SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5.

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

The transcription factor PAX5 activates human LINE1 retrotransposons to induce cellular senescence.

As a hallmark of senescent cells, the derepression of Long Interspersed Elements 1 (LINE1) transcription results in accumulated LINE1 cDNA, which triggers the secretion of the senescence-associated secretory phenotype (SASP) and paracrine senescence in a cGAS-STING pathway-dependent manner. However, transcription factors that govern senescence-associated LINE1 reactivation remain ill-defined. Here, we predict several transcription factors that bind to human LINE1 elements to regulate their transcription by analyzing the conserved binding motifs in the 5'-untranslated regions (UTR) of the commonly upregulated LINE1 elements in different types of senescent cells. Further analysis reveals that PAX5 directly binds to LINE1 5'-UTR and the binding is enhanced in senescent cells. The enrichment of PAX5 at the 5'-UTR promotes cellular senescence and SASP by activating LINE1. We also demonstrate that the longevity gene SIRT6 suppresses PAX5 transcription by directly binding to the PAX5 promoter, and overexpressing PAX5 abrogates the suppressive effect of SIRT6 on stress-dependent cellular senescence. Our work suggests that PAX5 could serve as a potential target for drug development aiming to suppress LINE1 activation and treat senescence-associated diseases.

Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop.

BACKGROUND: Existing studies have found that circular RNAs (circRNAs) act as sponges for micro RNAs (miRNAs) to control downstream genes. However, the specific functionalities and mechanisms of circRNAs in human clear cell renal cell carcinoma (ccRCC) have yet to be thoroughly investigated. METHODS: Patient cohorts from online databases were used to screen candidate circRNAs, while another cohort from our hospital was obtained for validation. CircSOD2 was identified as a potential oncogenic target, and its relevant characteristics were investigated during ccRCC progression through various assays. A positive feedback loop containing downstream miRNA and its target gene were identified using bioinformatics and validated by luciferase reporter assays, RNA pull-down, and high-throughput sequencing. RESULTS: CircSOD2 expression was elevated in tumor samples and significantly correlated with overall survival (OS) and the tumor stage of ccRCC patients, which appeared in the enhanced proliferation, invasion, and migration of tumor cells. Through competitive binding to circSOD2, miR-532-3p can promote the expression of PAX5 and the progression of ccRCC, and such regulation can be salvaged by miR-532-3p inhibitor. CONCLUSION: A novel positive feedback loop, PAX5/circSOD2/miR-532-3p/PAX5 was identified in the study, indicating that the loop may play an important role in the diagnosis and prognostic prediction in ccRCC patients.

MicroRNA-138 promotes the progression of multiple myeloma through targeting paired PAX5.

BACKGROUND: Multiple myeloma cancer stem cells (MMSC) have been considered as the leading cause of multiple myeloma (MM) drug resistance and eventual relapse, microRNAs (miRNAs) collectively participate in the progression of MM. However, the pathogenesis of miR-138 in MMSC is still not fully understood. OBJECTIVE: The intention of this study was to investigate the mechanism and role of miR-138 in multiple myeloma. METHOD: Bone marrow samples and peripheral blood from patients and normal controls were collected. Use Magnet-based Cancer Stem Cell Isolation Kit to separate and extract MMSC. Real-time quantitative PCR (RT-qPCR) was carried out to determine mRNA level. Western blot was applied to detect protein levels. MTT and flow cytometry were conducted to examine the proliferation and apoptosis of MMSC. Finally, dual-luciferase reporter gene assays were performed to confirm that paired box 5 (PAX5) is a direct target for miR-138. RESULTS: Compared with normal group, the expression of miR-138 in patients was significantly up-regulated, and the expression of miR-138 was in a negative correlation with PAX5. Additionally, downregulated miR-138 facilitated the apoptosis and inhibited the proliferation of MMSC in vitro and in vivo. Downregulated miR-138 moderated the expression of PAX5, Bcl-2, Bax, and Caspase-3. PAX5 was a direct target of miR-138. CONCLUSION: Taken together, miR-138 plays a carcinogenic role in MM, and miR-138 adjusted the proliferation and apoptosis of MMSC by targeting PAX5. miR-138 has the probability of becoming a new medicinal target for the treatment of MM.

Brevilin A Inhibits Prostate Cancer Progression by Decreasing PAX5-Activated SOX4.

Brevilin A possesses inhibitory effects on the development of prostate cancer (PCa); however, the underlying mechanism remains unclear. The present work aims to analyze how Brevilin A regulates PCa cell malignancy. RNA expression of paired box 5 (PAX5) and SRY-box transcription factor 4 (SOX4) was analyzed by quantitative real-time polymerase chain reaction. Protein expression of PAX5, SOX4, and nuclear proliferation marker (Ki67) was detected by western blotting or immunohistochemistry assay. The viability, proliferation, apoptosis, and migratory and invasive abilities of PCa cells were investigated by cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays, respectively. The association between PAX5 and SOX4 was identified by dual-luciferase reporter assay and chromatin immunoprecipitation assay. Xenograft mouse model assay was used to reveal the effect of Brevilin A on tumor tumorigenesis in vivo. PAX5 and SOX4 expression were upregulated in PCa tissues and cells relative to normal prostate tissues and human prostate epithelial cells. Brevilin A treatment inhibited PAX5 protein expression in PCa cells. Additionally, Brevilin A inhibited proliferation, migration and invasion and induced apoptosis of PCa cells, whereas these effects were attenuated after PAX5 overexpression. SOX4 was transcriptionally activated by PAX5, and its introduction partially relieved the inhibitory effects of PAX5 knockdown on PCa cell malignancy. Moreover, Brevilin A delayed tumor formation in vivo. Brevilin A inhibited PCa progression by regulating SOX4 expression in a PAX5-dependent manner, providing a promising anti-tumor drug for PCa.

PAX5 functions as a tumor suppressor by RB-E2F-mediated cell cycle arrest in Kaposi sarcoma-associated herpesvirus-infected primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a malignant B-cell lymphoma attributable to Kaposi sarcoma-associated herpesvirus (KSHV) infection. PEL is characterized by invasive behavior, showing recurrent effusions in body cavities. The clinical outcome and typical prognosis in patients with PEL are poor and potentially lethal. Clarification of the pathogenesis in PEL is urgently needed in order to develop novel therapies. PEL cells generally lack B-cell surface markers, and we therefore hypothesized that the B-cell transcription factor, PAX5, would be down-regulated in PEL. The expression of PAX5 is detected from the pro-B to the mature B-cell stage and is indispensable for the differentiation of B-cells. PAX5 was silenced in PEL cells via its promoter methylation. Up-regulation of PAX5 induced several genes coding for B-cell surface marker mRNA, but not protein level. PAX5 inhibited cell growth via G1 cell cycle arrest. PAX5 bound to RB and increased its protein expression. RB/E2F-regulated genes were significantly down-regulated in microarray analysis and PCR experiments. To elucidate the in vivo role of PAX5, we examined the restoration of PAX5 in a PEL mouse model. The ascites volume and organ invasions were significantly suppressed by PAX5 restoration. Reduction of PAX5 has played a crucial role in the oncogenesis of PEL, and PAX5 is a tumor suppressor in PEL. Targeting PAX5 could represent a novel therapeutic strategy for patients with PEL.

PAX5 Alterations in a Consecutive Childhood B-Cell Acute Lymphoblastic Leukemia Cohort Treated Using the ALL IC-BFM 2009 Protocol.

In this study, we aimed to identify patients within our B-ALL cohort with altered PAX5. Our objective was to use a comprehensive analysis approach to characterize the types of genetic changes, determine their origin (somatic/germline), and analyze the clinical outcomes associated with them. A consecutive cohort of 99 patients with B-ALL treated at the Children's Hospital of the UMC Ljubljana according to the ALL IC-BFM 2009 protocol was included in our study. We used RNA sequencing data for gene expression analysis, fusion gene detection and single nucleotide variant identification, multiplex-ligation dependent probe amplification for copy number variation assessment, and Sanger sequencing for germline variant detection. PAX5 was impacted in 33.3% of our patients, with the genetic alterations ranging from CNVs and rearrangements to SNVs. The most common were CNVs, which were found in more than a third of patients, followed by point mutations in 5.2%, and gene rearrangements in 4.1%. We identified eight patients with a PAX5-associated genetic subtype that were previously classified as "B-other", and they showed intermediate outcomes. We showed higher minimal residual disease values at the end of induction and poorer event-free survival in hyperdiploid cases carrying duplications in PAX5 compared to other hyperdiploid cases. We also report an interesting case of a patient with PAX5::FKBP15 and a pathogenic variant in PTPN11 who underwent an early relapse with a monocytic switch. In conclusion, this study provides valuable insights into the presence, frequency, and prognostic significance of diverse PAX5 alterations in B-ALL patients, highlighting the complexity of genetic factors and their impact on patient outcomes.

Case report: A familial B-acute lymphoblastic leukemia associated with a new germline pathogenic variant in PAX5. The first report in Mexico.

B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common childhood cancers worldwide. Although most cases are sporadic, some familial forms, inherited as autosomal dominant traits with incomplete penetrance, have been described over the last few years. Germline pathogenic variants in transcription factors such as PAX5, IKZF1, and ETV6 have been identified as causal in familial forms. The proband was a 7-year-old Mexican girl diagnosed with high-risk B-ALL at five years and 11 months of age. Family history showed that the proband's mother had high-risk B-ALL at 16 months of age. She received chemotherapy and was discharged at nine years of age without any evidence of recurrence of leukemia. The proband's father was outside the family nucleus, but no history of leukemia or cancer was present up to the last contact with the mother. We performed exome sequencing on the proband and the proband's mother and identified the PAX5 variant NM_016734.3:c.963del: p.(Ala322LeufsTer11), located in the transactivation domain of the PAX5 protein. The variant was classified as probably pathogenic according to the ACMG criteria. To the best of our knowledge, this is the first Mexican family with an inherited increased risk of childhood B-ALL caused by a novel germline pathogenic variant of PAX5. Identifying individuals with a hereditary predisposition to cancer is essential for modern oncological practice. Individuals at high risk of leukemia would benefit from hematopoietic stem cell transplantation, but family members carrying the pathogenic variant should be excluded as hematopoietic stem cell donors.

DNA methylation regulates B cell activation via repressing Pax5 expression in teleost.

In mammals, the transcription factor Pax5 is a key regulator of B cell development and maturation and specifically expressed in naive/mature B cells but repressed upon B cell activation. Despite the long-standing proposal that Pax5 repression is essential for proper B cell activation, the underlying mechanisms remain largely elusive. In this study, we used a teleost model to elucidate the mechanisms governing Pax5 repression during B cell activation. Treatment with lipopolysaccharide (LPS) and chitosan oligosaccharide (COS) significantly enhanced the antibody secreting ability and phagocytic capacity of IgM+ B cells in large yellow croaker (Larimichthys crocea), coinciding with upregulated expression of activation-related genes, such as Bcl6, Blimp1, and sIgM, and downregulated expression of Pax5. Intriguingly, two CpG islands were identified within the promoter region of Pax5. Both CpG islands exhibited hypomethylation in naive/mature B cells, while CpG island1 was specifically transited into hypermethylation upon B cell activation. Furthermore, treatment with DNA methylation inhibitor 5-aza-2'-deoxycytidine (AZA) prevented the hypermethylation of CpG island1, and concomitantly impaired the downregulation of Pax5 and activation of B cells. Finally, through in vitro methylation experiments, we demonstrated that DNA methylation exerts an inhibitory effect on promoter activities of Pax5. Taken together, our findings unveil a novel mechanism underlying Pax5 repression during B cell activation, thus promoting the understanding of B cell activation process.

Paired Box 5 (PAX5) Gene Has Diagnostic and Prognostic Potential in Nasopharyngeal Carcinoma.

Purpose: Paired Box 5 (PAX5) is a transcription factor that is widely associated with carcinogenesis. PAX5 can maintain Epstein-Barr virus (EBV) latency in B cells, while a close association exists between EBV infection and nasopharyngeal carcinoma (NPC). However, there are very few reports on the correlation between PAX5 and NPC development. The aim of this study was to investigate the role of PAX5 in NPC. Patients and Methods: The clinical value and prognostic significance of PAX5 in NPC and the association with PAX5 expression and immune cell infiltration were analyzed by multiple GEO datasets. In vivo and in vitro experiments including real-time PCR, Western blot, CCK-8 assay, and methylation sequencing were used to validate the results of bioinformatics analysis. Results: The expression of PAX5 was significantly reduced in NPC tissues, with the low expression being correlated with advanced clinical stage, low tumor mutation burden and immune activation, high relative expression of EBV, poor survival for NPC patients. PAX5 exhibited excellent diagnostic performance and had potential as a predictive factor for response to the immune checkpoint inhibitors therapy. Enrichment analysis suggested that the low expression of PAX5 was associated with the dysregulation of Hippo and Wnt signaling pathways. The promoter of PAX5 gene was hypermethylated in NPC tissues. Furthermore, the in vitro and in vivo experiments revealed that NPC tissue and cell lines had low mRNA expression levels of PAX5, the PAX5 promoter was hypermethylated in NPC cell lines, and PAX5 overexpression inhibited NPC cell proliferation and tumor growth in nude mice. Conclusion: PAX5 may be a tumor suppressor and serve as a novel potential diagnostic and prognostic marker for NPC.

EBF1, PAX5, and MYC: regulation on B cell development and association with hematologic neoplasms.

During lymphocyte development, a diverse repertoire of lymphocyte antigen receptors is produced to battle against pathogens, which is the basis of adaptive immunity. The diversity of the lymphocyte antigen receptors arises primarily from recombination-activated gene (RAG) protein-mediated V(D)J rearrangement in early lymphocytes. Furthermore, transcription factors (TFs), such as early B cell factor 1 (EBF1), paired box gene 5 (PAX5), and proto-oncogene myelocytomatosis oncogene (MYC), play critical roles in regulating recombination and maintaining normal B cell development. Therefore, the aberrant expression of these TFs may lead to hematologic neoplasms.

The prognostic significance of programmed cell death protein 1 and its ligand on lymphoma cells and tumor-immune cells in diffuse large B-cell lymphoma, not otherwise specified.

BACKGROUND: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is the most common type non-Hodgkin's lymphoma, where the treatment of relapsed/refractory cases is the major challenge. Programmed cell death protein 1 (PD-1) and its ligand PD-L1 play a crucial role in the negative regulation of the immune response against the disease. The aim of the study was to analyze the expression of PD-1 and PD-L1 on lymphoma cells (LCs) and tumor-immune cells (TICs) and to investigate their correlation with outcome. PATIENTS AND METHODS: Samples from 283 patients diagnosed with DLBCL, NOS (both germinal center B cell like [GCB] and non-GCB subtypes) were included in the study. Expression of PD-1 and PD-L1 was determined using double immunohistochemical staining (D-IHC) for PD-1/PAX5 and PD-L1/PAX5 on tissue microarrays. LCs were highlighted by D-IHC to obtain more accurate results. Clinical data and histologic diagnoses were obtained from electronic data records. We correlated clinical characteristics, and PD-1 and PD-L1 expression on LCs and TICs with progression-free survival (PFS) and overall survival (OS). RESULTS: Expression of PD-1 on TICs was observed in 38.4% and on LCs in 8.8% of cases, while PD-L1 was expressed on TICs in 46.8% and on LCs in 6.5% of cases. PD-L1 expression on LCs was more frequent in non-GCB subtype (p = 0.047). In addition, patients with PD-L1 expression on LCs had significantly shorter PFS (p = 0.015), and the expression retained significant in the multivariate model (p = 0.034). CONCLUSIONS: PD-L1 was more frequently expressed in LCs of the non-GCB subtype. Additionally, PD-L1 in LCs may predict shorter PFS time. D-IHC staining for PD-L1/PAX5 is a feasible method to assess PD-L1 expression on LCs of DLBCL, NOS patients and can be used to identify patients who may benefit from targeted immunotherapy with checkpoint inhibitors.

Cytokines-activated nuclear IKKα-FAT10 pathway induces breast cancer tamoxifen-resistance.

Endocrine therapy that blocks estrogen signaling is the most effective treatment for patients with estrogen receptor positive (ER+) breast cancer. However, the efficacy of agents such as tamoxifen (Tam) is often compromised by the development of resistance. Here we report that cytokines-activated nuclear IKKα confers Tam resistance to ER+ breast cancer by inducing the expression of FAT10, and that the expression of FAT10 and nuclear IKKα in primary ER+ human breast cancer was correlated with lymphotoxin ß (LTB) expression and significantly associated with relapse and metastasis in patients treated with adjuvant mono-Tam. IKKα activation or enforced FAT10 expression promotes Tam-resistance while loss of IKKα or FAT10 augments Tam sensitivity. The induction of FAT10 by IKKα is mediated by the transcription factor Pax5, and coordinated via an IKKα-p53-miR-23a circuit in which activation of IKKα attenuates p53-directed repression of FAT10. Thus, our findings establish IKKα-to-FAT10 pathway as a new therapeutic target for the treatment of Tam-resistant ER+ breast cancer.

IKZF1plus is a frequent biomarker of adverse prognosis in Mexican pediatric patients with B-acute lymphoblastic leukemia.

Background: Recurrent genetic alterations contributing to leukemogenesis have been identified in pediatric B-cell Acute Lymphoblastic Leukemia (B-ALL), and some are useful for refining classification, prognosis, and treatment selection. IKZF1plus is a complex biomarker associated with a poor prognosis. It is characterized by IKZF1 deletion coexisting with PAX5, CDKN2A/2B, or PAR1 region deletions. The mutational spectrum and clinical impact of these alterations have scarcely been explored in Mexican pediatric patients with B-ALL. Here, we report the frequency of the IKZF1plus profile and the mutational spectrum of IKZF1, PAX5, CDKN2A/2B, and ERG genes and evaluate their impact on overall survival (OS) in a group of patients with B-ALL. Methods: A total of 206 pediatric patients with de novo B-ALL were included. DNA was obtained from bone marrow samples at diagnosis before treatment initiation. A custom-designed next-generation sequencing panel was used for mutational analysis. Kaplan-Meier analysis was used for OS estimation. Results: We identified the IKZF1plus profile in 21.8% of patients, which was higher than that previously reported in other studies. A significantly older age (p=0.04), a trend toward high-risk stratification (p=0.06), and a decrease in 5-year Overall Survival (OS) (p=0.009) were observed, although heterogeneous treatment protocols in our cohort would have impacted OS. A mutation frequency higher than that reported was found for IKZF1 (35.9%) and CDKN2A/2B (35.9%) but lower for PAX5 (26.6%). IKZF1MUT group was older at diagnosis (p=0.0002), and most of them were classified as high-risk (73.8%, p=0.02), while patients with CDKN2A/2BMUT had a higher leukocyte count (p=0.01) and a tendency toward a higher percentage of blasts (98.6%, >50% blasts, p=0.05) than the non-mutated patients. A decrease in OS was found in IKZF1MUT and CDKN2A/2BMUT patients, but the significance was lost after IKZF1plus was removed. Discussion: Our findings demonstrated that Mexican patients with B-ALL have a higher prevalence of genetic markers associated with poor outcomes. Incorporating genomic methodologies into the diagnostic process, a significant unmet need in low- and mid-income countries, will allow a comprehensive identification of relevant alterations, improving disease classification, treatment selection, and the general outcome.

Understanding the role of Pax5 in development of taxane-resistant neuroendocrine like prostate cancers.

Resistance to the current Androgen Receptor Signaling Inhibitor (ARSI) therapies has led to higher incidences of therapy-induced neuroendocrine-like prostate cancer (t-NEPC). This highly aggressive subtype with predominant small cell-like characteristics is resistant to taxane chemotherapies and has a dismal overall survival. t-NEPCs are mostly treated with platinum-based drugs with a combination of etoposide or taxane and have less selectivity and high systemic toxicity, which often limit their clinical potential. During t-NEPC transformation, adenocarcinomas lose their luminal features and adopt neuro-basal characteristics. Whether the adaptive neuronal characteristics of t-NEPC are responsible for such taxane resistance remains unknown. Pathway analysis from patient gene-expression databases indicates that t-NEPC upregulates various neuronal pathways associated with enhanced cellular networks. To identify transcription factor(s) (TF) that could be important for promoting the gene expression for neuronal characters in t-NEPC, we performed ATAC-Seq, acetylated-histone ChIP-seq, and RNA-seq in our NE-like cell line models and analyzed the promoters of transcriptionally active and significantly enriched neuroendocrine-like (NE-like) cancer-specific genes. Our results indicate that Pax5 could be an important transcription factor for neuronal gene expression and specific to t-NEPC. Pathway analysis revealed that Pax5 expression is involved in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are critical for strong cellular communications. Further results suggest that depletion of Pax5 disrupts cellular interaction in NE-like cells and reduces surface growth factor receptor activation, thereby, sensitizing them to taxane therapies. Moreover, t-NEPC specific hydroxymethylation of Pax5 promoter CpG islands favors Pbx1 binding to induce Pax5 expression. Based on our study, we concluded that continuous exposure to ARSI therapies leads to epigenetic modifications and Pax5 activation in t-NEPC, which promotes the expression of genes necessary to adopt taxane-resistant NE-like cancer. Thus, targeting the Pax5 axis can be beneficial for reverting their taxane sensitivity.

Identificación de alteraciones moleculares en pacientes Venezolanos con diagnóstico de leucemia linfoblástica aguda / Identification of molecular alterations in Venezuelan patients with acute lymphoblastic leukemia diagnosis

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.

Expression and significance of BSAP(PAX-5)in anaplastic large cell lymphoma / 实用医学杂志

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.

A clinicopathological study of 10 cases of neoplastic cell rich Hodgkin's lymphoma / 白血病·淋巴瘤

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.

Importância da análise da proteína Pax-5 no diagnóstico diferencial entre linfoma de Hodgkin e linfoma difuso de grandes células B em biópsias de mediastino / The importance of Pax-5 protein immunostaining in the differential diagnosis between Hodgkin's lymphoma and diffuse large B-cell lymphoma in biopsies of the mediastinum

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.

Transcription factor PAX5 promotes anti-apoptosis and proliferation on multiple myeloma cell IM9 / 中国免疫学杂志

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.