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Background: Venetoclax in combination with hypomethylating agents (HMA) is revolutionizing the therapy of acute myeloid leukemia (AML). However, evidence on large sets of patients is lacking, especially in relapsed or refractory leukemia. Methods: AVALON is a multicentric cohort study that was conducted in Italy on patients with AML who received venetoclax-based therapies from 2015 to 2020. The study was approved by the ethics committee of the participating institution and was conducted in accordance with the Declaration of Helsinki. The effectiveness and toxicity of venetoclax+HMA in 190 (43 newly diagnosed, 68 refractory, and 79 relapsed) patients with AML are reported here. Results: In the newly diagnosed AML, the overall response rate and survival confirmed the brilliant results demonstrated in VIALE-A. In the relapsed or refractory AML, the combination demonstrated a surprisingly complete remission rate (44.1% in refractory and 39.7% in relapsed evaluable patients) and conferred to treated patients a good expectation of survival. Toxicities were overall manageable, and most incidents occurred in the first 60days of therapy. Infections were confirmed as the most common nonhematologic adverse event. Conclusions: Real-life data show that the combination of venetoclax and HMA offers an expectation of remission and long-term survival to elderly, newly diagnosed patients, and to relapsed or chemoresistant AML, increasing the chance of cure through a different mechanism of action. The venetoclax+HMA combination is expected to constitute the base for triplet combinations and integration of target therapies. Our data contribute to ameliorate the understanding of venetoclax+HMA effectiveness and toxicities in real life.

Omidubicel (nicotinamide-expanded cord blood) is a potential alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-identical donor is lacking. A phase I/II trial with standalone omidubicel HCT showed rapid and robust neutrophil and platelet engraftment. In this study, we evaluated the immune reconstitution (IR) of patients receiving omidubicel grafts during the first 6 months post-transplant, as IR is critical for favorable outcomes of the procedure. Data was collected from the omidubicel phase I-II international, multicenter trial. The primary endpoint was the probability of achieving adequate CD4+ T-cell IR (CD4IR: > 50 x 10(6)/L within 100 days). Secondary endpoints were the recovery of T-cells, natural killer (NK)-cells, B-cells, dendritic cells (DC), and monocytes as determined with multicolor flow cytometry. LOESS-regression curves and cumulative incidence plots were used for data description. Thirty-six omidubicel recipients (median 44; 13-63 years) were included, and IR data was available from 28 recipients. Of these patients, 90% achieved adequate CD4IR. Overall, IR was complete and consisted of T-cell, monocyte, DC, and notably fast NK- and B-cell reconstitution, compared to conventional grafts. Our data show that transplantation of adolescent and adult patients with omidubicel results in full and broad IR, which is comparable with IR after HCT with conventional graft sources.

Giacomo Andreani, Daniela Cilloni Department of Clinical and Biological Sciences, University of Turin, Turin, Italy Abstract: Currently, the post-remission treatment in acute leukemia is based on the genetic profile of leukemic cells at diagnosis (ie, FLT3 ITD positivity) and on the level of measurable residual disease (MRD) after induction and consolidation chemotherapy. Two methods are currently preferred for MRD evaluation in many centers: multiparameter flow cytometry and real-time quantitative PCR. Additional methods such as next-generation sequencing and digital PCR are under investigation, in an attempt to increase the sensitivity and thus allowing the detection of small clones. Many studies suggest that MRD positivity after chemotherapy is associated with negative prognosis, and the reappearance of MRD during follow-up allows impending relapse to be identified and consequently enables early intervention. Finally, MRD positivity before hematopoietic stem cell transplantation is predictive of the outcome. Although the significance of MRD in acute leukemia has been widely explored, the assessment of molecular MRD is not yet a routine practice. In this review, we describe the significanceof MRD in different settings and the main markers and methods used for MRD detection. Keywords: minimal residual disease, acute leukemia, multiparameter flow cytometry, real-time quantitative polymerase chain reaction, next-generation sequencing, digital PCR

Background Gram-negative bacteremia (GNB) is a major cause of illness and death after hematopoietic stem cell transplantation (HSCT), and updated epidemiological investigation is advisable. Methods We prospectively evaluated the epidemiology of pre-engraftment GNB in 1118 allogeneic HSCTs (allo-HSCTs) and 1625 autologous HSCTs (auto-HSCTs) among 54 transplant centers during 2014 (SIGNB-GITMO-AMCLI study). Using logistic regression methods. we identified risk factors for GNB and evaluated the impact of GNB on the 4-month overall-survival after transplant. Results The cumulative incidence of pre-engraftment GNB was 17.3% in allo-HSCT and 9% in auto-HSCT. Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were the most common isolates. By multivariate analysis, variables associated with GNB were a diagnosis of acute leukemia, a transplant from a HLA-mismatched donor and from cord blood, older age, and duration of severe neutropenia in allo-HSCT, and a diagnosis of lymphoma, older age, and no antibacterial prophylaxis in auto-HSCT. A pretransplant infection by a resistant pathogen was significantly associated with an increased risk of posttransplant infection by the same microorganism in allo-HSCT. Colonization by resistant gram-negative bacteria was significantly associated with an increased rate of infection by the same pathogen in both transplant procedures. GNB was independently associated with increased mortality at 4 months both in allo-HSCT (hazard ratio, 2.13; 95% confidence interval, 1.45–3.13; P Conclusions Pre-engraftment GNB is an independent factor associated with increased mortality rate at 4 months after auto-HSCT and allo-HSCT. Previous infectious history and colonization monitoring represent major indicators of GNB. Clinical Trials registration NCT02088840.

Background: ALL is a disease characterised by lack of differentiation and uncontrolled proliferation of lymphocyte precursor cells, driven by activation of oncogenes and inactivation of tumour suppressor genes. About 30% of B-cell ALL carries the Philadelphia chromosome (Ph), originated by the translocation t(9;22)(q34;q11), and thus express the fusion gene BCR-ABL. The constitutively active tyrosine kinase encoded by BCR-ABL blocks the differentiation of B precursor cells, prevents apoptosis and also causes genetic instability. This ultimately leads to the acquisition of new mutations, resistance to BCR-ABL inhibitors and disease progression. In particular BCR-ABL stimulates DNA repair by non-homologous end-joining and homologous recombination repair mechanisms after various genotoxic stimuli, but a lot of deletions and abnormalities are also generated. Methods and patients: In order to identify whether some genetic lesions could contribute to genetic instability in Ph+ ALL, we analyzed the genome of 18 Ph+ ALL patients using high-resolution single nucleotide polymorphism (SNP) array. 250 ng of genomic DNA were processed on 250K NspI array according to protocols provided by the manufacturer (Affymetrix Inc., Santa Clara, CA, USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH) and real-time quantitative polymerase chain reaction (PCR) were performed to validate our results. Results: We found a significant deletion on chromosome 22, cytoband q11.22, close to immunoglobulin lambda light chain genes, in the region 20913571-20931814, corresponding to the VpreB1 (CD179a, Pre-B-Lymphocyte gene 1) gene in about 30% of newly diagnosed Ph+ ALL patients. It is highly conserved among mammals and selectively expressed during the pre-B stage of B cells ontogenesis and together with IGLL1 encodes for the surrogate light chain of the pre-B cell receptor. In mice its monoallelic deletion is associated with an increase of lymphocytes in pre-B stage in bone marrow, while its complete absence causes the impossibility to generate mature B cells. Since Ph+ ALL cells are immunophenotypically blocked at the pre-B stage, we are investigating whether VpreB1 deletion could interfere with normal B cell development and thus could have a role in leukeamogenesis and disease progression. Conclusion: This study demonstrated that the use of high-resolution SNP array is a powerful technology to detect genetic anomalies not revealed by classical cytogenetics which may provide new insights into leukemogenesis.

Background: The Bcr-Abl kinase expressed in ALL drives malignant transformation of human pre-B cells and promote aberrant alternative splicing of genes involved in B-cell signaling and differentiation. The fact that Ikaros (Ik) transcription factor functions as a critical regulator of normal lymphocyte development and the observation of rapid development of leukaemia in mice expressing non-DNA binding isoforms, prompted our study to investigate whether normal Ik expression and function might be altered in Ph+ ALL. Patients and methods: Bone marrow and peripheral blood samples from 47 adult patients (median age 55 yrs, 18-76) with Ph+ ALL were collected after informed consent. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for exon 1 and exon 7 of Ik, cloning, nucleotide sequencing and western blot analyses were performed to identify the specific Ik isoforms. Results: In 43/47 (90%) patients the Ik6 isoform, lacking all N-terminal zinc-fingers responsible for DNA-binding, was detected and in 23 of them (54%) it was the predominant isoform. Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patients expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In the patients who seemed to express by RT-PCR only full-length Ik isoforms cloning and subsequent sequencing revealed clones harboring different transcripts. We detected an in frame deletion of 10 aa (ΔKSSMPQKFLG) at the end of ex6 due to the selection of an alternative donor splice site. The consequences were impaired activation of transcription and impaired ability to form dimers. We also observed isoforms with a 60-bp insertion (TYGADDFRDFHAIIPKSFSR) immediately upstream of exon 4 at the exon 2/exon 4 junction either alone or together with the in-frame deletion. Also in this in case the aberration is due to the selection of alternative splice site which determines the skipping of exon 3 and the insertion of a sequence inside the intron 3-4 which starts with an alternative acceptor splice site. Aberrant Ik isoforms had a cytoplasmatic localization as revealed by immunofluorescence and confocal laser scanning microscopy analyses. Conclusions: Our data demonstrated that aberrant splicing of Ikaros represents a frequent feature in Ph+ ALL patients and that dominant negative Ik6 splice variant correlates with BCR-ABL mRNA levels, disease progression and relapse.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.

Purpose: Most patients with chronic-phase chronic myeloid leukemia (CML) who receive imatinib achieve a complete cytogenetic remission (CCgR) and low levels of BCR-ABL transcripts. CCgR is durable in the majority of patients but relapse occurs in a subset. Experimental Design: To determine the potential of quantitative reverse transcription-PCR of BCR-ABL to predict cytogenetic relapse, we serially monitored residual disease in 97 CML patients with an imatinib-induced CCgR. Patients with late chronic phase CML after IFN-α failure were treated with imatinib (400 mg daily). Results: During the imatinib median follow-up time of 36 months (range, 12-54 months), disease monitoring occurred by cytogenetics and quantitative PCR. Twenty percent of patients experienced cytogenetic relapse at a median of 18 months after CCgR and a median of 24 months after starting imatinib. None of the possible prognostic factors studied in univariate and multivariate analyses seemed to predict for loss of cytogenetic response but the reduction of BCR-ABL transcript levels at the time of CCgR is an important prognostic factor. Conclusions: In our study, we showed not only that achieving a major molecular remission at 12 months is predictive of a durable cytogenetic remission but also that patients who achieved a major molecular remission (expressed both as the BCR-ABL/β2 microglobulin ratio %

Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL. Aim: To characterize mutations and rearrangements on 9p involving PAX5 in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of exons (2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions. Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, respectively, detecting transcripts from 62% and 64% of human annotated genes. Low RPKM estimates (0.01

Introduction: Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for its development. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and may be used to better classify the disease by dissection of genomic subsets. Methods: 105 adult patients with acute myeloid leukemia were analyzed. These cases included all French-American-British subtypes, miscellaneous cytogenetic abnormalities and normal karyotype subgroups. Among these, the M3 subtype included 28 patients, representing the 33% of the whole study population. Genomic DNA was isolated from blast cells and applied to Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) following the manufacturer's instructions. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different copy number alterations (CNAs) were identified in all cases and no significant difference in the average number of alterations was detected among different leukemia cytogenetic subgroups except for the complex subgroup, which had an average of 55 CNA/patient. In APL cases an average of 8 CNAs per case (range, 1-24) was found. The macroscopic alterations were rare, confirmed conventional cytogenetics and involved trisomy of chromosome 8 in 3 cases, loss of chromosome 6, loss of chromosome 20 and deletions on chromosome 9 and 7. Microscopic CNAs (< 1.5 Mbps) involved every chromosome at least once and predominantly chromosomes 1, 2, 9, 15 and 17. Genetic gains were more common than losses and their median size was 300 kb (range 0.2- 1.4 Mb). The majority of lesions were not recurrent, being identified in only a single patient. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes. Hemizygous deletions were identified at at 2q33.3-q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 3p11.2 (EPHA3) and at 12p13 (ETV6). Deletions also affected genes involved in cell regulations as CDKN2A (9p21) and RB1 (13q14.2). Most frequent gains affected the TP73 gene at 1p36.3, the oncogenes MYC and PVT1 at 8q24 (4.33 Mb), PRAME at the 22q11.22. Copy neutral loss of heterozygosity events affected 1p34.2-1p32.3, 10p11.2 (MLLT10), 11p11.2 (WT1, CDKN1C, HRAS). Patients with more than 10 CNAs were associated with a worse prognosis. Conclusions: These data demonstrate that different cooperating events may be involved in the generation of APL. These novel findings may be used to stratify patients according to genomic changes and to facilitate the screening for novel therapeutic targets. Supported by: AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo.

Background: BCR-ABL1-positive ALL is the most frequent and prognostically unfavorable subtype of adult ALL, mainly because of genetic instability. Activation-induced cytidine deaminase (AID) introduces single-strand breaks into target DNA and produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and Methods: We analyzed 61 adult de novo BCR-ABL1-positive ALL patients (pts) and 60 CML (chronic phase and myeloid/lymphoid blast crisis) pts. AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5#8217; end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. AID expression was also found in lymphoid blast crisis CML (50%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform; 19/61 (31%) co-expressed the wild-type and different AID splice variants with deaminase activity (AID#916;E4a, with a 30 bp deletion of exon 4; AID#916;E4, with the exon 4 deletion; AIDins3, with the retention of intron 3-4); 4/61 (7%) expressed the AID#916;E3-E4 isoform without deaminase activity (deletion of exons 2 and 3). To investigate whether AID introduces DNA-single strand breaks, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA). Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative (median copy number alteration of 14 versus 4. respectively, p

By genome-wide analyses of DNA copy number abnormalities in adult BCR-ABL1-positive ALL patients (pts) using single nucleotide polymorphism (SNP) microarrays (Affymetrix 500K and SNP 6.0), we identified a high frequency of genetic alterations of regulators of B lymphoid development (PAX5, EBF1, IKZF1) and cell cycle (CDKN2A, CDKN2B, BTG1). The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1 (68/106 patients, 64%), which encodes the transcription factor Ikaros required for the earliest stages of lymphoid lineage commitment. Here, we characterized and mapped all breakpoints in IKZF1 gene to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4-7 (44%) and due to breakpoints occurring in introns 3 and 7; type B characterized by removal of exons 2-7 (19%) and due to a variable pattern of breakpoints in introns 1 and 7. In two pts we had both \#916;2-7 and \#916;4-7 deletions and in one we identified a deletion of all IKZF1 gene and part of GRB10. A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of both deletions correlated with the expression of dominant-negative isoforms with cytoplasmatic localization. The IKZF1 alteration was also identified in B-negative ALL pts (41%) and at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML and chronic phase CML. Known DNA sequences were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH). Gene expression profiling analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3, BLK), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to an impaired B-cell differentiation. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.

Background: The Ph chromosome is the most frequent karyotypic aberration in adults with ALL and it represents the single most significant adverse prognostic marker. Despite the encouraging results achieved with imatinib, resistance develops rapidly and is quickly followed by disease progression. Recently, the introduction of array-based analysis of single nucleotide polymorphism (SNP) has allowed the rapid determination of genome-wide allelic information at high density for a DNA sample. Patients and Methods: In order to identify, at submicroscopic level, genetic lesions which can escape standard cytogenetic observations and may contribute to leukemogenesis and resistance, we profiled, until now, the genomes of 18 patients out of 55 Ph+ ALL patients treated in our institute with imatinib or dasatinib. 250 ng of genomic DNA were processed on 500K SNP array according to protocols provided by the manufacturer (Affymetrix Inc., Santa Clara, CA, USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals and a diploid reference set of samples obtained from acute leukaemia cases in remission using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH) and real-time quantitative PCR were performed to validate our results. Results: We identified multiple copy number alterations per case, with deletions outnumbering amplification almost 3:1. Lesions varied from loss or gain of complete chromosome arms (trisomy 4, monosomy 7, loss of 9p, 10q, 14q, 16q and gain of 1q and 17q) to microdeletions and microduplications targeting genomic intervals. The common microdeletions included 1p36.21 (PRAMEF), 3q29 (TFCR), 8p23 (DEFB105A), 14q11.2 (DAD1), 16p13.11 (PDXDC1, NTAN1, RRN3), 16p11.2 (SPN) and 19p13.2 (CARM1, SMARCA4). Some genomic alterations were identified in genes regulating B-lymphocyte differentiation, such as PAX5 (n=3), BLNK (n=1) and VPREB1 (n=6), IKZF1 (n=7) and in genes with an established role in leukemogenesis, such as MDS, BTG1, MLLT3, RUNX1. The most frequently affected genes were those controlling G1/S cell cycle transition (e.g. CDKN2A and CDKN2B). Furthermore, many of the deletions detected included genes encoded for phosphatase proteins (e.g. PTPRD, PPP1R9B, PTPN18) and for zinc-finger proteins. It is noteworthy that some lesions felt in regions lacking annotated genes. Discussion: Using high-resolution genome wide approach we showed that multiple sub-microscopic genomic anomalies, which may be suspected to contribute to leukemogenesis and used as targets for existing or novel drugs, frequently occur in Ph+ ALL

To detect factors associated with quality of life (QOL) of patients with myelodysplastic syndrome (MDS) and to compare the MDS patients’ self-assessed QOL with that perceived by their physicians. In an observational, non-interventional, prospective, multicentre study, QOL was evaluated in 148 patients with newly diagnosed low- and intermediate-risk IPSS MDS. QOL measures (QOL-E v.2, LASA and EQ-5D) and patient-related candidate determinants of QOL were assessed for up to 18 months. Patients' QOL scores were compared with those obtained by appointed hematologists’ assessment and with ECOG performance status (PS). Fatigue was not prevalent at diagnosis, though physical QOL and energy levels were low. Transfusion-dependent patients had worse QOL scores. In multivariate analysis, Hb levels and comorbidities were a major determinant of QOL. Physicians’ perception of patients’ well-being correlated with patients’ QOL. Physicians underestimated the impact of disturbances on patients’ QOL, mainly in the MDS-specific components. ECOG PS did not discriminate patients according to QOL status. In conclusion, the association of anemia with QOL is confirmed, while co-morbidities emerge as an independent predictor of QOL in MDS. Fatigue is not a major concern. ECOG PS is not a valuable surrogate of patient’s QOL, thus highlighting that physician’s judgment of patient’s well-being must not substitute patient-reported outcomes. Appropriate questionnaires should be used to assess MDS patients’ QOL in order to improve communication and therapeutic choice.

The FIP1L1-PDGFRA fusion gene is a recurrent molecular abnormality in patients with eosinophilia-associated myeloproliferative neoplasms. We characterized FIP1L1-PDGFRA junction sequences from 113 patients at the mRNA (n=113) and genomic DNA (n=85) levels. Transcript types could be assigned in 109 patients as type A (n=50, 46%) or B (n=47, 43%), which were created by cryptic acceptor splice sites in different introns of FIP1L1 (type A) or within PDGFRA exon 12 (type B). We also characterized a new transcript type C (n=12, 11%) in which both genomic breakpoints fell within coding sequences creating a hybrid exon without use of a cryptic acceptor splice site. The location of genomic breakpoints within PDGFRA and the availability of AG splice sites determine the transcript type and restrict the FIP1L1 exons used for the creation of the fusion. Stretches of overlapping sequences were identified at the genomic junction site, suggesting that the FIP1L1-PDGFRA fusion is created by illegitimate non-homologous end-joining. Statistical analyses provided evidence for clustering of breakpoints within FIP1L1 that may be related to DNA- or chromatin-related structural features. The variability in the anatomy of the FIP1L1-PDGFRA fusion has important implications for strategies to detect the fusion at diagnosis or for monitoring response to treatment.Leukemia advance online publication, 6 November 2008;(2008) 0, 000-000. doi:10.1038/leu.2008.310.

The introduction of the BCR-ABL kinase inhibitor, imatinib mesylate (Gleevec®, Novartis) led to significant changes in the treatment of chronic myeloid leukaemia (CML) patients. However, despite the impressive percentage of responding patients, some CML cases, particularly those in advanced phases of the disease, show primary resistance or relapse after the initial response. The second-generation BCR-ABL inhibitors nilotinib (Tasigna®, Novartis) and dasatinib (Sprycel®, Bristol-Myers Squibb) have shown significant activity in clinical trials in patients who failed imatinib therapy, but these agents are still incapable of inhibiting the T315I mutant of Bcr-Abl and present partial activity in advanced phases of CML. The acquired biological notions of the mechanisms of tyrosine kinase inhibitor (TKI) resistance has led to the development of new compounds, some of which have shown encouraging preliminary results in clinical trials, even against T315I mutants. In this paper we discuss the new emerging therapies which may overcome TKI resistance in CML patients. A introdução do inibidor de tirosino quinase BCR-ABL mesilato de imatinibe (Glivec®, Novartis) levou a significantes mudanças no tratamento da LMC. Entretanto, a despeito de impressionante porcentagem de pacientes que respondem, alguns casos de LMC, particularmente em fases avançadas da doença mostram resistência primaria ou recidivas após terapêutica inicial. Inibidores de tirosino quinases de segunda geração como o nilotinibe (Tasigna®, Novartis) e o dasatinibe (Sprycel®, Bristol Myers Squibb) têm mostrado significante atividade nos estudos clínicos em paciente onde o imatinibe falhou. Porém, estes agentes não são capazes de inibir a mutação T315I do Bcr-Abl e apresentam atividade parcial em fases avançadas da LMC. As noções biológicas adquiridas sobre os mecanismos de resistência aos inibidores de TK levaram ao desenvolvimento de novos compostos alguns dos quais têm resultados preliminares encorajadores incluindo a mutação T315I. Neste trabalho nós discutimos os novos agentes emergentes e qual o potencial poderão atingir para ultrapassar a resistência aos inibidores de TK em pacientes com LMC.

Purpose Imatinib mesylate (IM) has rapidly become the front-line treatment of Philadelphia-positive (Ph-pos) chronic myeloid leukemia, but the number of patients who were treated and are being treated with IM second-line is still substantial. Patients and Methods We have monitored and analyzed the cytogenetic and molecular response to IM 400 mg/d in a cohort of 277 late chronic phase (LCP) patients who were resistant or intolerant to interferon-α and were observed for 48 to 79 months (median, 72 months). Results One hundred fifty-three patients (55%) achieved a complete cytogenetic response (CCgR). Seventy-seven percent of them were still in CCgR after 5 years. The rate of response loss did not increase over time. The 6-year progression-free survival and overall survival of these 153 complete cytogenetic responders were 90% and 91%, respectively. Molecular response was less than major in 21%, major in 78%, and complete in one patient only. Conclusion These data confirm that, in LCP the CCgR rate to IM is 50% to 60%, and show that CCgR is stable and is associated with a prolonged survival, even if leukemia continues to be molecularly detectable.

To assess the effect of age on response and compliance to treatment in patients with chronic myeloid leukemia (CML) we performed a sub-analysis within a phase II trial of the GIMEMA CML Working Party (CML/002/STI571). Since the WHO cut-off age to define an older patient is 65 years, among the 284 patients considered, we identified 226 (80%) younger patients (below 65 years) and 58 (20%) older patients (above 65 years) before starting imatinib. Response rates (hematologic and cytogenetic) were lower in the older age group but the probabilities of progression-free survival and overall survival (median observation time 3 years) were the same. Moreover, among complete cytogenetic responders, no differences were found in the level of molecular response between the two age groups. As might be expected, older patients experienced more adverse events, both hematologic and non-hematologic: this worsened compliance did not, however, prevent a long-term outcome similar to that of younger patients.

Purpose Point mutations within the ABL kinase domain of the BCR-ABL gene have been associated with clinical resistance to imatinib mesylate in chronic myeloid leukemia (CML) patients. To shed further light on the frequency, distribution, and prognostic significance of ABL mutations, we retrospectively analyzed a homogeneous cohort of late chronic phase CML patients who showed primary cytogenetic resistance to imatinib. Patients and Methods Using denaturing high-performance liquid chromatography (D-HPLC) and sequencing, we screened for ABL mutations in a total of 178 bone marrow and/or peripheral blood samples from 40 late chronic phase CML patients homogeneously treated with imatinib 400 mg/d, who did not reach a major cytogenetic response at 12 months. Results Mutations were found in 19 of 40 patients (48%). Mutations were already detectable by D-HPLC at a median of 3 months from the onset of therapy. The presence of a missense mutation was significantly associated with a greater likelihood of subsequent progression to accelerated phase/blast crisis (P = .0002) and shorter survival (P = .001). Patients carrying mutations falling within the P-loop seemed to have a particularly poor outcome in terms of time to progression (P = .032) and survival (P = .045). Conclusion Our results show that, irrespective of the hematologic response, monitoring for emerging mutations in the first months of therapy may play a role in detecting patients with worse prognosis, for whom a revision of the therapeutic strategy should be considered.