Welbert Oliveira Pereira

BCR-ABL-mediated upregulation of PRAME is responsible for knocking down TRAIL in CML patients.

Tumor necrosis factor-related apoptosis-inducing ligand—TNFSF10 (TRAIL), a member of the TNF-α family and a death receptor ligand, was shown to selectively kill tumor cells. Not surprisingly, TRAIL is downregulated in a variety of tumor cells, including BCR–ABL-positive leukemia. Although we know much about the molecular basis of TRAIL-mediated cell killing, the mechanism responsible for TRAIL inhibition in tumors remains elusive because (a) TRAIL can be regulated by retinoic acid (RA); (b) the tumor antigen preferentially expressed antigen of melanoma (PRAME) was shown to inhibit transcription of RA receptor target genes through the polycomb protein, enhancer of zeste homolog 2 (EZH2); and (c) we have found that TRAIL is inversely correlated with BCR–ABL in chronic myeloid leukemia (CML) patients. Thus, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR–ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is upregulated in BCR–ABL cells and is associated with the progression of disease in CML patients. There is a positive correlation between PRAME and BCR–ABL and an inverse correlation between PRAME and TRAIL in these patients. Importantly, knocking down PRAME or EZH2 by RNA interference in a BCR–ABL-positive cell line restores TRAIL expression. Moreover, there is an enrichment of EZH2 binding on the promoter region of TRAIL in a CML cell line. This binding is lost after PRAME knockdown. Finally, knocking down PRAME or EZH2, and consequently induction of TRAIL expression, enhances Imatinib sensibility. Taken together, our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Development of plasma cell myeloma in a B-cell chronic lymphocytic leukemia patient with chromosome 12 trisomy

BackgroundCancer development results from the progressive accumulation of genomic abnormalities that culminate in the neoplastic phenotype. Cytogenetic alterations, mutations and rearrangements may be considered as molecular legacy which trace the clonal history of the disease. Concomitant tumors are reported and they may derive from a common or divergent founder clone. B-cell chronic lymphocytic leukemia (B-CLL) and plasma cell myeloma (PCM) are both mature B-cell neoplasms, and their concomitancy, albeit rare, is documented.Case presentationHere, we described a patient with prior B-CLL with secondary development of PCM. Cytogenetic and multi parametric flow cytometry analyses were performed. The B-CLL population presented chromosome 12 trisomy, unlikely the arisen PCM population.ConclusionThe close follow up of B-CLL patients is important for early intervention in case of development of other malignancy, such as myeloma. Our observation suggests these two diseases may have arisen from different clones. We understand that the investigation of clonal origin may provide important information regarding therapeutic decisions, and should be considered in concomitant neoplasm.

Granulocyte whole exome sequencing and endothelial JAK2V617F in patients with JAK2V617F positive Budd‐Chiari Syndrome without myeloproliferative neoplasm

Budd-Chiari Syndrome (BCS) is characterized by hepatic venous outflow obstruction. JAK2-positive myeloproliferative neoplasms (JAK2 MPN) are one of the most frequent thrombotic conditions underlying a diagnosis of BCS (Smalberg et al, 2012). A subset of BCS patients harbour the JAK2 mutation in peripheral blood (PB) granulocytes without evidence of overt MPN (JAK2 MPN-neg). While the occurrence of other myeloid-associated mutations in genes are common in patients with JAK2 MPN, it is unknown if such mutations are present in patients with JAK2 MPN-neg. In addition, it remains to be determined if there are differences in JAK2 allele burden between JAK2 MPN and JAK2 MPN-neg. Recently, it has been demonstrated that a subset of patients with JAK2 MPN have endothelial cells (ECs) that are positive for JAK2(Teofili et al, 2011) and that these cells may possibly contribute to the prothrombotic state. However, the presence of JAK2-positive endothelial colony-forming cells (E-CFC) in JAK2 MPN-neg patients with BCS has not been evaluated. The objective of this study was to assess the presence of JAK2 in BM E-CFC from JAK2 MPN-neg patients with BCS and to evaluate the JAK2 allele burden and the presence of additional mutations. We selected patients diagnosed with BCS and the presence of JAK2 mutation in the absence of other thrombophilic conditions at Hospital Israelita Albert Einstein, Sao Paulo, Brazil, between March 2013 and June 2015. Patients were investigated for the presence of an associated MPN and diagnosed according to the 2001 World Health Organization criteria. This study was approved by the Institutional Review Board and conducted according to the Principles of the Declarations of Helsinki. Paired DNA (sorted CD66b-granulocytes/skin biopsy) was subjected to whole exome sequencing (WES) on the HiSeq 2000 platform (Illumina Inc., San Diego, CA) using the SureSelect library preparation kit (Agilent, Santa Clara, CA). Somatic variant calls were generated by combining the output of SomaticSniper (http://gmt.genome.wustl.edu/packages/somatic-sniper/), MuTect (https://www.broadinstitute.org/cancer/cga/mutec) and Pindel (www.sanger.ac.uk/ science/tools/pindel) plus additional in-house criteria (minimum coverage at both tumour/germline ≥8 reads; ratio of allele fraction tumour:germline >2). Bone marrow (BM) mononuclear cells were obtained by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Approximately 20 ml of BM was washed twice with phosphate-buffered saline and 2% fetal bovine serum and then suspended in Endocult liquid medium (STEMCELL Technologies Canada Inc., Vancouver, BC, Canada), as previously described (Wu et al, 2012), but extending the culture time by 7 days. ECs grown in culture were then evaluated by flow cytometry (ECs were considered CD146+, CD144+, CD34 , CD117 ) (Ozdogu et al, 2007). ECs were isolated by fluorescence-activated cell sorting (FACS) in a FACSAria (BD Biosciences, San Jose, CA). DNA was extracted from sorted ECs and tested for the presence of the JAK2 mutation by an allele-specific polymerase chain reaction (PCR). Data on JAK2 allele burden was extracted from WES results obtained from a cohort of 77 patients with JAK2-positive MPN (including 4 patients with JAK2 MPN-neg) (Santos et al, 2014) and from 110 patients previously reported by Nangalia et al (2013). We identified 32 cases of BCS, 10 (31 2%) of who were positive for the JAK2 mutation. Three of these patients passed away before inclusion in the study and 7 patients were included; their baseline features are summarized in Table I. Two patients were diagnosed with primary myelofibrosis (PMF) (Patients 1 and 7) during evaluation and the remaining 5 cases were JAK2 MPN-neg. Samples from Patients 1–6) were analysed by WES. In the 5 JAK2MPN-neg patients (Patients 2–6), the only known pathogenic somatic mutation found was JAK2. We found no additional myeloid-associated mutations in these patients. The median JAK2 variant allele frequency Table I. Demographic features of BCS patients and JAK2 alleleburden.

PRAME/EZH2-Mediated Regulation of TRAIL: A New Target for Cancer Therapy

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts a cancer cell-specific pro-apoptotic activity. This property made the TRAIL associated pathway one of the most promising strategies aimed at inducing tumor-selective death. In fact, several approaches have been considered to explore this pathway for cancer therapy, such as recombinant TRAIL, agonist antibodies for TRAIL receptors, and adenoviral TRAIL. However, all of these approaches have certain disadvantages that limit their clinical use. Our recent discovery that the complex PRAME/EZH2 is able to repress TRAIL expression, in a cancer-specific manner, suggests an alternative approach for combined cancer therapy. A genetic or pharmacological inhibition of TRAIL repressors in cancer cells could restore endogenous TRAIL expression, thereby overcoming some of the limitations of and/or cooperating with previous approaches.

BCR–ABL1-induced downregulation of WASP in chronic myeloid leukemia involves epigenetic modification and contributes to malignancy

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR–ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR–ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott–Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR–ABL1 and is particularly low in blast crisis. Enforced expression of BCR–ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR–ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR–ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR–ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).

Identification of ANLN as ETV6 partner gene in recurrent t(7;12)(p15;p13): a possible role of deregulated ANLN expression in leukemogenesis

The ETV6 gene encodes an ETS family transcription factor that is involved in a myriad of chromosomal rearrangements found in hematological malignancies and other neoplasms. A recurrent ETV6 translocation, previously described in patients with acute myeloid leukemia (AML) (Genes Chromosomes Cancer 51:328–337,2012, Leuk Res 35:e212-214, 2011), whose partner has not been identified is t(7;12)(p15;p13). We herein report that the t(7;12)(p15;p13) fuses ETV6 to ANLN, a gene not previously implicated in the pathogenesis of hematological malignancies, and we demonstrate that this translocation leads to high expression of the fusion transcript in the myeloid and lymphoid lineages.

The lack of PI3Kγ favors M1 macrophage polarization and does not prevent kidney diseases progression

ABSTRACT Acute kidney injury (AKI) and chronic kidney disease (CKD) are major concerns in worldwide public health, and their pathophysiology involves immune cells activation, being macrophages one of the main players of both processes. It is suggested that metabolic pathways could contribute to macrophage modulation and phosphatidylinositol‐3 kinase (PI3K) pathway was shown to be activated in kidneys subjected to ischemia and reperfusion as well as unilateral ureteral obstruction (UUO). Although PI3K inhibition is mostly associated with anti‐inflammatory response, its use in kidney injuries has been shown controversial results, which indicates the need for further studies. Our aim was to unveil the role of PI3K&ggr; in macrophage polarization and in kidney diseases development. We analyzed bone‐marrow macrophages polarization from wild‐type (WT) and PI3K&ggr; knockout (PI3K KO) animals. We observed increased expression of M1 (CD86, CCR7, iNOS, TNF, CXCL9, CXCL10, IL‐12 and IL‐23) and decreased of M2 (CD206, Arg‐1, FIZZ1 and YM1) markers in the lack of PI3K&ggr;. And this modulation was accompanied by higher levels of inflammatory cytokines in PI3K KO M1 cells. PI3K KO mice had increased M1 in steady state kidneys, and no protection was observed in these mice after acute and chronic kidney insults. On the contrary, they presented higher levels of protein‐to‐creatinine ratio and Kim‐1 expression and increased tubular injury. In conclusion, our findings demonstrated that the lack of PI3K&ggr; favors M1 macrophages polarization providing an inflammatory‐prone environment, which does not prevent kidney diseases progression. HighlightsPI3K&ggr; absence favors M1 macrophage polarization.The lack of PI3K&ggr; promotes a more inflammatory environment.The absence of PI3K&ggr; does not prevent kidney from diseases.

Challenge, Discussion and Conclusion: an active teaching strategy to turn traditional lectures into collaborative classes

D2R: step by step Just as with other active strategies, a D2R interactive class requires some pillars that enable preparing the students (therefore it is necessary to provide study material in advance), group projects (it is fundamental to divide the students into teams before class), and practical application of the content (the teacher prepares and presents cases and issues that simulate the practical application of the class, avoiding the valid question of students “Why do I need to learn this?”). [...]

Immunophenotypic evolution of blast populations in pediatric acute myeloid leukemia

Hospital Israelita Albert Einstein, Sao Paulo, SP, Brazil.Corresponding author: Welbert Oliveira Pereira – Avenida Albert Einstein, 627/701 − Morumbi − Zip code: 05652-900 − Sao Paulo, SP, Brazil − Phone: (55 11) 2151-3512 – E-mail: welbert.pereira@einstein.brReceived on: Sep 8, 2015 – Accepted on: Nov 4, 2015DOI: 10.1590/S1679-45082016AI3516

Mutational Profiling of JAK2V617F vs. CALR mutated Primary Myelofibrosis

Background: The median survival of patients with primary myelofibrosis (PMF) is 5 to 7 years after diagnosis. In the majority of patients with PMF somatic mutations were detected either in Janus Kinase 2 (JAK2; in 60% of patients), Calreticulin (CALR; in 25% of patients) or MPL (in 5% of patients) genes. Neither mutation was detected 5% to 10% of PMF patients. Patients with mutated JAK2 are known to have a more aggressive disease compared to patients with mutated CALR. However patients with mutated JAK2 and high allele burden have a favorable outcome compared to patients with a low mutated JAK2 burden. Aim: To develop a model that uses genetic information to predict survival outcome of patients with PMF. Patients and Methods: Bone marrow samples were collected from 344 patients with PMF that were followed at MD Anderson Cancer Center between 2000 and 2013 (157 months). All samples were screened for JAK2 and for mutations in CALR. Patients who did not have a mutation in either gene were also screened for mutations in MPL. Results: In 226 patients (66%) JAK2 was detected and in 43 (13%) CALR was mutated. Of the 75 patients who did not have JAK2 or CALR mutations, 16 (21%) had mutated MPL. In 59 patients (17%), none of those mutations was detected. In the 226 patients who harbored the JAK2 mutation, a cut-point of 50% dichotomized patients into those with a high JAK2 burden and a favorable overall survival (OS; median OS: 80 months) and those with a low JAK2 burden and an adverse OS (median OS: 50 months). Age (above 65 years) and mutation status (low JAK2 burden or triple-negative) were independent risk factors. Patients with a favorable mutation status and age below 65 had a median survival of 126 months (n 1⁄4 82). Patients with either one risk factor, age above 65 (n 1⁄4 88) or adverse mutation status (n 1⁄4 87) had intermediate survival expectancy. The two risk factors were additive and patients age > 65 years and adverse mutation status (n 1⁄4 87) had a median survival of 35 months. Conclusions: Age and mutation status are independent predictors of survival in patients with PMF and stratify patients into 4 groups of equal size with very different survival outcome. 703 Mutational Profiling of JAK2V617F vs. CALR mutated Primary Myelofibrosis Fabio Santos, Renato Puga, Bianca Lisboa, Welbert Pereira, Mariana Miyagi, Evelyn Mata, Tarcila Datoguia, Isabel Bello, Michelli Diniz, Sandra Nakashima, Guilherme Perini, Ricardo Helman, Nelson Hamerschlak, Paulo Campregher Hematology/Oncology, Hospital Israelita Albert Einstein