RESUMO
Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.
Assuntos
Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Adolescente , Adulto , Criança , Deleção Cromossômica , Inversão Cromossômica , Feminino , Rearranjo Gênico do Linfócito T , Proteínas Homeobox A10 , Humanos , Imunofenotipagem , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/fisiopatologia , Masculino , Pessoa de Meia-Idade , Receptor Notch1/genética , Ativação Transcricional , Translocação GenéticaRESUMO
The orphan homeobox gene HOX11L2 was previously found to be transcriptionally activated as a result of the t(5;14)(q35;q32) translocation in three T-ALL cases. We now tested by RT-PCR Hox11L2 expression in 23 consecutive cases of T-ALL (15 children aged 0.8-14 years, eight adults aged 17-55 years) and as control 13 B-ALL patients from a single institution. Hox11L2 expression was undetectable in all patients with B-ALL, nor in adults with T-ALL. Nine children (60% of the cases), all boys, expressed Hox11L2. Blast cells from most of the latter patients carried surface CD1a, CD10 and not CD34 antigens, in contrast to the other children. FISH, M-FISH and IPM-FISH analysis failed to detect a t(5;14)(q35;q32) in one of them, which suggests a possible distinct genetic mechanism in Hox11L2 expression induction. Hence, Hox11L2 expression seems to be the most frequent abnormality in childhood T-ALL to date, comparable to the t(12;21) in child B-ALL.
Assuntos
Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Oncogênicas/genética , Adolescente , Adulto , Fatores Etários , Idoso , Antígenos CD/análise , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 5 , Feminino , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Incidência , Lactente , Leucemia-Linfoma de Células T do Adulto/mortalidade , Masculino , Proteínas Proto-Oncogênicas , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Taxa de Sobrevida , Translocação GenéticaRESUMO
Abnormal CCND1 expression is found in the majority of mantle cell lymphomas (MCL) and in a minority of other mature B cell malignancies. Its evaluation can therefore aid diagnostic classification, in conjunction with clinical, morphological, immunophenotypic and cytogenetic analysis. We describe a rapid slot-blot hybridization technique allowing quantitative assessment of CCND1 expression relative to beta-actin, with a sensitivity cut-off of approximately 10%. This allowed clear separation (P < 0.01) of CCND1 MCL (0.89 +/- 0.4; range 0.23-1.81; n = 25) from control samples (0.02 +/- 0.04; range 0-0.09; n = 22) on limited quantities of RNA (1-3.5 microg). Of nine samples in which a potential diagnosis of MCL lymphoma was based on morphological analysis of paraffin-embedded material, without adequate immunophenotype analysis, all were CCND1 negative and subsequent immunophenotype demonstrated features compatible with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) (CD5+, CD23+, FMC7-) in all cases tested. This study demonstrates the feasibility of slot-blot CCND1 quantification and the importance of the availability of cryopreserved material.
Assuntos
Ciclina D1/biossíntese , Linfoma não Hodgkin/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autorradiografia , Northern Blotting/métodos , Medula Óssea/patologia , Criopreservação , Ciclina D1/análise , Diagnóstico Diferencial , Feminino , Humanos , Imunofenotipagem , Linfonodos/patologia , Linfócitos/imunologia , Linfócitos/patologia , Linfoma não Hodgkin/sangue , Linfoma não Hodgkin/imunologia , Masculino , Pessoa de Meia-Idade , Derrame Pleural Maligno/imunologia , Derrame Pleural Maligno/patologia , Sensibilidade e EspecificidadeRESUMO
To accurately estimate the incidence of HOX11L2 expression, and determine the associated cytogenetic features, in T-cell acute lymphoblastic leukemia (T-ALL), the Groupe Français de Cytogénétique Hématologique (GFCH) carried out a retrospective study of both childhood and adult patients. In total, 364 patients were included (211 children =15 years and 153 adults), and 67 (18.5%) [47 children (22.4%) and 20 adults (13.1%)] were shown to either harbor the t(5;14)q35;q32) translocation or express the HOX11L2 gene or both. Most of the common hematological parameters did not show significant differences within positive and negative populations, whereas the incidence of CD1a+/CD10+ and cytoplasmic CD3+ patients was significantly higher in positive than in negative children. Out of the 63 positive patients investigated by conventional cytogenetics, 32 exhibited normal karyotype, whereas the others 31 showed clonal chromosome abnormalities, which did not include classical T-ALL specific translocations. Involvement of the RANBP17/HOX11L2 locus was ascertained by fluorescence in situ hybridization in six variant or alternative (three-way translocation or cytogenetic partner other than 14q32) translocations out of the 223 patients. Our results also show that HOX11L2 expression essentially occurs as a result of a 5q35 rearrangement, but is not associated with another identified T-ALL specific recurrent genetic abnormality, such as SIL-TAL fusion or HOX11 expression.
Assuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 5/genética , Proteínas de Homeodomínio/genética , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Oncogênicas/genética , Translocação Genética , Adolescente , Adulto , Criança , Pré-Escolar , Aberrações Cromossômicas , Células Clonais , Feminino , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Ploidias , Proteínas Proto-Oncogênicas , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
Much of our understanding of the molecular anomalies involved in the process of oncogenesis has resulted from research into malignant hematologic diseases, facilitated by the accessibility of hematopoietic cells. For example, in lymphoid tumors, rearrangement of the genomic DNA can lead to the juxtaposition of proto-oncogenes and the highly active sequences regulating synthesis of immunoglobulins or T-cell receptors. The subsequent malignancy results from an uncontrolled overexpression of a normal protein. This type of "quantitative" anomaly occurs in follicular lymphomas where B-cells overexpress the normal BCL2 protein which inhibits apoptosis, contributing to immortalization of the B done. The same type of rearrangement process can approach gene fragments which fusion and lead to production of a highly oncogenic chimerical or truncated abnormal protein. Such "qualitative" anomalies occur in myeloid hemopathies. Both types of anomalies involve genes controlling the cell cycle, cell differentiation or cell death (apoptosis), in particular transcription factors (for example, E2A, RARA, MYC) and molecules involved in signal transduction (for example RAS, ABL, LCK). A molecular anomaly can be detected in approximately 30% of all cases of acute leukemia and in up to 75% of the non-Hodgkin lymphomas. Analysis of the junction fragments of the different heavy chains of the immunoglobulins produced in these cases provides a specific marker for detecting the B or T-cell clone in digestive or skin biopsies. For example, detection of a BCR-ABL transcript in a patient with primary thrombocythemia or an atypical myeloproliferative syndrome can be diagnostic and detection of the donal immunoglobulin or T-cell receptor rearrangement can confirm the malignant nature of the lymphoid proliferation. Molecular markers also have prognostic value allowing patient stratification and more adapted therapy. Molecular anomalies detected in malignant hematologic diseases are thus examples of nearly perfect "tumor-specific" markers.
Assuntos
Neoplasias Hematológicas/genética , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/terapia , Humanos , Biologia Molecular , Reação em Cadeia da PolimeraseAssuntos
Fusão Gênica Artificial , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 9 , Proteínas de Homeodomínio/genética , Leucemia Mieloide/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Translocação Genética , Doença Aguda , Sequência de Aminoácidos , Sequência de Bases , DNA de Neoplasias , Humanos , Cariotipagem , Leucemia Mieloide/etiologia , Dados de Sequência MolecularAssuntos
Proteínas de Ligação a DNA/genética , Amplificação de Genes/fisiologia , Síndromes Mielodisplásicas/genética , Proteínas Repressoras/genética , Idoso , Crise Blástica , Aberrações Cromossômicas , Análise Citogenética , Humanos , Masculino , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETSAssuntos
Proteínas de Ligação a DNA/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Fatores de Transcrição/genética , Adolescente , Adulto , Sequência de Bases , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-ets , RNA Neoplásico/análise , Fatores de Transcrição/biossíntese , Transcrição Gênica , Variante 6 da Proteína do Fator de Translocação ETSAssuntos
Cromossomos Humanos Par 2 , Cromossomos Humanos Par 8 , Chaperonas Moleculares/genética , Doenças Mieloproliferativas-Mielodisplásicas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Translocação Genética , Feminino , Humanos , Pessoa de Meia-IdadeAssuntos
Antígenos CD/metabolismo , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/metabolismo , Células Neoplásicas Circulantes/metabolismo , Baço/metabolismo , Baço/patologia , Antígenos CD/genética , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Humanos , Imunofenotipagem , Linfoma de Zona Marginal Tipo Células B/genética , Transtornos Linfoproliferativos/metabolismoRESUMO
A cytological, immunophenotypical and cytogenetical study of 136 chronic B-cell proliferations (93 CLL, 43 B-cell lymphomas) was led in order to precise diagnosis and to characterize and appreciate chromosomal rearrangements. In this series, mainly selected on blood lymphocytosis criteria, B-CLL were twice more frequent than small B-cell lymphomas. Probes used revealed cryptic abnormalities, which remained unknown by conventional cytogenetics (CC). The frequency of clonal abnormalities (CC and FISH) was 74.8% for this series, with 74.4% for lymphomas and 75.3% for CLL, mainly of Binet stage A (69 A, 13 B, 1 C, 10 unspecified). Proportion was 88.4% in A stages and 84.6% in B stages. In CLL, 13q14 cryptic deletions and translocations were widely majority, 14q32 translocations and trisomy 12 being predominant in lymphoma series. Interphase FISH study of non-clonal metaphasic abnormalities with locus-specific probes often revealed unrecognised clones.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/genética , Aneuploidia , Cromossomos Humanos/ultraestrutura , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 13/ultraestrutura , Células Clonais/patologia , Estudos de Coortes , Feminino , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/patologia , Transtornos Linfoproliferativos/genética , Masculino , Estadiamento de Neoplasias , Deleção de SequênciaRESUMO
Molecular techniques are becoming increasingly important in the analysis of NHL, both for diagnostic purposes and in order to evaluate prognosis accurately. The increasing number of techniques available renders evaluation of their relative roles important and a review of their informativity in NHL at diagnosis timely. Molecular equivalents of chromosomal translocations generate either a qualitative change due to the expression of a chimaeric, relatively tumour specific, protein, such as the NPM-ALK associated with the t(2;5) in ALCL or a quantitative change in the extent, stage or site of expression of a full length protein, due to its juxtapositioning to and deregulation by an Ig or TCR gene. The latter represents errors of the somatic recombination process which lymphoid precursors undergo. In NHL, this category includes BCL1/CCND1, BCL2, BCL6 and MYC. The molecular characteristics, the functional consequences and the main clinical correlations of each of these abnormalities is reviewed. At diagnosis, immunological detection of the deregulated 'protooncogene' may well provide the simplest, most appropriate screening technique for CCND1 and NPM-ALK induced ALK expression. BCL6 abnormalities demonstrate similarities to BCL2 and MYC and a combination of immunophenotypic, FISH, Southern blot and PCR techniques are useful in their characterization. For the approximately 50% of NHL without one of the above markers, identification of a clonal Ig or TCR rearrangement can provide a useful 'pan' B or T molecular equivalent, provided that the limitations of the detection techniques are appreciated. Appropriate use of these techniques will transform our ability to classify, stratify and eventually treat in a risk adapted manner, patients with NHL.
Assuntos
Linfoma não Hodgkin/diagnóstico , Aberrações Cromossômicas , Linfoma não Hodgkin/genética , Recombinação GenéticaRESUMO
Both TCRA alleles are rearranged in mature T lymphocytes, as a result of a lack of allelic exclusion at the TRCA locus. We show in a series of T cell clones that the two TCRJA segments are not randomly, but rather coincidentally, rearranged in a given T cell. The TCRJA coincidence relies, in part, on the presence of "T early alpha" (TEA), a cis-regulatory genetic element located upstream of the TCRJA cluster. TEA promotes specific recombinational accessibility that targets primary TCRVAJA rearrangements on the 5' side of the TCRA locus. In a model of multiple waves of TCRVAJA recombination, this cis-regulatory effect of TEA allows for the scanning of the entire TCRJA cluster, thereby increasing the TCR alpha/beta diversity potential.
Assuntos
Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Linfócitos T/imunologia , Regiões 5' não Traduzidas , Animais , Células Clonais , Hibridomas , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Elementos de RespostaAssuntos
Linfócitos B/patologia , Interleucina-2/farmacologia , Cariotipagem/métodos , Ativação Linfocitária/efeitos dos fármacos , Transtornos Linfoproliferativos/patologia , Mitógenos/farmacologia , Oligonucleotídeos/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/ultraestrutura , Aberrações Cromossômicas , Bandeamento Cromossômico/métodos , Estudos de Coortes , Feminino , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Transtornos Linfoproliferativos/genética , Masculino , Metáfase , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Expression of NG2 has been reported in the majority of paediatric acute leukaemia (AL) cases with MLL rearrangement. We demonstrated 7. 1 positivity in 2/3 paediatric and 4/11 adult MLL rearranged acute myeloid leukaemia (AML) but in 0/28 adult AML without MLL rearrangement, thus extending the 100% specificity to adult cases. Positivity correlated with stage of maturation arrest since it was found in 0/6 immature AML but in 6/8 monoblastic cases. These data demonstrate that, if NG2 expression in AL is the (in)direct result of MLL rearrangement, such activation is restricted to a monoblastic population in AML. They also have practical implications for NG2 diagnostic screening strategies.
Assuntos
Antígenos/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Proteoglicanas/genética , Proto-Oncogenes , Fatores de Transcrição , Doença Aguda , Adolescente , Adulto , Antígenos/metabolismo , Pré-Escolar , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Imunofenotipagem , Leucemia Mieloide/imunologia , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide , Proteoglicanas/metabolismo , Células Tumorais CultivadasRESUMO
In order to characterize the genetic diversity in splenic lymphoma with villous lymphocytes (SLVL), we have undertaken cytogenetic and molecular analyses of CCND1 expression and BCL1-IgH PCR rearrangement in 76 cases diagnosed predominantly on morphological criteria. Cytogenetic abnormalities were detected in 19/44 (43%) of cases, including in 16/25 (64%) of cases with an absolute lymphocytosis. Abnormalities included those involving chromosome 14q32 (9/19, 47%), predominantly t(11;14)(q13;q32) (5/19, 26%), chromosome 3 (26%), predominantly 3q, chromosome 17p (26%) and trisomy 12 (3/19, 16%) and were thus suggestive of pathogenetic diversity. CCND1 was expressed in 8/30 (27%) cases, including in all t(11;14) cases, 5/10 (50%) CD5-positive cases and also in 3/20 (15%) CD5-negative cases. Three CCND1-positive SLVL demonstrated immunophenotypic features similar to mantle cell lymphoma (MCL) but the majority differed in their CD5 negativity or CD23 positivity. BCL1-IgH rearrangement was only seen in 1/62 (2%) of cases overall and in none of the t(11;14) cases, which demonstrated FISH breakpoints both centromeric and telomeric to the BCL1/MTC, suggesting that, if genomic clustering exists in t(11;14) SLVL, it differs from MCL. Although CCND1 expressing SLVL more commonly had marked lymphocytosis, they did not demonstrate a more aggressive clinical course than their negative counterparts, demonstrating that the detection of CCND1 expression or of a t(11;14) should not suffice to alter diagnostic classification in the absence of other criteria.
Assuntos
Linfoma de Células B/genética , Neoplasias Esplênicas/genética , Idoso , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 3/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Imunofenotipagem , Cariotipagem , Masculino , Pessoa de Meia-Idade , Monossomia , Reação em Cadeia da Polimerase/métodos , Translocação Genética , TrissomiaRESUMO
BACKGROUND & AIMS: The etiology of refractory sprue is unclear. To gain insight into its pathogenesis, the phenotype and T-cell receptor (TCR) gene rearrangement status of intestinal lymphocytes were analyzed in a group of patients with clinical or biological features of celiac disease but either initially or subsequently refractory to a gluten-free diet. METHODS: Intestinal biopsy specimens were obtained from 26 adults: 6 patients with refractory sprue, 7 patients with active celiac disease, and 13 normal controls. The phenotype of intestinal lymphocytes was studied by immunohistochemistry and, in 3 patients with refractory sprue, by cytometry of lymphocytes purified from intestinal biopsy specimens. TCR rearrangements were assessed by studying TCRgammaV-J junctional regions from DNA extracted from intestinal biopsy specimens and purified intestinal lymphocytes. RESULTS: In the 6 patients with refractory sprue, but not in normal controls or patients with active celiac disease, the intestinal epithelium was massively infiltrated by small lymphocytes that lacked CD8, CD4, and TCR, contained intracytoplasmic but not surface CD3epsilon chains, and exhibited restricted TCRgamma gene rearrangements. CONCLUSIONS: Refractory sprue is associated with an abnormal subset of intraepithelial lymphocytes containing CD3epsilon and restricted rearrangements of the TCRgamma chain but lacking surface expression of T-cell receptors.
Assuntos
Doença Celíaca/imunologia , Intestinos/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Antígenos CD/análise , Doença Celíaca/etiologia , Doença Celíaca/patologia , Feminino , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Humanos , Imunofenotipagem , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
AIMS: We recently showed that refractory sprue is distinct from coeliac disease, the former being characterized by abnormal intraepithelial T-lymphocytes expressing a cytoplasmic CD3 chain (CD3c), lacking CD3 and CD8 surface expression, and showing TCRgamma gene rearrangements. To take advantage of the abnormal phenotype of CD3c + CD8 - intraepithelial lymphocytes (IEL) in refractory sprue we developed a simple method to distinguish coeliac disease from refractory sprue. METHODS AND RESULTS: Comparative immunohistochemical studies using anti-CD3 and anti-CD8 antibodies were applied on paraffin-embedded and frozen biopsy specimens in refractory sprue (n = 6), coeliac disease (n = 10), healthy controls (n = 5) and suspected refractory sprue (n = 6). Comparable results were obtained on fixed and frozen biopsy specimens. In four of the six patients with suspected refractory sprue, abnormal CD3c + CD8 - IEL and TCRgamma gene rearrangements were found, as in refractory sprue; the remaining two patients had normal (CD3 + CD8 +) IEL and no TCRgamma gene rearrangements. Both patients had coeliac disease, as one failed to comply with a gluten-free diet, while the other was a slow responder. CONCLUSION: This simplified immunostaining method using anti-CD3 and anti-CD8 antibodies on paraffin sections can distinguish active coeliac disease from refractory sprue and should prove useful in clinical practice.