BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

5'-nucleotidase, cytosolic II genotype, and clinical outcome in patients with acute myeloid leukemia with intermediate-risk cytogenetics.

Acute myeloid leukemia (AML) is a complex disease, and its treatment needs to be adjusted to the risk, which is conferred by cytogenetics and molecular markers. Cytarabine is the main drug to treat AML, and it has been suggested that the genotype of cytarabine metabolizing enzymes may have a prognostic relevance in AML. Here we report the association between the 5'-nucleotidase, cytosolic II (NT5C2) rs10883841, cytidine deaminase (CDA) rs2072671 and rs532545 genotypes and the clinical outcome of 477 intermediate-risk cytogenetic AML patients receiving cytarabine-based chemotherapy. Patients younger than 50 years old with the NT5C2 rs10883841 AA genotype had lower overall survival (OS) (p: .003; HR 2.16, 95% CI 1.29-3.61) and lower disease-free survival (DFS) (p: .002; HR 2.45, 95% CI 1.41-4.27), associated to a higher relapse incidence (p: .010; HR 2.23, 95% CI 1.21-4.12). Interestingly, subgroup analysis showed that the negative effect of the NT5C2 rs10883841 AA genotype was detected in all subgroups except in patients with nucleophosmin mutation without high ratio FLT-3 internal tandem duplication. CDA polymorphisms were associated with the complete remission rate after induction chemotherapy, without influencing OS. Further studies are warranted to determine whether this pharmacogenomic approach may be helpful to individualize AML treatment.

Multilineage dysplasia is associated with a poorer prognosis in patients with de novo acute myeloid leukemia with intermediate-risk cytogenetics and wild-type NPM1.

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

LAG3 genotype of the donor and clinical outcome after allogeneic transplantation from HLA-identical sibling donors.

Introduction: The association of polymorphisms in molecules involved in the immune response (checkpoint inhibitors) with the clinical outcome after allogeneic transplantation (alloHSCT) has been described. Lymphocyte Activation 3 (LAG3) is a surface protein that plays a regulatory role in immunity as an inhibitory immune checkpoint molecule. Methods: To determine its role in the alloHSCT setting, we analyzed 797 patients transplanted from HLA-identical sibling donors. The LAG3 rs870849 C>T polymorphism was genotyped in donors. Results: We detected a higher incidence of severe acute GVHD in patients transplanted from donors with TT genotype (p: 0.047, HR 1.64; 95% CI 1.01 - 2.67). Overall survival (OS) was worse for patients transplanted from donors with the rs870849 CT/TT genotype (0.020; HR, 1.44; 95% CI 1.06 - 1.96), as well as disease-free survival (DFS) (p: 0.002; HR 1.58, 95%CI: 1.18 - 2.14) and transplant-related mortality (TRM) (p< 0.001; HR: 1.88, 95% CI 1.29 - 2.74). When combining the LAG3 rs870849 and the PDCD1 rs36084323 genotypes of the donor, three genetic groups were well defined, allowing a good stratification of the risk of acute GVHD, TRM, OS and DFS. Discussion: We conclude that the LAG3 genotype of the donor may be considered in donors' selection. As this selection may be limited in the HLA-identical sibling donor scenario, further studies exploring the impact of LAG3 genotype of the donor in unrelated transplantation are warranted.

Donor CTLA-4 genotype influences clinical outcome after T cell-depleted allogeneic hematopoietic stem cell transplantation from HLA-identical sibling donors.

CTLA-4 (cytotoxic T-lymphocyte antigen-4) plays a pivotal role in inhibiting T cell activation through competitive interaction with B7 molecules and interruption of costimulatory signals mediated by CD28. Polymorphisms on the CTLA-4 gene have been previously associated with autoimmune diseases, predisposition to leukemic relapse, and with graft-versus-host disease (GVHD) or relapse after allogeneic transplant. As CTLA-4 is expressed on T-lymphocytes, the aim of this study was to determine whether the donor CTLA-4 CT60 genotype also influences clinical outcome even after T cell depletion with CD34-positive selection. We studied 136 patient-donor pairs. Overall survival (OS) was worse for those patients who received grafts from a donor with the CT60 AA genotype rather than from a donor with the AG or GG genotype (35.6% vs 49.4%; P = .043). This association was confirmed through multivariate analysis, which identified the donor CT60 genotype as an independent risk factor for OS (P = .008; hazard ratio [HR]: 2.24, 95% confidence interval [CI]: 1.23-4.08). The donor CT60 AA genotype was also associated with lower disease-free survival, this being related to an increased risk of relapse (P = .001; HR: 3.41, 95% CI: 1.67-6.96) and a trend toward higher transplant-related mortality. These associations were stronger when considering only patients in the early stage of disease. Our results suggest that graft-versus-leukemia (GVL) activity after T cell depletion is conditioned by the donor CTLA-4 genotype.

Acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): Clinical and biological features and comparison with other acute myeloid leukemias with cytogenetic aberrations involving long arm of chromosome 3.

OBJECTIVES: To compare, from a biological and clinical perspective, a significant group of patients with AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) with another group of AML carrying different abnormalities of 3q at q21 or q26, the latter named as the AML abn(3q) group. METHODS: We developed a national survey with the participation of 13 Spanish hospitals, and retrospectively reviewed (from 1990 to 2010) these subtypes of AML. Fifty-five patients were collected: 35 with AML inv(3)/t(3;3) and 20 with AML abn(3q). A data collecting page that included main features at diagnosis, therapeutic approach and response, and survival variables, was distributed and completed. RESULTS: We did not find significant differences in sex, age, history of myelodysplastic syndrome or chemo-/radiotherapy, clinical presentation, WBC and platelet counts, hemoglobin level, blasts immunophenotype, serum lactatedehydrogenase, peripheral blood and bone marrow cellular dysplasia, and bone marrow biopsy findings. Although the association with monosomy 7 was significantly more frequent in AML inv(3)/t(3;3), this did not seem to influence outcome. The lack of response to the different modalities of treatment and the aggressive course of the disease were the standard in both cohorts of patients. DISCUSSION: Although not yet recognized by the World Health Organization classification, our results are in agreement with the findings of other authors, who include both subsets of AML together in the same group of adverse prognosis. CONCLUSION: In an attempt to simplify and bound entities with similar genetic background and clinical behavior, it would be desirable to bring together both subgroups of AML in a single section.

Kinetics of the CTLA-4 isoforms expression after T-lymphocyte activation and role of the promoter polymorphisms on CTLA-4 gene transcription.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) plays a key inhibitory role during T lymphocyte activation. The CTLA4 gene is translated into two proteic isoforms: a full-length protein (flCTLA-4) and a soluble counterpart. We explored the expression of both isoforms on healthy subjects. Whereas in non-stimulated cells the flCTLA-4 isoform is predominant, after stimulation the expression of the soluble form rapidly increases, reaching its maximum 24h after and falling again to the basal levels 72 h after stimulation. In contrast, the flCTLA-4 mRNA levels increase is slower, reaching the maximum level 72 h after stimulation. The presence of the T allele in the promoter positions -1722 and -318 is associated with an increased transcriptional activity and this effect seems to be synergic. We conclude that the kinetics of CTLA-4 isoform expression are sequential, and that the promoter polymorphisms -1722(C/T) and -318(C/T) are involved in the control of the CTLA4 transcription.

[Persistent polyclonal B-cell lymphocytosis: study of 35 cases]. / Linfocitosis B policlonal persistente: estudio de 35 casos.

BACKGROUND AND OBJECTIVES: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare entity, presenting especially in adult smoker women. It is characterized by an increase of serum IgM, DR7-HLA haplotype, cytogenetic abnormalities and multiple IgH/BCL-2 rearrangements. To date, it has not been elucidated whether this is a benign or premalignant disorder. We analyzed the PPBL characteristics with especial attention to its evolution. PATIENTS AND METHODS: Thirty-five PPBL patients from 5 hospitals in Catalonia were retrospectively analyzed. A simultaneous morphologic review of the blood smears was performed by members of the GCCH in a 16 multiple-observer optic microscope. Clinical and biological data were also analyzed. RESULTS: PPBL presents in the majority of cases with persistent polyclonal B-cell lymphocytosis and affects primarily smoker women. The morphologic hallmark, in absence of viral infections, is the presence of activated lymphocytes with bilobulated and/or cleaved nuclei, and nuclear pockets in the ultrastructural study. Increased serum IgM, HLA-DR7 haplotype, chromosomal abnormalities such as i(3)(q10) and multiple IgH/BCL-2 rearrangements were detected. Thirty-four out of 35 patients are alive after a median follow up of 70.7 months. One patient died because of lung adenocarcinoma and another developed a follicular lymphoma without relation to PPBL. CONCLUSIONS: PPBL has an asymptomatic and stable evolution, although it frequently presents genetic abnormalities. It remains unknown whether it is a premalignant entity, similar to monoclonal gammopathies of unknown significance. Hence, accurate cytologic diagnosis and follow-up are essential.

Genetic changes including gene copy number alterations and their relation to prognosis in childhood acute myeloid leukemia.

We studied a series of 68 subjects diagnosed with childhood acute myeloid leukemia (AML) using conventional cytogenetics and fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) to analyze mutations in FLT3 and NPM1 genes, and/or array comparative genomic hybridization (CGH). Cytogenetic/FISH abnormalities were observed in 71% of subjects, FLT3-ITD mutations in 15%, and NPM1 mutations in 13%. The array CGH alterations (average 3.6 per case) were observed in 96% of the tested subjects. The most frequent alterations were gains of 8q24.3 and 11p15.5-p15.4 in 16% of the samples. Six genes (AKT1, RUNX1, LTB, SDC1, RUNX1T1, and JAK2) from the imbalanced regions have been reported to be involved in AML, whereas other 30 cancer genes, not previously reported in an AML context, were identified as imbalanced. They probably correspond to non passenger alterations that cooperate with the recurrent translocations. The clinical data and genetic changes were tested to find out the possible association with prognosis. Genomic instability (four or more genomic imbalances) was correlated with poor patient outcome (p = 0.029).

Differential expression of genes mapping to recurrently abnormal chromosomal regions characterize neuroblastic tumours with distinct ploidy status.

BACKGROUND: Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status. METHODS: Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent in situ Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles. RESULTS: Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (P = 0.01) and 17p13-q21 (P < 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours. CONCLUSION: NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.

Idiopathic hypereosinophilic syndrome in children: report of a 7-year-old boy with FIP1L1-PDGFRA rearrangement.

Idiopathic hypereosinophilic syndrome (HES) in children is a very rare disorder; certain clinical differences with adult HES have been described, with no pediatric case with the imatinib-responsive FIP1L1-PDGFRA fusion gene reported to date. The authors describe the clinical course of three children with HES in whom FIP1L1-PDGFRA fusion gene was studied and report the first child with this rearrangement.

Acute erythroid neoplastic proliferations. A biological study based on 62 patients.

BACKGROUND AND OBJECTIVES: The terms acute erythroleukemia and AML-M6 are defined in the FAB classification as proliferations of dysplastic erythroid elements mixed with blasts of myeloid origin, but pure erythroid leukemias are not included. The recent WHO classification has a category of acute myeloid leukemia not otherwise categorized, which includes acute erythroid leukemia (M6) of two subtypes: M6a-erythroleukemia (erythroid/myeloid) and M6b-pure erythroid leukemia. The aims of this co-operative study were to discover the incidences of these different subtypes, and pay special attention to the morphology of these entities. DESIGN AND METHODS: We reviewed a series of 62 patients with erythroid neoplastic proliferations. Previous medical history, age, sex, peripheral blood and bone marrow cell counts, cytochemical stains, immunophenotype, and cytogenetics were evaluated at presentation. We analyzed the incidence of erythrocyte, leukocyte and platelet abnormalities in the peripheral blood. In bone marrow we analyzed dysplastic features of erythroblasts, granulocytic elements and the megakaryocytic lineage. RESULTS: Fifty-three patients met the criteria of M6a subtype of the WHO classification, and 2 were classified as having pure erythremia (M6b); 7 cases could not be classified according to the WHO criteria. Fifty-five patients presented with de novo acute leukemia, and seven patients had secondary acute leukemia. The most frequent dysplastic features in blood smears were: schistocytes, tear-drop and pincered cells in erythrocytes; hypogranulation and hyposegmentation in leukocytes; gigantism and hypogranulation in platelets. In bone marrow, megaloblastic changes, multinuclearity, karyorrhexis and basophilic stippling in erythroblasts; hypogranulation and gigantism in granulocytic series, and micromegakaryocytes and unconnected nuclei in megakarocytes were the most dysplastic features. A positive PAS reaction and increase of bone marrow iron with ring sideroblasts were common features. Trilineage dysplasia was present in 54% of cases. Dysplastic features in granulocytic elements were absent in 26% of patients and minimal erythroblastic dysplasia was observed in seven patients. A complex karyotype was seen in 27% of patients; chromosomes 5 and 7 were the most frequently involved. INTERPRETATION AND CONCLUSIONS: De novo acute erythroid leukemia was more frequent than secondary cases in our series. The most frequent type of acute erythroid proliferation was the WHO M6a subtype and the least the pure erythroid leukemia. We found a group of seven patients (11%) who could not be classified according to the WHO criteria. Morphologic findings of erythrocytes in peripheral blood, such as schistocytes, tear-drop and pincered cells, were outstanding features. Morphologic aspects remain one of the most important tools for diagnosing these entities.