Elena Ciabatti

Minimal residual disease after conventional treatment significantly impacts on progression-free survival of patients with follicular lymphoma: the FIL FOLL05 trial.

Purpose: The role of the minimal residual disease (MRD) in follicular lymphoma is still debated. In this study, we assessed whether the BCL2/IGH rearrangement could have a prognostic role in patients receiving R-CHOP, R-FM, or R-CVP. Experimental Design: DNAs from 415 patients among the 504 cases enrolled in the FOLL05 trial (NCT00774826) were centralized and assessed for the BCL2/IGH at diagnosis, at the end of treatment, and after 12 and 24 months. Results: At diagnosis, the molecular marker was detected in 53% of cases. Patients without molecular marker or with a low molecular tumor burden (<1 × 10−4 copies) showed higher complete remission (CR) rate and longer progression-free survival (PFS; 3-year PFS 80% vs. 59%; P = 0.015). PFS was significantly conditioned by the PCR status at 12 and 24 months, with 3-year PFS of 66% for MRD− cases versus 41% for those MRD+ at 12 months (P = 0.015), and 84% versus 50% at 24 months (P = 0.014). The MRD negativity at 12 and 24 months resulted in an improved PFS both in CR and in partial remission (PR) patients (3-year PFS = 72% for cases CR/PCR− vs. 32% for those CR/PCR+ vs. 62% for those PR/PCR− and 25% for patients in PR/PCR+; P = 0.001). The prognostic value of MRD at 12 and 24 months of follow-up was confirmed also in multivariate analysis. Conclusions: In this study, standardized molecular techniques have been adopted and applied on bone marrow samples from a large cohort. Data reported show that the MRD detection is a powerful independent predictor of PFS in patients with follicular lymphoma receiving conventional chemoimmunotherapy. Clin Cancer Res; 20(24); 6398–405. ©2014 AACR.

The c.480C>G polymorphism of hOCT1 influences imatinib clearance in patients affected by chronic myeloid leukemia.

The aim of the study was to investigate any possible influence of polymorphisms of transmembrane transporters human organic cation transporter 1 (hOCT1), ABCB1, ABCG2 on imatinib pharmacokinetics in 33 men and 27 women (median age and range, 56 and 27–79 years, respectively) affected by chronic myeloid leukemia. A population pharmacokinetic analysis was performed to investigate imatinib disposition in every patient and the role of transporter polymorphisms. Results showed that the α1-acid glycoprotein and the c.480C>G genotype of hOCT1 had a significant effect on apparent drug clearance (CL/F) being responsible, respectively, for a 20% and 10% decrease in interindividual variability (IIV) of CL/F (from 50.1 up to 19.6%). Interestingly, 25 patients carrying at least one polymorphic c.480 G allele had a significant lower CL/F value with respect to the 35 c.480CC individuals (mean±s.d., 9.6±1.6 vs 12.1±2.3 l h−1, respectively; P<0.001). In conclusion, the hOCT1 c.480C>G SNP may significantly influence imatinib pharmacokinetics, supporting further analyses in larger groups of patients.

Safety and efficacy of 90Yttrium‐Ibritumomab‐Tiuxetan for untreated follicular lymphoma patients. An Italian cooperative study

90Yttrium (90Y)‐Ibritumomab‐Tiuxetan combines the targeting advantage of a monoclonal antibody with the radiosensitivity of Follicular Lymphoma (FL). Previous studies showed that 90Y‐IT is safe and effective in relapsed/refractory indolent FL, irrespective of prior treatment with rituximab. This multicentre trial aimed to evaluate the safety and the efficacy of “upfront” single‐agent (90Y)‐Ibritumomab‐Tiuxetan in advanced‐stage FL. The primary objective was the incidence of responses in terms of complete (CR) and partial remission (PR). Fifty patients with stage II “bulky”, III or IV FL received a single treatment course with (90Y)‐Ibritumomab‐Tiuxetan as initial therapy. The median age was 60 years. Bone marrow involvement (<25%) was observed in 24 patients (48%) and 7 (14%) had an elevated lactate dehydrogenase level. The overall response (ORR) and CR rates were 94% and 86%, respectively with a median follow‐up of 38·8 months. The median progression‐free survival (PFS) was not reached, whereas the 3‐year estimated PFS and overall survival (OS) rate was 63·4% and 90%, respectively. Grade 3/4 neutropenia and thrombocytopenia occurred in 30% and 26% of patients respectively; none experienced grade 3/4 non‐haematological toxicity. No cases of secondary haematological malignancies were observed. (90Y)‐Ibritumomab‐Tiuxetan was demonstrated to be highly effective and safe as first‐line treatment for advanced‐stage FL.

Real-Time PCR and Droplet Digital PCR: two techniques for detection of the JAK2(V617F) mutation in Philadelphia-negative chronic myeloproliferative neoplasms.

Philadelphia‐negative chronic myeloproliferative neoplasms (MPNs) are clonal disorders that present JAK2V617F mutation in 50–95% of cases. The main objective of this study was the comparison of two PCR methods, real‐time (qPCR) and droplet digital PCR (DD‐PCR) for detection of the JAK2V617F mutation, to assess analytic sensitivity, specificity, and feasibility of the two methods.

WT1 expression levels at diagnosis could predict long-term time-to-progression in adult patients affected by acute myeloid leukaemia and myelodysplastic syndromes.

Mutations of Wilms’ tumour gene (WT1) are reported in 10% of acute myeloid leukemias (AML) with normal karyotype, with reduction in both relapse-free-survival and overall survival (Virappane et al, 2008). WT1 is highly expressed in acute leukemias and the myelodysplastic syndromes (MDS) (Rosenfeld et al, 2003) where it is associated with poorer prognosis (Cilloni et al, 2008; Candoni et al, 2009). About 65% of low-risk and up to 100% of high-risk MDS cases express high WT1 transcripts, correlated with higher risk of progression (Tamaki et al, 1999; Cilloni et al, 2003). To date, no studies have evaluated whether diagnostic WT1 mRNA levels influence the long-term time-to-progression (TTP) in MDS and AML. Moreover, no significant data have been produced concerning WT1 and MDS cases that have been classified according to the newer World Health Organisation Prognostic Scoring System (WPSS) score (Malcovati et al, 2007). Thus, in the present study, we evaluated the possible impact on long-term TTP exerted by WT1 mRNA levels measured at diagnosis in a series of 54 cases (24 AML and 30 MDS). WT1 transcript was quantified with the ProfileQUANT TM kit (Ipsogen, Marseille, France) on total RNA isolated using RNeasy Mini kit (QIAGEN, Valencia, CA, USA). This method estimates the ‘normal’ WT1 copies/ABL1 · 10 copies ratio to be between 3 and 180. Clinical and demographic characteristics of the entire series are reported in Table I. Patients were stratified in two categories (WT1-low and -high) when the WT1 copies/ABL1 · 10 copies ratio was lower or higher than 180 respectively; the chi-square and logistic regression tests were used to assess eventual differences in clinical and demographic data. t-test was adopted for comparing mean values; Kaplan–Meier life tables were constructed for survival data and compared by means of the logrank test, with surviving patients being censored at 15 June 2009. All statistical analyses were performed with the Statistical Package for the Social Sciences (spss) software, version 17.0 (SPSS Italia, Bologna, Italy). P values <0Æ05 were considered significant. All low-risk MDS patients received epoietins and/or additional blood transfusion support; the high-risk MDS group included patients who received azacitidine 75 mg/m, 6 d a week for almost four cycles. For AML cases, induction therapy included idarubicin, less often doxorubicin with aracytin, according to the ‘3 + 7’ or ‘2 + 5’ scheme, on the basis of age (£ or >65 years). Fourteen transplanted patients were censored before stem cell infusion. At diagnosis, WT1 expression was high in 9 out of the 30 MDS cases (30%) (four in low-risk and five in high-risk group), and in 15 of the 24 patients (62Æ5%) affected by AML. Mean and standard deviation values were: 333Æ19 WT1 copies/10 ABL1 copies ± 97Æ89 for low-risk MDS; 551Æ31 copies/10 ABL1 copies ± 72Æ02 for high-risk MDS; 2390Æ89 copies/10 ABL1 copies 10 ± 39Æ92 for AML. WT1 mRNAs were significantly higher in AML when compared to both low-risk (P = 0Æ02) and high-risk MDS (P = 0Æ04). On the contrary, no significant difference in WT1 expression was found between the two risk score groups in MDS (P > 0Æ05). In our series of 30 MDS patients, the 36-monthTTP was 65% (median not reached at 5 years); it was not significantly affected by age, sex, performance status, white blood cell count (WBC), haemoglobin level (Hb), and platelet count (PLT) at diagnosis, blast percentage, cytogenetic features, or spleen dimension. Even the WPSS risk score in our series did not affect the TTP (36-month TTP 71% for low-risk versus 61% high-risk patients, P = 0Æ71). Similarly, the probability of progression was also not significantly affected by these analysed parameters. In contrast, the probability of progression was influenced by WT1 level: it was 14% for patients expressing low WT1 levels versus 56% for those with high WT1 mRNA (P = 0Æ03). Moreover, WT1 expression levels at diagnosis also significantly affected the 36-month TTP (Fig 1B): 73% of patients with normal WT1 expression were progression-free versus 19% of cases with elevated WT1 (P < 0Æ01). Noteworthy, this prognostic role of WT1 high expression was evident both in WPSS low-risk (Fig 1C) and high-risk categories (Fig 1d) (36-month TTP 78% vs. 5% in low-risk cases and 67% vs. 37% in the high-risk group; P < 0Æ01). In AML, the 36-month TTP was 46% (median = 23 months) and was not significantly conditioned by performance status, sex, WBC, Hb, PLT at diagnosis, blast per centage, French-American-British (FAB) subtype, cytogenetic features, presence/absence of FLT3 mutations, or spleen dimension. TTP was much lower for older patients (36-month TTP 32% vs. 60% for younger patients), but it was not statistically significantly different (P = 0Æ12). Even in AML, the probability of progression was not significantly affected by the analysed demographic/clinical correspondence

The Efficacy of Rituximab plus Hyper-CVAD Regimen in Mantle Cell Lymphoma Is Independent of FCγRIIIa and FCγRIIa Polymorphisms

Abstract Mantle cell lymphoma (MCL) accounts for 3-10% of all non-Hodgkins lymphomas, with median overall survival not exceeding 3-4 years. Rituximab in combination with the Hyper-CVAD regimen appears the most promising regimen; thus, we adopted it as a first-line treatment strategy in a series of 24 patients. In addition to evaluation of clinical success of the regimen, we investigated a possible role of polymorphism in IgG Fc receptors, FCγRIIIa and FCγRIIa. The frequencies of FCγRIIIa-158 were as follows: V/V=4/24 (17%); V/F=16/24 (66%); F/F=4/24 (17%). Those of the FCγRIIa-131 polymorphism were H/H=11/24 (46%), H/R=9/24 (37%), R/R=4/24 (17%). The overall response rate was 62.5%, with 33% of complete responses (CRs) after four cycles of R-Hyper-CVAD. Two-year progression-free survival (PFS) was 78% for 158V/V patients vs 75% for cases carrying phenylalanine (p=0.88). When the FCγRIIa polymorphism was assessed, the 2-year PFS was 82% for 131H/H patients vs 75% for those carrying arginine (p=0.26). Eighty-three percent of cases achieved Polymerase Chain Reaction (PCR)-negativity: the progression rate was significantly influenced by the minimal residual disease clearance, with 12% progression in the subgroup of PCR-negative cases versus 67% progression in PCR-positive cases (p=0.008). The achievement of PCRnegativity was not significantly influenced by FCγR polymorphisms. Results confirm that rituximab plus Hyper-CVAD is an effective regimen for the induction of prolonged remission in patients with aggressive MCL and suggest that rituximab efficacy is independent of the FCγR polymorphisms.

Polycomb genes are associated with response to imatinib in chronic myeloid leukemia.

AIM Imatinib is a tyrosine kinase inhibitor that has revolutionized the treatment of chronic myeloid leukemia (CML). Despite its efficacy, about a third of patients discontinue the treatment due to therapy failure or intolerance. The rational identification of patients less likely to respond to imatinib would be of paramount clinical relevance. We have shown that transmembrane transporter hOCT1 genotyping predicts imatinib activity. In parallel, Polycomb group genes (PcGs) are epigenetic repressors implicated in CML progression and in therapy resistance. PATIENTS & METHODS We measured the expression of eight PcGs in paired pre- and post-imatinib bone marrow samples from 30 CML patients. RESULTS BMI1, PHC3, CBX6 and CBX7 expression was significantly increased during imatinib treatment. Post-treatment levels of CBX6 and CBX7 predicted 3-month response rate. Measurement of post-treatment BMI1 levels improved the predictive power of hOCT1 genotyping. CONCLUSION These results suggest that the expression levels of PcGs might be useful for a more accurate risk stratification of CML patients.

Evaluation of the MDR1, ABCG2, Topoisomerases IIα and GSTπ gene expression in patients affected by aggressive mantle cell lymphoma treated by the R-Hyper-CVAD regimen

The genomic profile of mantle cell lymphoma (MCL) has been reported to be significantly different from that of other indolent lymphoproliferative disorders, Topoisomerase IIα, glutathione-s-transferaseπ (GSTπ) and ABCG2 (BCRP) chemoresistance genes being over-expressed in MCL. In our study, expression levels of the above mentioned genes plus MDR1 were tested on bone marrow samples from 20 patients treated with Rituximab plus hyper-CVAD regimen, in order to evaluate a possible impact of the chemoresistance phenomenon on this promising treatment regimen. All patients expressed ABCG2 and MDR1 genes; 85% of cases expressed GSTπ and topoisomerase IIα. Only ABCG2 were over-expressed in comparison both with marrow from healthy donors and tonsilar CD5+/CD20+ lymphocytes (adopted as normal counterpart of the neoplastic population). The overall response rate of the entire series was 87.5%, with 44% of complete responses. Fifty-seven percent of patients achieved the clearance of minimal residual disease. Levels of tested genes did not condition either quality of clinical response or PFS (76% at 24 months). Nevertheless, an ABCG2 higher expression appeared associated with a worse PFS and levels of this gene paralleled the status of minimal residual disease. A further evaluation of ABCG2 expression in larger series of MCL patients would be suitable.

Boron nitride nanotube-functionalised myoblast/microfibre constructs: a nanotech-assisted tissue-engineered platform for muscle stimulation

In this communication, we introduce boron nitride nanotube (BNNT)‐functionalised muscle cell/microfibre mesh constructs, obtained via tissue engineering, as a three‐dimensional (3D) platform to study a wireless stimulation system for electrically responsive cells and tissues. Our stimulation strategy exploits the piezoelectric behaviour of some classes of ceramic nanoparticles, such as BNNTs, able to polarize under mechanical stress, e.g. using low‐frequency ultrasound (US). In the microfibre scaffolds, C2C12 myoblasts were able to differentiate into viable myotubes and to internalize BNNTs, also upon US irradiation, so as to obtain a nanotech‐assisted 3D in vitro model. We then tested our stimulatory system on 2D and 3D cellular models by investigating the expression of connexin 43 (Cx43), as a molecule involved in cell crosstalk and mechanotransduction, and myosin, as a myogenic differentiation marker. Cx43 gene expression revealed a marked model dependency. In control samples (without US and/or BNNTs), Cx43 was upregulated under 2D culture conditions (10.78 ± 1.05‐fold difference). Interactions with BNNTs increased Cx43 expression in 3D samples. Cx43 mRNA dropped in 2D under the ‘BNNTs + US’ regimen, while it was best enhanced in 3D samples (3.58 ± 1.05 vs 13.74 ± 1.42‐fold difference, p = 0.0001). At the protein level, the maximal expressions of Cx43 and myosin were detected in the 3D model. In contrast with the 3D model, in 2D cultures, BNNTs and US exerted a synergistic depletive effect upon myosin synthesis. These findings indicate that model dimensionality and stimulatory regimens can strongly affect the responses of signalling and differentiation molecules, proving the importance of developing proper in vitro platforms for biological modelling. Copyright

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup

In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 “new” unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a “clinical warning”; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).