Lorenza Borin

Valproic Acid at Therapeutic Plasma Levels May Increase 5-Azacytidine Efficacy in Higher Risk Myelodysplastic Syndromes

Purpose: Epigenetic changes play a role and cooperate with genetic alterations in the pathogenesis of myelodysplastic syndromes (MDS). We conducted a phase II multicenter study on the combination of the DNA-methyltransferase inhibitor 5-azacytidine (5-AZA) and the histone deacetylase inhibitor valproic acid (VPA) in patients with higher risk MDS. Experimental Design: We enrolled 62 patients with MDS (refractory anemia with excess blasts, 39 patients; refractory anemia with excess blasts in transformation, 19 patients; and chronic myelomanocytic leukemia (CMML), 4 patients) and an International Prognostic Scoring System (IPSS) rating of Intermediate-2 (42 patients) or high (20 patients). VPA was given to reach a plasma concentration of >50 μg/mL, then 5-AZA was added s.c. at 75 mg/m2 for 7 days in eight monthly cycles. Results: The median overall survival was 14.4 months. At a median follow-up of 12 months (range, 0.7-21.0), the disease progressed in 20 patients, with 21% cumulative incidence of progression. Of 26 patients who completed eight cycles, 30.7% obtained complete or partial remission, 15.4% had a major hematologic improvement, whereas 38.5% showed stable disease. Drug-related toxicity was mild. Favorable prognostic factors for survival were IPSS Intermediate-2 and plasma VPA of ≥50 μg/mL (log rank = 0.013 and 0.007, respectively). Analysis of polymorphisms important for the metabolism of the drugs used in the trial showed that carriers of the CYP2C19*2 variant of cytochrome P450 required higher VPA doses to achieve the target VPA plasma concentration of 50 μg/mL on day 1 of 5-AZA treatment (P = 0.0021). Conclusion: Our data show that the 5-AZA/VPA combination is active and safe in patients with MDS with a poor prognosis. Achievement of VPA therapeutic levels may indeed increase 5-AZA efficacy.

Deferasirox for transfusion-dependent patients with myelodysplastic syndromes: safety, efficacy, and beyond (GIMEMA MDS0306 Trial)

In the absence of randomized, controlled trial data to support iron chelation therapy in transfusion‐dependent patients with myelodysplastic syndromes (MDS), continued evidence from large prospective clinical trials evaluating the efficacy and safety of iron chelation therapy in this patient population is warranted.

Selective purging by human interleukin‐2 activated lymphocytes of bone marrows contaminated with a lymphoma line or autologous leukaemic cells

Summary The ability of recombinant interleukin 2 (rIL2) activated lymphocytes (LAK) to purge BM samples contaminated by tumour cells was evaluated. Human BM mononuclear cells were contaminated with 10% of the lymphoma line CA46 and then cultured in liquid medium containing 1000 U/ml of rIL2 and/or LAK autologous to the used BM. At the end of coculture the growth of residual tumour cells and of CFU‐GM were evaluated by clonogenic assay. No tumour cell growth was observed in 5/5 independent experiments after 18 h of coculture with LAK. No significant inhibition of CFU‐GM growth was also noted. Subsequently, the effect of LAK on BM obtained from four leukaemic patients and contaminated with 20–50% of their own AML and ALL cells was studied using MAb as a tool for identifying leukaemic cells. LAK eliminated 24–78% of contaminating cryopre‐served uncultured autologous leukaemic cells. In five cases the BM was contaminated by a low (2%) amount of ALL cells. In these patients the monoclonal heavy chain rearrangement typical of ALL was no longer visible after coculture with LAK. Evidence for selective tumour cytotoxicity by LAK was confirmed by using autologous BM cells as hot and cold targets in a 51Cr release assay. Finally, successful haematologic reconstitution of lethally irradiated BALB/c mice was obtained using syngeneic BM cocultured with LAK. These results support the investigational use of rIL2 and LAK in the treatment of human leukaemia.

Decision analysis of allogeneic hematopoietic stem cell transplantation for patients with myelodysplastic syndrome stratified according to the revised International Prognostic Scoring System.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Crucial questions in clinical decision-making include the definition of optimal timing of the procedure and the benefit of cytoreduction before transplant in high-risk patients. We carried out a decision analysis on 1728 MDS who received supportive care, transplantation or hypomethylating agents (HMAs). Risk assessment was based on the revised International Prognostic Scoring System (IPSS-R). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of different treatment policies on survival. Life expectancy increased when transplantation was delayed from the initial stages to intermediate IPSS-R risk (gain-of-life expectancy 5.3, 4.7 and 2.8 years for patients aged ⩽55, 60 and 65 years, respectively), and then decreased for higher risks. Modeling decision analysis on IPSS-R versus original IPSS changed transplantation policy in 29% of patients, resulting in a 2-year gain in life expectancy. In advanced stages, HMAs given before transplant is associated with a 2-year gain-of-life expectancy, especially in older patients. These results provide a preliminary evidence to maximize the effectiveness of allo-SCT in MDS.

Health-related quality of life in transfusion-dependent patients with myelodysplastic syndromes: a prospective study to assess the impact of iron chelation therapy

Objective The primary objective of this study was to evaluate the health-related quality of life (HRQOL) in lower-risk, transfusion-dependent patients with myelodysplastic syndromes (MDS) treated with deferasirox. A secondary objective was to investigate the relationship between HRQOL, serum ferritin levels and transfusion dependency. Patients and methods This was a prospective multicentre study enrolling 159 patients, of whom 152 received at least one dose of deferasirox. HRQOL was assessed with the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) at baseline and then at 3, 6, 9 and 12 months. Primary analysis was performed estimating mean HRQOL scores over time by a linear mixed model on selected scales. Results The median age of treated patients was 72 years (range 24–87 years). No statistically significant changes over time were found in mean scores for global health status/quality of life (p=0.564), physical functioning (p=0.409) and fatigue (p=0.471) scales. Also, no significant changes were found for constipation (p=0.292), diarrhoea (p=0.815) and nausea and vomiting (p=0.643). Serum ferritin levels were not associated with HRQOL outcomes. A higher patient-reported baseline pain severity was an independent predictive factor of an earlier achievement of transfusion independence with a HR of 1.032 (99% CI 1.004 to 1.060; p=0.003). Conclusions HRQOL of transfusion-dependent patients with MDS receiving deferasirox therapy remains stable over time. HRQOL assessment might also provide important predictive information on treatment outcomes. Trial registration number NCT00469560.

Patient-reported outcomes enhance the survival prediction of traditional disease risk classifications: An international study in patients with myelodysplastic syndromes

Current prognostic systems for myelodysplastic syndromes (MDS) are based on clinical, pathologic, and laboratory indicators. The objective of the current study was to develop a new patient‐centered prognostic index for patients with advanced MDS by including self‐reported fatigue severity into a well‐established clinical risk classification: the International Prognostic Scoring System (IPSS).

Wide-transcriptome analysis and cellularity of bone marrow CD34+/lin- cells of patients with chronic-phase chronic myeloid leukemia at diagnosis vs. 12 months of first-line nilotinib treatment

BACKGROUND Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. The biomolecular mechanisms of CML response to tyrosine-kinase inhibitors are not fully defined. OBJECTIVE We undertook a gene expression profiling (GEP) study of selected bone marrow (BM) CD34+/lin- cells of chronic-phase CML patients at diagnosis and after 12 months of TKI nilotinib to investigate molecular signatures characterizing both conditions.

RNA-seq is a valuable complement of conventional diagnostic tools in newly diagnosed AML patients.

p.Arg30Profs*3 in exon 2 [3]) and Patient 3 (p.Arg678Pro in exon 17 at the homozygous state) presented with amenorrhea due to pituitary hypogonadism and liver cirrhosis. In addition, Patient 3 developed severe arthropathy, whereas Patient 2 quickly developed heart failure and diabetes. In silico analysis revealed that all TFR2 mutations we reported are deleterious and lead to truncate or dysfunctional proteins. c.2039_2040insG insertion disrupts the RGD domain at amino acid Asp680 and creates a premature stop codon leading to a 790-amino acid protein. IVS131 1G>A affected a donor splicing site, and the substitution created an abnormal splicing site with a probability of 38% (Human Splicing Finder). p.Arg678Pro substitution affects the RGD domain likely limiting the binding of holo-transferrin to TFR2. It was predicted to be a deleterious mutation with PolyPhen score of 1.00, SIFT score of 0.04, and MutPred probability of 0.75. We performed functional in vitro studies to assess DAVAQ, p.Arg678Pro, and IVS131 1G>A mutations causality. We evaluated membrane expression of wild type and mutants DAVAQand p.Arg678Pro-TFR2 in both HuH7 (Fig. 1A) and HeLa (Supporting Information Fig. S1A) cells. In the wild-type construct, we found large amount of protein composed by two closely migrating bands that increased after holo-transferrin treatment, indicating protein stabilization at cell membrane, as expected. In DAVAQ mutant cells, only the lower form of TFR2 was evident, whereas in p.Arg678Pro mutants, both bands were visible, although in smaller amounts than the wild-type construct. Protein level did not change after holo-transferrin administration in both mutants, suggesting that they do not correctly interact with holo-transferrin. Moreover, in HuH7 (Fig. 1B,C), which are representative of the tissue (hepatocytes) where TFR2 is functionally expressed in vivo, we observed a different glycosylation pattern between wild-type and mutant proteins: although the majority of wild-type TFR2 was composed of complex oligosaccharides [6], mutant TFR2s were composed only of hybrid oligosaccharides, suggesting that they were not properly glycosylated. These results suggest that DAVAQ and p.Arg678Pro mutations alter the trafficking and function of TFR2. Only small amount of mutant proteins reached the cell membrane; however, they were unable to correctly act as holotransferrin sensor, likely due to an improper glycosylation. This is also confirmed by their inability to release TFR2 soluble form that originates from a cleavage of the cell surface protein (data not shown) [7]. The functional study performed on IVS131 1G>A mutation confirmed that the G>A substitutions abolished the splicing site at position 11 causing the skipping of exon 13 in both HuH7 (Fig. 1D) and HeLa (Supporting Information Fig. S1D) cells, suggesting a nonliver-specific splicing. Surprisingly, the analysis of the wild-type cDNA also revealed a shorter band of lower intensity, corresponding to the skipping of exon 13, in addition to the correct splicing fragment. This raises the possibility that alternative splicing of exon 13 might occur in the normal protein as well. Overall, our results indicate that the TFR2 mutations identified affect important functional domains of TFR2 or lead to dysfunctional or truncated protein. Type 3 HH is a fully penetrant disorder; however, our and previous findings suggest that expression may vary from adultto juvenile-like HH. Interestingly, a recent study described three Chinese subjects with adultonset HH due to mutations in HFE2 [8]. Although this could also be ascribed to different ethnicity and genetic background, all these findings suggest that the term juvenile hemochromatosis might indicate a phenotypic rather than a genetic entity (e.g., a form of HH exclusively caused by HAMP or HFE2 mutations) and that TFR2 mutations should be considered in the genetic workup of individuals presenting early in life with hemochromatosis.

Early detection of relapse in acute non-lymphoblastic leukaemia patients by cancer procoagulant assay.

The clinical usefulness of our assay can be demonstrated by two typical cases: Fig. 1 gives the course of a patient who was investigated three times during CR. While the proportion of leukaemic clones was below 20% during maintenance chemotherapy it had increased to 60% 10 months later during CR. 3 months later, the patient relapsed clinically. In contrast, another patient maintained a proportion of 5-30% of phenotypically leukaemic clones at repeated investigations over a period of nearly 3 years without relapsing clinically (Fig. 2). Our data clearly demonstrate that “complete remission” of adult AML represents a balance of leukaemic and normal hematopoiesis rather than eradication of leukaemia. They argue for the action of mechanisms which suppress the outgrowth of leukaemic progenitor cells in viva. Our in vitro culture system is applicable to all cases of AML and not restricted to certain immunological constellations as the investigation of uncultured bm cells in ALL [5] and much more sensitive than the Southern blot methodology in those cases which have a gene rearrangement as a clonal marker [4, 61. Since it is relatively easy to perform it can be a valuable tool in clinical trials of postremission therapy including alternative approaches (e.g. cytokines like interleukin-2 or autologous bone marrow transplantation) as well as for testing the effectivity of in vitro purging methods.

Lenalidomide treatment of myelodysplastic syndromes with chromosome 5q deletion: Results from the National Registry of the Italian Drug Agency

The most typical cytogenetic aberration in myelodysplastic syndromes is del(5q), which, when isolated, is associated with refractory anaemia and good prognosis. Based on high rates of erythroid response and transfusion independence, Lenalidomide (LEN) became the standard treatment. This multi‐centre study was designed to supplement Italian Registry data on LEN by addressing prescription, administration appropriateness, haematological and cytogenetic responses and disease evolution.