Allogeneic Stem Cell Transplantation for MDS

Myelodysplastic syndromes are clonal disorders with morphological dysplasia, a variable degree of cytopenia and a risk of transformation to acute myeloid leukemia. Prognosis is very variable and is defined by blast count, cytopenia, cytogenetics and more recently by somatic mutations, with IPSS or revised IPSS score being the most widely used to assess disease risk. HSCT remains the only curative treatment to date, with high-risk patients obtaining the biggest benefit. However, NRM should be carefully assessed before indicating the transplant in this usually old population, where organ toxicity and comorbid conditions are to be considered. Multi-domain assessment tools, such as CGA (comprehensive geriatric assessment) and EBMT score, are useful in this context and might guide physician decisions regarding the transplant. Indeed, with the development of reduced intensity conditioning regimens, the number of patient candidates for an HSCT has increased. Regarding pre-transplant treatment, patients with a blast excess > 10% might be treated with HMAs or chemotherapy, although there are no randomized trials confirming the benefit of this approach, even when achieving a complete response. Concerning donor choice, matched sibling donors continue to be the first option, although matched unrelated donors, and more recently haploidentical donors, have proven to be valid options and should be offered in the absence of a related donor. Relapse remains the main cause of transplantation failure. MRD assessment and pre-emptive or prophylactic use of HMA or other targeted inhibitors with or without DLI are accepted strategies to reduce relapse risk, but the prognosis in this context remains dismal, and is the subject for several ongoing clinical protocols.

The effect of children’s facial expressions in eliciting benevolent behavior for child sponsorships versus one-time donations

Purpose This paper aims to examine the influence of personal distress on donor choice of happy- or sad-faced child in two donation contexts, monetary donations and child sponsorships. Design/methodology/approach This research conducted two experimental studies, in which participants chose a child to benefit out of eight needy children. Findings More people chose sad-faced children than happy-faced children in monetary donations, whereas in child sponsorships, the preference for sad-faced over happy-faced children disappeared: people chose happy-faced children as often as they chose sad-faced children. Originality/value This research distinguishes between two types of personal distress, experienced and anticipated distress, explaining why donor choices of child differ between monetary donations (where only experienced distress is present) and child sponsorships (where both experienced and anticipated distress are present).

Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

Impact of Donor Search Strategy for Allogeneic Hematopoietic Stem Cell Transplantation from Haploidentical or Matched Unrelated Donor for Patients Older Than 55 Years with Hematological Malignancies: Randomized Prospective Phase III Study

Background Allogeneic stem cell transplantation (Allo-SCT) from a matched related donor (MRD) is rarely performed for older patients because of lack of such a donor. Matched unrelated donor (MUD) is considered as an alternative for this population with limited access due to excessive expected toxicity. Recently, the development of allo-SCT from haploidentical donors (Haplo) with the use of high dose post-transplant cyclophosphamide showed promising results with outspreading diffusion despite HLA disparity. Donor search is early implemented when patients are referred to transplant team. The impact of this strategy on patients with high risk malignancies outcomes is not well known. We propose to address this question through an intention-to-treat trial. We also hypothesized that Haplo-SCT could be a valid alternative to MUD-SCT for older patients with hematological disease whenever allo-SCT is recommended. Study design and methods We performed a prospective, multicenter, open-label, randomized controlled trial (NCT02623309) comparing two strategies of allo-HSCT from UD or Haplo. Patients older than 55 years with hematological malignancies were randomly assigned to a Haplo and a MUD search soon after the absence of MRD was established. The goals of the study were to prospectively evaluate feasibility, safety and efficacy of these approaches in an intent-to-treat analysis and a HSCT-performed analysis. Results From February 2016 to June 2018, 108 patients were enrolled. One hundred and six patients were analyzed. Median age was 65 years (range 55-70). Diseases were myeloid malignancies in 84 patients (79%).DRI was low, intermediate and high in 5(5%), 59(55%) and 42(40%),respectively. Fifty-five patients were assigned to Haplo group and 51 patients to MUD group. Fifteen patients in Haplo group could not proceed to allo-SCT because of progression (n=9), contraindication (n=5), no donor (n=1) and 14 patients in MUD group because of progression (n=8), contraindication (n=4) and loss of indication (n=2). Among 40 patients in Haplo group, 9 patients (22%) actually received allo-SCT from MUD because of donor contraindication (n=6), donor specific antibodies (n=1), no Haplo identified (n=2). Eleven patients out of 37 patients (30%) in MUD group received allo-SCT from Haplo because of no MUD identified (n=7), donor refusal (n=2), donor contraindication (n=1), excessive search delay to identify a MUD (n=1). After cross over, 42 and 35 patients actually underwent per protocol allo-HSCT from a Haplo and a MUD, respectively. Median time from randomization to allo-SCT was 3 months (range 0.7-10). In intention-to-treat analysis from date of randomization, 2-year PFS and OS did not differ between the two groups (Haplo vs MUD arm: PFS: 42 vs 48 %, p=0.463; OS: 44 vs 61%, p=0.126). Non-relapse mortality (NRM) at 2 years was 31% for both groups while the 2-year cumulative incidence of relapse (CIR) was 14% and 18 % (p=0.99) after Haplo and MUD arm, respectively. In per-protocol analysis, with a median follow up of 26 months (range 3-34) after transplant 2-year NRM was 37% and 35% (p=0.893) after Haplo and MUD SCT, respectively. The cumulative incidence of grades 3-4 acute GVHD at 100 days was 21% and 17% (p = 0.402) in the Haplo and MUD SCT, respectively. No difference was observed in 2-year extensive chronic GVHD (Haplo vs MUD: 10% vs 15%, p = 0.534). 2-year CIR was 17% and 18% (p=0. 952) after Haplo and MUD SCT, respectively. No significant difference in 2-year PFS (Haplo vs MUD: 46% vs. 47%, p = 0.948) and OS (Haplo vs MUD: 55% vs. 52%, p = 0.944) was observed. Conclusion In an intent-to-treat analysis from the time of randomization, outcomes do not differ between the 2 groups. However, we observed that almost half of the patients did not receive the randomly attributed treatment 1/ Twenty-nine (27%) patients did not succeed to go to transplant 2/ Twenty (19%) patients were transplanted from another donor source that the one initially randomized to. It notably indicates that defining an early donor choice may be somehow dogmatic and should invite to more flexibility. We were able to demonstrate for older patients with high risk malignancies (IR: 17% Haplo; 18% MUD) a good disease control. Our per protocol analysis prospectively confirm in randomized study that Haplo-HSCT is a valid alternative in older patients. This suggests that HLA matching should not be necessarily considered as the most important factor for donor choice Disclosures Harbi: Sanofi: Honoraria. Chevallier:Incyte Corporation: Honoraria. Malard:JAZZ pharmaceutical: Honoraria; Astellas: Honoraria; Theralos/Mallinckrodt: Honoraria; Keocyt: Honoraria; Sanofi: Honoraria; Biocodex: Honoraria; Janssen: Honoraria. Cluzeau:Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Menarini: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Rubio:Neovii: Research Funding; Novartis: Honoraria; MSD: Honoraria; Gilead: Honoraria; Medac: Consultancy. Mohty:Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Blaise:Jazz Pharmaceuticals: Honoraria.

Putative Germline Variants in the Predisposition Genes DDX41, ETV6 and GATA2 investigated in 1,228 Patients with Sporadic AML or MDS

Background: In the latest revision of WHO classification (2017) the new category `myeloid neoplasms with germline predisposition´ was introduced. Pathogenic germline (GL) mutations predisposing to hereditary hematological malignancies (HHMs) have been described in various genes and have largely been identified in families, but may also occur sporadically (Godley, Blood Adv 2019; Rio-Machin et al., Nat Commun 2020). It is estimated that 4-9% of adults with myeloid malignancies have GL predisposition and it is discussed that HHMs could even be more common (Sung and Babushok, Blood 2020). Here, we performed a screening for putative GL variants in the predisposition genes DDX41, ETV6 and GATA2 in a large cohort of 1,228 patients with sporadic AML or MDS. Aim: To determine the frequency of genetic variants in the predisposition genes DDX41, ETV6 and GATA2 in patients with AML or MDS and to characterize the mutational patterns in patients carrying putative GL variants in these genes. Patients and Methods: Between 02/2019 and 01/2020 a total of 1,228 patients were diagnosed with AML or MDS by cytomorphological analysis (475 de novo AML, 647 MDS, 60 s-AML from MDS, 46 MDS/AML; M: 753, F: 475; median age: 74 [20-96]) and analyzed by next-generation sequencing for DDX41, ETV6, GATA2 and additional genes associated with AML or MDS (Figure 1). DNA was isolated from bone marrow or peripheral blood; sequencing was performed on NovaSeq after NextFlex library preparation (Illumina, ILMN, San Diego, CA) and hybrid capture according to manufacturer's protocol (IDT Inc. Coralville, IA). Data was analyzed with Pisces and Pindel (for FLT3-ITD) (available via BaseSpace, ILMN) using a minimum of 3% sensitivity. All changes except for synonymous mutations and known polymorphisms were declared as genetic variants. Results: We identified 73/1,228 patients (5.9%) with genetic variants in DDX41, 28 (2.3%) with variants in ETV6 and 50 (4.1%) with variants in GATA2. Aiming to estimate the proportion of patients carrying a putative GL variant, a variant allele frequency (VAF) higher than 0.3 was considered to be presumptive of a GL origin. We identified 64/1,228 patients (5.2%) with a putative GL variant in DDX41. 37/64 patients harbored DDX41 variants that were previously described as causal GL variants, like for example p.M1I (12/37) or p.D140fs (4/37), and consistent with other studies 43/64 (67.2%) had an additional DDX41 mutation with a lower VAF (<0.3) suggesting somatic acquisition of the second mutation (27/43: p.R525H) (Sébert et al., Blood 2019). 13/1,228 patients (1.1%) were found to have a putative GL variant in ETV6. Two of them carried a p.R353Q mutation which was recently presumed to be a rare GL variant contributing to myeloid malignancy susceptibility (Li et al., Leukemia 2020). 26/1,228 patients (2.1%) carried a putative GL variant in GATA2, including the already in the context of HHMs described mutation p.P41A (3/26) (Holme et al., Br J Haematol 2012). In total, 102/1,228 patients (8.3%) diagnosed with AML or MDS carried a putative GL variant in one of the predisposition genes DDX41, ETV6 or GATA2. Patients with presumed GL variants were about the same age as patients without (mean: 72 vs. 71 years). The mutational patterns for the 102 patients are illustrated in Figure 1. Genes that were most often additionally mutated were ASXL1, DNMT3A, SRSF2 and TET2. For ETV6 and GATA2, all but one of the patients carrying a putative GL variant harbored at least one genetic variant in an additional gene. In contrast, 16/64 patients (25.0%) carrying a presumed GL variant in DDX41 (and in 12 cases a second somatic DDX41 mutation) had no further genetic variants in other well-known AML- or MDS-related genes indicating that DDX41 and especially the gain of a second somatic mutation drives leukemogenesis, while for ETV6 and GATA2 additional hits are required. Conclusions: Our data support the hypothesis that GL mutations in predisposition genes could be more common in sporadic cases of AML or MDS than anticipated highlighting the importance of systematic testing for these variants, irrespective of family history or age. Identifying GL mutations can be important for clinical management, including donor choice for allogeneic stem cell transplantation. Routine workflows should be adapted to improve the identification of variants in predisposition-associated genes and to facilitate confirmation of GL origin, e.g. by analyzing reference material. Figure 1 Disclosures No relevant conflicts of interest to declare.

Current Trends on Haemopoietic Stem Cells

: Advances in single cell technology and genetic mouse models have resulted in the identification of new types of haemopoietic stem cells(HSC) , resulting in baffling observations, suggesting a reconsideration of the long-held notion that all hematopoietic cells in the adult are derived from HSCs. The existence of long-lived HSC-independent hematopoiesis has led to the conclusion that despite the single hierarchical differentiation route that generates functional blood types, other differenciation routes exist invivo. Heterogeneity in the HSC population and the evolving knowledge around HSC has translated to it’s improved application as a therapeutic tool for various blood disorders. The reprogramming of non-hematopoietic somatic and mature blood cells to pluripotency with their subsequent differentiation into hematopoietic stem cells/progenitors cells and the introduction of new generation sequencing holds potential for the resolution of ambiguities involved in HSC bone marrow transplantation. There is a change in the paradigim for HSC transplantation donor selection. Donor choice favours haploidentical HCT than cord blood. This review provides a general overview of the current events around haemopoietic stem cells, with emphasis on the rising trend of HSC transplantation especially haploidentical stem cell transplantation.

Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

Impact of Type 2 Diabetes Mellitus on Human Bone Marrow Stromal Cell Number and Phenotypic Characteristics

Human bone marrow-derived mesenchymal stromal cells (MSCs) have been investigated in numerous disease settings involving impaired regeneration because of the crucial role they play in tissue maintenance and repair. Considering the number of comorbidities associated with type 2 diabetes mellitus (T2DM), the hypothesis that MSCs mediate these comorbidities via a reduction in their native maintenance and repair activities is an intriguing line of inquiry. Here, it is demonstrated that the number of bone marrow-derived MSCs in people with T2DM was reduced compared to that of age-matched control (AMC) donors and that this was due to a specific decrease in the number of MSCs with osteogenic capacity. There were no differences in MSC cell surface phenotype or in MSC expansion, differentiation, or angiogenic or migratory capacity from donors living with T2DM as compared to AMCs. These findings elucidate the basic biology of MSCs and their potential as mediators of diabetic comorbidities, especially osteopathies, and provide insight into donor choice for MSC-based clinical trials. This study suggests that any role of bone marrow MSCs as a mediator of T2DM comorbidity is likely due to a reduction in the osteoprogenitor population size and not due to a permanent alteration to the MSCs’ capacity to maintain tissue homeostasis through expansion and differentiation.

Utility of clinical comprehensive genomic characterization for diagnostic categorization in patients presenting with hypocellular bone marrow failure syndromes

Bone marrow failure (BMF) related to hypoplasia of hematopoietic elements in the bone marrow is a heterogeneous clinical entity with a broad differential diagnosis including both inherited and acquired causes. Accurate diagnostic categorization is critical to optimal patient care and detection of genomic variants in these patients may provide this important diagnostic and prognostic information. We performed real-time, accredited (ISO15189) comprehensive genomic characterization including targeted sequencing and whole exome sequencing in 115 patients with BMF syndrome (median age 24 years, range 3 months - 81 years). In patients with clinical diagnoses of inherited BMF syndromes, acquired BMF syndromes or clinically unclassifiable BMF we detected variants in 52% (12/23), 53% (25/47) and 56% (25/45) respectively. Genomic characterization resulted in a change of diagnosis in 30/115 (26%) including the identification of germline causes for 3/47 and 16/45 cases with pre-test diagnoses of acquired and clinically unclassifiable BMF respectively. The observed clinical impact of accurate diagnostic categorization included choice to perform allogeneic stem cell transplantation, disease-specific targeted treatments, identification of at-risk family members and influence of sibling allogeneic stem cell donor choice. Multiple novel pathogenic variants and copy number changes were identified in our cohort including in TERT, FANCA, RPS7 and SAMD9. Whole exome sequence analysis facilitated the identification of variants in two genes not typically associated with a primary clinical manifestation of BMF but also demonstrated reduced sensitivity for detecting low level acquired variants. In conclusion, genomic characterization can improve diagnostic categorization of patients presenting with hypoplastic BMF syndromes and should be routinely performed in this group of patients.