Michael O. Alberti

Childhood B-acute lymphoblastic leukemia: a genetic update

In the pediatric population, B-acute lymphoblastic leukemia (B-ALL) is the most prevalent childhood hematological malignancy, as well as the leading cause of childhood cancer-related mortality. Advances in cytogenetics utilizing array-based technologies and next-generation sequencing (NGS) techniques have revealed exciting insights into the genetic basis of this disease, with the hopes of developing individualized treatment plans for affected children. In this comprehensive review, we discuss our current understanding of childhood (pediatric) B-ALL and highlight the most recent genetic advances and their therapeutic implications.

The lncRNA CASC15 regulates SOX4 expression in RUNX1-rearranged acute leukemia

BackgroundLong non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia.ResultsCASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter.ConclusionsOur findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.

Antimicrobial Susceptibilities of Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans

ABSTRACT Nutritionally variant streptococci (NVS) are fastidious Gram-positive cocci comprised of the species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans. NVS are an important cause of bacteremia and infective endocarditis (IE) associated with significant morbidity and mortality. Antimicrobial susceptibility testing (AST) was performed for 14 antimicrobials using the broth microdilution MIC method described in the Clinical and Laboratory Standards Institute (CLSI) M45 guideline. A total of 132 clinical NVS blood isolates collected from 2008 to 2014 were tested. Species level identification of NVS isolates was achieved by 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Ninety isolates were identified as G. adiacens, 37 as A. defectiva, and 5 as G. elegans. All isolates were susceptible to vancomycin (MIC90 = 1 μg/ml), and none displayed high-level resistance to aminoglycosides. G. adiacens was considerably more susceptible to penicillin than A. defectiva (38.9% versus 10.8% of isolates susceptible) but was less susceptible to cephalosporins than was A. defectiva (43.3% versus 100% of isolates susceptible to ceftriaxone). Several isolates were resistant to levofloxacin (6%), erythromycin (51%), and clindamycin (10%). The MIC90 for daptomycin was ≥4 μg/ml for G. adiacens and A. defectiva. G. elegans isolates were 100% susceptible to all antimicrobials tested, with the exception of erythromycin, to which only 20% were susceptible. This study provides antimicrobial susceptibility data for a recent collection of NVS and demonstrates important NVS species-related differences with respect to susceptibility to penicillin, cephalosporins, carbapenems, and daptomycin. Species-level identification of NVS organisms when susceptibility testing is not readily available may aid in treatment decisions.

Regulation of Marginal Zone B-Cell Differentiation by MicroRNA-146a.

B-cell development in the bone marrow is followed by specification into functional subsets in the spleen, including marginal zone (MZ) B-cells. MZ B-cells are classically characterized by T-independent antigenic responses and require the elaboration of distinct gene expression programs for development. Given their role in gene regulation, it is not surprising that microRNAs are important factors in B-cell development. Recent work demonstrated that deficiency of the NFκB feedback regulator, miR-146a, led to a range of hematopoietic phenotypes, but B-cell phenotypes have not been extensively characterized. Here, we found that miR-146a-deficient mice demonstrate a reduction in MZ B-cells, likely from a developmental block. Utilizing high-throughput sequencing and comparative analysis of developmental stage-specific transcriptomes, we determined that MZ cell differentiation was impaired due to decreases in Notch2 signaling. Our studies reveal miR-146a-dependent B-cell phenotypes and highlight the complex role of miR-146a in the hematopoietic system.

The pH of chemistry assays plays an important role in monoclonal immunoglobulin interferences

Objectives Immunoglobulin paraproteins can interfere with multiple chemistry assays. We want to investigate the mechanisms of immunoglobulin interference. Design and methods Serum samples containing paraproteins from the index patient and eight additional patients were used to investigate the interference with the creatinine and total protein assays on the Beckman Coulter AU5400/2700 analyzer, and to determine the effects of pH and ionic strength on the precipitation of different immunoglobulins in these patient samples. Results The paraprotein interference with the creatinine and total protein assays was caused by the precipitation of IgM paraprotein in the index patients samples under alkaline assay conditions. At extremely high pH (12–13) and extremely low pH (1–2) and low ionic strength, paraprotein formed large aggregates in samples from the index patient but not from other patients. Conclusions The pH and ionic strength are the key factors that contribute to protein aggregation and precipitation which interfere with the creatinine and total protein measurements on AU5400/2700. The different amino acid sequence of each monoclonal paraprotein will determine the pH and ionic strength at which the paraprotein will precipitate.

BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia.

BackgroundA new class of non-coding RNAs, known as long non-coding RNAs (lncRNAs), has been recently described. These lncRNAs are implicated to play pivotal roles in various molecular processes, including development and oncogenesis. Gene expression profiling of human B-ALL samples showed differential lncRNA expression in samples with particular cytogenetic abnormalities. One of the most promising lncRNAs identified, designated B-ALL associated long RNA-6 (BALR-6), had the highest expression in patient samples carrying the MLL rearrangement, and is the focus of this study.ResultsHere, we performed a series of experiments to define the function of BALR-6, including several novel splice forms that we identified. Functionally, siRNA-mediated knockdown of BALR-6 in human B-ALL cell lines caused reduced cell proliferation and increased cell death. Conversely, overexpression of BALR-6 isoforms in both human and mouse cell lines caused increased proliferation and decreased apoptosis. Overexpression of BALR-6 in murine bone marrow transplantation experiments caused a significant increase in early hematopoietic progenitor populations, suggesting that its dysregulation may cause developmental changes. Notably, the knockdown of BALR-6 resulted in global dysregulation of gene expression. The gene set was enriched for leukemia-associated genes, as well as for the transcriptome regulated by Specificity Protein 1 (SP1). We confirmed changes in the expression of SP1, as well as its known interactor and downstream target CREB1. Luciferase reporter assays demonstrated an enhancement of SP1-mediated transcription in the presence of BALR-6. These data provide a putative mechanism for regulation by BALR-6 in B-ALL.ConclusionsOur findings support a role for the novel lncRNA BALR-6 in promoting cell survival in B-ALL. Furthermore, this lncRNA influences gene expression in B-ALL in a manner consistent with a function in transcriptional regulation. Specifically, our findings suggest that BALR-6 expression regulates the transcriptome downstream of SP1, and that this may underlie the function of BALR-6 in B-ALL.

Performance of Etest for Antimicrobial Susceptibility Testing of Abiotrophia defectiva and Granulicatella Species

ABSTRACT The Etest on chocolate Mueller-Hinton agar was compared to broth microdilution (BMD) for 125 isolates of nutritionally variant streptococci. Vancomycin Etests yielded 31.1% essential agreement (EA) and 20.0% categorical agreement (CA). Penicillin Etests yielded 86.0% EA and 85.6% CA, whereas ceftriaxone Etests yielded 73.6% EA and 68.0% CA.

Influence of serum separator tubes on mycophenolic acid concentrations determined by HPLC.

To the Editors: Serum separator tubes (SSTs) provide a number of advantages over nongel–containing blood collection tubes. One of the greatest advantages of SSTs is that they can be centrifuged at the collection site and are stable for longer periods of time when not aliquoted right away. However, SSTs cannot be used for monitoring of several therapeutic drugs because of absorption of the drug to the gel barrier and/or release of gel materials into the serum sample that may interfere with a specific test methodology (reviewed in Ref. ). Numerous studies have described the negative influence of SSTs when measuring a number of therapeutic drugs including phenytoin, phenobarbital, lidocaine, carbamazepine, and quinidine. For these reasons, SSTs are not recommended when monitoring therapeutic drugs. This practice has been extended when monitoring immunosuppressant drugs such as mycophenolic acid (MPA), a drug that is used to prevent organ transplant rejection because it selectively inhibits lymphocyte proliferation. In this regard, many laboratories, including reference laboratories such as Quest Diagnostics, Mayo Clinic Laboratories, and ARUP Laboratories, require blood samples to be collected in non-gel– containing collection tubes when testing for therapeutic drugs and list collection in SSTs as a cause for specimen rejection. However, to our knowledge, there are no published reports describing any adverse effects of SSTs on measured MPA concentrations. Thus, given the advantages of using SSTs, we believed that this discrepancy warranted further investigation to properly assess the clinical utility of SSTs in MPA monitoring. To evaluate the possible effects SSTs may have on measured MPA concentrations, we measured MPA concentrations by high-performance liquid chromatography with ultraviolet detection (HPLC/UV) in paired non-gel– containing collection tubes and SSTs. Whole blood was collected from a total of 71 patients receiving MPA (as the prodrugs mycophenolate mofetil or mycophenolate sodium) from December 2011 to February 2012 (41 patients) and February 2013 to March 2013 (30 patients). Institutional review board approval was not required because these samples were drawn for patient care and not for research purposes. For each patient, paired specimens were obtained from a single venipuncture in “Red top” BD Vacutainer Plus plastic serum tubes (Catalog # 367814; BD Diagnostics, Franklin Lakes, NJ) and “Gold top” BD Vacutainer Plus SST serum tubes (Catalog # 367977; BD Diagnostics). The study included multiple lots for each tube type. On the day of collection, total serumMPA concentrations were measured (without quantification of MPA-7-Oglucuronide [MPAG] or other MPA metabolites) from serum aliquots by HPLC/ UV (Shimadzu Prominence HPLC, S/N 51300; Shimadzu Scientific Instruments, Columbia, MD) based on the method described by Hosotsubo et al. The data were plotted using Prism 4.0 (GraphPad Software, La Jolla, CA) (Fig. 1) and linear regression statistics were calculated with EP Evaluator 7.0 (Data Innovations LLC, South Burlington, VT) (Table 1). Total serum MPA concentrations using Red top (non-gel–containing collection tubes) and Gold top (SSTs) tubes were highly correlated over the entire range of concentrations [r = 0.9942, Gold top = 1.029 (Red top) 2 0.10]. However, because the vast majority of the 71 paired samples had MPA concentrations less than 6.0 mcg/mL and correlation is dependent on the range of values tested, we also compared the data after exclusion of the 4 samples with concentrations greater than 6.0 mcg/mL. In this instance, there was a similar correlation observed between Red top and Gold top MPA levels [r = 0.9873, Gold top = 0.994 (Red top) 2 0.04], with