Francesca Guerrini

Quantitative molecular evaluation in autotransplant programs for follicular lymphoma: efficacy of in vivo purging by Rituximab

Summary:The main aim of this paper was to compare results of Genescan and real-time PCR methods in order to detect contamination in harvests from patients with follicular lymphoma. The secondary goal was to evaluate the efficacy of Rituximab as an in vivo purging agent. A total of 23 patients had been treated with CHOP followed by either high-dose therapy (12 patients) or high-dose plus Rituximab (11 patients), both followed by autologous transplantation. Results show that 86% of harvests from patients treated whith Rituximab were PCR-negative compared to 14.3% from controls. Real-time PCR was more sensitive than Genescan PCR; quantitative analysis revealed a correlation between the amount of contamination in the harvests and relapse after transplantation. Whereas all patients reinfused with negative aphereses achieved complete remission and showed a significantly better 5-year PFS (100%) compared to those reinfused with contaminated samples (41%), a very low amount of contamination does not appear to negatively affect outcome, suggesting that determination of a cutoff in the contamination level of harvests could be useful. Results suggest that real-time PCR is superior to Genescan PCR to select transplantable harvests and confirm the ability of Rituximab as an in vivo purging tool for follicular lymphoma.

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.

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frogRana esculenta and its parental species

Hybrid water frogsRana esculenta reproduce by hybridogenesis: one parental genome (ofRana lessonae) is excluded in the germ line, the other (ofRana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher inRana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed inRana ridibunda genomes by the restriction endonucleaseStul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1–5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes allRana ridibunda chromosomes. RrS1 represents about 2.5% of the genome ofRana ridibunda; it may represent as little as 0.2% of the genome ofRana lessonae, and cannot be detected inXenopus laevis frogs orSalamandra salamandra andTriturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

Peripheral blood stem cell contamination evaluated by a highly sensitive molecular method fails to predict outcome of autotransplanted multiple myeloma patients

Summary. To evaluate the clinical impact of minimal residual disease in multiple myeloma, apheretic products from 51 autotransplanted patients were tested by fluorescent (GeneScan) polymerase chain reaction (PCR). Sixty‐nine per cent of harvests were contaminated when evaluated for IgH rearrangement. Forty‐six patients responded to transplant, with 52·9% achieving complete response (CR). The clinical response of patients was significantly influenced by the number of re‐infused CD34+ cells. Positive PCR results of re‐infused harvests were not significantly related to patient outcome. Median overall survival (OS) was 33 months, and a significant advantage for patients transplanted by 12 months from diagnosis was observed. Moreover, OS was longer for patients receiving PCR‐negative stem cells, with 72% of patients surviving to 70 months in the group receiving PCR‐negative harvests vs 48% in the group transplanted with contaminated precursors (not statistically significant). Ex vivo purging caused a reduction of contamination of up to 3 logs; nevertheless, 80% of purged harvests remained PCR‐positive and the purging procedure did not alter response or survival rates. Thus, the failure of a predictive role for this highly sensitive molecular method could be explained by the assumption that in vivo persisting malignant cells are the true source of relapse in MM.

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.

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis

A high expression of Wilms’ tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon.

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.

Synergistic antiproliferative effect of arsenic trioxide combined with bortezomib in HL60 cell line and primary blasts from patients affected by myeloproliferative disorders

Both arsenic trioxide (ATO) and bortezomib show separate antileukemic activity. With the purpose of evaluating whether the combination of ATO and bortezomib would be an option for patients with acute leukemia, we incubated HL60 leukemic cells with ATO alone and in combination with bortezomib. ATO and bortezomib cooperated to induce cell death and to inhibit proliferation and apoptosis in a synergistic way. The combined treatment resulted in a stronger activation of caspase 8 and 9, moderate activation of caspase 3, and increased expression of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-DR5 receptors. When bortezomib was added, some proapoptotic genes (CARD9, TRAIL) were upregulated, and some antiapoptotic genes (BCL2, BCL3, FLICE) were downregulated. When coincubated, approximately 80% of cells showed altered mitochondrial membrane permeability. Moreover, ATO alone and in combination with bortezomib abrogated DNA-binding activity of nuclear factor kappa beta (NF-kappaB). Gene expression assays showed that more deregulated genes were related to proliferation of leukocytes, tumorigenesis, control of cell cycle, hypoxia and oxidative stress, cytokines, PI3K-AKT, ERK-MAPK, EGF pathways, and ubiquitination. Finally, in three cases of acute myeloid leukemia, the addition of bortezomib to ATO significantly increased cytotoxicity. We conclude that the combination of bortezomib and ATO may be efficacious in the treatment of myeloid disorders.

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 clinical relevance of the expression of several multidrug-resistant-related genes in patients with primary acute myeloid leukemia.

Abstract Multidrug resistance (MDR) is a complex phenomenon that includes the expression of many different genes regulating drug transport or metabolism, cellular repair or detoxification mechanisms. The co-expression of several genes could be at the basis of the resistant phenotype in vivo. In order to test a possible prognostic role of the expression and co-expression of several MDR-related genes (MDR1, topoisomerase IIα, topoisomerase IIβ, MRP, GSTπ, LRP), 35 patients affected by acute myeloid leukemia (AML) were tested by RT-PCR assays. In our series, topoisomerase IIβ was significantly co-expressed with MRP ( p=0.05), GSTπ (p=0.017) and LRP (p=0.005). GSTπ was co-expressed with LRP (p=0.03) and MRP (p=0.007); on the other hand, 53.8% of patients were LRP and MRP-positive (p=0.02). The PCR-positivity did not differ according to biological/clinical characteristics of patients, including age; this latter was the only parameter conditioning the response and overall survival. Neither the expression nor the co-expression of the tested genes was significantly correlated with the response to the induction treatment and long-term outcome.