Leucocytosis during induction therapy with all-trans-retinoic acid and arsenic trioxide in acute promyelocytic leukaemia predicts differentiation syndrome and treatment-related complications.

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) represent the standard of care for low-intermediate risk acute promyelocytic leukaemia (APL). Leucocytosis during induction with ATRA-ATO represents a common complication with an incidence of up to 60%. To identify predictive factors for this complication, we studied a cohort of 65 low-intermediate risk APL patients treated with ATRA-ATO in three highly specialized Italian centres. Overall, 39/65 (60%) patients developed leucocytosis, with a peak in leucocyte count being most frequent in the second week from diagnosis. All cases were successfully managed with hydroxyurea. Predictive factors for leucocytosis in univariate analysis were lower platelet counts (odds ratio [OR] 0.98, 0.97-1.00, p = 0.018), lower fibrinogen levels (OR 0.36, 0.17-0.66, p = 0.003), higher bone marrow blast infiltration (OR 1.03, 1.01-1.07, p = 0.021) and CD117 expression by flow (OR 1.04, 1.01-1.08, p = 0.012). Multivariate analysis confirmed lower levels of fibrinogen at diagnosis as the strongest predictive factor for the development of leucocytosis (OR 0.36, 0.15-0.72, p = 0.009). Differentiation syndrome (DS) occurred only in patients developing leucocytosis showing a strict correlation with rising leucocytes counts (16/39 vs. 0/26, p < 0.001). In addition, other treatment-related complications including QTc prolongation, cardiac events, liver, and haematological toxicities were significantly more frequent in patients experiencing leucocytosis (22/39 vs. 3/26, p < 0.001). In conclusion, APL patients undergoing ATRA-ATO therapy with lower fibrinogen levels and platelet counts at diagnosis and with a massive bone marrow blast infiltrate should be carefully monitored for the development of leucocytosis during induction. DS and other treatment-related complications seem to occur almost exclusively in patients developing leucocytosis, who should necessarily receive DS prophylaxis and more intensive monitoring and supportive therapy to prevent treatment complications.

Systemic, Mucosal Immune Activation and Psycho-Sexual Health in ART-Suppressed Women Living with HIV: Evaluating Biomarkers and Environmental Stimuli.

Gender medicine is now an approach that can no longer be neglected and must be considered in scientific research. We investigated the systemic and mucosal immune response in a population of women living with HIV (WLWH) who were receiving successful ART and the sexual and psychological repercussions of HIV infection on the women's health. As control group, healthy women (HW) matched for age and sex distribution, without any therapy, were included. In summary, our study highlighted the persistence of immune-inflammatory activation in our population, despite virological suppression and a normal CD4 cell count. We found a hyperactivation of the systemic monocyte and an increase in inflammatory cytokine concentrations at the systemic level. The analysis carried out showed a significantly higher risk of HPV coinfection in WLWH compared to HW. Furthermore, our data revealed that WLWH have a profile compatible with sexual dysfunction and generalized anxiety disorders. Our study underlines that patients living with HIV should be evaluated by multidisciplinary teams. These findings also support the idea that more and different immunological markers, in addition to those already used in clinical practice, are needed. Further studies should be carried out to clarify which of these could represent future therapy targets.

Consensus for Flow Cytometry Clinical Report on Multiple Myeloma: A Multicenter Harmonization Process Merging Laboratory Experience and Clinical Needs.

Flow cytometry is a highly sensitive and specific approach for discriminating between normal and clonal plasma cells in multiple myeloma. Uniform response criteria after treatment have been established by the International Myeloma Working Group and the EuroFlow Group; however, the way in which flow cytometry data are reported has suffered from no collaborative or multicentre efforts. This study, involving 8 expert laboratories and 12 clinical hematology units of the Lazio region in Italy, aims to produce a uniform and shared report among the various Centres. From the pre-analytical phase to sample processing, data acquisition, analysis, and evaluation of the potential limitations and pitfalls of the entire process, the study reaches a final conclusion shared by laboratories and clinicians according to the most updated principles and recommendations. The aim was to identify the necessary data to be included in the clinical report by using multiple-choice questionnaires at every single stage of the process. An agreement of more than 75% of the laboratories was considered mandatory for the data to be included in the report. By ensuring the operational autonomy of each laboratory, this study provides a clear report that limits subjective interpretations and highlights possible bias in the process, better supporting clinical decision-making.

Impact of gemtuzumab ozogamicin consolidation on hematopoietic stem cells (HSCs) mobilization in AML: analysis of 20 patients.

Gemtuzumab ozogamicin (GO), is an anti-CD33 monoclonal antibody, approved for AML CD33 + , those patients with low and intermediate-risk who obtain a complete response may also be candidated for consolidation with autologous stem cell transplantation (ASCT). However, there are scant data on the mobilization of hemopoietic stem cells (HSC) after fractionated GO. We retrospectively studied data from five Italian centers and identified 20 patients (median age 54 years, range 29-69, 15 female, 15 NPM1mutated) that attempted HSC mobilization after fractionated doses of GO + "7 + 3" regimen and 1-2 cycles of consolidation (GO + HDAC + daunorubicin). After chemotherapy and standard G-CSF, 11/20 patients (55%) reached the threshold of 20 CD34 + /µL, and HSC were successfully harvested, while 9 patients (45%) failed. The median day of apheresis was Day + 26 from the start of chemotherapy (range 22-39 days). In good mobilizer patients, the median circulating CD34 + cells were 35.9 cells/µL and the median CD34 + harvested were 4.65 × 106/kg of patients' body weight. With a median follow-up of 12.7 months, at 24 months from the first diagnosis, 93.3% of all 20 patients were alive and the median overall survival was 25 months. The 2-year RFS rate from the timepoint of the first CR was 72.6%, while the median RFS was not reached. However, only five patients underwent ASCT and achieved full engraftment.In conclusion, in our cohort of patients, the addition of GO reduced HSC mobilization and harvesting, which was reached in about 55% of patients. Nevertheless, further studies are warranted to evaluate the effects of fractionated doses of GO on HSC mobilization and ASCT outcomes.

A Relapsing Meningeal Acute Myeloid Leukaemia FLT3-ITD+ Responding to Gilteritinib.

A patient with a therapy-related acute myeloid leukaemia (AML), NPM1mut, and FLT3-ITD+ was treated with induction and consolidation with CPX-351, obtaining a complete response (CR) but minimal residual disease persisted positive. Later, she complained progressive burning leg pain, weakening of the right hand and leg muscles, associated with absence of osteotendinous leg reflexes. Examination of cerebrospinal fluid (CSF) showed a meningeal relapse of AML. Moreover, a magnetic resonance imaging (MRI) showed 2 right meningeal implants of myeloid sarcoma and bone marrow revealed haematologic relapse of disease. She was treated with medicated lumbar punctures (LPs) followed by an FLA-Ida scheme, and she achieved a 2nd CR. Unfortunately, the patient developed hyperleucocytosis and reappearance of meningeal myeloid sarcoma at MRI. For this reason, a monotherapy with gilteritinib (an FLT3 inhibitor) was started: after 3 months of therapy, central nervous system (CNS)-disease shrunken and then faded, while AML in the bone marrow achieved only a partial response. This is the 1st report of a positive biological effect of gilteritinib on CNS (meningeal) myeloid sarcoma. There are no studies of gilteritinib concentration into CSF and penetration of gilteritinib into the blood-brain barrier should be further studied, given the paucity of drugs active on CNS relapse of AML. In patients receiving CPX-351 only, diagnostic LP should be considered after induction.

A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant.

BACKGROUND: In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. STUDY DESIGN AND METHODS: This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay." RESULTS: The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%. CONCLUSION: The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory.

Optimization of the immunomagnetic selection in microcythemic donors enrolled for haploidentical transplantation.

BACKGROUND: Immunomagnetic cell selection (ICS) cells is increasingly used in allogeneic hematopoietic transplantation in order to reduce the T cells quantity. The aim of this study was to evaluate an protocol based on Ficoll method before ICS. STUDY DESIGN AND METHODS: The automated procedure was compared with the standard method. In the group 1 the cell processing involves the extraction of the buffy-coat by Ficoll before incubation with antibodies. This procedure was performed with the Sepax S-100 device. The efficacy of this automated procedure was compared with the group 2. In this group, the cell washing and the incubation were performed through the standard method. The CD34+ cells collected by apheresis (HPC-A) were selected with ICS. RESULTS: The results obtained after Ficoll procedure, showed a total nucleated cells (TNCs) and CD34+ cells recovery of 85.73% (75.90-90.63; SD 4.25) and 79.31% (51.77-112.31; SD 18.40), respectively. The TNC and CD34+ cells recovery after the pre-incubation washing performed through the standard method, was 75.54% (38.36-97.76; SD 22.5) and 61.51% (30.87-81.79; SD 19.3), respectively. The CD34+ cells recovery after ICS was 79% (51.77-100; SD 18.40) and 44% (15.57-88.24; SD 25.91) in the group 1 and the group 2, respectively. This difference was statistically significant (p=0.001). CONCLUSION: The efficacy of the ICS which resulted to be higher in the group 1 compared to the group 2. Overall, our data suggest that the Ficoll procedure before incubation is suitable for the clinical routine in the ICS for haploidentical transplantation in patients affected by thalassemia.

Cell processing for haplo-identical hematopoietic stem cell transplantation: automated washing and immunomagnetic-positive selection.

BACKGROUND AIMS: Immunomagnetic cell selection (ICS) of CD34(+) cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection. METHODS: The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method. RESULTS: The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34(+) cell recovery of 84.87% (71.80-105, SD 8.62; range, standard deviation) and 83.45% (47-109, SD 16.12), respectively. The NC and CD34(+) cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36-97.76, SD 22.5) and 61.51% (30.87-81.79, SD 19.3), respectively. The CD34(+) cell recovery after ICS was 51.27% (13.77-98.82, SD 24.97) and 48.89% (15.57-88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4-96.10, SD 13.07) and in group 2 84.97% (58.1-97.8, SD 15.58). CONCLUSIONS: The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML.

Evaluation of cell death after treatment with extracorporeal photopheresis.

The aim of our study is to assess the mortality of leukocytes during extracorporeal photopheresis. Sixty-three photopheresis performed on 13 patients affected by chronic GvHD were evaluated. Samples were analyzed using a FACSCalibur flow cytometer. Apoptosis and necrosis of limphomononuclear cells dramatically increased after the apheretic procedure. We found a further increase of apoptotic and necrotic limphomononuclear cells after treatment with 8-MOP and UVA (p≤0.05). Our data suggested that the immunomodulatory effects of extracorporeal photopheresis, triggered by circulating apoptotic or necrotic cells, could play an important role in the treatment of GvHD with this procedure.

Transplantation in the onco-hematology field: focus on the manipulation of αß and γδ T cells.

γ/δ T cells represent a subset of T cells expressing a T cell receptor (TCR) variant composed of gamma and delta chains. The γ/δ TCR is expressed by 2-10% of all T cells in human peripheral blood, whereas the majority of T cells express α/ß TCRs. γ/δ T cells display a range of innate effector functions including rapid secretion of chemokines and cytokines, as well as target cell lysis. Recent interest has focused on the function of γ/δ T lymphocytes in allogeneic transplantation in the onco-hematology field. Several studies, in vitro and in vivo, suggest that γ/δ T lymphocytes are potential beneficial effector cells in the context of hematopoietic stem cell transplantation (HSCT). In addition, in this review, we discuss the depletion of α/ß T lymphocytes in the graft for allogeneic transplantation. In fact, an efficient TCR α/ß cell depletion potentially reduces the risk of GvHD. Furthermore, TCR α/ß T cell depletion, especially with immunomagnetic negative selection, retains other potential beneficial effector cells in the graft, such as γ/δ T cells, NK cells, and stem cells. These "facilitating" cells might facilitate engraftment, exert GvL effects, and reduce the risk for infections.

Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system.

BACKGROUND: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. STUDY DESIGN AND METHODS: The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. RESULTS: The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. DISCUSSION: Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

T(reg) cells: collection, processing, storage and clinical use.

T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells.