Long term persistence of SARS-CoV-2 humoral response in multiple sclerosis subjects.

BACKGROUND & OBJECTIVES: The persistence of the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 pandemic, partly due to the appearance of highly infectious variants, has made booster vaccinations necessary for vulnerable groups. Here, we present data regarding the decline of the SARS-CoV-2 BNT162b2 mRNA vaccine-induced humoral immune response in a monocentric cohort of MS patients. METHODS: 96 MS patients undergoing eight different DMTs, all without previous SARS-CoV-2 infection, were evaluated for anti-Spike IgG levels, 21 days (T1) and 5-6 months (T2) after the second SARS-CoV-2 BNT162b2 mRNA vaccine dose. The anti-Spike IgG titre from MS subjects was compared with 21 age- and sex-matched healthy controls (HC). RESULTS: When compared with SARS-CoV-2 IgG levels at T2 in HC, we observed comparable levels in interferon-ß 1a-, dimethyl fumarate-, teriflunomide- and natalizumab-treated MS subjects, but an impaired humoral response in MS subjects undergoing glatiramer acetate-, cladribine-, fingolimod- and ocrelizumab-treatments. Moreover, comparison between SARS-CoV-2 IgG Spike titre at T1 and T2 revealed a faster decline of the humoral response in patients undergoing dimethyl fumarate-, interferon-ß 1a- and glatiramer acetate-therapies, while those receiving teriflunomide and natalizumab showed higher persistence compared to healthy controls. CONCLUSION: The prominent decline in humoral response in MS subjects undergoing dimethyl fumarate-, interferon-ß 1a- and glatiramer acetate-therapies should be considered when formulating booster regimens as these subjects would benefit of early booster vaccinations.

Interferon Beta-1a treatment promotes SARS-CoV-2 mRNA vaccine response in multiple sclerosis subjects.

BACKGROUND: Several concerns exist on the immunogenicity of SARS-CoV-2 vaccines in multiple sclerosis (MS) subjects due to their immunomodulating disease modifying therapies (DMTs). Here we report a comparison of the humoral response to BNT162b2-mRNA coronavirus (COVID)-19 vaccine and the immunological phenotype in a cohort of 125 MS subjects undergoing different DMTs, with no history of SARS-CoV-2 infection. METHODS: We collected serum and blood samples at the first day of vaccine (T0) and 21 days after the second vaccine dose (T1) from 125 MS subjects, undergoing eight different DMTs. Sera were tested using the Elecsys anti-SARS-CoV-2-IgG assay for the detection of IgG antibodies to SARS-CoV-2 spike protein. The anti-spike IgG titres from MS subjects were compared with 24 age- and sex-matched healthy controls (HC). Percentage and absolute number of B and T lymphocytes were evaluated by cytofluorimetric analysis in the same study cohort. RESULTS: When compared with SARS-CoV-2 IgG levels in HC (n = 24, median 1089 (IQR 652.5-1625) U/mL), we observed an increased secretion of SARS-CoV-2 IgG in interferon-beta 1a (IFN)-treated MS subjects (n = 22, median 1916 (IQR 1024-2879) U/mL) and an impaired humoral response in MS subjects undergoing cladribine (CLAD) (n = 10, median 396.9 (IQR 37.52-790.9) U/mL), fingolimod (FTY) (n = 19, median 7.9 (IQR 4.8-147.6) U/mL) and ocrelizumab (OCRE) (n = 15, median 0.67 (IQR 0.4-5.9) U/mL) treatment. Moreover, analysis of geometric mean titre ratio (GMTR) between different DMT's groups of MS subjects revealed that, when compared with IFN-treated MS subjects, intrinsic antibody production was impaired in teriflunomide (TERI)-, natalizumab (NAT)-, CLAD-, FTY- and OCRE-, while preserved in DMF- and GA-treated MS subjects. CONCLUSION: Humoral response to BNT162b2-mRNA-vaccine was increased in IFN-treated MS subjects while clearly blunted in those under CLAD, FTY and OCRE treatment. This suggests that the DMTs could have a key role in the protection from SARS-CoV-2 related disease and complication in MS subjects, underlying a novel aspect that should be considered in the selection of the most appropriate therapy under COVID-19 pandemic.

Electrochemiluminescence immunoassay method underestimates cortisol suppression in ulcerative colitis patients treated with oral prednisone.

AIM: To evaluate cortisolemia by using conventional electrochemiluminescence immunoassay (ECLIA) method compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in active ulcerative colitis (UC) patients treated with oral prednisone (PD). METHODS: Twenty patients (12 males) with acute relapse of UC started oral PD at a dose of 40 mg once a day, tapered of 10 mg every 2 wk. When a stable 2-wk daily dose of 30 mg was reached, blood samples for cortisol levels' measurement were drawn in the morning in fasting conditions to determine circulating cortisol by LC-MS/MS and ECLIA assay. RESULTS: Median interquartile range cortisolemia with ECLIA and LC-MS/MS method was 54.1 (185.8) nmol/L and 32.1 (124.0) nmol/L, respectively (P < 0.001). The within-patient median differences between the two methods was 23.2 (40.6) nmol/L, with higher cortisol levels for the ECLIA method. The estimated geometric mean ratio between methods was 1.85 (95%CI: 2.39-1.43) considering all data or 1.58 (95%CI: 2.30-1.09) considering only data above the limit of quantification (n = 12). The 95%CIs of the geometric mean ratio between methods confirm a statistically significant difference. CONCLUSION: Blood cortisol levels detected with ECLIA method seems to be higher than the ones measured by LC-MS/MS, indicating a possible overestimation of them in patients treated with PD. Therefore, the cortisol suppression in patients under treatment with oral PD should not be measured using ECLIA method.

Comparison of fixed dose pegfilgrastim and daily filgrastim after autologous stem cell transplantation in patients with multiple myeloma autografted on a outpatient basis.

Different authors have explored the feasibility of autografting patients with multiple myeloma (MM) on an outpatient basis. Peg-filgrastim (PEG), a long-acting recombinant G-CSF, has similar efficacy when compared to conventional G-CSF for chemotherapy-induced neutropenia, but little is known about its use in the autologous stem-cell transplantation (ASCT) setting, namely in patients programmed to be autografted on outpatient basis. In this study, we compared therapeutic results in terms of hematopoietic recovery, non-hematologic toxicity, duration of hospitalization and percentage of hospital readmission between patients receiving either conventional G-CSF or PEG. Thirty-eight MM patients (48 autografts) received PEG, given at a single dose of 6 mg at day +5 from stem cell infusion, while 81 (113 autografts) received G-CSF from day + 2 up to stable neutrophil recovery. The conditioning regimen was high dose melphalan in all patients. The median age and the median number of CD34 + cell infused were comparable between the two groups. Overall, a second hospital admission was required in 36 procedures out of 161 (32%). Febrile neutropenia (FN) and severe mucositis were the most frequent causes of hospitalization. There was no statistically significant difference as percentage of hospital readmission is concerned: in the PEG group readmission was needed in 6 out of 48 autografts (12%) as opposed to 30 out of 113 (26%) in the G-CSF subgroup, p: 0.06. The median time of hospital stay for readmitted patients was identical for the two subgroups (9 days vs. 9 days, p: 0.94). Finally, one case of transplant related mortality occurred in the whole patient series (0.6%). In conclusion, ASCT on an outpatient basis is feasible and safe in patients with MM, the majority of whom are manageable at home. The administration of single dose PEG results in no different outcome in terms of safety and efficacy as compared to 8 days of G-CSF.

Favorable outcome in patients with acute myelogenous leukemia with the nucleophosmin gene mutation autografted after conditioning with high-dose continuous infusion of idarubicin and busulfan.

Mutations of the nucleophosmin gene (NPM1), in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) have impressive prognostic value in patients with acute myelogenous leukemia (AML), carrying normal karyotype (NK). In this study we describe treatment results from a series of 19 patients with NPM+/FLT3- autografted in first complete remission (CR) after conditioning with a regimen, named BuI, based on high-dose continuous infusion of idarubicin and Busulfan. Ninety-nine consecutive patients (median age of 54 years) with NK AML autografted in first CR were analyzed. Nineteen of 99 patients (19%) had NPM1 mutation in the absence of FLT3 mutations. The control group, accounting for 80 patients, included 16 cases (15%) with both mutations, 10 (12%) with FLT3/ITD mutation and no NPM mutation, and 54 (68%) in whom neither NPM1 nor FLT3 mutations were detectable. The median overall survival (OS) for the whole patient population was 34 months, the median disease-free survival (DFS) was 22 months. Median OS and DFS were significantly longer for patients with isolated NPM1 mutation as opposed to controls (OS: not reached versus 25 months, P = .02; DFS: not reached versus 16 months, P = .007, respectively). Of interest, patients with isolated NPM1 mutation had a better outcome in terms of either OS or DFS compared to the group of 16 NMP1+/FLT3+ patients. In conclusion, our study suggest that BuI regimen results in favorable clinical outcome in patients with isolated NPM1 mutation, and could be investigated in a randomized study versus other regimes or repeated courses of high dose cytosine-arabinoside.

Total serum tryptase: a predictive marker for KIT mutation in acute myeloid leukemia.

Human tryptase is a serine protease expressed in mast-cells. We previously observed that AML blast cells, cultured in vitro from a KIT D816Y patient, give rise to adherent cells with mast-cell like phenotype and tryptase was released in the serum-free medium. To correlate total serum tryptase (ts-try) levels with cytogenetic features and KIT mutational status, we analyzed serum samples from AML patients at diagnosis. In 70 out of 155 patients (45%) we detected elevated ts-try (>15 ng/mL), significantly linked to t(8;21) (P < .001) and inv(16) (P = .007). In patients that achieved complete remission the ts-try decreased to normal values. In 75 patients screened for KIT mutation, we found a clear relationship between elevated ts-try and mutated patients with t(8;21) (P < .001). In conclusion, we propose that checking for ts-try at diagnosis of AML may be a simple tool to select patients to be addressed to KIT mutation screening.

Autologous stem cell transplantation for elderly patients with acute myeloid leukaemia conditioned with continuous infusion idarubicin and busulphan.

Different studies have suggested the potential utility of autologous stem cell transplantation (ASCT) in acute myeloid leukaemia (AML) of the elderly with encouraging results in selected patients. However, while the introduction of peripheral blood stem cells (PBSC) has consistently reduced morbidity and mortality of the procedure, relapse still represents the major cause of ASCT failure. One possibility to ameliorate therapeutic results could rely on the adoption of conditioning regimens specifically designed for AML. We report therapeutic results from a series of 40 AML patients older than 60 years (median age 67 years) autografted in first complete remission (CR), after conditioning with continuous infusion (c.i.) high dose idarubicin and busulphan. Fourty patients (median age: 67 years) received 2 days c.i. idarubicin at 20 mg/m(2)/day, followed by 3 days oral or intravenous busulphan (4 mg/kg/day) as conditioning. No case of transplant-related mortality occurred. Cardiac toxicity was absent, while 31 patients (77%) had grade 3-4 mucositis. After a median follow-up of 25 months, median disease free and overall survival (OS) for the whole patient population were 13 and 22 months, respectively. Three patients died while in CR from causes unrelated to AML. Better results were achieved in patients with intermediate karyotype as opposed to those with adverse cytogenetics. Our data confirm the feasibility of a conditioning regimen based on high-dose idarubicin plus busulphan in older selected AML patients and suggest clinical improvement in patients with normal cytogenetics.

Molecular analysis of PDGFRA and PDGFRB genes by rapid single-strand conformation polymorphism (SSCP) in patients with core-binding factor leukaemias with KIT or FLT3 mutation.

BACKGROUND: Mutations involving KIT and FLT3 genes, encoding tyrosine kinase (TK) membrane receptors, are detected in core-binding factor leukaemia (CBFL) patients. PDFGRA and PDGFRB encode class III TK receptors and are involved both in physiological processes and in the pathogenesis of haematological and solid tumours. The aim of this study was to investigate if PDGFR mutations are involved in CBFL. PATIENTS AND METHODS: In order to detect PDGFR mutations in CBFL, 35 patients without KIT or FLT3 mutations patients were screened by rapid and sensitive single-strand conformation polymorphism (SSCP) analysis. Sequence analysis was performed in polymerase chain reaction (PCR) products showing altered mobility in SSCP analysis in order to determine the nucleotide changes. RESULTS: Three types of single-nucleotide polymorphism (SNP) were detected in the PDGFRA gene (exon 12, exon 13 and exon 18) while no mutation of PDGFRB was detected in the tested CBFLs. CONCLUSION: These data showed that no pathogenic mutations in PDGFRA and PDGFRB were detected in the context of CBFL without KIT and FLT3 mutations. Thus, PDGFR genes do not seem to be involved in CBFL and future studies are needed to establish the genetic causes of the disease in these particular patients.

Neapolis (CD 126 beta+ GGT->GGG): a result of a screening in Campania, a region in Southern Italy.

Between January 1995 and December 2005, we conducted a screening program for the presence of Hb Neapolis, a rare abnormal Hb variant, in Campania, a region in Southern Italy. Nineteen patients with Hb Neapolis in heterozygosis and six patients with a genetic compound (Hb Neapolis/beta-thalassemia) were identified. Patients with Hb Neapolis in heterozygosis showed a slight alteration in HbA2 levels while compounds showed typical characteristics of thalassemia intermedia ranging from a non transfusion-dependent form for five patients to a transfusion-dependent form for one adult patient.

Age has no influence on mobilization of peripheral blood stem cells in acute myeloid leukemia.

The upper age limit for autologous stem cell transplantation (ASCT) in acute myeloid leukemia (AML) is increasing and peripheral blood (PB) represents the standard source of stem cell (SC). However, no data are available on the impact of age on SC mobilization in AML. We analyzed a cohort of 150 consecutive AML patients in first complete remission in order to make a comparison between patients up to 60 years and above 60 years, by evaluating CD34+ cells mobilization into PB and the number of leukapheresis needed to collect at least one single SC graft. The successful mobilization rate (>2 x 10(6) CD34+ cells/kg) was comparable between the two groups (87% vs. 80%, p = 0.29). In addition, no statistically significant difference was found in terms of either median number of CD34+ cells collected (p = 0.54) or CD34+ cells peak in PB (p = 0.70). Both groups of patients needed a median of two apheresis and no difference was found in the median number of CD34+ cells collected per single apheresis (p = 0.67). Finally, no correlation was found between age and total number of CD34+ cells collected (r = 0.003, p = 0.58). We conclude that age has no impact on mobilization of PBSCs in AML.

Myeloablative chemotherapy followed by autologous stem cell infusion may overcome the adverse prognostic impact of FLT3 (foetal liver tyrosine kinase 3) mutations in patients with acute myeloid leukaemia and normal karyotype.

In this study, we analysed the prognostic relevance of foetal liver tyrosine kinase 3 (FLT3) mutations in 73 patients with acute myeloid leukaemia (AML) with normal karyotype, who survived induction and consolidation and received autologous stem cell transplantation (ASCT) after successful mobilization of peripheral blood stem cell (PBSC). There were 44 males and 29 females with a median age of 54 years (range 20-77). Overall, 16 out of 73 autografted patients (22%) had FLT3 mutations. More in detail, FLT3/ITDs were detected in 10 out of 73 patients (14%), while FLT3 D835 mutations were detected in five cases (7%). One patient (1%) was found as having both abnormalities. White blood cell count (p=0.009), serum concentration of lactate dehydrogenase (p=0.01), and percentages of peripheral blood (p=0.002) and bone marrow blasts (p=0.03) were significantly higher in patients showing the FLT3 mutations. On the contrary, overall survival and disease-free survival were similar between patients with or without FLT3 mutations (p=0.73 and 0.78, respectively). In conclusion, our data suggest that myeloablative chemotherapy supported by auto-PBSCT may overcome the adverse prognostic implications of FLT3 mutations in AML. However, it is to consider that autografted patients are highly selected for best response to induction, consolidation and mobilization, as well as for minor non-haematologic toxicity.

An antecedent diagnosis of refractory anemia with excess blasts has no influence on mobilization of peripheral blood stem cells and hematopoietic recovery after autologous stem cell transplantation in acute myeloid leukemia.

Several studies have reported data on factors influencing mobilization of peripheral blood stem cells (PBSC) in non-myeloid malignancies. On the contrary, data from patients with acute myeloid leukemia (AML) are very limited, in particular, as the impact of an antecedent diagnosis of refractory anemia with excess blasts (RAEB) on mobilization of PBSCs as well as hematopoietic recovery after autologous stem cell transplantation (ASCT) is concerned. We retrospectively analyzed a cohort of 150 consecutive AML patients in first complete remission in order to make a comparison between patients with de novo AML and secondary AML (s-AML) in terms of CD34 positive (CD34+) cells mobilization and number of leukapheresis needed to collect at least one single stem cell graft. Data concerning hematopoietic recovery after ASCT were also compared. The successful mobilization rate (>2 x 10(6) CD34+ cells/kg) was comparable between de novo AML patients (87%) and those with s-AML (76%), P:0.21. No statistically significant difference was found in terms of either median number of CD34+ cells collected (P:0.44) or CD34+ cells peak in peripheral blood (P:0.28). Both groups of patients needed a median of two apheresis (P:0.45) and no difference was found on the median number of CD34+ cells collected per single apheresis (P:0.59). Finally, neutrophil and platelet recovery after ASCT were comparable between the two groups. An antecedent diagnosis of RAEB has no impact on mobilization and collection of PBSCs in AML as well as on hematopoietic recovery after ASCT.

HFE gene mutations in patients with acute leukemia.

An increased incidence of HFE gene mutations has been described in hematologic malignancies. In the present study, we investigated the allelic frequency of HFE gene mutations in 154 adult patients with acute leukemia (AL) [107 acute myeloid leukemia (AML), 20 acute promyelocytic leukemia (APL) and 27 acute lymphoblastic leukemia (ALL)]. The allelic frequency of the H63D mutation was 29% in AL patients and 25% in the healthy controls [P = 0.41; odds ratio (OR) = 1.20; 95% confidence interval (CI) = 0.77 - 1.93]. No difference was found between controls and AML or APL patients, whereas the H63D mutation was significantly more frequent in ALL than controls (44% vs. 25%, P = 0.04; OR = 2.37; 95% CI = 1.05 - 5.36). The overall comparison of the mutation among the three subtypes of AL demonstrated a higher allelic frequency in ALL (P = 0.02). In conclusion, our data demonstrate a correlation between the presence of the H63D mutation and the occurrence of ALL in adult patients.

Vinorelbine plus intermediate dose cyclophosphamide is an effective and safe regimen for the mobilization of peripheral blood stem cells in patients with multiple myeloma.

High dose cyclophosphamide (HD-Cy) is commonly used to mobilize stem cells in multiple myeloma (MM). However, timing of collection is variable and incidence of side effects is substantial. We evaluated a combination of vinorelbine (VNB) (25 mg/m(2) day 1) plus Cy (1.5 g/m(2) day 2) and G-CSF as mobilizing regimen in 37 patients with MM. Results were compared to those achieved in 41 previously diagnosed patients mobilized with Cy at 4 g/m(2). Overall, 36/37 patients receiving VNB-Cy (97%) mobilized, as opposed to 40/41 (97%) in the controls (p:0.51). Median CD34+ cells peak was 94/mul for VNB-Cy patients and 96 for controls, p=0.36; median number of CD34+ cells collected was 9.2x10(6)/kg and 8.7x10(6)/kg, respectively (p=0.85). Median number of days to the highest CD34 count was shorter for VNB-Cy patients (nine vs 11, p=0.001). No VNB-Cy patient experienced grade 3-4 neutropenia and thrombocytopenia, as opposed to 63 and 19% in the controls (p=0.001 and 0.01, respectively). Hospitalization from toxicity was never required in VNB-Cy patients as compared to 19% in control group (p=0.01). We conclude that an outpatient combination of VNB plus intermediate dose Cy plus G-CSF is a safe, predictable, and highly effective mobilization regimen for patients with newly-diagnosed MM.

Therapeutic results in patients with relapsed diffuse large B cell Non-Hodgkin's lymphoma achieving complete response only after autologous stem cell transplantation.

Forty patients with relapsed diffuse large B cell lymphoma (DLBCL) autografted in partial response (PR) (n = 23) or in refractory relapse (RR) (n = 17) achieved complete remission (CR) after autologous stem cell transplantation (ASCT). Salvage treatment consisted of ifosphamide, epirubicin and etoposide (IEV) in 33 patients and Cisplatinum, ARA-C and dexamethasone (DHAP) in 7 patients. All PR and 8 RR patients were conditioned with BEAM, while 9 RR cases received the BCV regimen. There were no significant differences between the two groups as age, serum LDH, duration of CR1 and IPI at relapse are concerned. Relapse rate after ASCT was 39% in PR group as opposed to 88% in RR group (p = 0.003). Median relapse free survival from ASCT was 6 months for RR patients as opposed to 34 months for PR patients (p = 0.003); median overall survival from ASCT was 10 months for RR subset as opposed to not reached for RR subgroup (p = 0.001). These data demonstrate that CR achieved after ASCT in DLBCL patients who are refractory to previous salvage therapy does not result in long-term disease control. Alternative preparative regimens, allogeneic SCT and/or monoclonal antibodies in the post-ASCT phase should be considered for RR patients despite CR achievement.

Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21).

High-dose idarubicin and busulphan as conditioning to autologous stem cell transplantation in adult patients with acute myeloid leukaemia.

Between 30 and 50% of patients with acute myeloid leukaemia (AML) relapse after autologous stem cell transplantation (ASCT). One possibility of reducing the relapse rate could be the adoption of conditioning regimens specifically designed for AML. We report treatment results achieved with a new conditioning for ASCT, based on high-dose idarubicin (IDA) plus oral busulphan. Patients (n = 40) were conditioned with a regimen consisting of 3 d continuous intravenous infusion IDA at 20 mg/m2, followed by 4 d conventional dose oral busulphan. Unpurged peripheral blood stem cells were used in all cases. All patients had non-M3-AML and were in first complete remission (CR). The median number of CD34+ cells infused was 6.9 x 10(6)/l (2.6-24). No case of transplant-related mortality occurred. In all cases, left ventricular ejection fraction remained unmodified after ASCT. Thirty-three of 40 patients (82%) had grade 3-4 mucositis requiring total parenteral nutrition in all cases. After a median follow up for surviving patients of 32 months from ASCT, 30 patients (75%) are alive and 26 (65%) are in continuous CR. Our data show that a conditioning regimen based on high-dose IDA plus busulphan results in an encouraging reduction of the relapse rate after ASCT in AML.

Ifosfamide, epirubicin and etoposide (IEV) regimen as salvage and mobilization therapy for refractory or early relapsing patients with aggressive non-Hodgkin's lymphoma.

The prognosis of early relapsing or refractory aggressive non-Hodgkin's lymphoma (NHL) is still poor. Effective salvage therapy should be able to induce high response rate as well as to mobilize hematopoietic precursors. A combination of ifosfamide, epirubicin and etoposide (IEV) was given to 28 patients with refractory or relapsing high grade NHL (4 lymphoblastic lymphoma and 24 large cell lymphoma). All patients were evaluated for response. After 2 courses of IEV, the overall and complete response rate were 64% and 39%, respectively. All patients were controlled for mobilization of peripheral blood stem cells, which was successful in 26 out of 28 (93%). Overall, 25 out of 26 patients proceeded to autologous stem cell transplantation (ASCT). Toxicity was mild, with no occurrence of severe persisting extra-hematologic side-effects. Following the entire therapeutic program, including IEV and ASCT, median progression free survival has not yet been reached and 21 patients are alive (18 in continuous complete remission) after a median follow-up of 18 months. Our results demonstrate that treatment with IEV regimen is effective in refractory or relapsing aggressive NHL, resulting in a high percentage of successful stem cell mobilization and feasibility of ASCT.

Hb Cardarelli [beta86(F2)Ala-->Pro]: a new unstable and hyperaffine variant in association with beta(+)-thalassemia.

Hb Cardarelli [beta86(F2)Ala-->Pro] is a new unstable and high oxygen affinity variant found in several members of a family from Naples, Southern Italy. A detailed structural and functional characterization of the variant was performed on two subjects, at both the protein and DNA level. The first patient exhibited 43% of the variant hemoglobin (Hb) without major hematological problems. The proband showed 82% of the abnormal Hb in association with beta(+)-thalassemia (thal) that caused relevant erythrocytosis requiring frequent phlebotomies. Structural investigation of the Hb variant by mass spectrometric methodologies identified the amino acid replacement as Ala-->Pro at beta86. The corresponding DNA mutation GCC-->CCC at codon 86 of the beta-globin gene was assessed by both DNA sequencing and amplification refractory mutation system (ARMS) techniques. Functional studies carried out on whole blood and diluted hemolysates from both patients demonstrated increased oxygen affinity, decreased Bohr effect, reduced heme-heme interaction and nearly halved 2,3-diphosphoglycerate (2,3-DPG) and chloride effects.