Expression of CD47 antigen in Reed-Sternberg cells as a new potential biomarker for classical Hodgkin lymphoma.

INTRODUCTION: CD47 over expression has been reported in several tumor subtypes. CD47 interacts with SIRPalpha on macrophages inhibiting phagocytic signal, providing a survival advantage to tumor. CD47, therefore, represents a valuable target for immunotherapy and is currently under clinical investigation. We aimed to study CD47 expression in Hodgkin Reed Sternberg cells (HRS). METHODS: We tested a polyclonal CD47 antibody (LifeSpan Biosciences, Seattle, WA) expression along with classical HRS cell markers on a tissue array of 16 classical Hodgkin Lymphoma (CHL) tumor biopsies obtained from newly diagnosed, non-selected patients (8 Female, 8 Male patients) in our institution from October 2016 to January 2018. Histologic subtypes were nodular sclerosis in 11 cases, mixed Cellularity in 3 cases and lymphocyte rich in 2 additional cases. Median age was 53 years (Range: 8, 74). Early stage disease was found in three patients without unfavorable prognostic factors according to EORTC and GHSG criteria, one patient with unfavorable prognostic factors and nine patients had advanced disease. Bulk disease was present in one patient. Normal lymphoid tissue and normal prostate epithelium were used as normal controls as recommended by manufacturer. Approval from the Local Ethical committee was obtained before any analysis. RESULTS: CD47 was overexpressed on all HRS cells with a characteristic dot-like pattern in 13/13 cases of CHL. HRS clearly expressed CD47 more intensely than infiltrating T and stromal cells. DISCUSSION: We propose that HRS cells, by up-regulating CD47, might avoid innate immunity check on tumor growth, which could be circumvented using blocking monoclonal antibodies.

Flow cytometry diagnosis in myelodysplastic syndrome: Current practice in Latin America and comparison with other regions of the world.

BACKGROUND: Flow cytometry (FC) is a valuable tool for the diagnosis of myelodysplastic syndromes (MDS). We present results of a survey carried out to evaluate FC current practice for MDS diagnosis in Latin America (LA), focusing on markers used and characteristics of the clinical diagnostic report. Compliance to IMDSflow recommendations was also evaluated. These practices were then compared with those used in other countries. METHODS: An online survey was sent through the Grupo Latino-Americano de Mielodisplasia to LA cytometrists and other international scientific societies. RESULTS: 91 responses from 15 LA countries were received. The median of the number of markers used was 20 ± 4.5, but only 8.1% of participants adopted the complete panel proposed by the International/European LeukemiaNet Working Group (IMDSflow). We received 140 eligible answers from regions other than LA (66 Europe, 59 USA-Canada, 8 Oceania, 6 Asia and 1 Africa). LA utilized more markers for MDS diagnosis than USA/Canada (p = 0.006), but similar to Europe. The use of MDS scoring systems differed among regions: 10.3% in LA, 0% USA/Canada and 25.7% Europe reported the "Ogata score". Finally, 52.0% of all participants included a general interpretation statement in the final report about the consistency of the FC results with MDS diagnosis, with no statistical differences between regions. CONCLUSIONS: This survey shows a low compliance with the IMDSflow recommendations and a scarce use of the scoring systems proposed in the literature. However, the number of surface markers used is high. We will work to develop a FC consensus for MDS diagnosis adapted to the clinical practice requirements in LA.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesis.

Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. Here, we investigated the clonal vs reactive nature of residual maturing bone marrow cells in 59 newly diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

The synergy of panobinostat plus doxorubicin in acute myeloid leukemia suggests a role for HDAC inhibitors in the control of DNA repair.

Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of myeloid blasts in the bone marrow. Here, we report the effects of the novel histone deacetylase inhibitor panobinostat (LBH589) in combination with doxorubicin on AML cells. Panobinostat exhibited potent anti-AML activity in all AML cell lines tested and in primary AML cells from patients (IC(50)<20 nM). In addition, panobinostat potentiated the action of several standard-of-care anti-AML compounds, particularly, doxorubicin. The molecular effects induced by panobinostat and doxorubicin treatment were investigated by analyzing gene expression, cell cycle, apoptosis and signaling pathways. Analyses of gene expression profiles identified 588 genes whose expression was exclusively affected by the combination of panobinostat and doxorubicin. The combination induced AML cell death by an increase in the mitochondrial outer membrane permeability and release of cytochrome c from the mitochondria, resulting in caspase-dependent apoptosis and accompanied by the upregulation of Bax, Bak and, particularly, Bad. The drug combination provoked a strong activation of a DNA damage response, indicating that this combination may trigger cell death by a mechanism that induced DNA double-strand breaks. These data indicate that the combination of panobinostat and doxorubicin may be an effective therapy for the treatment of AML.

An individualized pre-operative blood saving protocol can increase pre-operative haemoglobin levels and reduce the need for transfusion in elective total hip or knee arthroplasty.

We have prospectively evaluated the efficacy of an individualized pre-operative blood saving protocol in elective total hip arthroplasty (THA) or total knee arthroplasty (TKA). The primary aim was to obtain a pre-operative haemoglobin (Hb) level of > or =14 g dL(-1). A reduction in requirements for allogeneic transfusion was considered the second aim. Several strategies are available for increasing pre-operative Hb levels and reducing red blood cell (RBC) transfusions following THA or TKA, but the success of these programmes depends on selecting the most appropriate treatment for each patient. Three hundred and five patients with an indication of elective THA or TKA were individually assigned to the following strategies according to Hb and ferritin levels and medical conditions: (a) no pre-operative intervention, (b) oral iron therapy, (c) intravenous (i.v.) iron therapy, (d) recombinant human erythropoietin alpha with i.v. iron and (e) pre-operative autologous donation (PAD) plus oral iron. Eighty-two percent of the patients reached a pre-operative Hb level of > or =14 g dL(-1) compared with 62% of patients with Hb levels of > or =14 g dL(-1) at the baseline visit. Treatment with PAD showed a significant reduction in the pre-operative Hb levels. The rate of RBC transfusion was 18.8% compared with 31.5% of matched historic group (P < 0.001). In conclusion, all patients scheduled to undergo THA or TKA should be candidates for an individualized pre-operative blood salvage programme.

Optimización del uso de Agentes estimulantes del Receptor de la Eritropoyetina (AREs) en pacientes con neoplasias hematológicas basado en el nivel de hemoglobina reticulocitaria / Optimizing the use of Erythropoietin-Receptor stimulating Agents (ERAs) in patients with hematopoietic malignancies based on the level of reticulocyte hemoglobin

Los agentes estimulantes del Receptor de la Eritropoyetina (AREs) se usan en el tratamiento de la anemia de hemopatías malignas (HM). Sin embargo, la tasa de respuestas es variable por diversos factores como el déficit funcional de hierro (DFF). El nivel de hemoglobina (Hb) reticulocitaria es un parámetro fácil de obtener que ha mostrado su utilidad en el diagnóstico del DFF. El objetivo de este estudio fue identificar qué pacientes con HM tratados con AREs presentan DFF y si la Hb reticulocitaria predice la respuesta hemoglobínica en estos enfermos. Se incluyeron 42 pacientes con diagnostico de Mieloma Múltiple (n=17), Linfoma no Hodgkin (n=14), Linfoma de Hodgkin (n=3), Leucemia Linfática Crónica (n=4), Síndrome Mielodisplásico (n=2), Leucemia Linfoblástica Aguda (n=1) y Leucemia Mieloblástica Aguda (n=1). Veinticinco fueron tratados con Epoetina-beta, diez y seis con Darbepoetina y uno con Epoetina alfa.La respuesta fue favorable en el 28%, 53% y 58% de lospacientes a las 3, 6 y 12 semanas de tratamiento, respectivamente. Se detectó DFF en el 17% de los pacientes. En cuanto a la respuesta, no hubo diferencias estadísticamente significativas entre los pacientes con y sin DFF, si bien, a mitad de tratamiento, fue ligeramente superior en el grupo sin DFF (57% vs 43%, p>0.05). El nivel basal de Hb reticulocitaria se correlacionó con el grado de respuesta global a las 12 semanas de finalizar el tratamiento. Así, el 79% de los pacientes respondedores tenían una Hb reticulocitaria inicial >36·5 pg, mientras que este porcentaje bajó al 30% en los no respondedores (p = 0.024)En resumen, la Hb reticulocitaria es un método sensible ypreciso para detectar DFF en pacientes con HM bajo tratamiento con AREs. Además, un nivel elevado de Hb reticulocitaria basal es un parámetro que se asocia con respuesta favorable al tratamiento

The Erythropoietin-Receptor stimulating Agents (ERAs) areindicated in the supportive treatment of the anemia in hematological malignancies (HM). However, the response rate is variable due to factors such as the functional iron deficiency (FID). The level of the reticulocyte hemoglobin (RHb) is an easy-to-obtain parameter very useful for the diagnostic of the FID.The purpose of this study was to evaluate which patientswith HM treated with ERAs present FID. In addition, wetried to asses if the level of RH predicts the response in these patients.We included 42 patients with the following diagnostics:Multiple Myeloma (n= 17), Non Hodgkin Lymphoma(n=14), Hodgkin Lymphoma (n=3), Chronic lymphocyticLeukemia (n=4), Myelodisplastic syndrome (n=2), Acutelymphoblastic Leukemia (n=1), and Acute mieloblasticLeuKemia (n=1). Twenty five of them were treated withEpoetin beta, sixteen with darbepoetin alfa and one withEpoetin alfa at standard doses. The response was favorable in 28%, 53% and 58% of patients at the third, sixth and twelfth week of the treatment, respectively. FID was detected in 17% of the patients. Theresponse rate was not statistical significant different between patients with and without FID, although it was slightly superior in the group without FID (57% vs. 43%, p>0.05). Seventy nine percent of patients with a favorable response at twelve weeks showed an initial RH >36·5 pg, while this percentage was only 30% in patients who did not respond (p=0.024).In conclusion, RH is a sensitive and specific method to detect FID in patients with HM who are treated with ERAs.Furthermore, the high level of RH at the baseline is a predictor of favorable response of treatment, in these patients