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In newly diagnosed myeloma patients, upfront autologous transplant (ASCT) prolongs progression-free survival 1 (PFS1) compared with chemotherapy plus lenalidomide (CC+R). Salvage ASCT at first relapse may still effectively rescue patients who did not receive upfront ASCT. To evaluate the long-term benefit of upfront ASCT vs CC+R and the impact of salvage ASCT in patients who received upfront CC+R, we conducted a pooled analysis of 2 phase III trials (RV-MM-209 and EMN-441). Primary endpoints were PFS1, progression-free survival 2 (PFS2), overall survival (OS). A total of 268 patients were randomized to 2 courses of melphalan 200 mg/m2 and ASCT (MEL200-ASCT) and 261 to CC+R. Median follow-up was 46 months. MEL200-ASCT significantly improved PFS1 (median: 42 vs 24 months, HR 0.53; P

Multiple myeloma is a plasma cell disorder characterized by malignant plasma cell infiltration in the bone marrow, serum and/or urine monoclonal protein and organ damage. The aim of this study was to investigate the impact of chromosome 1 abnormalities in a group of elderly patients (65 years) with newly diagnosed multiple myeloma enrolled in the GIMEMA-MM-03-05 trial and treated with bortezomib, melphalan and prednisone or bortezomib, melphalan, prednisone and thalidomide followed by bortezomib and thalidomide maintenance. We also evaluated the link between chromosome 1 abnormalities and other clinical, genetic and immunophenotypic features by a multivariate logistic regression model. Interphase fluorescence in situ hybridization on immunomagnetically purified plasma cells and bone marrow multiparameter flow cytometry were employed. A multivariate Cox model showed that chromosome 1 abnormalities, age75 years and a CD19(+)/CD117(-) immunophenotype of bone marrow plasma cells were independent risk factors for overall survival in elderly patients with newly diagnosed multiple myeloma. Moreover, a detrimental effect of thalidomide, even when administered in association with bortezomib, was observed in patients with abnormal chromosome 1 as well as in those with 17p deletion, while the benefit of adding thalidomide to the bortezomib-melphalan-prednisone regimen was noted in patients carrying an aggressive CD19(+)/CD117(-) bone marrow plasma cell immunophenotype. This trial was registered at www.clinicaltri-als.gov as #NCT01063179.

Identification of patient sub-groups with smoldering multiple myeloma (SMM) at high risk of progression to active disease (MM) is an important goal. 18F-FDG PET/CT (positron emission tomography (PET) integrated with computed tomography (PET/CT) using glucose labelled with the positron-emitting radionuclide (18)F) allows for assessing early skeletal involvement. Identification of osteolytic lesions by this technique has recently been incorporated into the updated International Myeloma Working Group criteria for MM diagnosis. However, no data are available regarding the impact of focal lesions (FLs) without underlying osteolysis on time to progression (TTP) to MM. We hence prospectively studied a cohort of 120 SMM patients with PET/CT. PET/CT was positive in 16% of patients (1 FL: 8, 2 FLs: 3, >3 FLs: 6, diffuse bone marrow involvement: 2). With a median follow-up of 2.2 years, 38% of patients progressed to MM, in a median time of 4 years, including 21% with skeletal involvement. The risk of progression of those with positive PET/CT was 3.00 (95% confidence interval 1.58-5.69, P=0.001), with a median TTP of 1.1 versus 4.5 years for PET/CT-negative patients. The probability of progression within 2 years was 58% for positive versus 33% for negative patients. In conclusion, PET/CT positivity significantly increased the risk of progression of SMM to MM. PET/CT could become a new tool to define high-risk SMM.Leukemia advance online publication, 13 November 2015; doi:10.1038/leu.2015.291.

Introduction: Islet autotransplantation requires access to the portal vein or tributaries. We originally infused islets into the liver via the middle or right colic veins, but since 2005 we have used the recanalised umbilical vein. Here, we describe the technique, the advantages and the early results achieved. Materials and Methods: After removal of the pancreas and restoration of the biliary and enteric continuity, the ligamentum teres is transected. The obliterated umbilical vein is identified and recanalised with Bakes dilators giving access to the left portal vein. A Vygon® Nutricath ‘S’ 11-Fr catheter is inserted and used for the islet infusion. If the ligamentum teres is to be exteriorised for postoperative measurements or subsequent access, it is pulled through a 10-mm laparoscopic port in the epigastrium, sutured to the skin and covered with a dressing. Results: We have used this approach in 17 patients and exteriorised the falciform ligament in 4. There have been no intra- or postoperative complications. Conclusions: The recanalised umbilical approach is safe and allows for venous sampling and postoperative measurements of the portal pressure. Under local anaesthetic, the umbilical vein can be approached above the umbilicus and exteriorised if repeated transplants are required for allograft patients.

Background and study aims ThinPrep is often used for endoscopic ultrasound fine-needle aspiration (EUS-FNA) samples but the sensitivity of this method is unknown. The objective of the study was to compare sensitivity and accuracy of ThinPrep versus the smear method in pancreas and lymph node samples obtained by EUS-FNA. Patients and methods Patients with suspected malignancy in the pancreas or lymph node underwent EUS-FNA. On-site rapid assessment of all aspirates using the smear method was performed. After rapid assessment, three additional passes from each site were submitted into ThinPrep liquid medium. Cytopathologists interpreting the smear method and ThinPrep slides were blinded to each other. The gold standard was final cytology or pathology results. Results A total of 130 patients (36 % women, mean age 63 years) underwent EUS-FNA of 139 sites (50 pancreas, 89 lymph node). Malignancy was confirmed in 47 pancreas samples (94 %) and 48 lymph node samples (54 %). Mean +/- SD number of passes made for the smear method was 2.6 +/- 1.3. For pancreatic cancer, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the ThinPrep versus the smear method were: 62 % versus 98 %, 100 % versus 100 %, 100 % versus 100 %, 14 % versus 75 %, and 64 % versus 98 %, respectively. For lymph nodes the values were 67 % versus 92 %, 100 % versus 98 %, 100 % versus 98 %, 72 % versus 72 %, and 82 % versus 94 %, respectively. Conclusions The smear method is more sensitive and accurate than ThinPrep in detecting malignancy from EUS-FNA samples of the pancreas and lymph nodes. Smear method with on-site rapid assessment should be favored over ThinPrep in suspected malignancy.

Defibrotide is a polydisperse oligonucleotide with antiatherosclerotic, anti-inflammatory, anti-ischaemic, pro-fibrinolytic and antithrombotic actions without significant systemic anticoagulant effects. It has been used in the treatment of various cardiovascular disorders, and especially in endothelial complications of allogeneic stem-cell transplantation. We reviewed the published work for the mechanism of action and clinical use of defibrotide to consolidate data and to describe new applications of this drug. We reviewed the most relevant papers on defibrotide published from November 1982 to January 2008. (selected through PubMed), and used recent meeting abstracts as sources for this review. Reports have suggested that defibrotide has clinical efficacy for treatment and prophylaxis of hepatic sinusoidal obstruction syndrome occurring after stem-cell transplantation. Animal models have clearly shown the potential antineoplastic effect of this drug. Further clinical investigations are needed to clarify this new application.

This multicenter phase II trial evaluated the safety and efficacy of lenalidomide−prednisone (RP) induction, followed by lenalidomide−melphalan−prednisone (MPR) consolidation and RP maintenance in elderly unfit newly diagnosed myeloma patients. Patients received four 28-day RP induction courses (lenalidomide 25 mg/day on days 1–21 and prednisone 50 mg three times/week), followed by six 28-day MPR consolidation cycles (melphalan 2 mg, prednisone 50 mg three times/week and lenalidomide 10–15 mg/day on days 1–21), and maintenance with lenalidomide (10 mg/day on days 1–21 every 28 days) plus prednisone (25 mg three times/week). Forty-six patients were enrolled. Median age was 75 years, 59% of patients had at least one comorbidity and 35% at least two. Partial response rate was 80%, including 29% very good partial response. Median time to progression was 19.6 months, median progression-free survival was 18.4 months and 2-year overall survival was 80%. At the tolerated consolidation dose (melphalan 25 mg/month and lenalidomide 10 mg/day), the most frequent grade 3 adverse events were neutropenia (36.4%), anemia (12.1%), cutaneous reactions (18.2%) and infections (12.1%). Grade 4 neutropenia occurred in 12.1% of patients. In conclusion, RP induction followed by MPR consolidation and RP maintenance showed a manageable safety profile, and reduced the risk of severe hematological toxicity in unfit elderly myeloma patients.

Regulatory T-cells (Tregs) constitute a small subset of cells involved in antitumour immunity and are generally increased in patients with chronic lymphocytic leukemia (CLL). No data is available on Tregs in monoclonal B-cell lymphocytosis (MBL), a disease entity characterized by less than 5000/microL circulating clonal B-cells in absence of other features of lymphoproliferative disorders. We used multicolour flow cytometry to evaluate the number of circulating Tregs in 56 patients with#x0022;clinical#x0022; MBL, 74 patients with previously untreated CLL and 40 healthy subjects. MBL patients showed a lower absolute number of Tregs, compared to CLL patients, but slightly higher than controls. Moreover, the absolute cell number of Tregs directly correlated both with more advanced Rai/Binet clinical stages and peripheral blood B-cell lymphocytosis. Of note, the absolute number of Tregs was found lower in MBL patients than in CLL patients staged as 0/A Rai/Binet. The study showed that Treg increase gradually from normal subjects to#x0022;clinical#x0022; MBL patients and are significantly higher in CLL patients as compared to MBL patients. Moreover, a significant direct relationship was found between higher Treg values and a higher tumor burden expressed by B-lymphocytosis or more advanced clinical stages. In light of this data, MBL seems to be a preliminary phase preceding CLL. The progressive increase of Treg numbers might contribute both to the clinical evolution of MBL to overt CLL and to CLL progression.

Factor XI (FXI) deficiency is a rare bleeding disorder, resulting in a wide range of bleeding manifestations, from asymptomatic bleeding to injury-related bleeding. To identify mutations in FXI-deficient patients and to establish a possible relationship between clinical phenotype and genotype, we studied two patients from Southern Italy with FXI deficiency. They were identified by presurgical or routine laboratory screening. None of them showed bleeding. Three different mutations were detected (Glu117Stop, Cys118Arg and Trp497Gly); two of them were novel (Cys118Arg and Trp497Gly). One patient (with severe FXI levels) showed a compound heterozygosity (Glu117Stop with Cys118Arg). Two novel missense mutations were highly conserved among different species. In our patients, bleeding tendency did not appear to be correlated with FXI levels or with a single mutation in heterozygosis. On the other hand, the compound heterozygosis might explain low FXI levels, but it is not associated with bleeding. Our data confirm that a severe FXI deficiency is not necessarily associated with bleeding.

Total pancreatectomy is considered the final resort in the treatment of chronic pancreatitis; however, here we show that simultaneous islet autotransplantation can abrogate the onset of diabetes.: In Leicester, 46 patients have now undergone total pancreatectomy with immediate islet auto transplant, and they have received a median of 2246 islet equivalent (IEQ)/kg body weight (range, 405-20,385 IEQ/kg body weight).: Twelve patients have shown periods of insulin independence, for a median of 16.5 months (range, 2-63 months), and 5 remain insulin independent. Over the 10 years of follow-up, there has been a notable increase in insulin requirement per kilogram per day, and percentage of glycosylated hemoglobin levels have increased significantly (r = 0.66, P = 0.01). However, 100% of patients tested were C-peptide positive at their most recent assessment, and high fasting and stimulated C-peptide values recorded at 10 years after transplantation, 1.44 (range, 1.09-1.8 ng/mL) and 2.86 ng/mL (range, 1.19-4.53 ng/mL), respectively, suggest significant graft function in the long term. In addition, median serum creatinine has increased very little after the operation (71 nmol/L [range, 49-125 nmol/L] atpreoperation vs 76.5 nmol/L [range 72-81 nmol/L] at year 10), suggesting no diabetic nephropathy.: Although there is a notable decline in islet function after islet auto transplant, there is still evidence of significant long-term insulin secretion and possible protection against diabetic complications.

Bortezomib has shown significant activity in myeloma. In this multicenter trial, we assessed for the first time the combination of bortezomib, doxorubicin and low-dose dexamethasone (PAd) in the treatment of relapsed/refractory myeloma.Sixty-four patients were treated for a median of four 28-day cycles (1-6). Bortezomib was given at 1.3 mg/m(2) (days 1, 4, 8, 11) and dexamethasone at 40 mg (days 1-4); 34 patients receive doxorubicin at 20 mg/m(2) (days 1, 4) while 30 patients pegylated liposomal doxorubicin at 30 mg/m(2) (day 1).Fifty-eight percent of patients had undergone prior autologous transplantation, 70% prior anthracycline and 27% prior bortezomib-based regimens. Forty-three patients (67%) achieved at least a partial response including 16 (25%) with at least a very good partial response. One-year event-free survival was 34% after PAd and 31% after the previous line of therapy (hazard ratio 1.20, 95% confidence interval 0.76-1.90, P = 0.43). One-year overall survival from the start of PAd was 66%. Grade 3-4 toxic effects included thrombocytopenia (48%), neutropenia (36%), infections (15%), anemia (13%), gastrointestinal disturbances (11%) and peripheral neuropathy (10%). Two patients had grade 3-4 cardiac heart failure.PAd is an active salvage therapy with manageable toxicity in patients with relapsed/refractory myeloma.

To evaluate pressure control ventilation (PCV) delivered through a minitracheotomy in treating severe flail chest trauma.Case report.Intensive care unit of a trauma center.A 34-year-old woman affected by flail chest trauma and acute respiratory failure, who was initially treated with tracheal intubation to obtain internal pneumatic stabilization. The patient failed extubation and noninvasive mask treatment (pressure support ventilation plus PEEP) due to poor chestwall mechanics.Minitracheotomy was performed and ventilation was achieved with high levels of inspiratory pressure (PCV or assisted PCV) to overcome the resistance of the cannula (Mini-Trach II, Portex, ID 4 mm). Esophageal and carinal pressures were monitored. Ventilatory treatment was always performed with the full cooperation of the patient; the patient's glottic function was always intact. The patient was successfully treated with pressure control ventilation delivered through the Mini-Trach. After 7 days of PCV, the patient was switched to assisted PCV. On the 20th day after admission, she was weaned from mechanical ventilation.We conclude that a suitable gas exchange and pneumatic stabilization in a flail chest condition can be achieved using minitracheostomic ventilation. At the same time, this treatment could reduce some side effects of traditional tracheal intubation.

Background. Surgical resection of the pancreas is considered a final resort in the treatment of chronic pancreatitis. However, the opportunity to perform an islet autotransplant at the same time provides the potential to prevent the onset of diabetes. Methods. Pancreatectomy together with islet autotransplantation has been offered in our center since 1994. A total of 40 patients have now undergone this procedure. The follow-up times range from 6 months to 7 years. The data presented here include the annual postoperative oral glucose tolerance test and glycosylated hemoglobin (HbA 1c ) results, together with insulin and opiate requirements. Results. Nineteen male and 21 female patients (median age 44, range 21-65) have been transplanted. Pancreatitis was related to alcohol in 45% and was idiopathic in 40%. A median of 130,108 (24,332-1, 165,538) islet equivalent (IEQ) were transplanted, which related to 2,020 (320-23,311) IEQ per kilogram of body weight. At 2 years posttransplant, 18 patients had a median HbA 1c of 6.6% (5.2-19.3%), fasting C-peptide of 0.66 ng/mL (0.26-2.65 ng/mL), and required a median of 12 (0-45) units of insulin per day. At 6 years, these figures were 8% (6.1-11.1%), 1.68 ng/mL (0.9-2.78 ng/ml) and 43 U/day (6-86 U/day), respectively. The majority of patients no longer require opiate analgesia, 68% have been able to return to work, and one patient has had a baby. Conclusions. Islet autotransplantation offers a valuable addition to surgical resection of the pancreas, as a treatment for chronic pancreatitis; and even in cases in which insulin independence is not achieved, the potential beneficial effects of C-peptide make the procedure worthwhile.

We administered the anti-angiogenic drug thalidomide to 21 patients (12 men) with myelofibrosis with myeloid metaplasia (MMM), who were not responsive to standard treatment. Patients received thalidomide at an escalating dose from 100 to 400 mg/d. Administration of the drug was discontinued before the planned 6 months of treatment in 19 patients (90.5%), mainly because of somnolence and/or fatigue, neurological symptoms or neutropenia. Of the 13 evaluable patients (who received more than 30 d of therapy), anaemia improved in three out of seven (43%) who were treated because of anaemia; thrombocytopenia improved in two out of three (66.6%) who were treated because of thrombocytopenia; splenomegaly was reduced in four (30.8%). Undesired increases in white blood cell and platelet counts were observed in three (23.1%) and five (38.5%) patients respectively. A severity score, indexed on haematological and clinical parameters, improved in two patients (15.4%), but worsened in five (38.5%). In conclusion, standard-dose thalidomide in MMM patients is burdened with a high rate of side-effects, which prevent prolonged treatment. Because the drug is effective in improving anaemia and thrombocytopenia and in reducing splenomegaly, low-dose therapy warrants evaluation. The unexpected observation of leucocytosis and thrombocytosis suggests biological studies and better criteria for selection of patients for treatment.

Objective To assess the safety and efficacy of islet autotransplantation (IAT) combined with total pancreatectomy (TP) to prevent diabetes. Summary background data There have been recent concerns regarding the safety of TP and IAT. This is thought to be related to the infusion of large volumes of unpurified pancreatic digest into the portal vein. Minimizing the volume of islet tissue by purifying the pancreatic digest has not been previously evaluated in terms of the postoperative rate of death and complications, pain relief, and insulin independence. Method During a 54-month period, 24 patients underwent pancreas resection with IAT. Islets were isolated using collagenase and a semiautomated method of pancreas digestion. Where possible, islets were purified on a density gradient and COBE processor. Islets were embolized into the portal vein, within the spleen and portal vein, or within the spleen alone. The total median volume of digest was 9.9 mL. Results The median number of islets transplanted was 140,419 international islet equivalents per kilogram. The median increase in portal pressure was 8 mmHg. Early complications included duodenal ischemia, a wedge splenic infarct, partial portal vein thrombosis, and splenic vein thrombosis. Intraabdominal adhesions were the main source of long-term problems. Eight patients developed transient insulin independence. Three patients were insulin-independent as of this writing. Patients had significantly decreased insulin requirements and glycosylated hemoglobin levels compared with patients undergoing TP alone. Of the patients alive and well as of this writing, four had failed to gain relief of their abdominal pain and were still opiate-dependent. Conclusion Combined TP and IAT can be a safe surgical procedure. Unfortunately, almost all patients were still insulin-dependent, but they had decreased daily insulin requirements and glycosylated hemoglobin levels compared with patients undergoing TP alone. A prospective randomized study is therefore needed to assess the long-term benefit of TP and IAT on diabetic complications.

Recombinant erythropoietin (r-EPO) induces erythroid responses in patients affected by myelodysplastic syndromes (MDS). However, the response rate declines to 10-15% in MDS with substantial transfusion needs. Both in vitro and in vivo studies have suggested that the addition of growth factors (G-CSF, GM-CSF) or interleukin-3 (IL-3) may potentiate the effect of r-EPO on dysplastic erythropoiesis. The aim of this study was to evaluate the effects of the combination of r-EPO with G-CSF, GM-CSF or IL-3 on the anemia of heavily transfusion-dependent MDS patients, previously unresponsive to r-EPO alone.Sixty patients with transfusion-dependent MDS, already treated without significant erythroid response with r-EPO alone, were scheduled to receive, for at least 8 weeks, r-EPO subcutaneously at the dose of 300 U/kg t.i.w. in combination with G-CSF (300 microcg s.c. t.i.w., 27 patients), or GM-CSF (300 microcg s.c. t.i.w., 23 patients), or IL-3 (5 microcg/kg s.c. t.i.w., 10 patients), after a two-week pre-phase during which G-CSF, GM-CSF and IL-3 were administered daily at the same dose, as single drugs.Ten patients were not evaluable for erythroid response because of relevant side effects related to GM-CSF or IL-3 administration. Overall, among 50 patients who completed the study, there were 3 erythroid responses (as determined by complete abolition of red-cell transfusions): 1 (4%) in the G-CSF + r-EPO and 2 (10.5%) in the GM-CSF + r-EPO treated groups. No patient responded to the combination of r-EPO + IL-3. All responders had inappropriate serum levels of endogenous EPO and a relatively short disease duration. Both responders to GM-CSF + r-EPO developed acute myeloid leukemia 2-9 months after the start of the combined therapy. A third elderly patient, treated with the same association, developed marrow hypoplasia. A significant increase in leukocyte count occurred in 96% of patients who received r-EPO + G-CSF, 78.9% of those treated with r-EPO + GM-CSF and 66% of subjects receiving r-EPO + IL-3. A significant increase in platelet count was observed in a single patient receiving r-EPO and GM-CSF, while a slight decrease in platelet count with respect to baseline levels occurred in about 20% of patients.Our results suggest that the combination of r-EPO with G-CSF, GM-CSF or IL-3, at least at the doses and schedules employed in the present study, has limited efficacy on the anemia of heavily transfusion-dependent MDS patients previously unresponsive to r-EPO alone. However, in this setting of patients, the combination of G-CSF or GM-CSF + r-EPO may occasionally be effective in subjects with low circulating levels of serum EPO and short disease duration.

To evaluate the effect of recombinant human erythropoietin (rHuEpo) on the haemoglobin level and transfusion requirement in low-risk myelodysplastic syndromes (MDS), 87 patients were enrolled in a randomized double-blind placebo-controlled study, 44 patients were assigned to epoetin alpha (150 U/kg/d s.c. for 8 weeks) and 43 to placebo arms. MDS types were homogenous in both groups: refractory anaemia (RA) 47.7-48.8%. refractory anaemia with ringed sideroblasts (RAS) 20.5-25.6%, refractory anaemia with excess of blasts (RAEB) (blasts10%) 31.8-25.6%, 14/38 evaluable patients responded to epoetin alpha versus 4/37 to placebo (P=0.007). 50% of RA responded to epoetin alpha versus 5.9% to placebo (P=0.0072), RAS 37.5% v 18.2% (P=0.6) and RAEB 16.7% v 11.1% (P=1.00). 60% of non-pretransfused patients responded to epoetin alpha (Hb 8.35or = 0.73 to 10.07+/-1.87 g/dl), whereas a slight decrease was observed in the placebo group (8.4+/-0.66 to 8.19+/-0.92 g/dl) (P=0.0004). Percentage of transfused patients was similar in both arms. Basal erythropoietin (Epo) serum levels200 mU/l predicted for a non-response. At week 4 sTfR levels were increased50% in responders (P=0.013), whereas an increase18% predicted for non-response (P=0.006). Leucocyte and platelet counts were not influenced by epoetin alpha treatment. Adverse events occurred in 31.8% of the rHuEpo-treated versus 42.99%) of the placebo-treated patients (P=0.2), and seven patients did not complete the course. In conclusion, rHuEpo was effective in the treatment of low-risk MDS. RA subtype, no transfusions prior to rHuEpo therapy, and low basal Epo levels were associated with higher probability of response. Soluble transferrin receptor level at the fourth week was an early predictor of response.

CD34+ hematopoietic progenitor cells (HPCs) constitute a heterogeneous population both in size and in immunological features. Lack of CD38, HLA-DR and lineage committed antigens as well as the co-expression of Thy-1 (CDw90) and c-kit receptor (CD117), are able to identify the so-called stem cells. A flow cytometric study was carried out to investigate the co-expression of Thy-1 and c-kit receptors, both members of Ig superfamily adhesion molecules, involved in cell to cell and cell to stroma interactions, on bone marrow (BM), mobilized peripheral blood (PB) and human umbilical cord blood (HUCB) CD34+ HPCs.Lysed whole blood from 15 BM, 25 mobilized PB and 25 HUCB samples were used to perform a five-dimensional flow cytometric evaluation of both CDw90 and CD117 on CD34+ cells.Few CD34+ cells co-expressed Thy-1 antigen in all three compartments (BM: 11.2 +/- 7.2%; PB: 6.2 +/- 3.6%; HUCB: 6 +/- 2.9%; BM vs PB0.04; BM vs HUCB0.008; PB vs HUCB ns). c-kit receptor was detected on the majority of CD34+ HPCs, particularly in HUCB (HUCB: 80.7 +/- 8.2%; BM: 72.3 +/- 13.1%; PB: 64.2 +/- 17%; HUCB vs BM0.03; HUCB vs PB0.0001; BM vs PB ns). CD34+Thy-1+ and CD34+c-kit+ HPCs generally displayed HLA-DR antigen, as expression of early cell commitment. However, the most immature CD34+Thy-1+HLA-DR- (HUCB: 1 +/- 0.6%; BM: 0.4 +/- 03%; PB: 0.7 +/- 0.5%; HUCB vs BM0.0001; BM vs PB0.04; HUCB vs PB ns) and CD34+c-kit+HLA-DR- HPCs (HUCB: 6.5 +/- 4.4%; BM: 6.3 +/- 4.8%; PB: 2.2 +/- 1.8%; HUCB vs BM ns; BM vs PB0.0001; HUCB vs PB0.0001) were mainly detected in HUCB. Finally, the greatest percentage of CD34+Thy-1+c-kit+ cells was found in BM (6.9 +/- 4.1%) followed by leukapheretic samples (4.4% +/- 2.7) and then by HUCB (3.7 +/- 1.2%; BM vs PB ns; BM vs HUCB0.001; HUCB vs PB ns).Since the blood release or HPCs is probably due to a perturbation of the adhesive interactions between these cells and the marrow stroma, the different pattern of Thy-1 and c-kit receptor expression on CD34+ HPCs found in the three hemopoietic compartments evaluated can lead to new knowledge about the mobilization kinetics in which the Ig superfamily adhesion molecules are involved.

CD27, a transmembrane homodimer belonging to the nerve growth factor (NGF) receptor superfamily, is typically expressed on leukemic CD5+ cells in B-cell chronic lymphocytic leukemia (CLL) and found in soluble form in the serum of CLL patients. Therefore, we investigated clinico-biological implications of increased serum levels of sCD27 in an unselected series of B-CLL patients.Serum CD27 (sCD27) levels were determined at the time of diagnosis in 82 previously untreated B-CLL patients using a sandwich enzyme-linked immunoassay (ELISA). Results were correlated with either clinico-hematological or biological features. Finally, quantitative flow cytometric analyses of membrane CD27 (mCD27) expression were carried out on peripheral blood (PB) cells of 22 B-CLL patients and 5 healthy controls, respectively.CD27 was found to be expressed on the surface of both resting normal and leukemic B cells. sCD27 levels were significantly higher in B-CLL patients (median value 2150 U/mL) than in healthy controls (median value 220 U/mL) (p0.0001). There was a close relationship between sCD27 and soluble TNF-alpha, another molecule belonging to the NGF receptor superfamily. Changes in sCD27 level correlated with clinical stage, beta 2 microglobulin and LDH.These findings indicate that sCD27 is a reliable marker of tumor mass in B-CLL. Its potential prognostic value should be tested in prospective studies.

The CD117 molecule is an antigen more frequently found on early normal and leukemic hematopoietic cells, but its correlation with the FAB subtypes and with other lineage and stage associated antigens is still not well established. In this study we investigated the surface expression of CD117 antigen in 135 patients with acute leukemia in relationship to de novo or secondary origin of AML, subtypes of FAB classification, expression of other antigens such as CD34, HLA-DR, CD15, CD14, CD45RA, CD45RO, CD11b, CD11c, CD4, CD7, mixed antigen co-expression (LyAg + AML and MyAg + ALL) and features of leukemic mass.The CD117 antigen expression (clone 95C3) was determined by flow cytometry in a series of 135 patients with acute leukemia at diagnosis consecutively observed during the years 1995-1997: 82 AML (including 51 cases of de novo AML, 22 cases of AML following myelodysplastic syndromes (MDS), 9 cases of myeloid blastic crisis of chronic myeloid leukemia (BC-CML) and 53 ALL. All cases were stratified in CD117+ and CD117- groups and the differences were analyzed by using appropriate statistical analyses.CD117 antigen was found over 10% in 74% of AML without significant differences of positivity between AML after MDS or BC-CML and de novo AML. We did not note a significant correlation between FAB classification and CD117 which was expressed in 100% of M1 and M7 cases, in 80% of M0 cases, in 75% of M2 cases, in 70% of M3 cases and in 82% of M4 cases. Instead, in M5 subtype CD117 was strictly restricted to earlier stages: ten of the eleven M5b (91%) cases completely lacked CD117 antigen expression, whereas 100% of M5a cases were positive. The results of Pearson's coefficient showed: 1) a significant inverse relationship between CD117 and CD15, CD4 and CD14 (only in M5 subtypes) and CD11b, CD11c and CD45RO (in all cases); 2) a significant direct correlation between CD117 and CD34 and CD45RA (in all cases); and 3) an independent expression between CD117 and CD15 associated with a low correlation between CD117 and HLA-DR antigen (only in non-monocytic cases). In ALL, whether of B or T lineages, surface expression of CD117 was never observed.We conclude that the CD117 antigen shows a high specificity for AML, independently upon FAB classification, and represents a reliable marker in characterizing the differentiative degree of the monocytic blasts.

One of the most important potential advantages in the use of human umbilical cord blood (HUCB) for hematopoietic reconstitution after myeloablative therapy seems to be the lower occurrence of acute graft-versus-host-disease (GvHD) in recipients after allogeneic transplantation. Since mature T cells play an important role in GvHD pathogenesis, we tried to verify whether a different immunophenotypic pattern exists between HUCB and peripheral blood (PB) T cells.An immunophenotypic study on 40 HUCB and 40 PB samples from healthy adult volunteers was performed, by means of flow cytometry using a large panel of monoclonal antibodies in double labeling.The absolute lymphocyte count was greater in HUCB (5233 +/- 1808 microL) than in adult PB (1941 +/- 378 microL). Significant differences in percentage were found between cord and adult T-cells, respectively (CD3+: 59.9 +/- 12 vs 74.9 +/- 4.6%), CD3- CD16+ and/or CD56+ natural killer (NK) cells (23.8 +/- 10.1 vs 10.8 +/- 5.3%) and CD3+CD16+ and/or CD56+ cytotoxic T lymphocyte subset (0.3 +/- 0.3 vs 10.7 +/- 4.1%). There was no difference in CD4/CD8 ratio (1.7 +/- 0.5 vs 1.6 +/- 0.2%) between the two groups. In absolute terms, HUCB contained an higher number of all lymphocyte subsets, with the exception of CD3+CD16+ and/or CD56+ T lymphocyte subpopulation, CD3+CD25+ and CD3+HLADR+ activated T-cells. CD38, a marker of activation and immaturity, was present on virtually all cord T cells and on approximately half of the adult T cells. The large majority of HUCB T cells co-expressed CD45RA naive antigen (CD4+CD45RA+: 87.6 +/- 5.2%, CD8+ CD45RA+: 93.5 +/- 7.8%; CD4+CD45RO+: 12.3 +/- 5.2%; CD8+CD45RO+: 6.4 +/- 7.8%) whereas in adult PB T cells an higher number of CD45RO+ memory cells was detected (CD4+CD45RA+: 44.8 +/- 9.6%; CD8+CD45RA: 71.5% +/- 8.1%; CD4+CD45RO+: 55.2 +/- 9.6%; CD8+ CD45RO+: 28.5 +/- 8.1%). Finally, less than 14% of lymphocytes were shown to belong to B lineage in both sources, while, in absolute terms, they were more represented in HUCB with respect to adult PB.In the present study we found significant difference between HUCB and adult PB lymphocytes in their immunophenotypic profile. In particular HUCB showed T lymphocytes that appeared to be phenotypically immature. Indeed, as a likely consequence of poor antigenic experience during pregnancy, the majority of HUCB cells were naive, expressing the RA isoform of the CD45 molecule. These findings could justify the previously reported reduced cord blood lymphocyte alloreactivity when allogeneic transplantation is performed and require further functional studies in order to confirm the impairment of HUCB immune system response to alloantigens.

The in vitro inhibitory effect of all-trans retinoic acid (ATRA) on myeloma cell growth may be synergistically potentiated by the activity of dexamethasone (DEX) and alpha-interferon (IFN). We treated 10 patients with advanced, refractory multiple myeloma (MM) using a combination of ATRA (100 mg p.o., once a day for two weeks every month), DEX (40 mg i.v., for 4 days every 4 weeks) and IFN (3 MU s.c., three times a week). Eight patients completed at least three months of treatment and were evaluable for response. Two of them showed a partial response which persists after 15 to 17 months. Three patients experienced a stable plateau phase of 4 to +11 months, with a significant improvement in the performance status and bone pain. Progressive disease was seen in the remaining three patients. We conclude that the association of ATRA, DEX and IFN warrants further consideration in MM patients.

Although less specific than sCD23, sCD54 levels have clinico-prognostic relevance in B-cell chronic lymphocytic leukemia (CLL). Since serological markers are now emerging as potentially important in CLL, we tried to verify whether sCD54 might complement clinical stages.Serum levels of sCD54 were determined at the time of diagnosis in 115 previously untreated CLL patients. Results were correlated with clinicobiological parameters as well as with survival.Life-expectancy was significantly shorter in patients with higher serum levels of sCD54 (p0.001); however, in a Cox's multivariate survival analysis, the only variables which entered the regression model at a significant level were bone marrow (BM) histology (p = 0.03) and lymphocyte doubling time (LDT) (p = 0.04). Interestingly, when LDT was excluded from analysis the only significant variables were clinical stages (p0.05) and sCD54 (p0.05). These results suggest that sCD54 and LDT give similar prognostic information.In CLL, sCD54 is a reliable prognostic parameter whose value is independent of clinical stages. When investigated in relation to clinical outcome, serum levels of sCD54 were able to predict progression to a more advanced clinical stage. On the basis of these data, an integrated clinico-biological classification which separates intermediate risk into two prognostic subgroups is proposed.

The prognostic significance of CD34 antigen expression in acute lymphoblastic leukemias (ALL), especially in adult patients, is still not well established. In the present report we analyzed a series of biological and clinical findings from 128 ALL patients in order to evaluate the possible clinical significance of this marker.The clinical and biological significance of CD34 expression, an early marker of hemopoietic cells, was analyzed by flow cytometry in a series of 128 patients affected by ALL, including 78 adults and 50 children under 15 years old.Overall, 68.7% of patients showed significant (10%) CD34 expression. There was no difference between CD34+ and CD34- ALL with respect to age, sex, FAB morphology, hepatosplenomegaly, Plt count, Hb level, DNA index, P-170 expression. CD34+ ALL displayed a significantly lower frequency of extramedullary involvement, a lower LDH level and lower WBC count, lower proliferative activity (as evaluated by the Ki67 monoclonal antibody) than CD34- ALL. CD34 expression was also associated with early phenotypes in both B- and T-ALL, co-expression of myeloid antigens, and the presence of the Ph1 chromosome. Due to a different distribution of prognostic factors investigated, DFS and OS were both significantly better in CD34+ than in CD34- childhood ALL, whereas no statistical difference was found in adults. Multivariate analyses confirmed these data in children.Expression of the CD34 antigen is a positive prognostic factor in childhood ALL. In adult ALL the presence of this marker on leukemic cell does not seem to influence the clinical outcome of these patients.

CD23 is a functionally relevant molecule in B-cell chronic lymphocytic leukemia (CLL) which mediates growth and differentiation signals in B-cells. An intriguing feature of CD23 is its ability to be cleaved from the cell surface and released into the serum.Serum levels of soluble CD23 (sCD23) were determined with a sandwich enzyme immunoassay at the time of diagnosis in 90 previously untreated CLL patients, in order to evaluate whether they reflected disease activity and tumor load. Results were correlated with those dealing with CD23 expression on leukemic cells to verify whether the cellular counterpart determines serum levels.CD23 was detected on peripheral blood mononuclear cells (PBMC) from 78 out of 90 (86.6%) B-CLL patients, without correlation with clinical stage. Circulating levels of sCD23 in the serum of patients with CLL were highly elevated in comparison to 15 normal controls (p0.0005); this increase reflected tumor mass as defined by either clinical stage (p0.0005) or bone marrow (BM) histology (p0.0005). Neither percentage nor absolute number of CD23+ cells correlated with circulating levels. Interestingly, life expectancy was significantly shorter in patients with higher serum levels of sCD23 (p0.0005). When integrated into the Binet clinical staging system, sCD23 led to isolation of two subgroups with different prognosis among intermediate-risk patients. Furthermore, longitudinal studies support the idea that sCD23 can be utilized as an indicator of disease progression.sCD23 is a highly sensitive and suitable marker with prognostic potential in B-CLL.

Human umbilical cord blood (HUCB) is a possible alternative to bone marrow (BM) and mobilized peripheral blood (PB) for transplantation of hematopoietic progenitors. The aim of this study was to evaluate the phenotypic profile of CD34+ progenitors present in HUCB.A flow cytometric analysis was performed on 20 HUCB samples, using a large panel of monoclonal antibodies recognizing different lineage or activation antigens, in double labeling with CD34.A toal of 13,897 +/- 2,529 cells/microL, 0.84 +/- 0.83% of which were CD34+, was found. The large majority of CD34+ cells were committed toward initial myeloid differentiation (CD33+, CD13+) and expressed the transferrin receptor (CD71). A substantial proportion of these cells (about 40%) co-expressed CD45RA and CD117, while a very small number displayed markers of advanced myeloid commitment, such as CD14, CD15 and CD41 (less than 2%), or those of lymphoid differentiation: CD2, CD5, CD7, CD10 and CD19 (less than 6%). About 11% of HUCB CD34+ cells were primitive progenitors, as suggested by the absence of HLA-DR and CD38 on their surface.As previously observed in BM and mobilized PB, the phenotype of HUCB CD34+ cells is quite heterogeneous. In particular, HUCB contains subpopulations of both early and committed hematopoietic progenitors which may represent a valid source for transplantation.

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1 beta) and interleukin-2-(IL-2) were measured in 19 atopic subjects and in 8 healthy controls. TNF-alpha concentration was increased in atopic subjects and the increase was correlated with the severity of the disease in patients affected by atopic dermatitis. Increased plasma levels of IL-1 beta were found only in symptomatic asthmatic subjects. No increase of serum IL-2 concentration was found in atopic subjects. The data suggest a possible role of TNF- alpha and IL-1 beta in the pathophysiological mechanism of the allergic inflammation.

We report a case of de novo plasma cell leukemia, resistant to standard VMD (vincristine, mitoxantrone, dexamethasone) and CVP (cyclophosphamide, vincristine and prednisone) protocols, treated with a chemotherapy intensification regimen (high-dose cyclophosphamide, modified EDAP, Dexa-BEAM) and peripheral blood stem cell transplantation, performed using fractionated total body irradiation and high dose melphalan. The patient is currently alive and well, in very good partial remission 12 months after transplant and 22 months after diagnosis, disclosing a significant proportion of bone marrow and peripheral blood CD3+, CD8+, CD57+, HLA-Dr+ large granular lymphocytes with cytotoxic activity against neoplastic plasma cells.

Hematopoietic progenitor cells (HPC), identified by expression of the CD34 surface antigen, show morphological and phenotypic heterogeneity in bone marrow (BM) and peripheral blood (PB).CD34+ HPC subpopulations present in 18 PB leukaphereses after high-dose chemotherapy in cancer patients and in 11 BM samples from patients with stage IA lymphoma were characterized. In order to identify CD34+ HPC subsets within these two compartments, the expression of lineage- or activation-associated antigens and the c-kit gene product (CD117) was studied by flow cytometry, using a large panel of monoclonal antibodies (MoAb) in double labelling.We observed a higher proportion of CD34+/CD13+ and CD34+/CD33+ cells (myeloid commitment) in harvested leukapheresis products than in BM. On the contrary, a higher percentage of CD34+/CD10+ and CD34+/CD19+ cells (B-lymphoid commitment) was found in BM. The percentage of the most immature subset of CD34+ HPC (CD38- and HLA-DR-) was also higher in BM than in mobilized PB. No differences in proportions were found with respect to the expression of CD14, CD15, CD45RA (myeloid commitment), CD2, CD5, CD7 (T-lymphoid commitment), CD117, CD71 and CD45RO antigens. In terms of absolute values, however, significantly higher amounts of CD34+ HPC co-expressing CD13, CD33, CD5, CD7, CD71, CD117, CD45RA, CD45RO were detected in leukaphereses than in BM. The absolute number of immature HPC (CD34+/CD38- and CD34+/HLA-Dr-) was also significantly increased in mobilized PB.Our data confirm the heterogeneous phenotypic profile of HPC, thus supporting the hypothesis that different CD34+ subpopulations may have clinical relevance on the rapidity and long-term durability of engraftment in patients who undergo high-dose chemotherapy followed by rescue with HPC. We also demonstrated that mobilized PB is a particularly rich source of both primitive and committed HPC, more than BM.

alpha-IFN is reported to be an effective treatment for a number of lymphoproliferative diseases. Little information is available at present on its effect in unaggressive immunoproliferative disorders.In a prospective non randomized study, 57 patients with IgG or IgA MGUS, smouldering myeloma or stage I MM treated with alpha-IFN (3 MU 3 times a week for at least 6 months) were compared to 129 untreated similar patients. Four patients in the IFN group showed a monoclonal component reduction50% versus none in the control group, and 25% of patients suffered disease progression (MC increase50% and/or osteolytic lesions) in the IFN group as compared to 18% in the control group.alpha-IFN administration at the dose used is ineffective for the majority of patients with slowly proliferating immunoproliferative disorders; only a small subgroup of them may benefit from such a treatment.

A series of immunological abnormalities has been described in patients with beta-thalassemia. The aim of this study was to investigate whether the measurement of serum levels of selected cytokines and soluble molecules (deriving from cell membrane antigens) involved in the immune response could be useful for a better definition of such alterations.Serum levels of interleukin-2 (IL-2), IL-6, tumor necrosis factor (TNF), soluble (s) CD4, sCD8, sCD23 and sCD25 were measured using immunoenzymatic assays in 45 transfusion-dependent patients affected by beta-thalassemia major and correlated to conventional immunological indexes, such as peripheral lymphocyte subpopulations and circulating immunoglobulins.Patients with beta-thalassemia major showed increased TNF, sCD8, sCD23 and sCD25 and lower sCD4 values compared to normal controls. IL-2 and IL-6 were found to be undetectable or within the normal range in all patients. Splenectomized patients presented lower levels of sCD8 and sCD23 than those observed in unsplenectomized ones. A series of correlations involving TNF, sCD8, sCD23, sCD25, serum immunoglobulins and some lymphocyte subpopulations was observed. In addition, serum markers of immune activation (TNF, sCD23, sCD25) correlated directly with the annual blood transfusion requirement. Despite this series of immunological anomalies, no patient had a history of repeated infectious episodes.Polytransfused beta-thalassemic patients are characterized by a partial functional immunodeficiency determined by increased activity of CD8+ suppressor/cytotoxic lymphocytes and possibly reduced activity of the CD4+ helper/inducer subset. B-lymphocytes also appear highly activated. The allo-antigenic stimulation of transfusions seems to play a major role in the determination of these defects; however, this functional immunological imbalance does not seem to have any clinical relevance.

BACKGROUND. Tumor necrosis factor (TNF) and interleukin-1 beta (IL-1) are two cytokines with erythropoietic inhibitory activity which may be involved in the pathogenesis of some types of anemia that may respond to recombinant erythropoietin (r-EPO). The aim of the present study was to evaluate whether TNF and IL-1 serum levels are related to clinical response in patients with myelodysplastic syndromes (MDS) receiving r-EPO. TNF and IL-1 serum levels were measured by means of immunoenzymatic assays in 26 patients affected by MDS and treated with r-EPO administered subcutaneously at dosages up to 1050 U/kg a week, for at least two months. Four patients (15%) showed a significant response, with an increase of hemoglobin2 g/dL and complete suspension of transfusions. Higher mean serum levels of both TNF (54.2 +/- 93 vs 4.2 +/- 7.9 pg/mL, p0.001) and IL-1 (114 +/- 58.5 vs 36.1 +/- 21.7 pg/mL, p0.001) were measured in MDS patients than in a group of 42 normal controls. However, responders showed significantly lower mean levels of TNF (8.2 +/- 9.6 vs 58.5 +/- 65.2 pg/mL, p0.05) and IL-1 (30 +/- 24.8 vs 127.8 +/- 51.4 pg/mL, p0.001) than those of non responders. In terms of absolute values, all responders evidenced undetectable or normal levels of both cytokines. No relationship was found between TNF or IL-1 and values of hemoglobin, serum erythropoietin, ferritin, soluble transferrin receptor or transfusional requirements. MDS patients who respond to r-EPO have lower serum levels of TNF and IL-1 than those who do not respond.

We describe the case of an infant with immune thrombocytopenia whose bone marrow showed an increased percentage of CD10/TdT-positive lymphoid cells that resembled the onset of an acute lymphoproliferative disorder. Genotypic analysis of bone marrow, however, failed to reveal the malignant origin of these B cell precursors. After 8 months of follow-up, the child is alive and well, and shows a chronic form of ITP. Although a relation between this B cell proliferation and the onset of ITP cannot be excluded, it is important to consider this atypical pattern as a benign hematologic condition.

Neuroleptic malignant syndrome (NMS) in its most severe form ranging from disorders of consciousness to coma is now reported relatively frequently in intensive care journals. Having observed such a case in the intensive care ward of their hospital, the Authors investigate the possibility of differential diagnosis between this syndrome and other prevalently psychiatric and non-psychiatric types. It is underlined that the diagnosis of NMS is not straightforward taking into account both the type of patients who are mainly affected, that is to say psychopathic subjects undergoing prolonged therapy with heavy tranquillisers, in poor general condition, sometimes abandoned and then found in coma, and the fact that some psychiatric syndromes or drug overdoses may led to similar symptoms but require completely different treatment.

P-170 glycoprotein is the phenotypic marker of multidrug resistance (MDR), and its detection may have relevance in identifying patients at risk of chemo-resistance. The expression of P-170 glycoprotein has been analyzed by the APAAP technique and monoclonal antibody C219 (which recognizes a cytoplasmic epitope of P-170) on bone marrow smears from 20 patients affected by responsive multiple myeloma. The study was performed longitudinally in the different phases of the disease, with specific regard to the remission phase. One of the patients evidenced a small number of P-170 positive plasma cells at diagnosis. Three of the patients showed scattered P-170 positive plasma cells during remission, which were also often positive for the nuclear proliferation-associated antigen recognized by monoclonal antibody Ki 67, as demonstrated by double immunostaining; all these subjects rapidly relapsed, expressing a MDR phenotype and resistant disease. Among the remaining patients, 5 are still in remission phase, 6 have relapsed without the MDR phenotype, achieving a second response to chemotherapy, 6 have had a resistant relapse with more than 80% of P-170 positive plasma cells in 3 cases. The presence of P-170 positive plasma cells during remission phase in multiple myeloma might identify a group of patients with high risk of early, resistant relapse.

The use of monoclonal antibodies has been recently proposed as a more sensitive technique than conventional cytochemistry in recognizing myeloperoxidase (MPO) in leukemic cells.In order to verify the diagnostic relevance of such an approach, the immunocytochemical detection of MPO by means of the APAAP technique and a monoclonal antibody directed against an antigenic determinant of MPO (MPO7) was performed in 10 patients with undifferentiated or biphenotypic (myeloid and lymphoid) acute leukemia. All the cases were negative for conventional cytochemical staining of MPO.Positive cells (15 to 65%) were found in three patients. Some clinical data and biomolecular studies of T-cell receptor and immunoglobulin genes, available in five patients, indirectly confirmed these findings.Although some technical and biological aspects remain to be better characterized, immunocytochemical methods which utilize monoclonal antibodies against MPO may represent a simple and reliable tool for improving the diagnostic capacity to identify undifferentiated myeloid blasts in difficult cases of acute leukemia.