A), exon 6 (625C>T, 716G>A), exon 7 (836C>A), exon 8 (955G>A, 964insC, 971A>G), exon 9 (1058C>T), exon 10 (1133G>A). There was no correlation between the presence of mutations and the listed genetic subtypes. Pax-5 alternative splicing was observed in 47/95 cases (49%): 29/45 adults (64%) and 18/50 children (36%) (p=n.s.). Alternative splicing resulted in increased frequencies of the Pax-5b isoform and the deletion of exons 8 and/or 9; furthermore, a novel isoform resulting from the skipping of exon 5 was documented. On the contrary, only the fulllenght isoform was present in CD34+ progenitors and CD34+CD19+ cells from healthy donors, in which deleted isoforms were detected only at the stage of mature B-lymphocytes.
Conclusion: Pax-5 mutations are frequent in childhood and adult ALL and are scattered all over the gene. We extend the current knowledge on their pathogenic role by demonstrating that alternative splicing is a common event in these patients. Imbalance between the fulllength and the Pax-5b isoforms is expected to affect the maturation of the B-cell and its propensity to apoptosis (Robichaud Nucleic Acids Res 2008), thus contributing to the process of leukemogenesis. Based on our findings, this mechanism acts broadly and is independent of the most common ALL genetic translocations.
FHL is a rare fatal disease of early infancy characterized by hyperinflammatory sindrome, fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, central nervous system ...alteration. Defective cellular cytotoxicity results in pathogen persistence, hypercytokinemia, disseminated infiltration of lymphocytes and histiocytes and hemophagocytosis. Differential diagnosis between FHL due to PRF1 (FHL2) or MUNC13–4 (FHL3) mutations, and additional unknown genetic subtypes is not easy unless mutation analysis is performed. Furthermore, some infection-associated cases of HLH (“secondary”) may mimic FHL.
We investigated natural killer (NK) cells function and phenotype in patients with FHL2 (n=5) or FHL3 (n=8). NK cells were cultured in IL-2 prior to their use in the various assays.
B-EBV cell lines and dendritic cells are suitable targets in 51Cr-release assays to reveal the FHL3 NK cell defect. Tumor targets were partially lysed by FHL3 NK cells expressing even trace amounts of Munc13–4 protein. FHL2 NK cells were completely unable to lyse target cells.
Cytokine production induced by mAb-crosslinking of triggering receptors was similar in patients and controls; under co-culture with 721.221 B-EBV cells, FHL NK cells were high producers, whereas control cells were almost ineffective. This could reflect persistence versus elimination of the source of stimulation in patients vs healthy controls, mimicking the pathophysiology of FHL. Finally, we tested cell surface CD107a expression in a degranulation assay, co-culturing 2x105 polyclonal NK cell or 24-hr IL-2 activated PBMCs with 2x105 target cells (K562, FO-1, 721.221 or P815 and anti-NKp30). CD107a expression was tested in CD56+ cell of either CD3− PBMC or polyclonal activated NK cell populations. CD107a was expressed in 11.8±7.5% cells in 6 FHL3 patients vs 48.5±11.5% in 9 controls (p<0.001). MFI was also lower in FHL3 patients vs. controls (9.3±3.1 vs. 42.6±21.9; p<0.001). Differently from FHL3, mean values of CD107a expression in 3 FHL2 patients were even higher than controls both in terms of percentage (63.3%) and MFI (86.6).
Altogether, these flow-cytometry data show that the pattern of CD107a expression represents a novel tool to identify the defect of degranulation characteristic of Munc 13–4 deficiency. Importantly, in FHL2 NK cells, whose granules lack perforin, degranulation pattern is normal. Although use of purified activated NK cells may increase discrimination power of the test, even PBMC (24-hr IL-2 activated) could be used to reveal a defect of granule exocytosis.
In addition to providing information useful for a better understanding of the pathophysiology of FHL and functional correlation with different gene mutations, our data may impact on FHL diagnosis. Combined use of surface expression of CD107a, together with intracytoplasmic staining with anti-perforin mAb, allows to promptly dissect FHL3 and FHL2 defects by the simple analysis of PBMC, so directing genetic analysis.
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