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The breakpoint cluster region–abelson 1 p190 fusion transcript is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit-of-detection determination, showing a reliability, sensitivity, and precision of the assay of up to 0.001%. Comparison of results obtained with qualitative PCR and ddPCR in 117 follow-up samples from 16 of 26 Ph+ ALL patients showed discordant results in 27% of cases (32 of 117). Real-time quantitative PCR analysis of 19 ddPCR-positive samples with a low tumor burden failed to provide quantitative results in 63% of cases (12 of 19). These results highlight that in p190+ ALL the ddPCR method has a sufficient analytical performance for very low MRD monitoring and for predicting molecular relapse several months before hematologic relapse. In conclusion, MRD monitoring by ddPCR may better stratify Ph+ ALL patients at risk of disease progression.

Nested RT-PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared. ddPCR showed good linearity and efficiency and reached an LOD comparable or even superior to nPCR and qPCR. When tested on primary samples, ddPCR exhibited a sensitivity and specificity of ≥95% and ≥91% for bcr1 and bcr3 transcripts and displayed a significant concordance with both techniques, particularly with nPCR. The peculiar advantage of ddPCR-based monitoring of MRD is represented by absolute quantification, which provides crucial information for the management of patients whose MRD fluctuates under the LOD of qPCR and is detectable, but not quantifiable, by nPCR. Our findings highlight ddPCR as a reliable complementary approach to monitor MRD in APL, and suggest its advantageous application, particularly for the molecular follow-up of patients at high risk of relapse.

No relevant conflicts of interest to declare.

Introduction.The JAK2haplotype known as “GGCC or 46/1 haplotype” consists of a germline combination of single nucleotide polymorphisms (SNPs) that are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPNs) positive for both JAK2V617 and exon 12 mutations. It has been reported a significant association between JAK2negative erythrocytosis and the simultaneous occurrence of JAK2haplotype GGCC_46/1 and CALRrs1049481_G allele, mapping in the 3' UTR of the gene. In the present study, we investigated the presence of JAK2haplotype GGCC_46/1 and CALRrs1049481_G allele in a more extensive series of erythrocytosis patients and evaluated a possible correlation with serum erythropoietin (EPO) level.

No relevant conflicts of interest to declare.

Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements.

No relevant conflicts of interest to declare.

Introduction.BCR-ABL1tyrosine kinase inhibitors (TKIs) are considered an important component of treatment for adult patients affected by Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). In fact, recent studies reported that treating Ph+ ALL with the combination of imatinib and multi-agent chemotherapy improved the overall outcome. Currently, no data are available on the impact of TKIs on minimal residual disease (MRD) in Ph+ ALL. In fact, although the real-time quantitative PCR (RQ-PCR) method, usually employed for monitoring the BCR-ABL1residual transcript, is sensitive and easy to perform, it lacks a full standardization and international quality validation. Here, we describe a highly sensitive and reproducible droplet digital PCR (ddPCR) test to monitor BCR-ABL1transcript level in Ph+ ALL.

Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/ β-catenin signaling pathway, which controls cell growth and differentiation. In normal hematopoiesis, LEF1 plays a pivotal role in the development of B- and T-lymphocytes as well as granulocyte progenitor cells. High LEF1 expression has been reported as a favorable prognostic marker in cytogenetically normal acute myeloid leukemia whereas it is associated with poor prognosis in adult B precursor acute lymphoblastic leukemia and in chronic lymphocytic leukemia. Moreover, marked downregulation of LEF1 is associated with disease progression in myelodysplastic syndromes. Given the functional role of LEF1 in hematopoiesis and its putative prognostic impact in several hematological malignancies, we evaluated the prognostic significance of LEF1 expression in adult acute promyelocytic leukemia (APL).One hundred and three consecutive patients with newly diagnosed APL were observed and treated with ATRA-based regimens (AIDA04937 and AIDA2000 GIMEMA protocols), between January 1996 and December 2012. LEF1 expression was measured by real-time qPCR in 78 patients (median age 45 years, range 16 to 88 years) with sufficient available material from the primary cohort. Advanced relative quantification analysis was performed using LightCycler 480 Software 1.5.1, based on the ΔΔCt method. APL samples were dichotomized at the median value and divided into two expression groups: low LEF1 (39 patients) with LEF1 values below the median value (LEF1low) and high LEF1 (39 patients) with LEF1 values above the median value (LEF1high). Clinical and biological features of the two groups were compared. A p value <0.05 was considered significant. Multivariable Cox proportional hazards models were used to study factors (LEF1 expression, FLT3 mutation status, and relapse risk grade) associated with survival endpoints. To evaluate the biological impact of the differential expression of LEF1 in APL, a dataset (GSE13159, including 37 samples of APL patients ) was identified and downloaded from the Gene Expression Omnibus.Patients with LEF1high expression had lower white blood cell counts at baseline (1.8 vs 12.0 x109/L; p < 0 .0001), and were less likely to carry a FLT3-ITD than LEF1low patients (12.8% vs 35.9%, respectively, p=0.02). The association between LEF1low and the presence of FLT3-ITD was also confirmed when the 11 patients with FLT3-TKD were included among patients with FLT3 mutations (p=0.03), or the group of FLT3 wild type patients, as compared to those bearing FLT3-ITD (p=0.03). Early death (defined as death during induction treatment) occurred in nine cases (23%) of the LEF1low group versus no case in the LEF1high group (OR = 0.04; p= 0.002). LEF1low expression was associated with a high relapse risk score (53.9% vs 7.7%, OR=0.07; p < 0.0001). The LEF1high group showed a trend toward a statistically significant association with a lower median age (p = 0.08). No significant differences were observed regarding CD34, CD2, bcr3 positivity and LEF1 expression. Survival analysis of 61 APL patients < 60 years revealed that the LEF1high group had significantly longer overall survival (OS) (p = 0.03), whereas no differences were observed between the two groups in terms of relapse-free survival. Cox analysis for OS confirmed only LEF1 expression as an independent prognostic factor (HR=5.4; 95% CI, 1.0 – 27.5, p =0.04). Among the 17 patients over the age of 60, those with LEF1high expression showed a higher median survival than those in the LEF1low group (6.5 vs 0.04 y.rs, p = 0.05). In silico analysis of the differential expression of LEF1 in APL identified 9 differentially expressed, up-modulated genes associated with a high expression of LEF1 (ETS1, FAIM3, CCR7, IL7R, LCK, IL2RB, ITK, RASGRP1, TRBC1); the majority of these genes is involved in the regulation of apoptosisOur study provides, for the first time, evidence that LEF1 expression is an independent prognostic factor in APL and that it could be used in patients risk stratification. The observation provided by in silico gene expression analysis that LEF1 expression in APL is associated with biologic changes, mostly in terms of apoptosis regulation, will need to be confirmed experimentally.No relevant conflicts of interest to declare.

Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/ β-catenin signaling pathway, which controls cell growth and differentiation. In normal hematopoiesis, LEF1plays a pivotal role in the development of B- and T-lymphocytes as well as granulocyte progenitor cells. High LEF1expression has been reported as a favorable prognostic marker in cytogenetically normal acute myeloid leukemia whereas it is associated with poor prognosis in adult B precursor acute lymphoblastic leukemia and in chronic lymphocytic leukemia. Moreover, marked downregulation of LEF1is associated with disease progression in myelodysplastic syndromes. Given the functional role of LEF1in hematopoiesis and its putative prognostic impact in several hematological malignancies, we evaluated the prognostic significance of LEF1expression in adult acute promyelocytic leukemia (APL).

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 5058

Abstract 1674

Tribbles homolog gene 2 (TRIB2) is a pseudokinase gene belonging to a three member gene family. Tribbles gene was first identified in Drosophila melanogaster where is involved in the regulation of morphogenesis and mitosis. Likewise, mammalian homologous genes of Tribbles (TRIB1, TRIB2, TRIB3) promotes the degradation of specific transcription factors and interacts with several cell signaling mediators and modulators. Recently it has been demonstrated that TRIB2 was able to induce AML in bone marrow transplanted mouse by inducing C/EBPα proteasome-dependent degradation. Moreover, gene expression profiles data from AML patients revealed that patients carrying C/EBPα mutations clustered with those patients with up-regulated TRIB2.Aims. To test the hypothesis that TRIB2 expression was related to a differentiation degree in AML, we evaluated several AML cases based on FAB cytotype.We performed quantitative Real Time-PCR (qRT-PCR) analysis on AML patients (15 AML-M0, 14 AML-M2, 5 AML-M3, 2 M4 and 3 M5b bone marrow aspirate samples) to measure the expression level of TRIB2 in different FAB AML subtype. Four healthy bone marrows were used as reference samples. qRT-PCR was conduct using SYBR green chemistry and specific primers for TRIB2 transcript and for two housekeeping genes (B2M and IPO8) previously tested for their stability and efficiency. All AML sample were tested by conventional cytogenetic analysis and by FISH with TRIB2 specific probe. We performed, also, methylation analysis of a CpG island located in the TRIB2 promoter region by methylation sensitive restriction enzyme (MSRE) and qPCR. Briefly, DNA samples were digested with MSRE and were quantified by qPCR using specific primer pairs surrounding restriction enzymes recognition sites.qRT-PCR experiments revealed that TRIB2 expression was higher (from 3 to 20 fold) in AML-M0 respect to the AML-M2 (p= 0.02), AML-M3 (p=0.01), AML-M5 (p=0.006) FAB subtypes, and references (p=0.003), respectively. Moreover, the AML-M0 cases showed a TRIB2 overexpression compared to that observed in the two AML-M4 cases (about 4 fold changes) but this difference there was not statistically significant probably because of M4 cases paucity in our series. Therefore qRT-PCR displayed a progressive decreasing TRIB2 expression along FAB subtypes. Conventional cytogenetic and FISH analysis did not show any kind of rearrangement involving TRIB2 gene. MSRE experiments revealed an higher methylation degree of a CpG island located in the TRIB2 promoter region in AML-M0 cases respect to the others FAB subtypes; moreover, methylation was significantly correlated with TRIB2 expression (r = 0.9; p = 0.0002).Our data showed that the TRIB2 gene overexpression was associated to AML-M0. In our cases TRIB2 dysregulation was not due to gene amplification as in other cancers. Surprisingly we observed that AML-M0 samples showed an higher methylation degree in the TRIB2 promoter region. Usually methylation cause gene silencing but our results showed that TRIB2 promoter region methylation was associated to the overexpression of the gene. Maybe methylation inhibits binding of some unknown transcriptional repressor as already seen for hTERT gene in others tumors cells. In conclusion, our data revealed that TRIB2 dysregulation seems to be linked to AML-M0 phenotype. Further studies are needed to clarify the reasons for this association.No relevant conflicts of interest to declare.

Abstract 4628

No relevant conflicts of interest to declare.

Abstract 3261