Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

HLA-G molecules and clinical outcome in Chronic Myeloid Leukemia.

The human leukocyte antigen-G (HLA-G) gene encodes a tolerogenic protein known to promote tumor immune-escape. We investigated HLA-G polymorphisms and soluble molecules (sHLA-G) in 68 chronic myeloid leukemia (CML) patients. Patients with G*01:01:01 or G*01:01:02 allele had higher value of sHLA-G compared to G*01:01:03 (109.2±39.5 vs 39.9±8.8 units/ml; p=0.03), and showed lower event free survival (EFS) (62.3% vs 90.0%; p=0.02). The G*01:01:03 allele was associated with higher rates and earlier achievement of deep molecular response (MR)4.5 (100% vs 65%, median of 8 vs 58 months, p=0.001). HLA-G alleles with higher secretion of sHLA-G seem associated with lower EFS, possibly because of an inhibitory effect on the immune system. Conversely, lower levels of sHLA-G promoted achievement of MR4.5, suggesting increased cooperation with immune system.

T cell tyrosine phosphorylation response to transient redox stress.

Reactive Oxygen Species (ROS) are crucial to multiple biological processes involved in the pathophysiology of inflammation, and are also involved in redox signaling responses. Although previous reports have described an association between oxidative events and the modulation of innate immunity, a role for redox signaling in T cell mediated adaptive immunity has not been described yet. This work aims at assessing if T cells can sense redox stress through protein sulfhydryl oxidation and respond with tyrosine phosphorylation changes. Our data show that Jurkat T cells respond to -SH group oxidation with specific tyrosine phosphorylation events. The release of T cell cytokines TNF, IFNγ and IL2 as well as the expression of a number of receptors are affected by those changes. Additionally, experiments with spleen tyrosine kinase (Syk) inhibitors showed a major involvement of Syk in these responses. The experiments described herein show a link between cysteine oxidation and tyrosine phosphorylation changes in T cells, as well as a novel mechanism by which Syk inhibitors exert their anti-inflammatory activity through the inhibition of a response initiated by ROS.

Early Death in Two Patients with Acute Promyelocytic Leukemia Presenting the bcr3 Isoform, FLT3-ITD Mutation, and Elevated WT1 Level.

Despite major advances in the treatment of acute promyelocytic leukemia (APL), the problem of early death (ED) remains unsolved. Alongside the currently known clinical and hematological risk factors, prognostic significance has been attributed to internal tandem duplication mutations of the fms-like tyrosine kinase-3 (FLT3-ITD), hypogranular variant morphology, and the bcr-3 isoform of PML-RAR α . We describe premature death of two patients with the hypogranular variant of APL who presented remarkably high expression levels of Wilms' tumor gene (WT1). Our results point to WT1 as an important prognostic factor of ED that needs to be promptly evaluated in all newly diagnosed cases of APL.

Absence of activating killer immunoglobulin-like receptor genes combined with hepatitis C viral genotype is predictive of hepatocellular carcinoma.

Killer immunoglobulin-like receptors and their human leukocyte antigen class I ligands have a critical role in natural killer cell response to viral pathogens and tumors. To investigate whether killer immunoglobulin-like receptor genes could influence the chronic course of hepatitis C virus infection and/or progression to hepatocellular carcinoma we retrospectively analyzed a cohort of 228 patients transplanted for hepatitis C virus-induced cirrhotic end stage liver disease, combined or not with hepatocellular carcinoma. We found that patients completely lacking activating killer immunoglobulin-like receptor genes had a high risk of developing hepatocellular carcinoma. Hepatitis C viral genotype and viral load are other risk factors that can influence the course of chronic hepatitis C virus infection. In our study, the risk conferred by hepatitis C viral genotypes was enhanced in patients lacking activating killer immunoglobulin-like receptors. These results point to an important role for activating killer immunoglobulin-like receptors in the control of hepatitis C virus infection and progression to hepatocellular carcinoma. In clinical practice, assessment of killer immunoglobulin-like receptor and hepatitis C viral genotype combinations should allow for more accurate monitoring of patients with chronic hepatitis C virus infection.

Final results of a multicenter trial addressing role of CSF flow cytometric analysis in NHL patients at high risk for CNS dissemination.

This prospective study compared diagnostic and prognostic value of conventional cytologic (CC) examination and flow cytometry (FCM) of baseline samples of cerebrospinal fluid (CSF) in 174 patients with newly diagnosed aggressive non-Hodgkin lymphoma (NHL). FCM detected a neoplastic population in the CSF of 18 of 174 patients (10%), CC only in 7 (4%; P < .001); 11 patients (14%) were discordant (FCM(+)/CC(-)). At a median follow-up of 46 months, there were 64 systemic progressions and 10 CNS relapses, including 2 patients with both systemic and CNS relapses. Two-year progression-free and overall survival were significantly higher in patients with FCM(-) CSF (62% and 72%) compared with those FCM(+) CSF (39% and 50%, respectively), with a 2-year CNS relapse cumulative incidence of 3% (95% confidence interval [CI], 0-7) versus 17% (95% CI, 0-34; P = .004), respectively. The risk of CNS progression was significantly higher in FMC(+)/CC(-) versus FCM(-)/CC(-) patients (hazard ratio = 8.16, 95% CI, 1.45-46). In conclusion, FCM positivity in the CSF of patients with high-risk NHL is associated with a significantly higher CNS relapse risk and poorer outcome. The combination of IV drugs with a higher CNS bioavailability and intrathecal chemotherapy is advisable to prevent CNS relapses in FCM(+) patients.

Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes.

Allogeneic bone marrow transplantation (BMT) from a genotypically identical family donor is an accepted therapeutic option for homozygous beta-thalassemia. However, only a minority of patients have access to this curative procedure. The aim of this study is to explore the feasibility of matched unrelated transplants in thalassemia and the possibility of reducing the risk of immunologic complications through careful selection of donor/recipient pairs. Since November 1992, 32 patients (age range, 2-28 years) have been enrolled. There were 4 patients assigned to risk-class I, 11 to risk-class II, and 17 to risk-class III of the Pesaro classification. Extended haplotype analysis and family segregation studies were employed for identification of suitable donors. Of the 32 donor/recipient pairs, 24 were identical for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci; 7 pairs were identical for 2 extended haplotypes, and 15 pairs shared one extended haplotype. Grade II-IV acute graft-versus-host disease (GVHD) developed in 11 cases (41%) and chronic GVHD in 6 (25%) out of 24 patients at risk. There are 22 patients (69%) who are alive and transfusion-independent after a median follow-up of 30 months (range, 7-109 months). There were 6 patients (19%) who engrafted and subsequently died from transplant-related complications. In 4 cases (12.5%) graft rejection was observed within 30 days and it was followed by autologous reconstitution. Out of 22 patients with a donor identical for at least one extended haplotype, there are 19 who survived, 17 of them being transfusion-independent. Among the 10 recipients who did not share any extended haplotype with the donor, only 5 are alive without thalassemia and 3 patients died. Of the 6 patients who died, 5 belonged to risk-class III and only 1 to risk-class II. BMT from well-selected unrelated donors may offer results comparable to those obtained in transplantations using HLA-identical family donors, especially for patients with lesser iron overload.