ALBERT

Description: CD200 is a transmembrane glycoprotein expressed on several tissues in rats and humans. It plays an immunoregulatory function by switching cytokine production from a TH1 to TH2 pattern, thus reducing cytotoxic response while indirectly enabling tumor escape and growth. Interestingly, CD200 is also a target for a novel humanized monoclonal antibody (Anti-CD200 MoAb, Alexion Pharmaceuticals, Cheshire, CT, USA). Anti-CD200 inhibits CD200 binding to its receptor, so modifying cytokine production and improving T-cell mediated cytotoxic response. Expression of CD200 has been already described in chronic lymphocytic leukemia/small lymphocyte lymphoma (CLL/SLL), multiple myeloma (MM) and acute myeloid leukemia (AML). Moreover, CD200 expression is considered an unfavorable prognostic factor in MM and AML. The goal of this work was to study, using flow cytometry, CD200 expression on a large number of onco-hematological samples, with the aim to exactly describe conditions in which anti-CD200 MoAb could be a therapeutic option. Analysis was conduced using a six-color FACSCanto II cytometer (Becton Dickinson, BD, San Jose, CA, USA) equipped with the FACSDiva software (BD). CD200 expression was evaluated by using CD200-PE-conjugated antibody (BD/Pharmingen). During the last year we analyzed CD200 expression in 184 samples: 131 bone marrow aspirates (BM), 33 peripheral blood specimens (PB) and 20 fine needle aspiration cytology samples (FNAC). One hundred and four were lymphoproliferative disorders, 12 MM, 16 myelodysplastic syndromes (MDS) and 52 acute leukemias. CD200 positivity was assigned to every single case when CD200 mean fluorescence intensity (MFI) was higher then 256 arbitrary units, a channel close to the cut-off point between positive and negative cells, in our experience. All results concerning our analysis are showed in the table. Three classes of hematologic neoplasms displayed a constant positivity for CD200 with a high level of MFI: CLL/SLL, hairy cell leukemia (HCL) and B-cell acute lymphoblastic leukemia (B-ALL). Lymphoplasmacytic lymphoma (LPL) was positive in 100% of cases but with lower MFI as compared to CLL/SLL, HCL and B-ALL (p

Description: The 67-kDa laminin receptor (67LR) is a nonintegrin cell-surface receptor with high affinity for laminin, which plays a key role in tumor invasion and metastasis. We investigated the role of 67LR in granulocyte colony-stimulating factor (G-CSF)–induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 35 healthy donors. G-CSF–mobilized HSCs, including CD34+/CD38– cells, showed increased 67LR expression as compared with unstimulated marrow HSCs; noteworthy, also, is the fact that the level of 67LR expression in G-CSF–mobilized HSCs correlated significantly with mobilization efficiency. During G-CSF–induced HSC mobilization, the expression of laminin receptors switched from α6 integrins, which mediated laminin-dependent adhesion of steady-state human marrow HSCs, to 67LR, responsible for G-CSF–mobilized HSC adhesion and migration toward laminin. In vitro G-CSF treatment, alone or combined with exposure to marrow-derived endothelial cells, induced 67LR up-regulation in marrow HSCs; moreover, anti-67LR antibodies significantly inhibited transendothelial migration of G-CSF–stimulated marrow HSCs. Finally, G-CSF–induced mobilization in mice was associated with 67LR up-regulation both in circulating and marrow CD34+ cells, and anti-67LR antibodies significantly reduced HSC mobilization, providing the first in vivo evidence for 67LR involvement in stem-cell egress from bone marrow after G-CSF administration. In conclusion, 67LR up-regulation in G-CSF–mobilized HSCs correlates with their successful mobilization and reflects its increase in marrow HSCs, which contributes to the egress from bone marrow by mediating laminin-dependent cell adhesion and transendothelial migration.

Description: Acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in severe aplastic anemia (SAA), or may selectively involve single lineages, as in pure red cell aplasia (PRCA) or in agranulocytosis (AGR). All these conditions share a cellular immune-mediated pathophysiology, which is supported by many experimental data; thus, various immunosuppressive (IS) strategies have been exploited. Alemtuzumab (MabCampath®) (ALE) is a lympholytic MoAb with strong and prolonged IS activity, more reliable than ATG or ALG as for activity, dosing and commercial availability. Here we report a phase II/III pilot study with ALE followed by low-dose cyclosporine A (CsA) on 11 patients suffering from SAA (n=4), PRCA (n=5) or AGR (n=2). All patients received ALE as s.c. injection premedicated by betamethasone, clorpheniramine and paracetamol, with a dose escalating schedule of 3-10-30-30-(30) mg in consecutive days; the total dose was 103 mg for SAA, and 73 for PRCA and AGR. Six patients received one or more additional courses as a result of relapse, so a total of 18 courses were administered. All patients on day 7 started oral low dose CsA (1 mg/kg). Valgancyclovir 450 mg bi-daily and trimethoprim-sulphamethoxazole bi-daily thrice a week were administered as anti-CMV and anti-Pneumocystis Carinii prophylaxis, respectively. All patients completed the treatment, with severe or moderate infusion-related side effect (fever, chills and/or injection site reaction) occurring in 1 (not premedicated) and 3 cases, respectively. No significant abnormality of routine biochemical testing, nor other medically significant adverse events were reported. A complete lympho-ablation was observed in all patients within 2–3 days, which persisted for several weeks; in addition, transient worsening of neutropenia (managed by occasional G-CSF support) and of thrombocytopenia were observed in some patients. At a median follow-up of 6 months, infectious events were irrelevant: in cumulative 75 patient-months, 1 HSV and 1 flu have been recorded (globally 1 day of fever), all resolving quickly. No CMV reactivation was demonstrated. Immune reconstitution was delayed up to several months, with absolute lymphocyte count ranging between 30–200/uL, 100–400/uL, 250–800/uL and 500–2000/uL at months +1, +3, +6 and +12 from the treatment. The CD4+ compartment was significantly more affected than the CD8+, with a persistent inversion of the CD4/CD8 ratio. As for efficacy, the 4 SAA patients showed 1 CR, 1 PR (both relapsing at 6 months and re-treated with additional ALE courses), 1 NR at 3 months (addressed to early stem cell transplantation due to life-threatening hemorrhages); 1 is not evaluable yet. In the 5 PRCAs, there were 4 CR and 1 NR (at 3 months); 3 responding patients relapsed and were successfully managed by further courses of ALE. The 2 AGRs showed both CR, followed by late relapse (at 18 months) in one case (now receiving a second course). In conclusion, ALE administered as subcutaneous injection is a feasible and safe IS regimen for patients suffering from immune-mediated marrow failure syndromes. Infectious complications were unremarkable, and preliminary results suggest good efficacy, especially in lineage-restricted forms; as with other IS regimens, the hematological response is late (3–4 months) and relapses may occur, which are sensitive to further ALE courses. Such favorable risk-to-benefit ratio predicts for this regimen a leading position in the future IS strategies.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by intravascular hemolysis, often resulting in the need for red blood cell (RBC) transfusions. PNH RBCs lack two complement regulatory molecules - CD59, a terminal complement inhibitor, and CD55, a C3 convertase inhibitor. Eculizumab, a humanized monoclonal antibody that inhibits terminal complement by binding to C5, effectively controls intravascular hemolysis as determined by a dramatic reduction in lactate dehydrogenase (LDH) to levels in or just above the normal range. Control of intravascular hemolysis in these patients led to a reduction in, or cessation of, RBC transfusions. During eculizumab treatment, a majority of patients demonstrate evidence of residual, low-level hemolysis; LDH levels remain slightly elevated, haptoglobin levels are low or undetectable, and bilirubin levels are above normal. We hypothesized that this low-level residual hemolysis may be due to clearance of PNH RBCs through a C3b-mediated mechanism. Therefore we investigated C3 deposition on RBC in PNH patients before and on eculizumab. A direct antiglobulin test (DAT) using monoclonal anti-C3d was positive in 29 out of 39 PNH patients on eculizumab. Of these 29 DAT-positive patients, who were all receiving transfusions, 25 had DAT testing prior to eculizumab therapy and only one of these was positive. DAT was negative in all of 8 normal volunteers. By two-color flow cytometric analysis with anti-CD59 and anti-C3, the majority of patients on eculizumab demonstrated three distinct RBC populations: CD59+/C3− (normal RBCs); CD59-/C3− (PNH RBCs without C3 coating); and CD59-/C3+ (PNH RBCs coated by C3). No CD59+/C3+ RBCs were observed. Of 21 DAT positive eculizumab treated patients tested, the median proportion of total RBCs that were C3b positive was 17.6%. 18 of 29 [62%] eculizumab patients with a positive DAT received at least one transfusion during eculizumab therapy compared with 1 of 10 [10%] for DAT negative patients (p=0.01), although even patients who did not become transfusion independent during eculizumab treatment showed a marked reduction in transfusion requirement. The median hemoglobin value for the 29 DAT positive eculizumab patients was 9.8 g/dL compared with 11.3 g/dL in the 10 DAT negative eculizumab patients (p= 0.08). No apparent relationship between LDH and DAT positivity was observed. It is proposed that resolution of intravascular hemolysis in PNH patients on eculizumab results in deposition of C3b on the surface of PNH RBCs which may explain, at least in part, the residual low level hemolysis occurring in some patients. This appears to be a previously undescribed mechanism of RBC clearance in PNH, most likely obscured by the rapidity of intravascular hemolysis in the absence of eculizumab therapy. Despite the low-level residual hemolysis, patients continue to receive significant benefit from eculizumab treatment.

Description: We investigated the involvement of the urokinase-type plasminogen-activator receptor (uPAR) in granulocyte–colony-stimulating factor (G-CSF)–induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 16 healthy donors. Analysis of peripheral blood mononuclear cells (PBMNCs) showed an increased uPAR expression after G-CSF treatment in CD33+ myeloid and CD14+ monocytic cells, whereas mobilized CD34+ HSCs remained uPAR negative. G-CSF treatment also induced an increase in serum levels of soluble uPAR (suPAR). Cleaved forms of suPAR (c-suPAR) were released in vitro by PBMNCs and were also detected in the serum of G-CSF–treated donors. c-suPAR was able to chemoattract CD34+ KG1 leukemia cells and CD34+ HSCs, as documented by their in vitro migratory response to a chemotactic suPAR-derived peptide (uPAR84-95). uPAR84-95 induced CD34+ KG1 and CD34+ HSC migration by activating the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR). In addition, uPAR84-95 inhibited CD34+ KG1 and CD34+ HSC in vitro migration toward the stromal-derived factor 1 (SDF1), thus suggesting the heterologous desensitization of its receptor, CXCR4. Finally, uPAR84-95 treatment significantly increased the output of clonogenic progenitors from long-term cultures of CD34+ HSCs. Our findings demonstrate that G-CSF–induced upregulation of uPAR on circulating CD33+ and CD14+ cells is associated with increased uPAR shedding, which leads to the appearance of serum c-suPAR. c-suPAR could contribute to the mobilization of HSCs by promoting their FPR-mediated migration and by inducing CXCR4 desensitization.

Description: Acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in aplastic anemia (AA), or may selectively involve single lineages, as in pure red cell (PRCA) or in pure white cell aplasias (PWCA). Because of their common cellular immune-mediated pathophysiology, standard treatment for these conditions includes immunosuppression (IS), which may differ according to the specific disease. We investigated an experimental IS regimen based on the anti-CD52 antibody alemtuzumab (MabCampath®, ALE); the study included a phase II/III prospective trial, as well as a collection of retrospective cases. A total of 32 patients have been treated by ALE (18 SAA, 10 PRCA and 4 PWCA), fourteen of them (mostly PRCA) having not received previous IS. The most utilized schedule (as defined in the prospective trial) was 3,10,30,30,30 mg (total dose 103), administered subcutaneously in consecutive days, with adequate premedication; the last dose was amended in PRCA and PWCA patients (total dose 73 mg). In the prospective trial, all patients also received oral low dose cyclosporine A (1 mg/kg) from day 7, and an intensive anti-infectious prophylaxis, which included oral valgancyclovir and cotrimoxazol. All patients completed the treatment with unrelevant injection-related side effect (fever and/or rash in some cases) and absence of laboratory abnormalities. Complete lympho-ablation was observed in all patients within 2–3 days, which persisted for several weeks; transient worsening of neutropenia and/or thrombocytopenia were observed in some cases. The median follow up was 12 months; there were 5 deaths, only one was possibly related to the treatment. In the prospective trial (n=23), infectious events were rare: a single FUO, associated with fatal complication of an underlying atrial fibrillation, other four viral infections (1 VZV with shingles, 2 HSV and 1 flu), all resolving quickly. No CMV or EBV disease was observed, even if 3 border-line CMV reactivations were documented (after discontinuation of the antiviral prophylaxis), promptly resolved by preemptive valganciclovir. One HBV reactivation without hepatitis required lamivudine. The response rate was globally 61% (42% CR and 19% PR), which raised to 73% (50% CR and 23% PR) when only patients with a follow up of at least 4 months were considered. In the more homogeneous cohort of the prospective trial, response rate was analyzed according to the underlying disease. Among 10 AA treated (5 as first line), 7 had an adequate follow up and showed 4 CR (57%) and 2 PR (29%). Response rate was even higher in 10 PRCA (8 as first line): 7 were evaluable for response, with 5 CR (71%) and 1 PR (14%); the 1 non responding patient subsequently showed evolution to MDS. Finally, 2 of 3 PWCA achieved a CR (66%), with the remaining showing early progression to MDS. Among the responding patients, relapses were quite frequent, even while on cyclosporine: 3/6 SAAs, 5/6 PRCAs and 1/2 PWCA. Relapses were successfully treated by additional ALE (as single shoots or complete courses). Immune reconstitution was delayed up to several months, especially affecting the CD4+ compartment; this was also due to additional ALE needed to treat or to prevent relapses. In conclusion, subcutaneous ALE is a feasible and safe IS regimen for patients suffering from immune-mediated marrow failure syndromes. Preliminary results suggest excellent efficacy, even if responses may be quite late (3–4 months); relapses often occur, but can be easily managed by ALE retreatment. ALE is an excellent alternative to standard IS regimen, and deserves systematic investigation in bone marrow failure patients.

Description: GATA1, founding member of the GATA transcription factor family, is essential for cell maturation and differentiation within the erythroid and megakaryocyte (MK) lineages. Disruption of DNA- or protein-binding capacity of GATA1 causes severe hematopoietic dysfunction and plays a role in blood disorders such as thrombocytopenia, anemia or leukemia. GATA1 expression seems to be related to the MK commitment both in mice and in humans; indeed, similarly to the murine myeloid M1 cell line, in which the enforced expression of GATA1 induces the c-Mpl appearance and MK differentiation, transduction of human hematopoietic stem cells with a GATA1 highly expressing vector results in self-renewal block and in the exclusive generation of Meg-E lineages. More recently, a role for GATA1 also in myeloproliferative disorders (MPDs) was indicated by the “GATA1-low” mouse model which develop a disease closely resembling the human idiopathic myelofibrosis. Interestingly, patients affected by myelofibrosis was also shown to express decreased GATA1 levels by immunostaining of BM sections. In this study, we investigated by Real Time PCR the levels of GATA1 in a myeloproliferative disorders such as essential thrombocytemia (ET) tipically characterized by a neoplastic megakaryocitic proliferation. We have studied BM samples of 40 newly diagnosed patients (M:F ratio 1:1 - median age 53 years, range 18–84) affected by ET, as for the PVSG group criteria. These patients were selected from a cohort of 65 ET patients considering a similar erythroid/myeloid ratio at the FACS analysis to reduce a possible bias for the RT-PCR results due to the erythroid compartment interference. The median platelets count of the selected patients was 670,000/mL (range 493,000–1,400,000/mL), myelofibrotic index 0/1, and 18 out of 40 patients (45%) showed mild splenomegaly both at the physical examination and US scan (median spleen vol 550 ml - range 430–1400 mL). No chromosomal abnormalities were detected by cytogenetic analysis. JAK2 sequencing in 21/40 patients indicated that 9/21 patients (43%) were positive for the JAK2 V617F genomic mutation. At the end of observation time (median 18 mo.) no patients had evidence or signs of thrombotic or hemorrhagic complications. BM cells from six healthy donors were used as normal controls in the study. The relative GATA1 quantification was calculated in according to the DCt method with GAPDH as internal control. The results showed a significant increase of GATA1 expression in BM cells from ET patients (median DCt + 6,11 ; range −0,41/+18,11) compared to the controls (median DCt + 0,172 ; range −4,03/+1,7) (p 〈 0,003). Interestingly, the GATA1 overexpression is not a mere consequence of the proliferation and activation of MKs, indeed samples from three patients affected by idiopathic thrombocytopenic purpura, whose BM smears had the typical secondary megakaryocytic hyperplasia, showed GATA1 levels much lower than the ET patients (median DCt − 0,6 ; range − 3,21/−0,9). No significant difference in GATA1 level was found between patients harbouring a JAK mutation (median DCt +5,86 ; range 0,85/16,12) and those with wild type alleles (median DCt +4,75 ; range −0,41/10,21). In conclusion, our results suggest that GATA1 overexpression could be a trigger for MK neoplastic commitment and proliferation and, consequently, seems to have a central role for ET pathogenesis both in JAK2 mutated and in JAK2 WT patients.

Description: Severe aplastic anemia (SAA) is a typical acquired marrow failure syndrome, presenting with pancytopenia associated to an empty marrow. Less frequent forms of mono-lineage cytopenia may be due to selective aplasia, as in pure red cell aplasia (PRCA) or in agranulocytosis. The link among these conditions is their cellular immune-mediated pathophysiology, which is supported by many experimental data; for these conditions different immunosuppressive strategies have been employed. Here we report our preliminary experience with alemtuzumab as alternative immunosuppressive therapy in patients failing or not eligible for standard immunosuppression. Three patients have been treated: (i). a 39 year-old male affected by SAA, who had failed to respond to two lines of therapy; (ii). a 55 year-old female, suffering from recurrent agranulocytosis, responding to methylprednisone (MP) for 4 years but then refractory to MP, cyclosporine A (CsA) and G-CSF; (iii). a 60 year-old male affected by PRCA, who developed significant early toxicity by steroid treatment. All patients received alemtuzumab as subcutaneous injection, with a dose escalating schedule of 3–10–30–30–(30) mg in consecutive days; the total dose was 103 mg for SAA, and 73 for PRCA and agranulocytosis. The patients received MP 1 mg/kg in concomitance with alemtuzumab, with the exception of the PRCA patient, due to his previous steroid toxicity; all patients on day 7 started low dose CsA (1 mg/kg). Valgancyclovir 450 mg bi-daily and trimethoprim-sulphamethoxazole bidaily twice a week were administered as anti-CMV and anti-Pneumocystis Carinii prophylaxis, respectively. Two of the patients completed the treatment without any side effect, while the PRCA patient experienced injection-related febrile reactions, managed by doubling the dose interval. No significant abnormality of routine biochemical tests was reported. A complete lympho-ablation was observed in all patients within 3–4 days, which persisted for 3 months. No infectious event was observed at a median follow-up of 6 months, nor other medical complications. After 6 months, flow cytometry documented a partial T cell recovery, with persistence of CD4+ cells reduction. No GPI-anchored protein deficient clone was documented within each blood cell lineage. As efficacy of the treatment is concerned, the patients will be described individually. The SAA patient did not show any clinical improvement at +8 months from treatment, with persistent platelet and red cell transfusion requirement. In contrast, the women affected by agranulocytosis showed early and stable response, with absolute neutrophil counts raising from 0 to 3,000/uL (15,000/uL during G-CSF administration); at +6 months from treatment, she is well, free from any treatment, with the exception of a weekly dose of G-CSF. Finally, the PRCA patient did not show any improvement for 3 months, still requiring transfusions. In the fourth month, he suddenly showed a marked rise of reticulocyte count (130,000/uL), which leaded in few weeks to transfusion independence and hemoglobin stabilization; at +6 months, he is on low dose CsA with hemoglobin 〉12 g/dL. In conclusion, alemtuzumab administered as subcutaneous injection is a feasible and safe treatment which may be indicated for patients suffering from immune-mediated acquired marrow failure syndromes. Its positioning in the setting of immunosuppression is still to be established, and promises to be pivotal if the favourable risk-to-benefit profile shown in this preliminary experience will be confirmed.

Description: In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59−). The proportion of C3+ RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom 51Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess 51Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.

Description: Chuvash polycythemia (MIM 263400) is an autosomal recessive disorder characterized by a high hemoglobin level, relatively high serum erythropoietin, and early death. It results from a Von Hippel-Lindau (VHL) gene mutation (C598T) that causes increased HIF-1α activity and erythrocyte production in the face of normoxia. This polycythemia is endemic in Chuvashia, whereas its worldwide frequency is very low. We investigated the incidence of the Chuvash-type VHL mutation in Campania (South Italy) and identified 14 affected subjects (5 families). Twelve live on the island of Ischia (Bay of Naples). From analysis of the mutated allele, we found that the disease was more frequent on Ischia (0.070) than in Chuvashia (0.057). The haplotype of all patients matched that identified in the Chuvash cluster, thereby supporting the single-founder hypothesis. We also found that nonaffected heterozygotes had increased HIF-1α activity, which might confer a biochemical advantage for mutation maintenance. In conclusion, we have identified the first large cluster of Chuvash erythrocytosis outside Chuvashia, which suggests that this familial polycythemia might be endemic in other regions of the world.