Predicting which subsets of patients with myelodysplastic neoplasms are more likely to progress to overt chronic myelomonocytic leukemia.

The boundary between myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) has been revised in the latest World Health Organization classification of myeloid malignancies. These changes were motivated by the description of a subgroup of MDS patients identified as oligomonocytic chronic myelomonocytic leukemia (OM-CMML) at risk of evolving into overt CMML. Various studies will be reviewed describing the clinical and biological features of MDS patients evolving to CMML. The efforts to discover biomarkers enabling the identification of these patients at the time of MDS diagnosis will be discussed. Finally, the molecular landscape of these patients will be presented with a specific focus on the biallelic inactivation of TET2 in light of its functional impact on hematopoietic stem cells, granule-monocytic differentiation, and its tight interplay with inflammation.

Effective use of intensive treatment against multiple myeloma of recipient origin after allogeneic transplantation for acute myeloid leukemia.

We describe here a 56-years -old woman cured in our institution for an acute myeloid leukemia (AML) and a monoclonal gammopathy of undetermined significance (MGUS). In order to treat AML, underwent allogeneic stem cell transplantation in second complete remission. Four years after transplant, MGUS evolved to multiple myeloma and was intensively treated with "autologous" transplant after successful mobilization. This report illustrates: (i) a lack of efficacy of graft versus myeloma effect in a patient probably cured of AML by graft versus leukaemia effect; (ii) the ability to mobilize peripheral blood stem cells in order to perform "autologous" transplantation after allogeneic transplantation.

[Myeloid neoplasms associated with rearrangement of PDGFRB: A rare and tricky disease]. / La néoplasie myéloïde associée à un réarrangement de PDGFRB : une pathologie rare de diagnostic difficile.

In the latest World Health Organization classification (WHO), eosinophilic disorders represent a group of rare pathologic conditions with highly heterogeneous pathophysiology. In this report, we describe a case of myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRB gene in a 67-year-old-male patient hospitalized with cerebellous ataxia. Initial investigations showed a bicytopenia with hypereosinophilia varying from 1.1 to 1.6×109/L. Bone marrow aspiration was rich and showed a heterogeneous distribution of myeloid cells with clusters of promyelocytes and proerythroblasts associated with numerous eosinophils and spindle-shaped mast cells but without excess of blasts, dysplasia nor maturation skewing. These aspects suggested an atypical myeloproliferative neoplasm. Bone marrow biopsy was performed showing also a very high cellularity with area of myeloid and erythroid precursors associated with numerous spindle-shaped mast cells. Diagnoses of unclassified myeloid neoplasm and/or systemic mastocytosis were then proposed. Further chromosome analysis showed a t(5;8) translocation with PDGFRB rearrangement revealed in fluorescent in situ hybridization. Patient was treated with imatinib and intravenous immunoglobulin therapy allowing a significant improvement in neurological symptoms and biological results. Patient condition is currently stable after six lines of treatment. This rare hematopoietic neoplasm displays unusual histological and cytological features and can mimic other myeloproliferative neoplasm. Specific cytogenetics analysis should be considered for such cases with hypereosinophilia to select patients that may benefit from targeted therapy.

Evaluation of two new highly multiplexed PCR assays as an alternative to next-generation sequencing for IDH1/2 mutation detection.

IDH1 and IDH2 somatic mutations have been identified in solid tumors and blood malignancies. The development of inhibitors of mutant IDH1 and IDH2 in the past few years has prompted the development of a fast and sensitive assay to detect IDH1R132 , IDH2R140 and IDH2R172 mutations to identify patients eligible for these targeted therapies. This study aimed to compare two new multiplexed PCR assays - an automated quantitative PCR (qPCR) on the PGX platform and a droplet digital PCR (ddPCR) with next-generation sequencing (NGS) for IDH1/2 mutation detection. These assays were evaluated on 102 DNA extracted from patient peripheral blood, bone marrow and formalin-fixed paraffin-embedded tissue samples with mutation allelic frequency ranging from 0.6% to 45.6%. The ddPCR assay had better analytical performances than the PGX assay with 100% specificity, 100% sensitivity and a detection limit down to 0.5% on IDH1R132 , IDH2R140 and IDH2R172 codons, and a high correlation with NGS results. Therefore, the new highly multiplexed ddPCR is a fast and cost-effective assay that meets most clinical needs to identify and follow cancer patients in the era of anti-IDH1/2-targeted therapies.

Fusion Gene Detection and Quantification by Asymmetric Capture Sequencing (aCAP-Seq).

Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARA are now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, µBCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.

High prevalence of unusual KRAS, NRAS, and BRAF mutations in POLE-hypermutated colorectal cancers.

Exonucleasic domain POLE (edPOLE) mutations, which are responsible for a hypermutated tumor phenotype, occur in 1-2% of colorectal cancer (CRC) cases. These alterations represent an emerging biomarker for response to immune checkpoint blockade. This study aimed to assess the molecular characteristics of edPOLE-mutated tumors to facilitate patient screening. Based on opensource data analysis, we compared the prevalence of edPOLE mutations in a control group of unselected CRC patients (n = 222) vs a group enriched for unusual BRAF/RAS mutations (n = 198). Tumor mutational burden (TMB) and immune infiltrate of tumors harboring edPOLE mutations were then analyzed. In total, 420 CRC patients were analyzed: 11 edPOLE-mutated tumors were identified, most frequently in microsatellite (MMR)-proficient young (< 70 years) male patients, with left-sided tumors harboring noncodon 12 KRAS mutation. The prevalence of edPOLE-mutated tumors in the control vs the experimental screening group was, respectively, 0.45% (n = 1) vs 5.0% (n = 10). Among the 11 edPOLE-mutated cases, two had a low TMB, three were hypermutated, and six were ultramutated. EdPOLE-mutated cases had a high CD8+ tumor-infiltrating lymphocyte (TIL) infiltration. These clinicopathological and molecular criteria may help to identify edPOLE mutations associated with a high TMB in CRC, and improve the selection of patients who could benefit from immunotherapy.

Genomic analysis of primary and secondary myelofibrosis redefines the prognostic impact of ASXL1 mutations: a FIM study.

We aimed to study the prognostic impact of the mutational landscape in primary and secondary myelofibrosis. The study included 479 patients with myelofibrosis recruited from 24 French Intergroup of Myeloproliferative Neoplasms (FIM) centers. The molecular landscape was studied by high-throughput sequencing of 77 genes. A Bayesian network allowed the identification of genomic groups whose prognostic impact was studied in a multistate model considering transitions from the 3 conditions: myelofibrosis, acute leukemia, and death. Results were validated using an independent, previously published cohort (n = 276). Four genomic groups were identified: patients with TP53 mutation; patients with ≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1, IDH2, NRAS, or KRAS (high-risk group); patients with ASXL1-only mutation (ie, no associated mutation in TP53 or high-risk genes); and other patients. A multistate model found that both TP53 and high-risk groups were associated with leukemic transformation (hazard ratios [HRs] [95% confidence interval], 8.68 [3.32-22.73] and 3.24 [1.58-6.64], respectively) and death from myelofibrosis (HRs, 3.03 [1.66-5.56] and 1.77 [1.18-2.67], respectively). ASXL1-only mutations had no prognostic value that was confirmed in the validation cohort. However, ASXL1 mutations conferred a worse prognosis when associated with a mutation in TP53 or high-risk genes. This study provides a new definition of adverse mutations in myelofibrosis with the addition of TP53, CBL, NRAS, KRAS, and U2AF1 to previously described genes. Furthermore, our results argue that ASXL1 mutations alone cannot be considered detrimental.

Aryl hydrocarbon receptor (AHR) is a novel druggable pathway controlling malignant progenitor proliferation in chronic myeloid leukemia (CML).

Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.

Whole-genome analysis reveals unexpected dynamics of mutant subclone development in a patient with JAK2-V617F-positive chronic myeloid leukemia.

We report here the first use of whole-genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML), who presented in chronic phase (CP) with doubly marked BCR-ABL1+/JAK2V617F-mutant cells and, over a 9-year period, progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS revealed that the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1, and RREB1 as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only six additional coding somatic mutations, despite retention by the hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells revealed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2 of 101 cases of myeloproliferative neoplasms, but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.

Leukemic stem cell persistence in chronic myeloid leukemia patients in deep molecular response induced by tyrosine kinase inhibitors and the impact of therapy discontinuation.

During the last decade, the use of tyrosine kinase inhibitor (TKI) therapy has modified the natural history of chronic myeloid leukemia (CML) allowing an increase of the overall and disease-free survival, especially in patients in whom molecular residual disease becomes undetectable. However, it has been demonstrated that BCR-ABL1- expressing leukemic stem cells (LSCs) persist in patients in deep molecular response. It has also been shown that the discontinuation of Imatinib leads to a molecular relapse in the majority of cases. To determine a possible relationship between these two phenomena, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL1-expressing LSCs in marrow samples from 21 patients in deep molecular response for three years after TKI therapy (mean duration seven years). LSCs were detected in 4/21 patients. Discontinuation of TKI therapy in 13/21 patients led to a rapid molecular relapse in five patients (4 without detectable LSCs and one with detectable LSCs). No relapse occurred in the eight patients still on TKI therapy, whether LSCs were detectable or not. Thus, this study demonstrates for the first time the in vivo efficiency of TKIs, both in the progenitor and the LSC compartments. It also confirms the persistence of leukemic stem cells in patients in deep molecular response, certainly at the origin of relapses. Finally, it emphasizes the difficulty of detecting residual LSCs due to their rarity and their low BCR-ABL1 mRNA expression.