Susanna Grassi

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.

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

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).

Association of the hOCT1/ABCB1 genotype with efficacy and tolerability of imatinib in patients affected by chronic myeloid leukemia

PurposeThe present study was aimed at investigating whether imatinib pharmacogenetics is related to its pharmacodynamics in patients affected by chronic myeloid leukemia.MethodsThrough a procedure based on a sequence of classical statistics methods, we investigated the possible relationships between treatment efficacy/tolerability and combinations of time-independent variables as gender and genetic covariates in the form of single nucleotide polymorphisms (SNPs) or combinations thereof. Moreover, since the drug tolerability has a strong incidence on the discontinuation of the therapy, we investigated whether the time of manifestation of the most frequent toxic effects can be related to time-independent patients’ characteristics or not.ResultsWe found that a combination of two polymorphisms, namely hOCT1 c.480C>G (rs683369) and ABCB1 c.3435C>T (rs1045642), seems to play the role of predictor for imatinib in both efficacy and toxicity. Furthermore, the time of manifestation of edema toxicity is found to be associated to a combination of gender and ABCB1 c.3435C>T, whereas the time of manifestation of cramp toxicity appears related to gender.ConclusionsThe novelty of this study is dual: the achievement of results that potentially have a significant clinical interest and the demonstration that the adoption of composed covariates may represent a unique tool to study different aspects of the treatment with imatinib.

The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment of B-RAF V600E Mutation in Hairy Cell Leukemia

Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by “classic” HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to “classic” quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5 vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected the B-RAF mutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up.

The hOCT1 and ABCB1 polymorphisms do not influence the pharmacodynamics of nilotinib in chronic myeloid leukemia

First-line nilotinib in chronic myeloid leukemia is more effective than imatinib to achieve early and deep molecular responses, despite poor tolerability or failure observed in one-third of patients. The toxicity and efficacy of tyrosine kinase inhibitors might depend on the activity of transmembrane transporters. However, the impact of transporters genes polymorphisms in nilotinib setting is still debated. We investigated the possible correlation between single nucleotide polymorphisms of hOCT1 (rs683369 [c.480C>G]) and ABCB1 (rs1128503 [c.1236C>T], rs2032582 [c.2677G>T/A], rs1045642 [c.3435C>T]) and nilotinib efficacy and toxicity in a cohort of 78 patients affected by chronic myeloid leukemia in the context of current clinical practice. The early molecular response was achieved by 81% of patients while 64% of them attained deep molecular response (median time, 26 months). The 36-month event-free survival was 86%, whereas 58% of patients experienced toxicities. Interestingly, hOCT1 and ABCB1 polymorphisms alone or in combination did not influence event-free survival or the adverse events rate. Therefore, in contrast to data obtained in patients treated with imatinib, hOCT1 and ABCB1 polymorphisms do not impact on nilotinib efficacy or toxicity. This could be relevant in the choice of the first-line therapy: patients with polymorphisms that negatively condition imatinib efficacy might thus receive nilotinib as first-line therapy.

A 14.8 Mb 12p Deletion Disrupting ETV6 in a Patient with Myelodysplastic Syndrome

We present on a new case of myelodysplastic syndrome characterized by array Comparative Genomic Hybridization. This technique confirmed the monosomy 7, detected by conventional cytogenetics, and revealed also a deletion on the short arm of chromosome 12. This deletion extends for about 14.8 Mb and breaks ETV6 gene. 12p deletion extents in hematological malignancies may vary, but the minimally deleted region almost invariably contains ETV6, that is considered the main candidate tumor suppressor genes within the region for tumor progression. It has been shown that levels of ETV6 were significantly decreased in cases with 12p13 deletions, whereas expression of other genes in the deleted region, like BCL2L14, LRP6, DUSP16 and GPRC5D, did not show any variation, independently of their copy number status. This observation strengthens the fact that ETV6 may play a potential role in the tumorigenesis process. The role of ETV6 in our patient myelodysplastic syndrome is showed by his clinical history and his poor prognosis.

The combination of rituximab and bendamustine as first-line treatment is highly effective in the eradicating minimal residual disease in follicular lymphoma: An Italian retrospective study

R-Bendamustine is an effective treatment for follicular lymphoma (FL). Previous large trials demonstrated the prognostic role of the molecular minimal residual disease (MRD) during the most frequently adopted chemotherapeutic regimens, but there are not yet conclusive data about the effect of combination of rituximab (R) and bendamustine in terms of MRD clearance. Thus, the aim of this retrospective study was to assess if and in what extent the combination of rituximab and bendamustine would exert a significant reduction of the molecular disease in 48 previously untreated FL patients. The molecular marker at baseline was found in the 62.5% of cases; no significant differences were observed between patients with or without the molecular marker in respect of the main clinical features. Moreover, the quantization of the baseline molecular tumor burden showed a great variability: the median value was 1.4 × 10−2 copies, ranging from 3 × 10−5 to 4 × 104. The initial molecular tumor burden did not correlate with clinical features and did not impact on the subsequent quality of response. After treatment, 93% of cases became MRD-negative; the median reduction of the BCL2/JH load was 4 logs. The 2-years PFS was 85%; it was significantly longer for patients in complete than for those in partial response (91 vs. 57%; p = 0.002), and for cases with lower FLIPI-2 score (88 vs. 60%; p = 0.004). On the contrary, PFS did not differ between patients with or without the molecular marker at baseline; a molecular tumor burden 15 times higher was observed in the relapsed subgroup in comparison to the relapse-free one, but this difference did not change the PFS length. The 2-years OS was 93.6%; the only variable that significantly impacted on it was the FLIPI-2 score; the presence of the molecular marker at baseline or its behavior after treatment did not impact on survival. This study, even if retrospective and conducted on a small series of patients, would represent a proof of concept that R-bendamustine is able to so efficaciously eradicate MRD that it could be able to by-pass the prognostic significance of MRD already demonstrated for other chemotherapeutic regimens in FL.