Assessing Measurable Residual Disease in Chronic Myeloid Leukemia. BCR-ABL1 IS in the Avant-Garde of Molecular Hematology

Chronic myelogenous leukemia (CML) is a malignancy of the myeloid cell lineage characterized by a recurrent chromosomal abnormality: the Philadelphia chromosome, which results from the reciprocal translocation of the chromosomes 9 and 22. The Philadelphia chromosome contains a fusion gene called BCR-ABL1. The BCR-ABL1 codes for an aberrantly functioning tyrosine kinase that drives the malignant proliferation of the founding clone. The advent of tyrosine kinase inhibitors (TKI) represents a landmark in the treatment of CML, that has led to tremendous improvement in the remission and survival rates. Since the introduction of imatinib, the first TKI, several other TKI have been approved that further broadened the arsenal against CML. Patients treated with TKIs require sensitive monitoring of BCR-ABL1 transcripts with quantitative real-time polymerase chain reaction (qRT-PCT), which has become an essential part of managing patients with CML. In this review, we discuss the importance of the BCR-ABL1 assay, and we highlight the growing importance of BCR-ABL1 dynamics. We also introduce a mathematical correction for the BCR-ABL1 assay that could help homogenizing the use of the ABL1 as a control gene. Finally, we discuss the growing body of evidence concerning treatment-free remission. Along with the continuous improvement in the therapeutic arsenal against CML, the molecular monitoring of CML represents the avant-garde in the struggle to make CML a curable disease

Microbial community dynamics in mother's milk and infant's mouth and gut in moderately preterm infants

Mother's own milk represents the optimal source for preterm infant nutrition, as it promotes immune defenses and gastrointestinal function, protects against necrotizing enterocolitis, improves long-term clinical outcome and is hypothesized to drive gut microbiota assembly. Preterm infants at birth usually do not receive their mother's milk directly from the breast, because active suckling and coordination between suckling, swallowing and breathing do not develop until 32-34 weeks gestational age, but actual breastfeeding is usually possible as they grow older. Here, we enrolled moderately preterm infants (gestational age 32-34 weeks) to longitudinally characterize mothers' milk and infants' gut and oral microbiomes, up to more than 200 days after birth, through 16S rRNA sequencing. This peculiar population offers the chance to disentangle the differential contribution of human milk feeding per se vs. actual breastfeeding in the development of infant microbiomes, that have both been acknowledged as crucial contributors to short and long-term infant health status. In this cohort, the milk microbiome composition seemed to change following the infant's latching to the mother's breast, shifting toward a more diverse microbial community dominated by typical oral microbes, i.e., Streptococcus and Rothia. Even if all infants in the present study were fed human milk, features typical of healthy, full term, exclusively breastfed infants, i.e., high percentages of Bifidobacterium and low abundances of Pseudomonas in fecal and oral samples, respectively, were detected in samples taken after actual breastfeeding started. These findings underline the importance of encouraging not only human milk feeding, but also an early start of actual breastfeeding in preterm infants, since the infant's latching to the mother's breast might constitute an independent factor helping the health-promoting assembly of the infant gut microbiome

Next-generation sequencing for BCR-ABL1 kinase domain mutation testing in patients with chronic myeloid leukemia: A position paper

BCR-ABL1 kinase domain (KD) mutation status is considered to be an important element of clinical decision algorithms for chronic myeloid leukemia (CML) patients who do not achieve an optimal response to tyrosine kinase inhibitors (TKIs). Conventional Sanger sequencing is the method currently recommended to test BCR-ABL1 KD mutations. However, Sanger sequencing has limited sensitivity and cannot always discriminate between polyclonal and compound mutations. The use of next-generation sequencing (NGS) is increasingly widespread in diagnostic laboratories and represents an attractive alternative. Currently available data on the clinical impact of NGS-based mutational testing in CML patients do not allow recommendations with a high grade of evidence to be prepared. This article reports the results of a group discussion among an ad hoc expert panel with the objective of producing recommendations on the appropriateness of clinical decisions about the indication for NGS, the performance characteristics of NGS platforms, and the therapeutic changes that could be applied based on the use of NGS in CML. Overall, these recommendations might be employed to inform clinicians about the practical use of NGS in CML

Next-generation sequencing for BCR-ABL1 kinase domain mutation testing in patients with chronic myeloid leukemia: A position paper

BCR-ABL1 kinase domain (KD) mutation status is considered to be an important element of clinical decision algorithms for chronic myeloid leukemia (CML) patients who do not achieve an optimal response to tyrosine kinase inhibitors (TKIs). Conventional Sanger sequencing is the method currently recommended to test BCR-ABL1 KD mutations. However, Sanger sequencing has limited sensitivity and cannot always discriminate between polyclonal and compound mutations. The use of next-generation sequencing (NGS) is increasingly widespread in diagnostic laboratories and represents an attractive alternative. Currently available data on the clinical impact of NGS-based mutational testing in CML patients do not allow recommendations with a high grade of evidence to be prepared. This article reports the results of a group discussion among an ad hoc expert panel with the objective of producing recommendations on the appropriateness of clinical decisions about the indication for NGS, the performance characteristics of NGS platforms, and the therapeutic changes that could be applied based on the use of NGS in CML. Overall, these recommendations might be employed to inform clinicians about the practical use of NGS in CML

Application of the whole-transcriptome shotgun sequencing approach to the study of Philadelphia-positive acute lymphoblastic leukemia

Although the pathogenesis of BCR–ABL1-positive acute lymphoblastic leukemia (ALL) is mainly related to the expression of the BCR–ABL1 fusion transcript, additional cooperating genetic lesions are supposed to be involved in its development and progression. Therefore, in an attempt to investigate the complex landscape of mutations, changes in expression profiles and alternative splicing (AS) events that can be observed in such disease, the leukemia transcriptome of a BCR–ABL1-positive ALL patient at diagnosis and at relapse was sequenced using a whole-transcriptome shotgun sequencing (RNA-Seq) approach. A total of 13.9 and 15.8 million sequence reads was generated from de novo and relapsed samples, respectively, and aligned to the human genome reference sequence. This led to the identification of five validated missense mutations in genes involved in metabolic processes (DPEP1, TMEM46), transport (MVP), cell cycle regulation (ABL1) and catalytic activity (CTSZ), two of which resulted in acquired relapse variants. In all, 6390 and 4671 putative AS events were also detected, as well as expression levels for 18 315 and 18 795 genes, 28% of which were differentially expressed in the two disease phases. These data demonstrate that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations potentially involved in ALL

Case Report: A Novel Activating FLT3 Mutation in Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) is among the most common driver genes recurrently mutated in acute myeloid leukemia (AML), accounting for approximately 30% of cases. Activating mutations of the FLT3 receptor include internal tandem duplications (ITD) that map to the auto-inhibitory juxtamembrane (JM) domain or point mutations within the tyrosine kinase domain (TKD). Several FLT3 tyrosine kinase inhibitors have been developed in the last few years, but midostaurin is currently the only one approved for the treatment of newly diagnosed patients harboring FLT3 mutations. Here we describe for the first time a novel in-frame deletion in exon 14 (JM domain) of the FLT3 gene, that we identified in a young woman with CBFb-MYH11-positive AML. We demonstrated that this novel FLT3 variant is pathogenic, since it is responsible for constitutive activation of FLT3 receptor. Finally, ex-vivo studies demonstrated that this novel mutation is sensitive to midostaurin

Identification of Two DNMT3A Mutations Compromising Protein Stability and Methylation Capacity in Acute Myeloid Leukemia

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795 17; NM-022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM-022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586-2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862-Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse

Rotation of nilotinib and imatinib for first-line treatment of chronic phase chronic myeloid leukemia.

he introduction of second-generation tyrosine-kinase inhibitors (TKIs) has generated a lively debate on the choice of first-line TKI in chronic phase, chronic myeloid leukemia (CML). Despite the TKIs have different efficacy and toxicity profiles, the planned use of two TKIs has never been investigated. We report on a phase 2 study that was designed to evaluate efficacy and safety of a treatment alternating nilotinib and imatinib, in newly diagnosed BCR-ABL1 positive, chronic phase, CML patients. One hundred twenty-three patients were enrolled. Median age was 56 years. The probabilities of achieving a complete cytogenetic response, a major molecular response, and a deep molecular response (MR 4.0) by 2 years were 93%, 87%, and 61%, respectively. The 5-year overall survival and progression-free survival were 89%. Response rates and survival are in the range of those reported with nilotinib alone. Moreover, we observed a relatively low rate of cardiovascular adverse events (5%). These data show that the different efficacy and toxicity profiles of TKIs could be favorably exploited by alternating their us