Paola Casieri

SETBP1 and miR_4319 dysregulation in primary myelofibrosis progression to acute myeloid leukemia

The molecular pathogenesis underlying the primary myelofibrosis (PMF) progression to acute myeloid leukemia (AML) is still not well defined. The involvement of microRNA (miRNA) is actually helping to shed light on an important issue in the occurrence of myeloproliferative neoplasms (MPNs). However, the role of intronic miRNA, derived from the intron regions of gene transcripts, has never been reported in MPNs. In this study, we describe a PMF case evolved to AML with a t(12;18)(p13;q12) rearrangement showing the downregulation of the intronic miR_4319 and the overexpression of its host gene, SET binding protein (SETBP1). A possible molecular mechanism regulating the PMF progression to AML is discussed.

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the “watch and wait” interval and after standard chemotherapy.

Droplet digital PCR assay for quantifying of CALR mutant allelic burden in myeloproliferative neoplasms

Dear Editor, Calreticulin (CALR) gene mutations (CALR) have recently been discovered in about 20–35 % of patients affected by essential thrombocythemia (ET) and primarymyelofibrosis (PMF) [1, 2]. Several molecular assays have been developed to detect the most frequentCALR (type 1 consisting of a 52bp deletion, and type 2 of a 5-bp insertion) [3, 4]. All these techniques are useful for identifyingCALR at the diagnosis, but they are not suitable for minimal residual disease (MRD) monitoring, since the maximum sensitivity is 1 %. The droplet digital PCR (ddPCR) technology is a third-generation PCR method that started to be used in hematological malignancies [5–7].We describe a ddPCR assay with a sensitivity of 0.01% developed for the absolute quantification of CALR type 1 and 2 mutations and analyze a cohort of 57 JAK2V617F-negative myeloproliferative neoplasm patients. ddPCR experiments were performed using the QX-200 instrument (BioRad) and specific primers and probes were designed for both type 1 and type 2 mutations (see Supplementary Files). CALR load in each sample was expressed as fractional abundance (FA, mutant allele/mutant allele + wild-type allele). The CALR allelic burden resulted heterogeneous in both ET (min.13.8 %– max. 51%) and PMF (min. 34.5%–max. 51.3%) patients.We show that the medianCALR allelic burden at diagnosis was significantly higher in PMF patients as compared to ET case (47.9 vs 43.8 %, p = 0.008) whereas no significant difference was observed between the type 1 and 2 mutations (Fig. 1a). Moreover, no relationship between the genemutation type and the CALR amount was observed within each group of ET and PMF patients. In our ET series, there were 14 (29.7 %) patients with a very low FA, <30 %; this group was not statistically different in terms of hemoglobin, white blood cells and platelet counts, age, sex, thrombosis and/or hemorrhage, and CALR type compared to those with FA >30 %. The PMF group included ten patients, too few for any type of statistical considerations. Sequential evaluations by ddPCR experiments were performed in three patients to monitor the CALR load during treatment. CALR load at diagnosis was 15.8 and 48 % in two ET patients. The former patient was treated with interferon-α (IFN-α) and after 5 years from diagnosis the FA was 7.7 %. The latter was also treated with IFN-α and after 2 years from diagnosis, the CALR load was 14.7 %. Both patients had stable disease and a well-controlled platelet count. A 44-year-old man at PMF diagnosis showed a FA of 49.7 %; 8 years later, we observed a leukemic transformation. At the time of the AML evolution, theCALR load was 0 %; this finding was also confirmed by PCR qualitative analysis. The patient underwent induction chemotherapy, achieving complete remission, then allogeneic bone marrow transplantation (ABMT) from a HLA-matched related donor. Two months later, the FA observed by ddPCR analysis was 0.01 %; 7 months after, the ABMT and AML relapsed and at this time, the CALR load was 13.5 % (Fig. 1b, c). Although the importance of the CALR allelic burden determination has not yet been defined at the disease onset, the utility of and need for a sensitive method, like our ddPCR assay, are unquestionable for the purposes of MRD monitoring [8–10]. Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2739-2) contains supplementary material, which is available to authorized users.

Gene expression profiling of chronic myeloid leukemia with variant t(9;22) reveals a different signature from cases with classic translocation

BackgroundThe t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5–10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified.FindingsIn this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed.ConclusionsOur results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.

FLAG-Ida Regimen as Bridge Therapy to Allotransplantation in Refractory/Relapsed Acute Myeloid Leukemia Patients

Background Patients with primary refractory or first relapse acute myeloid leukemia (AML) are considered to have worse clinical outcomes after treatment. For these patients, the achievement of complete remission appears crucial for them to be able to undergo allotransplantation, which might be the only possible treatment. Patients and Methods We used the FLAG‐Ida (fludarabine, cytarabine [cytosine arabinoside], granulocyte colony‐stimulating factor, idarubicin) regimen in patients with primary refractory/first relapse AML as a bridge to transplantation. We studied its efficacy in terms of overall response and overall survival to assess which variables (age, lactate dehydrogenase, bone marrow blast count, peripheral blood blast count, platelet count, white blood cell count, de novo or secondary AML, molecular‐cytogenetic risk, duration of response, and relapsed or refractory disease) might have an effect on outcome. Results We analyzed the data from 108 consecutive adult patients (52 males, 66 females; median age, 49 years; range, 17‐72 years) with newly diagnosed AML refractory to standard induction regimens or relapse after first complete remission, who had received the FLAG‐Ida protocol as salvage therapy from January 2005 to December 2015. An overall response was achieved in 48 patients (44%). On multivariate analysis, the variables with a positive effect on the response rate were molecular‐cytogenetic risk (P = .009), duration of first response in relapsed AML (P = .003), AML status (relapsed or refractory; P = .047), and peripheral blood blast count (P = .016). On multivariate analysis, overall survival was significantly associated with FLAG‐Ida response (hazard ratio, 0.343; P = .001) and receipt of allotransplantation (hazard ratio, 0.277; P < .001). Conclusion Our data seem to confirm the value of FLAG‐Ida in this setting and might suggest its best usage as bridge therapy for patients awaiting allotransplantation. Micro‐Abstract Allotransplant is crucial for improving survival in refractory/first relapsed AML patients. An overall response was achieved in 48 patients (44% of the whole group) with FLAG‐Ida chemotherapy approach. 24 of 48 responders underwent allotransplantation obtaining a median OS of 60 months.

Overexpression of the LSAMP and TUSC7 genes in acute myeloid leukemia following microdeletion/duplication of chromosome 3

The 3q13.31 microdeletion syndrome is characterized by developmental delay, postnatal growth above the mean, characteristic facial features, and abnormal male genitalia. Moreover, a frequent deletion in the 3q13.31 chromosome region has been identified in patients who are affected by osteosarcomas. Among the genes located within the deleted region, the involvement of the limbic system-associated membrane protein gene (LSAMP), together with a non-coding RNA tumor suppressor candidate 7 gene (TUSC7), has been suggested. We describe the case of an adult acute myeloid leukemia (AML) patient with a novel chromosomal rearrangement characterized by a 3q13.31 microdeletion and an extra copy of the 3q13.31-q29 chromosomal region translocated to the long arm of the Y chromosome. This karyotypic aberration seems to cause LSAMP and TUSC7 gene expression dysregulation. In conclusion, we report the first case of LSAMP and TUSC7 gene overexpression, possibly due to a position effect in an AML patient bearing a 3q13.31 cryptic deletion.

A new recurrent chromosomal translocation t(3;11)(q13;q14) in myelodysplastic syndromes associated with overexpression of the ILDR1 gene

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by ineffective hematopoiesis and an increased risk of evolution to acute myeloid leukemia (AML). In this study, the combination of conventional cytogenetic, FISH studies and molecular techniques allowed us to unveil a novel recurrent t(3;11)(q13;q14) causing the overexpression of the immunoglobulin-like domain-containing receptor (ILDR1) gene. The analysis of gene expression was extended to Refractory Anemia (RA) and Refractory Anemia with excess blasts (RAEB) cases revealing ILDR1 overexpression in 36% of RAEB subgroup. The biological implications of the ILDR1 overexpression in MDS pathogenesis and its potential prognostic significance should be further investigated.

A novel t(4;16)(q25;q23.1) associated with EGF and ELOVL6 deregulation in acute myeloid leukemia.

About 50% of acute myeloid leukemia (AML) patients show the occurrence of non-random chromosome rearrangements. Most of the recurrent karyotypic rearrangements in AML have been defined as distinct disease entities in the 2008 World Health Organization (WHO) classification. In this paper we report an AML case showing a novel t(4;16)(q25;q23.1) rearrangement causing the activation of epidermal growth factor (EGF) and elongation of long-chain fatty acids family member 6 (ELOVL6) genes, rather than the generation of a novel fusion gene.

Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia

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.

5'RUNX1-3'USP42 chimeric gene in acute myeloid leukemia can occur through an insertion mechanism rather than translocation and may be mediated by genomic segmental duplications

BackgroundThe runt-related transcription factor 1 (RUNX1) gene is a transcription factor that acts as a master regulator of hematopoiesis and represents one of the most frequent targets of chromosomal rearrangements in human leukemias. The t(7;21)(p22;q22) rearrangement generating a 5‘RUNX1-3’USP42 fusion transcript has been reported in two cases of pediatric acute myeloid leukemia (AML) and further in eight adult cases of myeloid neoplasms. We describe the first case of adult AML with a 5‘RUNX1-3’USP42 fusion gene generated by an insertion event instead of chromosomal translocation.MethodsConventional and molecular cytogenetic analyses allowed the precise characterization of the chromosomal rearrangement and breakpoints identification. Gene expression analysis was performed by quantitative real-time PCR experiments, whereas bioinformatic studies were carried out for revealing structural genomic characteristics of breakpoint regions.ResultsWe identified an adult AML case bearing a ins(21;7)(q22;p15p22) generating a 5‘RUNX1-3’USP42 fusion gene on der(21) chromosome and causing USP42 gene over-expression. Bioinformatic analysis of the genomic regions involved in ins(21;7)/t(7;21) showed the presence of interchromosomal segmental duplications (SDs) next to the USP42 and RUNX1 genes, that may underlie a non-allelic homologous recombination between chromosome 7 and 21 in AML.ConclusionsWe report the first case of a 5‘RUNX1-3’USP42 chimeric gene generated by a chromosomal cryptic insertion in an adult AML patient. Our data revealed that there may be a pivotal role for SDs in this very rare but recurrent chromosomal rearrangement.