Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring 13C incorporation from 1-13C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, 13C-citrate, 13C-malate, and 13C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5 mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30–50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated 13C-accumulation into citrate and malate increased 1.5–2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. 13C–2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal 12C–2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and 13C–2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.

La variabilidad clínica y la incertidumbre profesional en la prevención y tratamiento de las úlceras venosas de la extremidad inferior (UV) traen, como principal consecuencia, el hecho de que los pacientes puedan verse sometidos a pruebas diagnósticas y terapéuticas, a veces de dudosa utilidad, o incluso que estas puedan resultar nocivas para la salud del paciente o que, en otras ocasiones, puedan llegar a omitirse determinados procedimientos o procesos que sí podrían resultar adecuados a la situación y necesidades del paciente.Es por ello que surgen una serie de documentos específicos denominados guías de práctica clínica para el abordaje de las UV (GPC-UV), con la finalidad de mejorar la efectividad y la calidad de los cuidados, disminuir la variabilidad injustificada y establecer criterios homogéneos para su manejo. Sin embargo, la literatura recoge que no todas las GPC tienen el mismo criterio metodológico y de elaboración de la evidencia, por lo que muchas de ellas son de escasa calidad científica y rigor editorial. Esto implica que las GPC deben ser revisadas y actualizadas periódicamente en función de la evidencia más actual, y su calidad contrastada con instrumentos validados como el AGREE-II.Tras un análisis de la calidad de 6 GPC-UV disponibles en la actualidad, se ha podido identificar qué guías son recomendables para su implementación en la práctica asistencial y cuáles deberían modificarse para mejorar su aplicabilidad y desarrollo de las evidencias.The clinical variability and professional uncertainty in the prevention and treatment of lower extremity venous ulcers (VU) has as a main consequence, the fact that patients can be subjected to diagnostic and therapeutic tests, sometimes of dubious utility, these may even be harmful to the health of the patient and that, at other times, certain procedures or processes that may be appropriate to the patient's situation and needs may be omitted.It is for this reason that a series of specific documents called clinical practice guidelines for the approach of VU (CPG-VU) have been created, with the aim of improving the effectiveness and quality of care, reducing unjustified variability and establishing homogeneous criteria for its handling. Nevertheless, the literature shows that not all CPGs have the same methodological and evidence-drawing criteria. Many of them are of poor scientific quality and editorial rigor. This implies that CPGs should be periodically reviewed and updated based on the most current evidence and their quality contrasted with validated instruments such as AGREE-II.After an analysis of the quality of six CPG-VU available today, it has been possible to identify what guidelines are recommended for its implementation in the practice of care, which should be modified to improve their applicability and development of the evidence.

22006 Background: Although gene amplification is the mechanism of HER2 over-expression in most breast carcinomas, discordance does occur between results of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Testing algorithms reflex screening IHC 2+/equivocal cases to FISH resulting in delays in reporting of HER2 status. Furthermore, 0 or 1+ IHC staining in overtly HER2 gene amplified cases is well-documented, as are cases that are not amplified for HER2 but over-express the encoded protein. We assessed the analytical performance of a combined immunogenotypic bright field assay for determination of HER2 status. Methods: 53 invasive ductal carcinomas of the breast for which HER FISH results as assessed by the HER2/CEP17 PathVysion assay (Abbott Molecular), and HER2/CHR17 status as assessed by Silver In Situ Hybridization (SISH) (Ventana), were evaluated using a novel immunogenotypic assay. This approach utilized HER2 antibody clone 4B5 IHC with alkaline phosphatase fast red K for detection of protein over-expression (red) and SISH (metallic silver; black) to simultaneously evaluate the expression and copy number of the HER2 gene. SISHPro slides were scored independently without knowledge of historical FISH, SISH, or IHC results using conventional bright field microscopy. Results: Overall concordance of the SISH component of SISHPro with FISH was excellent (94%). Three cases (6%) of gene/protein discordance attributable to immunogenotypic heterogeneity were readily identified on a cell by cell basis using SISHPro. Conclusions: Concomitant immunogenotyping for HER2 status was readily accomplished using a bright field assay that simultaneously assessed gene copy number by SISH and HER2 protein expression by IHC using peroxidase and alkaline phosphatase systems respectively. This approach allows more efficient utilization of limited needle biopsy material and provides immediate internal reinforcement between immunotype and genotype for HER2 testing. Gene/protein discordances, and genotypic and phenotypic heterogeneity, are readily identified for use in future outcome studies that will determine the clinical significance of discordant gene and protein status in breast carcinoma. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Ventana Medical Systems International, Ventana Medical Systems, Inc. Ventana Medical Systems International, Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems International, Ventana Medical Systems, Inc.

11080 Background: HER2 dual color FISH is a ‘gold standard’ quantitative assay for selecting breast cancer patients for trastuzumab therapy. Current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists. Also, a specialized fluorescence microscope with oil immersion objectives in a dark room is necessary for the analysis and FISH slides are difficult to archive. Our objectives were: 1) to develop an automated BDISH application for HER2 and CEN17 that produces permanent slides and 2) to compare the assay performance with HER2 FISH tested breast carcinomas cases. Methods: BDISH assay was developed with a nick translated HER2 DNA probe and a CEN17 oligoprobe on an automated slide stainer. Detection of HER2 and CEN17 signals was accomplished with the silver, hydroquinone, and H2O2 reaction and the fast red and naphthol phosphate reaction, respectively. BDISH specificity was optimized with xenograft tumors [MCF7 (non-amplified HER2) and BT-474 (amplified HER2)]. BDISH performance was evaluated with 237 routinely processed breast carcinoma cases. Interpretation was conducted with a conventional brightfield microscope without oil immersion objectives. Data were analyzed for the concordance between BDISH and FISH scores for HER2 gene status assessment. Results: Specific signals for single HER2 copies were visualized as discrete black dots while single CEN17 copies were detected as slightly larger red dots in the same cells of xenograft tumor sections. Overall concordance was 93% between BDISH and FISH of breast carcinoma cases. Interobserver concordance was 97%. Investigation for the discordant cases with BDISH slides revealed that the heterogeneity of tumor cell populations within the same tissue section was a significant reason for the discordance. Conclusions: We successfully developed an automated BDISH assay that is specific for HER2 and CEN17 targets. Our study demonstrated a high concordance between observers interpreting BDISH results of clinical breast carcinoma cases. The majority of BDISH discordance with FISH was due to the tumor cell heterogeneity in the tumor sample. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc.

Summary.CD38 expression on chronic lymphocytic leukaemia (CLL) cells is a poor prognostic factor, however, methods for measuring this vary. The QuantiBRITETMflow cytometry (FC) system yields an absolute antigen expression value (antibodies bound/cell, ABC) and may be useful in standardizing CD38 expression analysis. We evaluated cryopreserved pretreatment CLL cells for CD20 ABC, CD38 ABC, and percentage of CD38+B cells from 131 patients requiring therapy. The 92 patients (70%) with ≥ 100 CD38 ABC had worse overall survival (OS; 34% at 5 years) compared with those with < 100 CD38 ABC (70% at 5 years, mortality hazard ratio 2·30, 95% confidence interval 1·28–4·12; two‐tailedP = 0·003). Among the 64 patients with < 30% CD38+cells, OS of the 25 with ≥ 100 ABC was worse than that of the 39 with < 100 ABC (P = 0·018). OS of patients with < 30% CD38+cells and ≥ 100 ABC was actually similar to that of patients with ≥ 30% CD38+cells.BrightCD20 expression (≥ 20 000 ABC) was not associated with a worse OS (P = 0·10). The presence of ≥ 100 CD38 ABC in CLL patients requiring therapy is an unfavourable prognostic factor for OS and quantitative FC may be superior to percentage CD38+cell assessment. Prospective trials are required to determine more precisely the prognostic significance of absolute expression levels in fresh CLL cells.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)