Matthew J. Oberley

High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays.

Publisher Summary Sequencing of the human genome provides a wealth of information that allows rapid advances in the understanding of gene regulation. For example, 5′ exon prediction programs provide estimates of the number of promoters in the human genome. Similarly, scanning the human genome for Myc binding sites identifies a large set of promoters that contain Myc consensus sites. Follow-up analyses of the predicted E2F and Myc target promoters using chromatin immunoprecipitation (ChIP) assays indicate that many of the putative targets are in fact bound by E2F or Myc family members in living cells. However, there are two major concerns that arise when one relies solely on a bioinformatics approach. First, many consensus sites may reside within inactive chromatin and may not be available for interaction with a factor due to inaccessibility. A major problem in studying human transcription factors is the fact that no appropriate microarrays are widely available that can be used to study intergenic DNA; that is, all commercially available microarrays are allowed only for the detection of transcribed sequences. It can be hoped that the technologies and products arising from such projects allow much greater access of all investigators to the required reagents needed for a thorough analysis of the regulatory elements encoded in the human genome. Similarly, a binding site may reside within active (euchromatin) but be inaccessible due to specific nucleosomal positioning.

Probing Chromatin Immunoprecipitates with CpG-Island Microarrays to Identify Genomic Sites Occupied by DNA-Binding Proteins

Publisher Summary This chapter discusses the chromatin immunoprecipitation (ChIP) assay to a high-throughput microarray–based method for discovering genomic regions occupied by human DNA-binding proteins. Others, primarily using yeast model systems, have also explored the use of coupled-chromatin immunoprecipitation and microarrays to identify large sets of binding sites of DNA-binding proteins. ChIPs and microarrays are used to identify the global binding locations of the majority of known yeast transcription factors and use the resulting data along with data from mRNA expression array analysis to define the regulatory networks that exist in yeast. The chapter discusses the protocol to immunoprecipitate sequences, specifically associated with E2F family members, pocket proteins (Rb, p107, and p130), β-catenin, TCF family members, RNA polymerase, Myc family members, histones, and so forth. This protocol is applicable to a wide variety of different types of DNA-binding factors and it provides a good starting point for most transcription factors. The identification of positive clones is a multistep procedure that first require normalization of the two arrays to be compared (that is, the one hybridized with chromatin immunoprecipitated with an antibody to a DNA-binding protein and the one hybridized with the no-antibody or preimmune control), followed by clone selection, confirmation, and ultimately, functional analysis.

Immunogold analysis of antioxidant enzymes in human renal cell carcinoma

Analysis of activities of the antioxidant enzyme manganese superoxide dismutase in human renal cell carcinomas often showed greatly altered enzyme levels (either elevated or depressed) compared to the cell of origin, the kidney proximal tubule. In order to better understand the variability observed, immunogold studies were performed on human renal cell carcinomas using a polyclonal antibody to human kidney manganese superoxide dismutase. For comparison, studies were also performed using antibodies to other antioxidant enzymes. For histologic studies, renal cell carcinomas were subclassified on the basis of light microscopy and ultrastructural analysis into clear cell, granular cell, or mixed clear and granular cell variants. In all three types of tumor, immunogold studies showed little staining using antibodies to copper, zinc superoxide dismutase or glutathione-dependent enzymes. However, intensity of labeling for manganese superoxide dismutase and catalase depended on the cell type(s) in the tumor. Clear cell variants demonstrated trace staining for manganese superoxide dismutase and catalase, while granular cell variants exhibited heavy staining for both of these enzymes. Mixed types of tumors showed clear cells with trace staining for all antioxidant enzymes examined, while granular cells again showed intense labeling for manganese superoxide dismutase and catalse. Using normal kidney proximal tubule as a comparison, immunogold ultrastructural analysis using antibody to manganese superoxide dismutase demonstrated infrequent small lightly labeled mitochondria in clear cell variants, while granular cell variants exhibited numerous medium-sized heavily labeled mitochondria. These data suggest that: 1) the variability in activity values for manganese superoxide dismutase may be due to heterogeneity of cell types in these tumors and 2) manganese superoxide dismutase immunoreactive protein was elevated in granular cells both because of an increase in number of mitochondria and because the labeling density in mitochondria was increased compared to mitochondria in clear cell types or in normal proximal tubular cells.

Immunohistochemical evaluation of MYC expression in mantle cell lymphoma

To assess the validity and potential clinical utility of evaluating MYC expression by immunohistochemistry (IHC) in mantle cell lymphoma (MCL).

Laboratory testing for cobalamin deficiency in megaloblastic anemia.

Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations. Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalamin and folate status. Current total serum cobalamin measurements are performed in the clinical laboratory with competitive binding luminescence assays, whose results may not always accurately reflect actual cobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocysteine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by themselves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, has been proposed as a replacement for current total cobalamin assays. Although holotranscobalamin measurements appear to have slighter better sensitivity, the specificity of this assay remains to be determined. The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patients with suspected megaloblastic anemia are discussed. Am. J. Hematol. 88:522–526, 2013.

Outcomes of adults and children with primary mediastinal B-cell lymphoma treated with dose-adjusted EPOCH-R

Treatment with dose‐adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA‐EPOCH‐R) has become the standard of care for primary mediastinal B‐cell lymphoma (PMBCL) at many institutions despite limited data in the multi‐centre setting. We report a large, multi‐centre retrospective analysis of children and adults with PMBCL treated with DA‐EPOCH‐R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA‐EPOCH‐R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA‐EPOCH‐R. Radiation therapy was administered in 14·9% of patients. With median follow‐up of 22·6 months, the estimated 3‐year event‐free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3–91·5] and overall survival was 95·4% (95% CI 91·8–99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy‐five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG‐PET) scan at the completion of DA‐EPOCH‐R, defined as Deauville score 1–3. Negative FDG‐PET at end‐of‐therapy was associated with improved EFS (95·4% vs. 54·9%, P < 0·001). Our data support the use of DA‐EPOCH‐R for the treatment of PMBCL in children and adults. Patients with a positive end‐of‐therapy FDG‐PET scan have an inferior outcome.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Infection with this parasite should be included in differential diagnosis of fever and anemia after blood transfusion or organ transplantation.

Polymorphic electrophile response elements in the mouse glutathione S-transferase GSTa1 gene that confer increased induction

Induced transcription of a battery of stress response genes in mammals, including several phase I and phase II drug-metabolizing enzymes, is regulated by the electrophile responsive element (EpRE). Because previous directed mutagenesis of nucleotide motifs within the large, composite EpRE were shown to affect transcription factor binding and associated induced expression of dependent genes, we hypothesized that naturally-occurring variation or polymorphism in the EpRE sequence, if found, could affect the induced expression of important protective genes like glutathione S-transferases, and that this could be an important determinant of cancer risk in humans and other mammals. To determine whether this occurred in nature, 32 strains and species of inbred mice were screened to examine the EpRE sequence present in the mGSTa1 promoter. Two species, Mus caroli and Mus spretus, showed TGAC-->TGGC mutations in the tandem TGAC motif. Inducibility (15-fold) of the variant Mus spretus EpRE sequence in a reporter gene construct in HepG2 cells was significantly increased versus the wild-type EpRE sequence (8-fold). A comparison of mGSTa1-induced expression in the livers of Mus spretus, Mus caroli, and BALB/cJ mice showed the highest level of mGSTa1 mRNA in livers from the Mus spretus and Mus carolimice. This naturally-occurring polymorphism within the EpRE domain is the first mutation with an associated phenotype to be reported within a promoter regulatory element of a drug metabolizing gene.

Clinical Significance of Isolated Myeloperoxidase Expression in Pediatric B-Lymphoblastic Leukemia

Objectives Diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) requires immunophenotypic evidence of B-lineage and absence of specific myeloid or T-lineage markers. Rare cases of otherwise typical B-ALL express myeloperoxidase (MPO) detectable by flow cytometry with an absence of other myeloid markers, but the clinical significance of this finding is not well studied. Methods A retrospective cohort analysis of flow cytometry and clinical data was performed to investigate the clinical outcome of this specific group of patients. Results Twenty-nine cases of otherwise typical B-ALL that expressed MPO by flow cytometry (B-ALL-isoMPO) without expression of other myeloid markers were identified. The B-ALL-isoMPO group had a significantly increased incidence of relapse (univariate log rank P  = .0083; multivariate hazard ratio, 2.50; 95% confidence interval, 1.07-5.85; P  = .034) and significantly worse event-free survival by univariate analysis (log rank P  = .0066) compared with a reference group of patients with B-ALL from the same time period (n = 264). Conclusions To our knowledge, this is the first report to document the clinical outcomes in a group of pediatric patients with B-ALL that expresses MPO in the absence of other myeloid markers. This group had an increased rate of relapse and a worse event-free survival than the patients with B-ALL who did not express MPO.

Value-Based Flow Testing of Chronic Lymphoproliferative Disorders A Quality Improvement Project to Develop an Algorithm to Streamline Testing and Reduce Costs

OBJECTIVES Flow cytometry is essential for the evaluation of lymphoproliferative disorders (LPDs) and their classification. Flow panels routinely incorporate a large array of antibodies, making testing complex and expensive; such panels are likely unnecessary in benign cases or those with straightforward diagnoses. Our aim was to develop a more cost-effective testing strategy based on a retrospective analysis of flow studies for possible LPDs in blood. METHODS We identified LPD frequencies and types, as well as associated results with patient age and absolute lymphocyte count. RESULTS We found that the likelihood of LPDs increased with patient age and absolute lymphocyte count and that CD5-positive LPD was the most common LPD diagnosed in our institution (71% of LPDs). Using these data, we devised flow-testing algorithms with a screening test for patients at low risk of disease and a focus on CD5-positive LPD detection, with reflexing as needed. CONCLUSIONS We project this approach will result in a 40% decrease in antibody utilization.