Marciano Reis

Fli-1, an Ets-Related Transcription Factor, Regulates Erythropoietin-Induced Erythroid Proliferation and Differentiation: Evidence for Direct Transcriptional Repression of the Rb Gene during Differentiation

ABSTRACT Erythropoietin (Epo) is a major regulator of erythropoiesis that alters the survival, proliferation, and differentiation of erythroid progenitor cells. The mechanism by which these events are regulated has not yet been determined. Using HB60, a newly established erythroblastic cell line, we show here that Epo-induced terminal erythroid differentiation is associated with a transient downregulation in the expression of the Ets-related transcription factor Fli-1. Constitutive expression of Fli-1 in HB60 cells, similar to retroviral insertional activation of Fli-1 observed in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia, blocks Epo-induced differentiation while promoting Epo-induced proliferation. These results suggest that Fli-1 modulates the response of erythroid cells to Epo. To understand the mechanism by which Fli-1 regulates erythropoiesis, we searched for downstream target genes whose expression is regulated by this transcription factor. Here we show that the retinoblastoma (Rb) gene, which was previously shown to be involved in the development of mature erythrocytes, contains a Fli-1 consensus binding site within its promoter. Fli-1 binds to this cryptic Ets consensus site within the Rb promoter and transcriptionally represses Rb expression. Both the expression level and the phosphorylation status of Rb are consistent with the response of HB60 cells to Epo-induced terminal differentiation. We suggest that the negative regulation ofRb by Fli-1 could be one of the critical determinants in erythroid progenitor cell differentiation that is specifically deregulated during F-MuLV-induced erythroleukemia.

Recombinant factor VIIa is associated with an improved 24-hour survival without an improvement in inpatient survival in massively transfused civilian trauma patients.

OBJECTIVE: To determine whether recombinant factor VIIa (rFVIIa) is associated with increased survival and/or thromboembolic complications. INTRODUCTION: Uncontrollable hemorrhage is the main cause of early mortality in trauma. rFVIIa has been suggested for the management of refractory hemorrhage. However, there is conflicting evidence about the survival benefit of rFVIIa in trauma. Furthermore, recent reports have raised concerns about increased thromboembolic events with rFVIIa use. METHODS: Consecutive massively transfused (≥ 8 units of red blood cells within 12 h) trauma patients were studied. Data on demographics, injury severity scores, baseline laboratory values and use of rFVIIa were collected. Rate of transfusion in the first 6 h was used as surrogate for bleeding. Study outcomes included 24‐hour and in‐hospital survival, and thromboembolic events. A multivariable logistic regression analysis was used to determine the impact of rFVIIa on 24‐hour and in‐hospital survival. RESULTS: Three‐hundred and twenty‐eight patients were massively transfused. Of these, 72 patients received rFVIIa. As expected, patients administered rFVIIa had a greater degree of shock than the non‐rFVIIa group. Using logistic regression to adjust for predictors of death in the regression analysis, rFVIIa was a significant predictor of 24‐hour survival (odds ratio (OR) =  2.65; confidence interval 1.26–5.59; p = 0.01) but not of in‐hospital survival (OR = 1.63; confidence interval 0.79–3.37; p = 0.19). No differences were seen in clinically relevant thromboembolic events. CONCLUSIONS: Despite being associated with improved 24‐hour survival, rFVIIa is not associated with a late survival to discharge in massively transfused civilian trauma patients.

Flow cytometry in the diagnosis of peritoneal carcinomatosis

OBJECTIVES:Peritoneal carcinomatosis is the second major cause of ascites. Because of its frequency and poor prognosis, it is important to establish an accurate diagnosis. The aim of this study was to analyze the use of a DNA index, detemined by flow cytometry in the differential diagnosis of ascites, and to compare it to the cytopathological examination.METHODS:A prospective analysis was carried out on 67 patients (39 female, 28 male; mean age, 53 ± 14 yr [range, 5–82]) with ascites of various etiologies. Peritoneal carcinomatosis was detected in 21 patients, whereas in 46 the ascites was of noncarcinomatosis origin.RESULTS:The sensitivity of the cytopathological examination for the diagnosis of peritoneal carcinomatosis was 42.9%, and the specificity was 100%. The mean DNA index determined by flow cytometry was similar for peritoneal carcinomatosis and noncarcinomatosis patients, being 1.28 versus 1.01, respectively, in the preparations without control lymphocytes and 1.28 versus 1.04, respectively, when control lymphocytes were added. The sensitivity of DNA index cytometry was 57.1% and specificity, 93.5%. The combined use of the DNA index and cytopathological examination did not show an advantage over the use of any of the tests individually, although the DNA index was able to detect half of the cases of peritoneal carcinomatosis in which cytopathological examination was negative. Although the sensitivity was higher when the parameters were associated, the DNA index did not offer a statistically significant advantage over the use of cytopathological examination alone, which in turn had higher specificity.CONCLUSION:The DNA index presented lower sensitivity for the diagnosis of peritoneal carcinomatosis when used alone, showing no advantage over conventional cytopathological examination. However, the DNA index was able to detect 50.0% of peritoneal carcinomatosis cases whose conventional cytopathological examinations were negative, and could be valuable in these situations.

Imaging genetics in multiple sclerosis: A volumetric and diffusion tensor MRI study of APOE ε4

Evidence linking the ε4 allele of APOE to more severe brain MRI abnormalities in multiple sclerosis (MS) has been conflicting and limited to studies of lesion load and whole brain atrophy. The purpose of the present study was to determine whether the ε4 allele of APOE is associated with more extensive brain pathology in MS using structural and diffusion tensor MRI. Using a case-control design, 43 MS patients with the ε4 allele and 47 ε4 negative MS patients underwent structural and diffusion tensor imaging (DTI) at 3T. Hypo- and hyperintense lesion volumes, whole brain and medial temporal volumes, and DTI parameters (fractional anisotropy (FA) and mean diffusivity (MD)) in normal-appearing brain tissue and lesions were compared between the groups. ε4+ and ε4- MS patients were well-matched on demographic characteristics, disease variables, and proportions receiving disease-modifying therapy. ε4+ and ε4- patients did not differ on any MRI or DTI measure. This study refutes a role for the ε4 allele in MRI abnormalities in MS, particularly those linking ε4 to greater T1 hypointense lesion volume and brain atrophy. Previous work on this putative gene-MRI relationship is extended by comparing DTI measures within lesions and normal-appearing brain tissue. A lack of differences in medial temporal regions, areas that have been linked to ε4-associated changes in health and disease, further supports the conclusion that that ε4 is not associated with more subtle MRI markers of brain pathology in MS.

Clinical Utility of Flow Cytometry in the Study of Erythropoiesis and Nonclonal Red Cell Disorders

Erythropoiesis involves proliferation and differentiation of small population of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of diseases and monitoring of therapy. Flow cytometric analysis is increasingly being used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. Here we discuss the role of flow cytometry in the study of erythropoiesis and nonclonal red blood cell disorders. First, we discuss flow cytometric analysis of reticulocytes. Next, we review salient quantitative methods that can be used for detection of fetal-maternal hemorrhage (FMH). We also discuss flow cytometric analysis of high hemoglobin F (HbF) in Sickle Cell Disease (SCD), hereditary spherocytosis (HS), red cell survival and red cell volume. We conclude by discussing cell cycle of erythroid cells.

Management of newly diagnosed high-risk and intermediate-risk follicular lymphoma with 90Y ibritumomab tiuxetan in a phase II study

Five‐year overall survival for high‐risk Follicular Lymphoma International Prognostic Index follicular lymphoma is only approximately 50% compared with 90% for low risk. To evaluate an approach to improve upon this poor outcome, we completed an exploratory phase II trial of intensified treatment for patients with intermediate and high‐risk follicular lymphoma. Front‐line treatment with chemo‐immunotherapy consisting of rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone was followed by radio‐ immunotherapy with 90‐Yttrium ibritumomab tiuxetan consolidation, and 2 years of rituximab maintenance. The 5‐year overall survival for intermediate and high‐risk patients was 88% and 83%, respectively. Of 33 enrolled patients, 3 were off study before receiving radio‐immunotherapy. Three months post radio‐immunotherapy, 28/33 (85%) patients had achieved complete response including 6 patients who had only a partial response to chemo‐immunotherapy and converted to complete response after radio‐immunotherapy. The 5‐year progression‐free survival for intermediate and high risk was 79% and 58%, respectively. Nine of 19 patients with molecular markers patients remain in molecular and clinical complete remission with a median follow‐up of 48 months (range 3‐84 months). Post radio‐immunotherapy, hematologic toxicities were mostly grade 1 and 2. However, asymptomatic grade 3 or 4 thrombocytopenia and neutropenia occurred in 11%‐36% and 10%‐24% of patients, respectively. Myelodysplastic syndrome occurred in 1 patient 4 years post treatment. Whereas many patients had prolonged B‐cell reduction and low immunoglobulin levels post treatment, previous immunities to rubella were maintained. More aggressive upfront approaches such as this may benefit higher risk follicular lymphoma, but confirmatory trials are required. http://www.clinicaltrials.gov: NCT01446562.

PROLONGED MOLECULAR AND CLINICAL REMISSIONS IN FOLLICULAR LYMPHOMA PATIENTS TREATED WITH HDT/ASCT AND COMBINATION IMMUNOTHERAPY WITH RITUXIMAB AND INTERFERON α

underwent 1 line of therapy prior to R‐DHAP/Ox, 11 (22.9%) underwent 2 lines of therapy, and 3 (6.3%) had 3 or more lines before R‐DHAP/Ox. The median TTNT from first‐line was 14.3 months (range 10.6‐19.4). With a median follow‐up of 66 months, the 5‐year OS was 73.1% (95% CI, 61‐85%). Among the 34 patients that underwent ASCT, the 5‐year OS was 84% (95% CI, 72‐98%). Time to next treatment (TTNT) after DHAP/Ox was 30.9 months (95% CI, 18‐47%). Stem cell collection failure was reported in 6 cases (12.5%). Four cases (8.3%) of transformation to DLBCL and 3 (6.2%) secondary malignancies (2 myeloid disorders, 1 bladder carcinoma) were reported. Conclusions: In this population of high‐risk, early relapsing FL, R‐ DHAP/Ox +/− ASCT was found to be active, with aTTNT significantly longer than that experienced following first‐line therapy. This population had very similar FLIPI risk scores compared to the LymphoCare study population but appears to have experienced better survival outcomes. Late events continue to occur, and cure appears unlikely. Prospective studies of R‐DHAP/Ox +/− ASCT are warranted in this high‐risk FL population.