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SummaryWhat is known and objective Bacterial infections are the leading causes of morbidity and mortality in haematologic patients with chemotherapy-induced neutropenia. The only strategy shown to be effective in reducing febrile neutropenia incidence is fluoroquinolone prophylaxis, but the safety of this class of drugs in patients with glucose-6-phosphate dehydrogenase deficiency (G6PD−), the most common human enzyme defect, is still controversial because of the claimed association with acute haemolytic anaemia. Methods We retrospectively analysed 242 patients treated with 628 intensive chemotherapy courses. Of these, 59 patients were with G6PD−. All patients underwent fluoroquinolone prophylaxis and were transfused according to our single-unit transfusion policy. The principal endpoint was the incidence of acute haemolytic anaemia. Secondary endpoints included the incidence of febrile neutropenia, microbiologically and clinically documented infection (MDI and CDI) and the incidence of Gram-positive or Gram-negative infections. Results and discussions No episode of acute haemolytic anaemia was observed in the entire cohort. The incidence of MDI and CDI was similar, but the incidence of invasive fungal disease (IFD; P

Invasive fungal diseases (IFD) are still a leading cause of morbidity and mortality in patients with acute myeloid leukemia (AML). Glucose-6-phosphate dehydrogenase is an enzyme that leads to the production of NADPH, required to destroy microorganisms in the respiratory burst reaction of white blood cells. We evaluated the role of G6PD deficiency in susceptibility of IFD in 108 AML patients undergoing intensive chemotherapy. In all, 28 patients harbored G6PD deficiency (G6PD−), whereas 80 were normal (G6PD +). Incidence of IFD was significantly higher in G6PD− patients compared to G6PD + patients (35.7% vs. 5%, p = .0002, OR = 10, 95% CI = 2.96–37.5). Higher risk of mold infections (17.9% vs. 5%, p = .048, OR = 4.1, 95% CI = 1.0–16.6) and Candida sepsis (17.9% vs. 0%, p = .0009, OR = 37.68, 95% CI =2.0–707.1) was observed in G6PD − patients. The evaluation of G6PD activity may help to identify AML patients at higher risk of IFD, allowing to design more intensive surveillance and therapeutic strate...

Although a number of studies suggest that different immune pathways may play a role in the pathogenesis of non-Hodgkin's lymphomas (NHL), the shape of the T-cell compartment has been only superficially explored in these patients. In our study, we analyzed the peripheral T-cell receptor (TCR) repertoire and the distribution of different T-cell subsets - including regulatory T cells (Treg) - in 30 patients with NHL, by combining flow cytometry and spectratyping. We first demonstrated by flow cytometry an increased frequency of expanded T-cell subpopulations expressing the same TCR beta variable (BV) subfamilies in CD8+ cells from NHL patients when compared with healthy controls, beside a higher frequency of Treg. Moreover, NHL patients were characterized by a higher percentage of BVs showing a skewed CDR3 profile both in CD4+ and CD8+ cells when analyzed by spectratyping. Our data suggest that the T-cell branch of the immune system of patients with B-cell NHL is deeply deranged, as witnessed by the increased degree of activation and skewing of their TCR repertoire along with the higher frequency of Treg.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant disorder associated with a progression to multiple myeloma (MM). MM is a plasma cell malignancy with a chronic lymphoprol...

Because different findings suggest that an immune dysregulation plays a role in the pathogenesis of myelodysplastic syndrome (MDS), we analyzed a large cohort of patients from a homogeneous Sardinian population using ImmunoChip, a genotyping array exploring 147,954 single-nucleotide polymorphisms (SNPs) localized in genomic regions displaying some degree of association with immune-mediated diseases or pathways. The population studied included 133 cases and 3,894 controls, and a total of 153,978 autosomal markers and 971 non-autosomal markers were genotyped. After association analysis, only one variant passed the genome-wide significance threshold: rs71325459 ( p = 1.16 × 10 −12 ), which is situated on chromosome 20. The variant is in high linkage disequilibrium with rs35640778, an untested missense variant situated in the RTEL1 gene, an interesting candidate that encodes for an ATP-dependent DNA helicase implicated in telomere-length regulation, DNA repair, and maintenance of genomic stability. The second most associated signal is composed of five variants that fall slightly below the genome-wide significance threshold but point out another interesting gene candidate. These SNPs, with p values between 2.53 × 10 −6 and 3.34 × 10 −6 , are situated in the methylene tetrahydrofolate reductase ( MTHFR ) gene. The most associated of these variants, rs1537514, presents an increased frequency of the derived C allele in cases, with 11.4% versus 4.4% in controls. MTHFR is the rate-limiting enzyme in the methyl cycle and genetic variations in this gene have been strongly associated with the risk of neoplastic diseases. The current understanding of the MDS biology, which is based on the hypothesis of the sequential development of multiple subclonal molecular lesions, fits very well with the demonstration of a possible role for RTEL1 and MTHFR gene polymorphisms, both of which are related to a variable risk of genomic instability.

Even though the pathogenesis of myelodysplastic syndromes (MDS) is dominated by specific molecular defects involving hematopoietic precursors, also immune mechanisms seem to play a fundamental functional role. In this review we will first describe the clinical and laboratory autoimmune manifestations often detectable in MDS patients. We will then focus on studies addressing the possible influence of different immune cell subpopulations on the disease onset and evolution. We will finally consider therapeutic approaches based on immunomodulation, ranging from immunosuppressants to vaccination and transplantation strategies.

In the GIMEMA LAL 0904 protocol, adult Philadelphia positive acute lymphoblastic leukemia patients were treated with chemotherapy for induction and consolidation, followed by maintenance with imatinib. The protocol was subsequently amended and imatinib was incorporated in the induction and post-remission phase together with chemotherapy. Due to the toxicity of this combined approach, the protocol was further amended to a sequential scheme based on imatinib plus steroids as induction, followed by consolidation with chemotherapy plus imatinib and, when applicable, by a hematopoietic stem cell transplant. Fifty-one patients (median age 45.9 years) were enrolled in the final sequential protocol. At the end of induction (day +50), 96% of evaluable patients (n=49) achieved a complete hematologic remission; after consolidation, all were in complete hematologic remission. No deaths in induction were recorded. Overall survival and disease-free survival at 60 months are 48.8% and 45.8%, respectively. At day +50 (end of imatinib induction), a more than 1.3 log-reduction of BCR-ABL1 levels was associated with a significantly longer disease-free survival (55.6%, 95%CI: 39.0-79.3 vs. 20%, 95%CI: 5.8-69.1; P=0.03), overall survival (59.1%, 95%CI: 42.3-82.6 vs. 20%, 95%CI: 5.8-69.1; P=0.02) and lower incidence of relapse (20.5%, 95%CI: 7.2-38.6 vs. 60.0%, 95%CI: 21.6-84.3; P=0.01). Mean BCR-ABL1 levels remained significantly higher in patients who subsequently relapsed. Finally, BCR-ABL1p190 patients showed a significantly faster molecular response than BCR-ABL1p210 patients (P=0.023). Though the study was not powered to evaluate the role of allogeneic stem cell transplant, allografting positively impacted on both overall and disease-free survival. In conclusion, a sequential approach with imatinib alone in induction, consolidated by chemotherapy plus imatinib followed by a stem cell transplant is a feasible, well-tolerated and effective strategy for adult Philadelphia positive acute lymphoblastic leukemia, leading to the best long-term survival rates so far reported. (clinicaltrials.gov identifier: 00458848).

From 1980 to 1991, 6.3% of the adult population of the province of Sassari, Northern Sardinia, underwent voluntary beta-thalassemia screening. Of the 28,000 subjects examined, 15.7% proved to be heterozygotes for beta-thalassemia. In addition, the screening of 7500 students in 26 villages in Sassari province fixed the frequency of beta-thalassemia in this part of Sardinia at 10.4%. Of the 539 couples at risk to be expected from this figure, the screening detected 43% (234). The data suggest that inductive screening played a major role in the efficiency of this preventive beta-thalassemia program. Follow up of 221 pregnancies found to be at risk for homozygous beta-thalassemia and referred to the Antenatal Diagnosis Service, Cagliari, Southern Sardinia, showed that antenatal diagnosis was carried out in 80% of them. The overall percentage of couples refusing antenatal diagnosis was 10.8%, but over the years the acceptance rate for the procedure increased from 87% to 96%. Atypical hematological findings in 1.5% of 468 members of the couples at risk required globin chain synthesis and molecular analyses to define the precise beta-thalassemia genotype. Heterogeneous "mild" beta-thalassemia mutations as well as coexisting delta-thalassemia were found in silent type I and type II beta-thalassemia carriers which, without chain synthesis and DNA investigations, would have escaped detection.

The T-cell receptor (TCR) is the key player within the so called immunological synapse and the analysis of its repertoire offers a picture of both versatility and wideness of the whole immune T-cell compartment. Among the different approaches applied to its study the so-called spectratyping identifies the pattern of the third complementarity determining region (CDR3) length distribution in each one of the beta variable (TRBV) subfamilies encoded by the corresponding genes. This technique consists in a CDR3 fragment analysis through capillary electrophoresis, performed after cell separation, RNA extraction and reverse transcriptase PCR. This review will run through the most relevant studies which have tried to dissect the TCR repertoire usage in patients with different immune-mediated and infective diseases as well as solid or haematologic malignancies.

Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia show several immunological abnormalities. In this clinical setting, by combining flow cytometry and CDR3 spectratyping we monitored the kinetic of the T-cell repertoire during Azacitidine treatment, in order to explore its potential ability to reverse the immune derangement typical of these disorders. We firstly demonstrated by flow cytometry an increase in both CD4+ and CD8+ T-cell frequencies after starting treatment. Moreover, when monitored by spectratyping our patients showed significant changes in their T-cell receptor (TCR) CDR3 profiles, which were much more evident in helper T-cells. In fact, the frequency of BV (beta variable) subfamilies showing a skewed CDR3 profile significantly decreased from baseline to the following evaluations in CD4+ T-cells (81% vs. 70%). This pattern was even more pronounced in patients responding to Azacitidine (90% vs. 61%). Our data show that the overall derangement of the T-cell repertoire detectable in patients with MDS and AML with multilineage dysplasia gradually improves during Azacitidine treatment. These findings therefore suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these hematologic disorders.

BACKGROUND: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. PATIENTS AND METHODS: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CML WP over a 40-year period. RESULTS: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P < 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P < 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P < 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). CONCLUSIONS: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome. CLINICAL TRIAL NUMBERS: NCT00510926, NCT00514488, NCT00769327, NCT00481052. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Fludarabine and Cytarabine Combination in the Induction of Adult Patients with Acute Myeloid Leukaemia Francesco Longu;Claudio Fozza;Laura Dessi;Maurizio Longinotti;Silvana Bonfigli;Maria Careddu;Lorenzo Coppola;Domenica Giannico;Rosa Nieddu;Luigi Podda;Simonetta Pardini;Fausto Dore;Simone Dore;Giovanni Sotgiu; Acta Haematologica

The T-cell receptor (TCR) Vβ gene usage of CD4 + and CD8 + T-cell subpopulations was evaluated by flow cytometric analysis and by CDR3 spectratyping in healthy subjects belonging to Sardinian population, which is ethnically homogeneous and genetically distant from all other Italian and Caucasoid groups. As described in healthy Caucasian subjects, we found a nonrandom Vβ gene usage and in some Vβ families a significant skewed reactivity toward CD4 + T cells. Moreover, different subjects showed expansions in some Vβ subfamilies, mainly in the CD8 + T cells. By CDR3 spectratyping analysis we found a significantly higher degree of skewness in the TCR Vβ repertoire of CD8 + than in that of CD4 + T cells. The similarity found in the TCR Vβ gene usage between the population examined and other Caucasoid groups suggest that the shape of the TCR repertoire is more influenced by rearrangement processes than ethnic background. However, genetic polymorphisms may condition the expression levels of some Vβs, determining the variability of the TCR repertoire between different populations. Finally, the profound perturbations evidenced in the CD8 + T cell subpopulation could be related to a different response to the antigenic stimulation between CD8 + and CD4 + T lymphocytes.

Clinical data suggest that in 8-thalassemia-intermedia patients, higher levels of circulating fetal hemoglobin (HbF) are associated with greater disease severity at comparable degrees of anemia. We assessed the influence of the amount of circulating HbF on serum erythropoietin (S-Epo) levels and on serum transferrin receptor, a measure of erythropoiesis, in 30 8-thalasemia-intermedia patients. Twenty-four showed more than 40% HbF (21 of whom with B-thalassemia) and 6 presented lower HbF levels (p+ -thalassemia). The two groups of patients did not differ in age (1 5.3 v 19 years, respectively) or degree of anemia (Hb = 8.8 g/dL in both groups). Log (S-Epo) was correlated inversely with Hb (r = - 0.47; P < .Ol), and directly with HbF (r = .55; P < .OOl). Multivariate regression analysis showed that Hb and HbF were independently correlated ETA-THALASSEMIA intermedia is a genetically heterogeneous disorder defined according to degree of anemia that allows patients to survive without blood transfusions and grow without severe bone deformities or impairment of sexual development.’,* Nevertheless, the phenotype of this disorder varies with respect to age of first symptoms, splenomegaly, jaundice, cardiomegaly, and the incidence of extramedullary erythropoiesis.“’ The expansion of erythropoiesis, resulting from an erythropoietin drive secondary to chronic anemia, is recognized as the most important determinant of the phenotypic expression of the disease; however, it has also been claimed that increased amounts of circulating fetal hemoglobin (HbF) may exert an independent effect on erythroid expansion.’-5 HbF, in fact, shifts the oxygen dissociation curve of the blood towards higher affinit~,~.~ thus potentially producing a functional anemia and an adjunctive stimulus to erythropoiesis. Even though patients with high HbF have been reported to have earlier disease presentation3 and higher incidences of extramedullary erythropoiesis4 and splenomegaly3 than those with low HbF at comparable levels of anemia, the influence of HbF on the regulation of erythropoiesis in @-thalassemia intermedia has never been quantitatively investigated. This is of overwhelming importance in view of the recent introduction of therapies aimed at increasing the production of HbF to ameliorate the anemia and the symptoms of &globin In this study we assessed the influence of high HbF levels on the regulation of erythropoiesis in p-thalassemia intermedia. We evaluated serum erythropoietin (S-Epo) in relation to the degree of anemia in 30 patients with different HbF concentrations. Moreover, because measurement of serum transfemn receptor (S-TfR) has recently been proposed as a convenient method for monitoring erythropoiesis,” we determined S-TfR levels and correlated them with that of s-Epo. The results of this investigation show that, in &thalassemia intermedia patients, HbF values greater than 40% are responsible for higher S-Epo activity than expected for the degree of anemia. This produces an oversetting of erythropoiesis to a degree that may be clinically significant.

In this hospital-based, multicenter case-control study we investigated the prevalence of hepatitis B virus (HBV)-related markers and HBV/hepatitis C virus (HCV) co-infection among B-cell non-Hodgkin's lymphoma (B-NHL) cases and controls. Four hundred newly diagnosed B-NHL cases and 392 controls from other departments of the same hospitals were studied. The prevalence of positivity for hepatitis B surface antigen (HBsAg) was 8.5% among B-NHL cases and 2.8% among controls (adjusted odds ratio, 3.67; 95% confidence interval, 1.75-7.66). HBV/HCV co-infection was found in four cases, but in no controls. The finding of a positive association between HBV infection and B-NHL raises the possibility that HBV may play an etiologic role in the induction of B-NHL.

Neurological and skin involvements usually dominate the clinical presentation of intravascular lymphomatosis (IL), while fever is the most frequent general sign. However, an onset only characterized by fever of unknown origin (FUO) has been rarely reported. We would like to describe a further case of IL, which presented a long-lasting FUO before the diagnosis. At admission, physical examination detected hepatosplenomegaly without lymph nodes enlargement or dermatological or neurological abnormalities. Significant laboratory data included severe anemia, leukopenia, thrombocytopenia, and increased serum LDH. Moreover, a chest CT evidenced bilateral multiple pulmonary infiltrates and pleural effusion. After the development of proteinuria, a diagnosis of large B-cell intravascular lymphoma was made with a renal biopsy 10 months after the onset of the clinical manifestations. So far, more than 100 cases of IL have been reported and the diagnosis often turned out to be difficult, as clinical signs did not point to a lymphoproliferative disorder. This report confirms that FUO is not only frequently associated with IL but that it even marks the real onset of the disease. We are then tempted to conclude that undiagnosed fever is not so rare in IL and if we call it FUO, it is only because diagnosis is necessarily elusive and hence time-consuming.

We describe 2 patients with multiple myeloma who had horn-like filiform spicules in the follicular orifices of the face, particularly on the nowe, and multiple small ulcerations on the trunk. In the first patient, histopathologic study of a specimen from the nose showed follicular plugs of compact homogeneous eosinophilic material filling the intercellular spaces surrounding the keratinocytes. The same eosinophilic deposits were seen in the ulcer. In the second patient, biochemical investigation revealed that skin matter from spicules and ulcers were made up of monoclonal dysprotein with electrophoretic characteristics identical to those found in patient serum.

The existence of an association between infection with hepatitis C virus (HCV) and B-cell non-Hodgkin lymphoma (B-NHL) remains controversial, largely because previous studies were based on prevalent case series or comparisons with less than optimal control groups. This hospital-based case-control study was conducted from January 1998 through February 2001 to evaluate the association between HCV infection and B-NHL of different types. Cases were consecutive patients with a new diagnosis of B-NHL; controls were patients from other departments of the same hospitals. Both groups were interviewed using a standardized questionnaire. The prevalence of HCV infection was calculated by histologic type of B-NHL and clinical behavior (indolent or aggressive). Adjusted odds ratio (OR) and HCV-attributable risk (AR) were estimated. HCV prevalence was 17.5% among the 400 lymphoma patients and 5.6% among the 396 controls. The OR of B-NHL (patients vs controls), adjusted by age, sex, level of education, and place of birth, was 3.1 (95% confidence interval [CI], 1.8-5.2); an OR indicative of positive association was found for indolent and aggressive B-NHL. The estimated AR was 4.6%. This study confirms an association between HCV and B-NHL. In Italy, 1 of 20 instances of B-NHL may be attributable to HCV infection and may, thus, benefit from antiviral treatment.

Repeated PCR analysis was performed on bone marrow and/or peripheral blood samples from 4 CML patients in complete cytogenetic remission during treatment with IFN-alpha. Two patients became PCR-negative. One was negative for the analyses carried out from the 9th to the 30th months, but reverted to PCR positivity 10 months after IFN was reduced from 1.5 x 10(6) IU/day to 1 x 10(6) IU and given on alternate days. Although the dose was again raised to 3 x 10(6) IU/day, 8 months later her peripheral blood cells were still PCR-positive, but remained persistently Ph'-negative. Another patient became PCR-negative at the 42nd month and remained so at the last analysis performed 3 months later. Two patients were persistently PCR-positive. Cytogenetic relapse was documented in both, in one while still on full therapy. Ph'-positive metaphases reappeared in the other patient 7 months after discontinuing IFN-alpha therapy.

Serum concentrations of erythropoietin (EPO) were determined by immunoassay in 45 patients with thalassemia intermedia (TI). The mean serum level of EPO was significantly higher in the thalassemic patients than in the controls, but transfused subjects had lower pretransfusional serum concentrations of EPO than untransfused ones. An inverse relationship between the serum values of EPO and total hemoglobin was observed only in the untransfused thalassemic patients. These data suggest that in TI, even a low transfusional regimen may cause a decrease in serum concentration of EPO, independent of the level of total Hb.

In this study, we investigated the clinical and hematological features and carried out alpha- and beta-globin gene analyses in 11 Sardinian adult beta zero-thalassemia homozygotes from Northern Sardinia who were not transfusion-dependent. Oligonucleotide analysis revealed in nine out of 11 patients the nonsense mutation at codon 39, which was associated either with haplotype II or IX (14/16 and 2/16 chromosomes, respectively). Haplotype II was linked to the A gamma T mutation. The G gamma globin level ranged from 50 to 70%. Four out of nine patients (44%) were heterozygous and 3/9 (33%) homozygous for the rightward deletional type of alpha-thalassemia; two (22%) had the normal alpha-gene complement. Patients who were alpha-thalassemia homozygotes (-alpha/-alpha) showed a more balanced globin chain synthesis ratio. This study confirms that alpha-thalassemia may ameliorate the clinical picture of homozygous beta zero-thalassemia.