P. R. G. Ribeiro

Adult B-cell acute lymphoblastic leukemia cells display decreased PTEN activity and constitutive hyperactivation of PI3K/Akt pathway despite high PTEN protein levels

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

Polymerase chain reaction screening for fungemia and/or invasive fungal infections in patients with hematologic malignancies

IntroductionInvasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool.Patients and methodsBetween June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. Results: PCR screening predicted the development of IFI to be 17xa0days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. Discussion: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes

AbstractGold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible.n Graphical abstractGold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome

A double Philadelphia chromosome-positive chronic myeloid leukemia patient, co-expressing P210BCR-ABL1 and P195BCR-ABL1 isoforms

The Philadelphia chromosome (Ph; t(9;22)(q34;q11)) and the associated BCR-ABL1 gene are unique markers for chronic myeloid leukemia (CML). The chimeric gene is remarkably homogenous in CML, as nearly all patients express the P210BCRABL1 protein originated, in the majority of cases, by the e14a2 or

A novel mutation in CEBPA gene in a patient with acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by clonal expansion of myeloid progenitor cells accounting for the majority of acute leukemia cases in adults, despite the high level of cell and molecular heterogeneity [1]. Karyotyping at diagnosis provides for the most important prognosis information in AML. Approximately 40–50% of AML pa tients do not show any clonal chromosomal aberrations and the most prominent sign of the disease is a blockage in granulocytic blasts differentiation [2]. Cytogenetically normal AML patients are now classified into molecularly defined subgroups with distinct clinical outcomes. For example, mutations within the transcription factor CCAAT/enhancer binding protein a (C/EBPa) gene (CEBPA) have been associated with prognosis [3,4]. CEBPA, located on chromosome 19q13.1, is a member of the basic region leucine zipper family of transcription factors. The gene encodes a 42-kDa protein with a Cterminal region (bZIP), consisting of a leucine zipper domain and an adjacent basic region, which modulates C/EBPa dimerization, DNA binding and triggers the two transactivation domains (TAD) in the N-terminal region [1]. In hematopoiesis, C/EBPa plays a central role during granulocyte differentiation, where a diminishing activity is associated to the reduction of the myeloid progenitors’ differentiation potential, thus deregulating proliferation of hematopoietic cells [1,5]. Mutations in CEBPA have been identified in 7–15% of AML patients, correlating to a favorable clinical outcome [4,6]. These mutations may alter bZIP or actuate TAD domains [7,8]. AML patients usually exhibit biallelic CEBPA mutations, comprising an N-terminal frame-shift mutation and a C-terminal insertion mutation, which result in complete abrogation of wild-type C/EBPa expression [4,9]. Single CEBPA mutations also occur, in which expression of the wild-type product is retained [10]. Screening for mutations in CEBPA constitute an useful tool for monitoring minimal residual disease in follow-up samples, to detect molecular relapse and may contribute to a more direct and personalized treatment. Here we report a new CEBPA mutation c.1067A4T; p.Asn356Ile in the bZIP region of the protein. The 67year-old male patient was referred to our hematology department with generalized unspecified pain and leucocytes of 100 000/mL. Bone marrow (BM) morphology showed 96% blast count and together with imunophenotyping analysis classified as AML without maturation (WHO 2008). The G-Band karyotyping was normal, t(9:22) was negative by FISH, and molecular analysis for NPM1 and FLT3 mutations were also negative (See supplementary material, available online). CEBPA molecular analysis was performed on RNA extracted from BM aspirate using TRIsure reagent, according to manufacturer’s instructions (Bioline, London, UK). Total RNA was reverse transcribed into cDNA using the NZY M-MuLV First-Strand cDNA Synthesis kit (Nzytech, Lisbon, Portugal). PCR amplification of CEBPA was performed in three fragments, each one comprising a different domain of the encoded protein: TAD-1; TAD-2 and bZIP (for specific conditions on the N-terminal domain amplification see supplementary material). The C-terminal bZIP coding region of the gene (Figure 1A; reference sequence NM_004364.4) was amplified using forward and reverse primers (CEBPA-F 50CAAGGCCAAGAAGTCGGTGGACA-30 and CEBPA-R 50CACGGCTCGGGCAAGCCTCGAGAT-30, respectively) and the following conditions: initial denaturation at 95 C for 10 min; 40 cycles of 95 C for 1 min, 61 C for 40 s, 72 C