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La Leucemia Linfoblástica Aguda (LLA) es la neoplasia más frecuente en edad pediátrica. En los últimos años, entre el 15 y 20% de los pacientes fracasan en el tratamiento. Conocimientos en citogenética y biología molecular repercuten de manera importante en la determinación del pronóstico y del esquema de tratamiento adecuado. En Venezuela existe un conocimiento limitado en cuanto a la genética molecular de esta alteración onco-hematológica. El objetivo del trabajo fue evaluar las alteraciones genéticas más frecuentes en pacientes venezolanos con diagnóstico clínico de leucemia linfoblástica aguda. Se realizó un estudio transversal, descriptivo y prospectivo de 2006 a 2014, en el que se evaluaron las translocaciones ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, así como las mutaciones en los genes PAX5 y FLT3 mediante el uso de diferentes tipos de PCR. Ciento treinta pacientes con diagnóstico clínico de leucemia linfocítica aguda fueron incluidos en el estudio. Se identificaron alteraciones moleculares en 56 pacientes (43,1%), en los que observamos la presencia de una o varias alteraciones en conjunción en un mismo paciente. Las alteraciones identificadas fueron t(12;21) (11,5%), t(4;11) (8,5%), t(1;19) (10%), t(9;22) (20,8%), ITD-FLT3 (14,8%), mutación P80S (4,2%) y S77del (4,2%) en el gen PAX5. La prevalencia de BCR/ ABL, es una de las más altas que ha sido descrita hasta ahora en casos de LLA donde la mayor parte de la población está conformada por pacientes pediátricos. Estos resultados representan el primer estudio molecular de la LLA en Venezuela, sentando las bases para el diagnóstico y seguimiento de la enfermedad en su población.
Acute Lymphoblastic Leukemia (ALL) is the most common neoplasm in pediatric age. In recent years, between 15 and 20% of patients failed in their treatments. Knowledge on cytogenetics and molecular biology has an important impact on the determination of the prognosis and the appropriate treatment scheme. In Venezuela there is limited knowledge regarding the molecular genetics of this onco-hematological alteration. The aim of this work was to evaluate the most frequent genetic alterations in Venezuelan patients with a clinical diagnosis of acute lymphoblastic leukemia. A cross-sectional, descriptive and prospective study was carried out from 2006 to 2014, in which the translocations ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, as well as mutations in the PAX5 and FLT3 genes were evaluated through the use of different types of PCR. One hundred and thirty patients with a clinical diagnosis of acute lymphocytic leukemia were included in the study. Molecular alterations were identified in 56 patients (43.1%), in which we observed the presence of one or several alterations in conjunction in the same patient. The alterations identified were t(12; 21) (11.5%), t(4; 11) (8.5%), t(1; 19) (10%), t(9; 22) (20.8%), ITD-FLT3 (14.8%), P80S mutation (4.2%) and S77del (4.2%) in the PAX5 gene. The prevalence of BCR/ABL is one of the highest described so far in cases of ALL where most of the population is made up of pediatric patients. These results represent the first molecular study of ALL in Venezuela, laying the foundations for the diagnosis and monitoring of the disease in its population.
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Objective To investigate the expression of PAX-5 in anaplastic large cell lymphoma, discuss the value and pitfalls of PAX-5 in the diagnosis and differential diagnosis of lymphomas. Methods A total of 269 T lymphomas were studied retrospectively.PAX-5 positive anaplastic large cell lymphoma was confirmed by immo-munohistochemistry and gene rearrangement analysis. Literature review was performed and the clinicopathological features were discussed.Results Two cases of anaplastic large cell lymphoma were found with aberrant expression of PAX-5. Conclusions As a relatively specific B cell marker, PAX-5 can also express in anaplastic large cell lymphoma. In the diagnosis of lymphomas, especially the differential diagnosis between classical Hodgkin lympho-ma and Anaplastic large-cell lymphoma, diagnosis errors should be avoided by interpreting PAX-5 immunohisto-chemistry in the context of clinical features, morphology, a panel of B- and T-lineage-associated antibodies, and gene rearrangement analysis.
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Background & objectives: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin’s lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. Methods: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, Western blot analysis, and lactate dehydrogenase (LDH) specific staining. Results: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, ZAP70, HIF1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. Interpretation & conclusions: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy.
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Objective To clarify clinical and morphological features and immunophenotype and Epstain-Barr virus infection of neoplastic cell rich Hodgkin's lymphoma (NCRHL)and to further improve our knowledge and pathological diagnosis for NCRHL. Methods 10 cases of NCRHL were analyzed for clinical features, morphology, immunophenotype, Epstein-Barr virus infection using routine eosin and haematoxylin stain, immunohistochemistry, Epstain-Barr virus encoded small RNA (EBER) in situ hybridization and combining clinical data. Results (1)NCRHL were more common in young people. The median age of the patients was 25.5 years old. The ratio of male to female was 1:2.3. Superficial lymph nodes were most frequently involved. Masses of mediastinum were seen commonly. Clinical manifestation of the patients included B symptom (6 cases), pruitus (5 cases) and anemia (1 case). (2)Architecture of lymph nodes were effected. Necrosis was seen in some cases. There were more tumor cells in NCRHL than that in the classical Hodgkin's lymphoma. The tumor cells were distributed in piece or patch or diffuse. The morphology of neoplastic cells was wore variable including Hodgkin-like cells, lacunar cell-like, mummy cell-like and anaplastic large cell-like, singular nucleated cells, and multinucleated giant cell-like cells. Numerous neutrophils and eosinophils were present in a few cases. Focal sheet, necrosis granulomatosis-like and diffuse growth pattern were found in NCRHL. (3)All of the cases were positive for CD30 and PAX-5.2/10 (20%) cases were CD15 positive. LCA, CD20 and CD3 were negative. (4)EBER was not detected in all 6 tested cases. (5)Follow up data was obtained in 8/10 cases, in which one patient was dead, one case relapsed in half a year,and the other 6 cases reached complete regression. Conclusion NRCHL is characterized mainly by neoplastic cell rich morphologically and focal sheet, necrosis granulomatosis-like and diffuse growth pattern.EBER was not detected in this tumor. Some cases have aggressive clinic process with a unfavourable prognosis. New treatment regimen should be explored.
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O linfoma de Hodgkin (LH) e o linfoma difuso de grandes células B (LDGCB) são neoplasias derivadas de células B, que apresentam marcadores imunofenotípicos em comum, e, por isso, em alguns casos onde ocorre sobreposição morfológica torna-se difícil o diagnóstico diferencial entre eles. O grau de dificuldade aumenta quando o tumor é localizado no mediastino. O fato de a biópsia de mediastino ser um procedimento ainda mais invasivo que a maioria das outras biópsias, muitas vezes faz com que sejam coletadas quantidades insuficientes de amostra, o que dificulta a análise das características morfológicas da linfoproliferação. Considerando tais dificuldades, neste trabalho investigaremos a expressão imuno-histoquímica da proteína Pax-5 no diagnóstico diferencial de LH e LDGCB. Foram analisadas amostras de 14 pacientes, sendo dez com LDGCB e quatro com LH. O estudo imuno-histoquímico com Pax-5 mostrou 100 por cento de positividade nuclear para LDGCB. Porém, destes, 30 por cento mostraram positividade forte (+++); 50 por cento moderada (++); 20 por cento fraca (+). Já nas amostras de LH, 50 por cento obtiveram marcação focal e fraca nas células de Reed/Sternberg (CRS) e 50 por cento foram negativas. A marcação focal e fraca, ou até negativa, do Pax-5 em LH pode ser um ponto importante para o diagnóstico diferencial, uma vez que difere muito da marcação visualizada no LDGCB. Assim, é possível concluir que o anticorpo Pax-5 é um bom marcador para ser usado em conjunto com outros marcadores no painel de rotina e, quando associado à clínica, pode ser muito útil no diagnóstico diferencial entre LDGCB e LH em linfoma de mediastino.
Hodgkin's Lymphoma (HL) and diffuse large B-cell Lymphoma (DLBCL) are neoplasias derived from B cells that present the same immunophenotypic markers, making the differential diagnosis between them very difficult particularly in cases where morphological overlapping occurs. An extra difficulty exists when the tumor is located in the mediastinum, thereby demanding a more invasive biopsy. In addition, frequently an excessively small sample is collected, putting the analysis of the morphological characteristics of lymphoproliferation at risk. The goal of this study was to evaluate PAX-5 as a marker to distinguish HL from DLBCL. Overall, 10 out of 14 studied cases had a diagnosis of DLBCL and 4 patients of HL. The Pax-5 immunohistochemical staining showed 100 percent positivity in the LBCL study group. However, 30 percent showed strong positivity (+++), 50 percent moderate (++) and 20 percent weak (+). On the other hand, 50 percent of the HL group showed a weak focal pattern of staining of the Reed/Sternberg Cells (RSC), whereas the other 50 percent showed negativity. The weak focal pattern as well as the negative staining pattern of PAX-5 in HL may be an important key in the differential diagnosis as it is very different from DLBCL. Hence, we suggest that the PAX-5 antibody is a good immunomarker and may be used with other markers in the laboratory routine. It is also very useful when associated to clinical features of patients to reach a differential diagnosis between DLBCL and HL mediastinal lymphoma.
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Humans , Biopsy , Diagnosis, Differential , Hodgkin Disease , Lymphoma, Large B-Cell, Diffuse , MediastinoscopyABSTRACT
Objective:To evaluate the biological functions and underlying mechanisms of transcription factor PAX5 in promoting cell proliferation and anti-apoptosis in multiple myeloma cells.Methods:a PAX5 knockdown cell line was established by stable transfection of vector-based PAX5-siRNA into multiple myeloma IM9 cells.The expressing level of PAX5,p53,XBP-1 and c-Myc was examined by either or both Western blot and RT-PCR for the targets.Cell proliferation and apoptosis were assayed by MTT and flow cytometry,respectively.Results:PAX5 was expressed selectively in IM9 cells,but neither in another common used multiple myeloma KAS6 cells nor in the prostate cancer cells DU145 and PC3.Knockdown PAX5 led to a significant up-regulation of p53 and XBP1,but decreased c-Myc expressions in IM9 cells that were correlated with the increased sensitivity of drug-induced apoptosis and decreased proliferation rates when compared to the control cells.Conclusion:PAX5 is specifically expressed in IM9 cells,which promotes cell proliferation and anti drug-induced apoptosis.In addition to inhibiting the expression of p53 and XBP-1,PAX5 is found to induce expression of oncogene c-Myc in IM9 cells,and this finding indicates an undiscovered signal pathway that may contributes to the malignancy of Multiple Myeloma cells.
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Abstract Objective:To better understand the regulatory mechanism of Pax-5 gene expression on B-cell differentation and development.Methods:The isolation and genomic colning of the 5'-flanking region of Pax-5 gene were carried out according to established protocols: molecu-lar cloning and subcloning The nucleotide sequences were determined by a double-stranded dideoxy sequencing method. Sequence analysis wasperformed on line programs. Results:Analysis of tha total sequence(6 671 bp) shows the proximal promoter include 3 CAT boxes, 1SP1 and 1Ebox. However, there was no consensus sequence for a TATA box in the 5'-flanking region. Putative regulatory sites of further upstream in thesequence revealed 6LMO2 COM, 5NFAT, 2LPOLYA-B, 3GATA1, 2AP4, 10MZF1, 1ets1B, 1GATA3, 1NKX-25, 2RORA1, 1LYF1, 2Ikaros2,2TCF11 , 1GATA-C and 1FREAC7. Conclusion: The 5'-flanking regionn of human Pax-5 gene exon1B could be involved in regulating the expres-sion of human Pax5 and B-cell differentiation and development.