Attilio Gabbas

Rabbit antithymocyte globulin (r‐ATG) plus cyclosporine and granulocyte colony stimulating factor is an effective treatment for aplastic anaemia patients unresponsive to a first course of intensive immunosuppressive therapy

About 30% of patients with severe aplastic anaemia (SAA) unresponsive to one course of immunosuppressive (IS) therapy with antithymocyte or antilymphocyte globulin can achieve complete or partial remission after a second IS treatment. Among various second‐line treatments, rabbit ATG (r‐ATG) could represent a safe and effective alternative to horse ALG (h‐ALG). In a multicentre study, 30 patients with SAA (17 males and 13 females, median age 21 years, range 2–67) not responding to a first course with h‐ALG plus cyclosporin (CyA) and granulocyte colony stimulating factor (G‐CSF), were given a second course using r‐ATG (3.5 mg/kg/d for 5 d), CyA (5 mg/kg orally from day 1 to 180) and G‐CSF (5 μg/kg subcutaneously from day 1 to 90). The median interval between first and second treatment was 151 d (range 58–361 d). No relevant side‐effects were observed, but one patient died early during treatment because of sepsis. Overall response, defined as transfusion independence, was achieved in 23/30 (77%) patients after a median time of 95 d (range 14–377). Nine patients (30%) achieved complete remission (neutrophils 2.0 × 109/l, haemoglobin 11 g/dl and platelets 100 × 109/l). The overall survival rate was 93% with a median follow‐up of 914 d (range 121–2278). So far, no patient has relapsed. Female gender was significantly associated with a poorer likelihood to respond (P = 0.0006). These data suggest that r‐ATG is a safe and effective alternative to h‐ALG for SAA patients unresponsive to first‐line IS treatment.

AIDA 0493 protocol for newly diagnosed acute promyelocytic leukemia: very long-term results and role of maintenance

All-trans-retinoic acid (ATRA) has greatly modified the prognosis of acute promyelocytic leukemia; however, the role of maintenance in patients in molecular complete remission after consolidation treatment is still debated. From July 1993 to May 2000, 807 genetically proven newly diagnosed acute promyelocytic leukemia patients received ATRA plus idarubicin as induction, followed by 3 intensive consolidation courses. Thereafter, patients reverse-transcribed polymerase chain reaction-negative for the PML-RARA fusion gene were randomized into 4 arms: oral 6-mercaptopurine and intramuscular methotrexate (arm 1); ATRA alone (arm 2); 3 months of arm1 alternating to 15 days of arm 2 (arm 3); and no further therapy (arm 4). Starting from February 1997, randomization was limited to ATRA-containing arms only (arms 2 and 3). Complete remission was achieved in 761 of 807 (94.3%) patients, and 681 completed the consolidation program. Of these, 664 (97.5%) were evaluated for the PML-RARA fusion gene, and 586 of 646 (90.7%) who tested reverse-transcribed polymerase chain reaction-negative were randomized to maintenance. The event-free survival estimate at 12 years was 68.9% (95% confidence interval, 66.4%-71.4%), and no differences in disease-free survival at 12 years were observed among the maintenance arms.

Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias

In this study, gradient echo T2* magnetic resonance imaging provided a rapid and reproducible method for detecting myocardial iron overload which developed after a heavy transfusion burden equal to or greater than 290 mL/kg of packed red blood cell units. Only limited data are available regarding myocardial iron overload in adult patients with transfusion dependent acquired anemias. To address this topic using MRI T2* we studied 27 consecutive chronic transfusion dependent patients with acquired anemias: (22 myelodysplastic syndrome, 5 primary myelofibrosis). Cardiac MRI T2* values obtained ranged from 5.6 to 58.7 (median value 39.8) milliseconds. Of the 24 analyzable patients, cardiac T2* correlated with transfusion burden (p=0.0002). No patient who had received less than 290 mL/kg of packed red blood cells (101 units=20 grams of iron) had a pathological cardiac T2* value (< 20 ms). All patients who had received at least 24 PRBC units showed MRI T2* detectable hepatic iron (liver T2* value ≤6.3 ms). Only patients with severe hepatic iron overload (T2* <1.4 ms) showed cardiac T2* value indicative of dangerous myocardial iron deposition. Serum ferritin was not significantly correlated with cardiac T2* (p=0.24). Gradient echo T2* magnetic resonance imaging provides a rapid and reproducible method for detecting myocardial iron overload which developed after a heavy transfusion burden equal to or greater than 290 mL/kg of packed red blood cell units.

Interleukin‐1B (IL1B) and interleukin‐6 (IL6) gene polymorphisms are associated with risk of chronic lymphocytic leukaemia

Common polymorphisms in genes encoding for cytokines implicated in the inflammatory response and Th1/Th2 balance might play a role in the development and prognosis of chronic lymphocytic leukaemia (CLL). To test the hypothesis, we investigated 13 single nucleotide polymorphisms (SNPs) in nine of such genes in a population‐based case‐control study, conducted in the Italian region of Sardinia in 1999–2003. Forty incident CLL cases and 113 population controls were available for study. The following SNPs were selected: IL1A‐889C > T, IL1RN 9589A > T, IL1B‐31C > T, IL1B‐511C > T, IL2‐384T > G, IL6‐174G > C, IL6‐597G > A, IL10‐1082A > G, IL10‐3575T > A, TNF‐308G > A, LTA‐ 91A > C, LTA 252A > G and CARD15 nt1007. After adjusting by age and gender, individuals homozygous for the IL1B‐511T allele run a lower risk of CLL (OR = 0.1, 95% CI 0.0, 0.8, p = 0.032), while risk showed a 4.5‐fold increase associated with the genotype homozygous for the IL6‐174C allele (OR = 4.5; 95% CI 1.1, 19.3, p = 0.041). Individuals homozygous for the IL6‐174C allele and carrying the homozygous IL1B‐511C allele showed an 11‐fold increase in CLL risk (OR = 11.4, 95% CI 1.9, 69.4, p = 0.008). None of the other interleukin SNPs evaluated showed any association with CLL risk. Large multicentre pooled studies are warranted, achieving the statistical power required to confirm whether IL6 and IL1B gene polymorphisms might play a role in CLL development and prognosis, as well as the null associations herein reported. Copyright

Genetic analysis of the 2q33 region containing CD28-CTLA4-ICOS genes: association with non-Hodgkin's lymphoma

There is strong evidence that altered immunological function entails an increased risk of lymphoma, although the current knowledge of aetiological factors for lymphomas is limited. The CTLA4 gene encodes a receptor that provides a negative signal to the T‐cell once an immune response is initiated and completed. We analysed the 2q33 chromosomal region harbouring CD28, CTLA4 and ICOS genes, which are closely linked and have related functions in immune regulation, for association in 100 non‐Hodgkins lymphoma (NHL) patients and in 128 healthy controls; both groups originated from Sardinia. There was a strong association of the CTLA4 49A and the 3′‐untranslated region (AT)82 alleles with NHL [odds ratio (OR) = 2, 95% confidence interval (CI) = 1·2–3·2, and OR = 1·6, 95% CI = 1·1–2·4 respectively]. CTLA4‐318C:49A:(AT)82 was the most represented haplotype in the studied population and was associated with NHL (P = 0·0029, OR = 1·76, 95% CI = 1·2–2·5). Strong linkage disequilibrium was detected between CD28, CTLA4 and ICOS and a ‘common’ haplotype was found very frequently among NHLs. However, no independent association between CD28, ICOS, D2S72 markers and NHL was observed. Our findings enable CTLA4 from adjacent functionally related genes as the true causative risk gene for NHL susceptibility at least in Sardinian patients.

Risk of malignant lymphoma following viral hepatitis infection

We investigated lymphoma risk following hepatitis infection in a case-control study of 274 incident lymphoma cases, defined according to the WHO classification, and 336 population controls in Sardinia, Italy. Part of our study population (198 cases and 219 controls) was included in the EPILYMPH study of Hepatitis C virus (HCV) infection in relation to non-Hodgklin’s lymphoma risk. Based on questionnaire information on whether and at what age a diagnosis of hepatitis was posed by a physician, systematic anti-HCV antibodies testing in cases and controls by enzyme-linked immunoassay, and HCV-RNA assessment by PCR analyses in positive samples, we investigated more in detail whether hepatitis non-C is also associated with lymphoma risk, and whether risk varies by clinical form of hepatitis (acute or chronic infection). After adjusting by age, gender, education, and area of birth whether from the study area or elsewhere in Italy, a previous generic diagnosis of hepatitis was associated with a significantly elevated lymphoma risk [odds ratio (OR) = 1.8; 95% CI 1.1, 2.8], which was equally increased for hepatitis B (OR = 1.8; 95% CI 0.9, 3.5), for HCV positive subjects overall (OR = 2.0; 95% CI 0.8, 4.8), and for hepatitis non-B non-C (OR = 1.6; 95% CI 0.7, 3.9). Once concurrent infection from other hepatitis viruses was excluded, acute or chronic hepatitis C was the only one showing a consistent risk increase in all lymphoma subtypes, but follicular lymphoma. Some indications of an excess risk of lymphoma were observed also for acute, but not chronic forms of hepatitis B and hepatitis non-B, non C. Self-limited hepatitis C did not show an association. No significant heterogeneity in the risk of major lymphoma subtype was observed. Our results confirm a role of either acute or chronic active HCV infection in lymphomagenesis. Further studies are warranted to test the hypothesis that acute infection from other hepatitis viruses might also increase lymphoma risk.

A role of BRCA1 and BRCA2 germline mutations in breast cancer susceptibility within Sardinian population

BackgroundIn recent years, numerous studies have assessed the prevalence of germline mutations in BRCA1 and BRCA2 genes in various cohorts. We here extensively investigated the prevalence and geographical distribution of BRCA1-2 mutations in the entire genetically-homogeneous Sardinian population. The occurrence of phenotypic characteristics which may be predictive for the presence of BRCA1-2 germline mutations was also evaluated.MethodsThree hundred and forty-eight breast cancer patients presenting a familial recurrence of invasive breast or ovarian carcinoma with at least two affected family members were screened for BRCA1-2 mutations by DHPLC analysis and DNA sequencing. Association of BRCA1 and BRCA2 mutational status with clinical and pathological parameters was evaluated by Pearsons Chi-Squared test.Results and ConclusionOverall, 8 BRCA1 and 5 BRCA2 deleterious mutations were detected in 35/348 (10%) families; majority (23/35;66%) of mutations was found in BRCA2 gene. The geographical distribution of BRCA1-2 mutations was related to three specific large areas of Sardinia, reflecting its ancient history: a) the Northern area, linguistically different from the rest of the island (where a BRCA2 c.8764_8765delAG mutation with founder effect was predominant); b) the Middle area, land of the ancient Sardinian population (where BRCA2 mutations are still more common than BRCA1 mutations); and c) the South-Western area, with many Phoenician and Carthaginian locations (where BRCA1 mutations are prevalent). We also found that phenotypic features such as high tumor grading and lack of expression of estrogen/progesterone receptors together with age at diagnosis and presence of ovarian cancer in the family may be predictive for the presence of BRCA1-2 germline mutations.

Patients with Early-Stage Myelodysplastic Syndromes Show Increased Frequency of CD4+CD25high+CD127low Regulatory T Cells

Regulatory T cells (Treg) have often been ascribed a role in the pathophysiology of several neoplastic diseases considering their potential ability to suppress anti-tumor immunity. This is particularly the case in myelodysplastic syndromes (MDS), which are clonal hematologic disorders characterized by marked immune dysregulation. We analyzed Treg frequencies in a cohort of 36 patients with early-stage MDS using a flow-cytometric approach based on the concomitant expression of CD25 and CD127. MDS patients showed a higher frequency of CD4+CD25high+CD127low Treg than healthy controls (1.51 vs. 1.14%), with no specific effect of patient- and disease-related factors. Our data point to impaired anti-tumor immunity in patients with MDS, even in the early stage, which has already been noted in other clonal disorders.

Methylation analysis of the phosphates and tensin homologue on chromosome 10 gene (PTEN) in multiple myeloma

BackgroundAberrant DNA methylation of promoter region CpG islands is an alternative mechanism that leads to genetic defects in the inactivation of tumor suppressor genes during myelomagenesis. The aim of this study was to examine the promoter methylation status of the phosphates and tensin homologue on chromosome 10 (PTEN) gene in a cohort of multiple myeloma patients.FindingsThe PTEN gene was hypermethylated in 7 out of 58 (12%) primary myeloma samples. The correlation between functional inactivation and PTEN mRNA levels was not statistically significant. The multiple myeloma subgroup with an aberrant PTEN status had a prevalence of the component IgG, Salmon Durie stage I, lower lactate dehydrogenase levels, intermediate-standard cytogenetic risk and longer overall survival with the respect to the unmethylated subgroup.ConclusionsThis is the first report demonstrating the presence of PTEN promoter hypermethylation in multiple myeloma.

Risk of lymphoma subtypes and dietary habits in a Mediterranean area

BACKGROUND Previous studies have suggested that diet might affect risk of lymphoma subtypes. We investigated risk of lymphoma and its major subtypes associated with diet in the Mediterranean island of Sardinia, Italy. METHODS In 1998-2004, 322 incident lymphoma cases and 446 randomly selected population controls participated in a case-control study on lymphoma etiology in central-southern Sardinia. Questionnaire interviews included frequency of intake of 112 food items. Risk associated with individual dietary items and groups thereof was explored by unconditional and polytomous logistic regression analysis, adjusting by age, gender and education. RESULTS We observed an upward trend in risk of lymphoma (all subtypes combined) and B-cell lymphoma with frequency of intake of well done grilled/roasted chicken (p for trend=0.01), and pizza (p for trend=0.047), Neither adherence to Mediterranean diet nor a frequent intake of its individual components conveyed protection. We detected heterogeneity in risk associated with several food items and groups thereof by lymphoma subtypes although we could not rule out chance as responsible for the observed direct or inverse associations. CONCLUSIONS Adherence to a Mediterranean diet does not seem to convey protection against the development of lymphoma. The association with specific food items might vary by lymphoma subtype.