Francisco J. Novo

Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci.

Many B‐cell malignancies bear chromosomal translocations juxtaposing immunoglobulin (IG) genes with oncogenes, resulting in deregulated expression of the latter. Translocations affecting the IG heavy chain (IGH) locus in chromosomal region 14q32 are most prevalent. However, variant translocations involving the IG kappa (IGK) locus in 2p12 or the IG lambda (IGL) locus in 22q11 occur recurrently in B‐cell neoplasias. No routine methods for the detection of all breakpoints involving IG light chain loci independently of the translocation partner have been described. For this reason, we have designed 2 novel interphase fluorescence in situ hybridization (FISH) assays using differentially labeled probes flanking the IGK and IGL locus, respectively. Based on extensive control studies, the diagnostic thresholds for the detection of breakpoints were set at 0.3% for IGK and 1.4% for IGL. Fifteen cases of B‐cell malignancies with cytogenetically detectable chromosomal abnormalities in 2p11‐14 were investigated with the FISH assay for IGK. Breakpoints affecting the IGK locus were detected in 7 cases including all 4 variant Burkitts translocations t(2;8)(p12;q24) and a variant BCL2‐associated translocation t(2;18)(p12;q21). Other translocation partners were chromosome bands 7q21 and 16q24. Ten cases with abnormalities in 22q11‐12 were investigated with the FISH assay for IGL. Breakpoints in the IGL locus were diagnosed in 7 cases including both variant Burkitts translocations t(8;22)(q24;q11) and a t(3;22)(q27;q11) involving the BCL6 locus. Other translocation partners were 2p13‐14, 4q13 and 16p12. Our results show that these FISH assays provide flexible, simple and reliable tools in the diagnosis and characterization of genetic changes in B‐cell malignancies.

SETBP1 overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia

Acute myeloid leukemias (AMLs) result from multiple genetic alterations in hematopoietic stem cells. We describe a novel t(12;18)(p13;q12) involving ETV6 in a patient with AML. The translocation resulted in overexpression of SETBP1 (18q12), located close to the breakpoint. Overexpression of SETBP1 through retroviral insertion has been reported to confer growth advantage in hematopoietic progenitor cells. We show that SETBP1 overexpression protects SET from protease cleavage, increasing the amount of full-length SET protein and leading to the formation of a SETBP1-SET-PP2A complex that results in PP2A inhibition, promoting proliferation of the leukemic cells. The prevalence of SETBP1 overexpression in AML at diagnosis (n = 192) was 27.6% and was associated with unfavorable cytogenetic prognostic group, monosomy 7, and EVI1 overexpression (P < .01). Patients with SETBP1 overexpression had a significantly shorter overall survival, and the prognosis impact was remarkably poor in patients older than 60 years in both overall survival (P = .015) and event-free survival (P = .015). In summary, our data show a novel leukemogenic mechanism through SETBP1 overexpression; moreover, multivariate analysis confirms the negative prognostic impact of SETBP1 overexpression in AML, especially in elderly patients, where it could be used as a predictive factor in any future clinical trials with PP2A activators.

Multicolor-FICTION: Expanding the Possibilities of Combined Morphologic, Immunophenotypic, and Genetic Single Cell Analyses

Phenotypic and genotypic analyses of cells are increasingly essential for understanding pathogenetic mechanisms as well as for diagnosing and classifying malignancies and other diseases. We report a novel multicolor approach based on the FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) technique, which enables the simultaneous detection of morphological, immunophenotypic, and genetic characteristics of single cells. As prerequisite, multicolor interphase fluorescence in situ hybridization assays for B-cell non-Hodgkins lymphoma and anaplastic large-cell lymphoma have been developed. These assays allow the simultaneous detection of the most frequent primary chromosomal aberrations in these neoplasms, such as t(8;14), t(11;14), t(14;18), and t(3;14), and the various rearrangements of the ALK gene, respectively. To establish the multicolor FICTION technique, these assays were combined with the immunophenotypic detection of lineage- or tumor-specific antigens, namely CD20 and ALK, respectively. For evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes. Thus, three-dimensional information was obtained. The multicolor FICTION assays were applied to well-characterized lymphoma samples, proving the performance, validity, and diagnostic power of the technique. Future multicolor FICTION applications include the detection of preneoplastic lesions, early stage and minimal residual diseases, or micrometastases.

Methylation of CpG dinucleotides and/or CCWGG motifs at the promoter of TP53 correlates with decreased gene expression in a subset of acute lymphoblastic leukemia patients

It has been shown that methylation of CpG dinucleotides located in the promoter region of TP53 is associated with low expression levels of this gene. We have analysed the methylation status of one CpG dinucleotide and of three CCWGG motifs, also located in the promoter region of the gene, in bone marrow samples obtained from patients with acute lymphoblastic leukemia (ALL). Eight out of 25 samples analysed showed methylation of either the CpG dinucleotide, the CCWGG motifs or both. Relative to nonmethylated leukemia samples, TP53 expression levels were decreased in all methylated samples in which TP53 expression could be measured. Methylation of CpG and CCWGG motifs in the promoter of TP53 could represent a novel mechanism leading to functional impairment of this tumor suppressor gene in ALL.

NIN, a gene encoding a CEP110-like centrosomal protein, is fused to PDGFRB in a patient with a t(5;14)(q33;q24) and an imatinib-responsive myeloproliferative disorder

We describe a new PDGFRB fusion associated with a t(5;14)(q33;q24) in a patient with a longstanding chronic myeloproliferative disorder with eosinophilia. After confirmation of PDGFRB involvement and definition of the chromosome 14 breakpoint by fluorescence in situ hybridization, candidate partner genes were selected on the basis of the presence of predicted oligomerization domains believed to be an essential feature of tyrosine kinase fusion proteins. We demonstrate that the t(5;14) fuses PDGFRB to NIN, a gene encoding a centrosomal protein with CEP110-like function. After treatment with imatinib, the patient achieved hematological and cytogenetical remission, but NIN-PDGFRB mRNA remained detectable by reverse transcription-PCR.

TP53 is frequently altered by methylation, mutation, and/or deletion in acute lymphoblastic leukaemia.

Different mechanisms, such as chromosomal rearrangements, deletions, mutations, and methylation/demethylation of the promoter regions of genes, have been shown to be involved in acute lymphoblastic leukaemia (ALL). These genetic and epigenetic alterations lead to the activation of protooncogenes or to inactivation of tumour supressor genes promoting cell proliferation. One of the most frequently inactivated tumour supressor genes is TP53, which is altered in 50% of human tumours. In this study, we have analysed: (1) the complete coding region, all intron‐exon junctions and noncoding regions of exons 1–11 of TP53 by lexon‐DGGE; (2) the methylation status of the 5′ region of TP53 and (3) the deletion of one or both alleles of the gene by fluorescence in situ hybridisation (FISH) in 57 ALL patients. Using these techniques, we have found promoter methylation in 32% of the cases, missense mutations in 8.8%, and deletion of one allele in 7.5% of the samples, with TP53 being altered in 40% of the ALL samples studied in this series.

Loading of plasmid DNA into PLGA microparticles using TROMS (Total Recirculation One-Machine System): evaluation of its integrity and controlled release properties

Loading plasmid DNA into poly(ester) microparticles usually involves the formation of a multiple emulsion, using homogenisation techniques such as sonication or Ultra-Turrax. These procedures may negatively affect the integrity of the macromolecule and consequently its activity. The aim of this study was to prepare and evaluate DNA-loaded microparticles by TROMS (Total Recirculation One-Machine System), a new procedure that is based on the formation of a multiple emulsion by the injection of the phases under a turbulent regime. Microparticles were prepared with either Resomer) RG 502 (MP 502) or RG 756 (MP 756) and DNA loading was quantified fluorimetrically. DNA loading in MP 756 was almost twice as high as in MP 502 (510 vs. 285 ng/mg, respectively). Under both formulations, the loaded plasmid was released while maintaining its integrity for at least 24 days (MP 502) and 40 days (MP 756). Finally, the transfection efficiency was studied after injection of the microparticles (MP 502) into rat skeletal muscle and compared with naked DNA injection. Injection of naked DNA (150 microg DNA per muscle) achieved higher but variable expression levels that decreased after 3 weeks. In contrast, the muscles injected with microparticles (6.8 microg DNA per muscle) showed lower but homogeneous expression values, which were maintained for at least 3 weeks.

Amplification of IGH/MYC fusion in clinically aggressive IGH/BCL2-positive germinal center B-cell lymphomas

Activation of an oncogene via its juxtaposition to the IGH locus by a chromosomal translocation or, less frequently, by genomic amplification is considered a major mechanism of B‐cell lymphomagenesis. However, amplification of an IGH/oncogene fusion, coined a complicon, is a rare event in human cancers and has been associated with poor outcome and resistance to treatment. In this article are descriptions of two cases of germinal‐center‐derived B‐cell lymphomas with IGH/BCL2 fusion that additionally displayed amplification of an IGH/MYC fusion. As shown by fluorescence in situ hybridization, the first case contained a IGH/MYC complicon in double minutes, whereas the second case showed a BCL2/IGH/MYC complicon on a der(8)t(8;14)t(14;18). Additional molecular cytogenetic and mutation analyses revealed that the first case also contained a chromosomal translocation affecting the BCL6 oncogene and a biallelic inactivation of TP53. The second case harbored a duplication of REL and acquired a translocation affecting IGL and a biallelic inactivation of TP53 during progression. Complicons affecting Igh/Myc have been reported previously in lymphomas of mouse models simultaneously deficient in Tp53 and in genes of the nonhomologous end–joining DNA repair pathway. To the best of our knowledge, this is the first time that IGH/MYC complicons have been reported in human lymphomas. Our findings imply that the two mechanisms resulting in MYC deregulation, that is, translocation and amplification, can occur simultaneously.

A novel gene, MDS2, is fused to ETV6/TEL in a t(1;12)(p36.1;p13) in a patient with myelodysplastic syndrome

ETV6/TEL is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners of which 16 have been cloned. Fluorescence in situ hybridization (FISH) analysis in a patient with myelodysplastic syndrome (MDS) revealed a t(1;12)(p36;p13) involving ETV6, with the breakpoint in this gene between exon 2 and exon 3. We report here the cloning of a novel ETV6 partner located on 1p36.1, involved in the t(1;12). 3′ RACE‐PCR from RNA identified a novel sequence fused to exon 2 of ETV6. Database searches localized this sequence in a bacterial artificial chromosome (BAC) mapped to 1p36 by fingerprint analysis. This result was confirmed by FISH using this BAC as probe. 5′ and 3′ RACE experiments with primers from this novel sequence were carried out on RNA from a healthy donor and identified a novel full‐length mRNA, which we named MDS2 (myelodysplastic syndrome 2). RT‐PCR experiments were performed on a panel of human cDNAs to analyze the expression pattern of this gene and they revealed four splicing variants. RT‐PCR analysis showed that ETV6‐MDS2, but not the reciprocal MDS2‐ETV6 fusion transcript, was expressed in the bone marrow of the patient. The product of the ETV6‐MDS2 fusion transcript predicts a short ETV6 protein containing the first 54 amino acids of ETV6 plus four novel amino acids, lacking both the PTN and the DNA‐binding domains. Possible mechanisms to account for the development of MDS in this patient are discussed.

t(10;16)(q22;p13) and MORF-CREBBP fusion is a recurrent event in acute myeloid leukemia

Recently, it was shown that t(10;16)(q22;p13) fuses the MORF and CREBBP genes in a case of childhood acute myeloid leukemia (AML) M5a, with a complex karyotype containing other rearrangements. Here, we report a new case with the MORF‐CREBBP fusion in an 84‐year‐old patient diagnosed with AML M5b, in which the t(10;16)(q22;p13) was the only cytogenetic aberration. This supports that this is a recurrent pathogenic translocation in AML.