Progressive Bone Marrow Failure Research Articles

FANCA c.3624C>T (p.Ser1208=) is a hypomorphic splice variant associated with delayed onset of Fanconi anemia

AbstractFanconi anemia (FA) is a hereditary, DNA repair deficiency disorder caused by pathogenic variants in any 1 of 22 known genes (FANCA-FANCW). Variants in FANCA account for nearly two-thirds of all patients with FA. Clinical presentation of FA can be heterogeneous and include congenital abnormalities, progressive bone marrow failure, and predisposition to cancer. Here, we describe a relatively mild disease manifestation among 6 individuals diagnosed with FA, each compound heterozygous for 1 established pathogenic FANCA variant and 1 FANCA exon 36 variant, c.3624C>T. These individuals had delayed onset of hematological abnormalities, increased survival, reduced incidence of cancer, and improved fertility. Although predicted to encode a synonymous change (p.Ser1208=), the c.3624C>T variant causes a splicing error resulting in a FANCA transcript missing the last 4 base pairs of exon 36. Deep sequencing and quantitative reverse transcription polymerase chain reaction analysis revealed that 6% to 10% of the FANCA transcripts included the canonical splice product, which generated wild-type FANCA protein. Consistently, functional analysis of cell lines from the studied individuals revealed presence of residual FANCD2 ubiquitination and FANCD2 foci formation, better cell survival, and decreased late S/G2 accumulation in response to DNA interstrand cross-linking agent, indicating presence of residual activity of the FA repair pathway. Thus, the c.3624C>T variant is a hypomorphic allele, which contributes to delayed manifestation of FA disease phenotypes in individuals with at least 1 c.3624C>T allele.

Complete Mouth Rehabilitation in a Patient with Fanconi Anaemia: A Rare Genetic Disorder

Fanconi anaemia is a rare, progressive congenital bone marrow failure syndrome characterised by autosomal recessive inheritance and clinical manifestations, including growth retardation, bone marrow failure leading to pancytopenia, an elevated risk of malignancy, skin pigmentation, and skeletal malformations. Patients who have undergone bone marrow transplantation can be treated comprehensively in the dental clinic like other routine patients, provided their blood indices are within normal limits. Its complications include aplastic anaemia, malignancies like acute myeloid leukaemia, liver tumours, and squamous cell carcinoma, often developing post-bone marrow transplantation. Dental anomalies, altered salivary flow, and increased susceptibility to cancer necessitate early diagnosis and specialised dental care. Hereby, the authors present a case of 15-year-old male with Fanconi Anaemia who had undergone bone marrow transplantation and was treated comprehensively in a hospital setting. The present case report aimed to equip healthcare practitioners, especially dentists, with the knowledge needed for accurate diagnosis and effective management.

Radiation and Busulfan-Free Hematopoietic Stem Cell Transplantation Using Briquilimab (JSP191) Anti-CD117 Antibody-Conditioning, Transient Immunosuppression and TCRαβ + T-Cell/CD19 + B-Cell Depleted Haploidentical Grafts in Patients with Fanconi Anemia

Introduction: Fanconi anemia (FA) is a rare, genetic disorder clinically characterized by congenital abnormalities, progressive bone marrow failure (BMF), and a predisposition to malignancies. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for BMF. In patients with FA who do not have a matched sibling donor, HSCT conditioning regimens typically use reduced doses of cyclophosphamide, fludarabine and anti-thymocyte globulin (ATG) with total body irradiation (TBI) or busulfan. However, treatment is complicated by conditioning related toxicities, graft vs host disease (GvHD) and increased predisposition to malignancies later in life. Prior attempts to remove TBI from FA conditioning regimens have been unsuccessful due to increased risk of graft rejection - and alternative approaches have replaced TBI with busulfan which is still genotoxic. Objective: To reduce acute and long-term treatment-related toxicities, we have developed a first of its kind treatment intended to improve the safety of allo-HSCT through: 1) a TBI- and busulfan-free conditioning regimen consisting of briquilimab, rabbit ATG (rATG - Thymoglobulin), fludarabine, cyclophosphamide and rituximab - briquilimab (formerly called JSP191) is a monoclonal antibody (mAb) that targets human CD117 to deplete host HSCs enabling blood and immune reconstitution with minimal toxicity with the other agents being used for transient immune suppression to prevent immunologic rejection; 2) transplantation of TCRαβ + T-cell/CD19 + B-cell hematopoietic grafts - a stem cell therapy that enhances donor hematopoietic and immune reconstitution while decreasing GvHD; and 3) a pre- and post-transplant monitoring protocol to maximize engraftment. This is the first HSCT regimen to use anti-CD117 mAb-based conditioning in immunocompetent patients without concurrent use of irradiation. Methods: Patients with FA in BMF were eligible to be enrolled in this Phase 1b/2a trial to receive allo-HSCT with TCRαβ + T-cell/CD19 + B-cell depleted hematopoietic grafts from 10/10 unrelated, 9/10 unrelated or haploidentical family donors. Per the treatment schema, in this regimen a fixed dose of briquilimab was administered at 0.6 mg/kg in combination with standard FA dosing of rATG, fludarabine, cyclophosphamide and rituximab as lymphodepletion (Figure 1). Prospective pharmacokinetic measurements of briquilimab, rATG and fludarabine are included in the protocol. Results: Treatment of all of the patients on the Phase 1b portion of the study has been completed with each of the three patients showing promising safety and efficacy results (Table 1). Importantly, briquilimab treatment was well tolerated without any complications with appropriate antibody clearance of < 500 ng/ml prior to HSCT in all three patients. Each of the patients achieved full blood count recovery by Day +14 post HSCT with haploidentical grafts that were within specifications of the protocol. Total, CD34 +, and CD15 + donor chimerism of > 98% in the bone marrow has been observed in all patients with > 91% chimerism in all immune subsets (CD3, CD19 and CD56) at the last follow-up with 1 year of follow-up post HSCT in the first patient. None of the patients have developed acute or chronic GvHD at the time of reporting. Conclusions: All three patients in the Phase 1b portion of the study show early safety and efficacy of this approach. Briquilimab was well tolerated and all patients have shown prompt and durable donor engraftment. This data indicates that it might be possible to remove irradiation or alkylator chemotherapy from the conditioning regimen in patients with FA without matched sibling donors thus decreasing the chances for cancer predisposition in these patients that have inherited DNA repair defects.

Hematopoietic Stem Cell Transplantation for Fanconi Anemia: Outcome and Prognostic Factors for Survival and Subsequent Neoplasms

Introduction Fanconi anemia (FA) is an inherited disorder of impaired DNA repair, associated with progressive bone marrow failure (BMF), leukemia, and cancer predisposition. Hematopoietic cell transplantation (HCT) may be curative for FA associated BMF, and changes in HCT practice over the past decades may have impacted outcomes in FA patients. Methods We used CIBMTR database to perform a retrospective cohort study of children and adults undergoing first allogeneic HCT for FA BMF (patients with MDS/AML excluded) from 2000-2018. We hypothesized that contemporary HCT approaches, including decreases in radiation and increase in use of serotherapy and ex-vivo graft manipulation, would associate with improved outcomes in FA patients receiving HCT. The primary endpoints were overall survival (OS) and early subsequent malignant neoplasm (SMN; by 5 yr post-HCT). Secondary endpoints included event free survival (EFS; death, SMN or graft failure), treatment related mortality (TRM), graft failure, and acute (grade II-IV) and cGVHD. Univariate and multivariate Cox regression models were created to evaluate the impact of conditioning, donor and graft source, treatment era, and patient characteristics on outcomes, adjusting for other significant covariates. Due to confounding factors, we were not able to create a single multivariate model that included radiation, ex-vivo graft manipulation, and donor and graft source; therefore, we performed subgroup univariate analysis for patients receiving unrelated donor bone marrow (BM) or peripheral blood stem cell (PBSC) HCT. Results We analyzed 652 HCTs from 2000-07 (n=353) and 2008-18 (n=293). 223 (35%) patients had a matched related donor (MRD) with any graft type, 244 (38%) had an unrelated donor BM or PBSC grafts, 123 (19%) had an unrelated cord blood graft, and 44 (7%) had a related haploidentical donor with any graft type. Between the two treatment eras, 5-yr OS has dramatically improved (78.8% 2008-18 v 62.5% 2000-07, p<0.01; figure 1) driven by lower 5-yr TRM (15.2% v 25.5%, p=0.004) and lower rates of primary (7.9% v 12.9% at 42 days, p=0.038) and secondary (4.1% v 8.9% at 1 year, p=0.014) graft failure. No statistically significant change was observed for aGVHD, cGVHD, or early SMN. Assessing the entire cohort, use of total body irradiation (TBI) >300cGy was associated with lower OS (HR=1.75, 95% CI 1.24-2.47) and increased TRM (HR=3.36, 95% CI 1.54-7.34; figure 2) on multivariable analysis. For patients that received unrelated BM or PBSC HCT, use of TBI was associated with inferior OS survival (HR=1.93, 95% CI 1.18-3.17), EFS (HR=1.78, 95% CI 1.13-2.80), and TRM (HR=2.31, 95% CI 1.20-4.46) on univariate analysis. Among all patients, CD34 selection or T-cell depletion ( ex vivo depletion) were not associated with better OS (95% HR=1.014, 95% CI 0.84-1.56), EFS (HR=1.163, 95% CI 0.86-1.58), or TRM (HR=0.79, 95% CI 0.52-1.19) but was associated with less aGVHD (HR=0.35 95% CI 0.22-0.57) and lower but not-statistically significant cGVHD (HR=0.54, 95% CI 0.29-1.01) on multivariable analysis. Within the unrelated donor setting, univariate analysis showed reduced aGVHD (HR=0.39 95% CI 0.23-0.68) and cGVHD (HR=0.47 95% CI 0.28-0.79) but did not have a significant effect on OS, EFS, or TRM. Early SMN occurred in 3.2% of participants by 5-years post-HCT and was associated with cGVHD occurrence (HR=2.14, 95% CI 1.06-4.33) and older age (>18 v <9 years, HR=6.35, 95% CI 2.66-15.14), but not ex-vivo depletion or use of ATG or alemtuzumab. Early SMN risk was not impacted by the use of TBI (HR=1.60, 95% CI 0.77-3.34), but secondary analysis demonstrated a trend towards a relationship with doses >300 cGy (HR=2.24, 95% CI 0.99-5.08). Conclusions Outcomes for patients with FA BMF have improved significantly in more recent HCT eras, driven by reductions in TRM and graft failure risk. Although radiation doses >300cGy were associated with greater toxicity, doses of 100-300cGy were not associated with inferior outcomes in our cohort. Use of CD34 selection or T-cell depletion may reduce the risk of GVHD in transplant FA patients, but does not appear to impact 5-yr OS. Risk for early SMN development in FA patients receiving HCT associates with older age and cGVHD.

Blood Count Recovery and Telomere Elongation in a TINF2 Patient Under Treatment with a Gonadotropin-Releasing Hormone (GnRH) Agonist

Telomere biology disorders (TBDs) caused by germline variants in TINF2 are a common cause of severe disease presenting in early childhood. Progressive bone marrow failure in patients with TBDs is often treated with androgens, known to improve hematopoiesis but with a controversial impact on telomere elongation. Here, we report a unique case of a young TINF2 male patient that had a striking recovery of blood counts and robust telomere elongation in peripheral blood cells during treatment with histrelin, a gonadotropin-releasing hormone (GnRH) agonist and inhibitor of testosterone production. We investigated whether underlying clonal events were linked to blood counts and telomere length (TL) dynamics. We extracted medical information and collected peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) over an 10-yr period beginning at diagnosis (age 4), which was based on cytopenias, marked marrow hypocellularity, very low TL in all leukocyte subsets by Flow-FISH, and a de novo TINF2 p.K302Rfs*17 germline variant. Following 2 yrs of observation, he was started on danazol for progressive cytopenias but it was discontinued after 21 months due to the onset of danazol-related precocious puberty, for which histrelin was administered via subcutaneous implants over 4.7 yrs. Hemoglobin levels normalized while on danazol and remained stable at last follow-up. Although platelet counts increased and stabilized at ~ 40,000 on danazol, they steadily increased after initiation of histrelin up to 156,000; they then gradually declined to 108,000 over the 18 months following removal of the last histrelin implant (Fig. 1A). The absolute neutrophil count normalized while on histrelin and has remained in normal range. While TLs remained very low in all leukocyte subsets while on danazol, they either stabilized or minimally changed (+/- 0.2 kb) in lymphocytes, CD45RA+, CD45RA-, and CD57+ cells or increased by 2.8 kb or 1 kb in granulocytes and B cells, respectively. While on histrelin, TL further increased in the lymphocytes, granulocytes, and B cells to max gains of 0.4 kb, 3 kb, and 2 kb, respectively. Fourteen months after discontinuing histrelin, TL shortened in all subsets. Screening of PBMC at 8.5 yrs of follow-up for clonal hematopoiesis (CH) in myeloid and telomere-related genes by error-corrected DNA sequencing identified POT1 p.D291Y and TINF2 p.W20* variants at variant allele frequencies (VAFs) of 20% and 4%, respectively, as likely somatic genetic rescue (SGR) mechanisms (Fig. 1B). POT1 D291Ywas retrospectively tracked at a VAF of 0.7% in total DNA before any treatment and found at a VAF of 8% in PBMC collected during danazol treatment, 27% during histrelin treatment, and 14% after histrelin discontinuation. TINF2 W20* emerged while on histrelin at a VAF of 1% in PBMC; this mutation VAF slightly increased during histrelin treatment but robust clonal expansion up to a VAF of 21% was not seen until off histrelin. Hematologic improvement, particularly thrombocytopenia, and continued telomere elongation in granulocytes and B cells (CD20+) coincided with clonal expansion of the POT1 D291Y variant. Single-cell proteogenomic sequencing of BMMNCs evidenced POT1 D291Y and TINF2 W20* as independent clonal events that emerged in CD34+ hematopoietic stem cells (HSC) and were present both in the megakaryocytic and erythroid, and myeloid and lymphoid progenitor cells. During histrelin treatment, POT1 D291Y dominated the landscape and associated with increased lymphocyte counts, being present in 30% of HSC and progenitor cells, myeloid cells, and T lymphocytes. When histrelin was discontinued, hematopoiesis greatly shifted toward myeloid production, which coincided with TINF2 W20*clonal expansion and a substantial reduction of progenitors and CD8+ cells. Single-cell RNAseq of BM HSC revealed similar transcriptional profiles while on danazol and histrelin compared to baseline, with up and downregulation of inflammatory pathways and MYC targets, respectively; these programs were enhanced while on histrelin compared to danazol. In summary, we characterized a unique combination of SGR events in a patient with a TINF2 germline mutation, which can guide future research on new therapeutic strategies. As opposed to androgens, histrelin promoted the specific expansion of POT1 clones with proliferation of HSC and progenitor cells, improved hematopoiesis, and increased TL.

Lnk/Sh2b3 Deficiency Increases DNA Damage Tolerance to Promote Hematopoietic Stem Cell Fitness

DNA repair deficiency depicted by the syndrome Fanconi Anemia (FA), results in progressive bone marrow failure (BMF), increased leukemia incidence and cancer susceptibility. Out of the 23 causative genes for FA, FANCD2 is crucial for its central role in the regulation of DNA repair and replication. The FA pathway plays an important role in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis and genome integrity. Our previous studies revealed that loss of the adaptor protein LNK (SH2B3), a negative regulator of cytokine signaling, restores hematopoietic stem cell (HSC) function and genome integrity in Fancd2 deficient FA mouse models and primary human FA progenitors. Notably, mice deficient in Lnk harbor a remarkable 14-fold expansion of HSCs with superior self-renewal capability. However, the protective mechanisms that LNK controls to prevent stem cell attrition and increase stem cell fitness are not established. Here, we show that Lnk deficiency reduced DNA damage in Fancd2-/- HSCs at the steady-state in vivo, as well as in forced proliferation upon transplantation. Lnk deficiency reduced HSPC DNA damage in replication stress upon cytokine-induced proliferation in ex vivo culture, accompanied by reduced p53 activation. One injection of pIpC induces HSPC into the cell cycle within 24hr, thus we used this as a model to study replication stress in vivo. We found that Lnk deficiency reduced ATR but not ATM pathway activation in Fancd2-/- HSCs in pIpC-induced proliferation in vivo. ATR is activated by single-strand DNA breaks (SSBs) during replication stress; thus we directly examined the amount of single-strand DNA (ssDNA) and the level of ssDNA binding protein RPA (Replication Protein A) in HSCs during the cell cycle. Of note, Lnk deficiency reduced ssDNA as well as chromatin-bound RPA2 in Fancd2-/- HSCs in replication in vivo. Lnk deficiency also reduced RPA2 phosphorylation in HSPCs under Camptothecin (CPT) and hydroxyurea (HU) induced replication stress conditions. Mechanistically, loss of Lnk protected stalled replication fork from degradation, suppressed ssDNA gaps, and promoted replication fork restart in Fancd2-/- HSPCs upon HU-induced replication stress. In light of our data suggesting a new role for LNK in replication stress-induced DNA damage, we examined the DNA damage tolerance (DDT) mechanism that enables DNA replication to circumvent the lesions, thereby allowing the completion of DNA replication, increasing the survival of HSPCs and preventing genome instability. One of the major DDT pathways is translesion synthesis (TLS) in which the replicative DNA polymerase is temporarily replaced by special TLS polymerases pol ζ or η that can replicate across DNA lesions. Indeed, we found that Lnk deficient HSCs have increased chromatin-bound TLS polymerase eta (Pol ƞ) and were resistant to shRNA-mediated knockdown of PolH (the gene encodes polymerase eta) compared to wildtype HSCs in transplant assays. Strikingly, Lnk deficient HSCs were more resistant to the inhibitor of Rev1 that disrupts Rev1/ pol ζ interaction than wildtype HSCs in transplanting assays. These findings highlight a previously-unappreciated role of LNK in limiting DNA damage tolerance mechanisms in both normal and diseased replicative states. The novel link between LNK-controlled cytokine signaling and TLS sheds light on our understanding of replication stress mitigation to promote HSC fitness that can be explored for future regeneration medicine.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure.

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.

New era for myelofibrosis treatment with novel agents beyond Janus kinase-inhibitor monotherapy: Focus on clinical development of BCL-XL /BCL-2 inhibition with navitoclax.

Myelofibrosis is a heterogeneous myeloproliferative neoplasm characterized by chronic inflammation, progressive bone marrow failure, and hepatosplenic extramedullary hematopoiesis. Treatments like Janus kinase inhibitor monotherapy (e.g., ruxolitinib) provide significant spleen and symptom relief but demonstrate limited ability to lead to a durable disease modification. There is an urgent unmet medical need for treatments with a novel mechanism of action that can modify the underlying pathophysiology and affect the disease course of myelofibrosis. This review highlights the role of B-cell lymphoma (BCL) protein BCL-extra large (BCL-XL ) in disease pathogenesis and the potential role that navitoclax, a BCL-extra large/BCL-2 inhibitor, may have in myelofibrosis treatment.

Clinical case: allogeneic transplantation of hematopoietic stem cells for Fanconi anemia in the onset of refractory anemia with excess blasts

Fanconi anemia (AF) is a hereditary genetic disease characterized by developmental abnormalities, progressive bone marrow failure, hypersensitivity to alkylating agents, and a tendency to hematological and solid tumors throughout life. The only curative option in the treatment of bone marrow failure in patients with AF is allogeneic hematopoietic stem cell transplantation (allo-HSCT). There are no detailed descriptions of allo-HSCT in patients with AF in the Russian-language literature. On the example of a clinical case with AF at the onset of myelodysplastic syndrome, a choose of method for treating bone marrow failure is presented.

Rare cytogenetic abnormalities in MDS evolving from fanconi anemia-A case report

Fanconi anemia (FA) is a genetically heterogenous rare autosomal recessive disorder. Mutations in FANCA gene are the most frequent among FA patients accounting for 60-65%. FA is characterised by congenital malformations, progressive bone marrow failure (BMF) and increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The risk of developing hematological abnormalities in FA patients is around 98% by 40 years of age. The risk of clonal cytogenetic abnormalities during BMF is around 67% by 30 years of age and risk of developing MDS or AML is 52% by 40 years of age. The frequent chromosomal abnormalities are 1q+, monosomy 7 and gains of 3q. Partial duplications/triplications of chromosome 1q are known to represent a nonrandom chromosomal anomaly in myeloid disorders.

Reduced-intensity conditioning is effective for allogeneic hematopoietic stem cell transplantation in infants with MECOM-associated syndrome

Mutations in the MECOM encoding EVI1 are observed in infants who have radioulnar synostosis with amegakaryocytic thrombocytopenia. MECOM-associated syndrome was proposed based on clinical heterogeneity. Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for progressive bone marrow failure. However, data regarding allogeneic HSCT for this rare disease are limited. We retrospectively assessed overall survival, conditioning regimen, regimen-related toxicities and long-term sequelae in six patients treated with allogeneic HSCT. All patients received a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, cyclophosphamide or melphalan, and rabbit anti-thymocyte globulin and/or low-dose total body/thoracic-abdominal/total lymphoid irradiation, followed by allogeneic bone marrow or cord blood transplantation from unrelated donors between 4 and 18 months of age. All patients survived and achieved stable engraftment and complete chimerization with the donor type. Moreover, no patient experienced severe regimen-related toxicities, and only lower grades of acute graft-versus-host disease were observed. Three patients treated with low-dose irradiation had relatively short stature compared to three patients not treated with irradiation. Therefore, allogeneic HSCT with RIC is an effective and feasible treatment for infants with MECOM-associated syndrome. Future studies are needed to evaluate the use of low-dose irradiation to avoid risks of other long-term sequelae.

Real-World Outcomes of Patients with Lower-Risk Myelodysplastic Syndromes (LR-MDS) Treated with Luspatercept: An Evaluation of US Clinical Practice Utilization and Treatment Patterns

Introduction: LR-MDS is characterized by ineffective hematopoiesis, cytogenic and molecular abnormalities, progressive bone marrow failure, ≥ 1 cytopenias, and variable risk of progression to acute myeloid leukemia. Patients (pts) may be classified as having LR-MDS based on an International Prognostic Scoring System (IPSS) score of Low or Intermediate risk and/or an IPSS-Revised (IPSS-R) score of Very Low, Low, or Intermediate risk. Erythropoiesis-stimulating agents (ESAs) are the first line of treatment for most pts with LR-MDS. However, many pts experience ESA failure and have limited subsequent treatment options. Luspatercept is a first-in-class erythroid maturation agent approved by the US FDA in April 2020 for the treatment of anemia following ESA failure and requiring ≥ 2 red blood cell units over 8 weeks in adult pts with LR-MDS with ring sideroblasts (RS) or with MDS/myeloproliferative neoplasms with RS and thrombocytosis. Limited evidence exists on treatment patterns and outcomes among pts with LR-MDS treated with luspatercept in routine clinical practice. The aim of this study was to examine pt characteristics, treatment patterns, and transfusion outcomes among pts with LR-MDS treated with luspatercept in the real-world setting. Methods: This was a retrospective, observational cohort study using physician-abstracted data from the charts of adults diagnosed with LR-MDS on or after January 1, 2015, who received luspatercept treatment in US clinical practice. Pts ≥ 18 years old with LR-MDS who received luspatercept for ≥ 3 months and had ≥ 3 months of follow-up after starting luspatercept (or until death if within 3 months of luspatercept initiation) were included. Pts were classified as having LR-MDS based on their IPSS-R or IPSS scores at diagnosis. Pts who participated in a clinical trial were excluded. All data were summarized using descriptive statistics. Results: A total of 76 eligible pts were included; 52.6% were male, 72.4% were White, and the mean age at luspatercept treatment initiation was 72.0 years (Table 1). Median follow-up time from luspatercept initiation was 5.6 months. Nearly all pts (92.1%) received only 1 line of systemic treatment for LR-MDS before receiving luspatercept. All pts initiated luspatercept treatment at the approved starting dose of 1 mg/kg every 3 weeks, and 75.0% of pts received luspatercept according to the approved US FDA indication. Based on Kaplan-Meier estimates, the median duration of luspatercept treatment was 13.3 months. Twenty-two pts (29.0%) discontinued luspatercept treatment during the follow-up period, of whom 54.5% discontinued on the same dose as their starting dose, ie, 1 mg/kg every 3 weeks (68.2% discontinued at a dose < 1.75 mg/kg) (Table 1). Disease progression to higher-risk MDS was the most common reason for luspatercept discontinuation (n = 10/22, 45.5%). No pts discontinued treatment due to adverse events. At luspatercept initiation, 85.5% of pts had low transfusion burden (TB), of whom 7.7% maintained low TB and 92.3% converted from low TB to transfusion independence (TI) during the first 24 weeks of luspatercept treatment (Table 2). Moderate TB was present in 13.2% of pts at luspatercept initiation, all of whom transitioned to having lower TB during the first 24 weeks of luspatercept treatment. Conclusions: This real-world chart review study showed > 90% of pts with low TB prior to luspatercept initiation achieved TI within the first 24 weeks of luspatercept treatment. All pts with moderate TB prior to luspatercept had a decrease in level of TB within the first 24 weeks of starting luspatercept treatment. The MEDALIST trial showed 38% of pts treated with luspatercept- with IPSS-R Very Low to Intermediate risk reached TI within 24 weeks (Fenaux P, et al. N Engl J Med 2020;382:140-151). The present study suggests that initiating luspatercept for low or moderate TB may have a substantial impact on TB and merits continued attention as experience with luspatercept in clinical practice accumulates. Among those who discontinued luspatercept treatment, more than half discontinued at the starting dose. Luspatercept dose titration in real-world clinical practice may not align with recommended dose increases according to the approved US indication. This discrepancy represents an unmet clinical need and potential area for further clarification and education in the community setting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cytogenetics in Fanconi Anemia: The Importance of Follow-Up and the Search for New Biomarkers of Genomic Instability.

Fanconi Anemia (FA) is a disease characterized by genomic instability, increased sensitivity to DNA cross-linking agents, and the presence of clonal chromosomal abnormalities. This genomic instability can compromise the bone marrow (BM) and confer a high cancer risk to the patients, particularly in the development of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). The diagnosis of FA patients is complex and cannot be based only on clinical features at presentation. The gold standard diagnostic assay for these patients is cytogenetic analysis, revealing chromosomal breaks induced by DNA cross-linking agents. Clonal chromosome abnormalities, such as the ones involving chromosomes 1q, 3q, and 7, are also common features in FA patients and are associated with progressive BM failure and/or a pre-leukemia condition. In this review, we discuss the cytogenetic methods and their application in diagnosis, stratification of the patients into distinct prognostic groups, and the clinical follow-up of FA patients. These methods have been invaluable for the understanding of FA pathogenesis and identifying novel disease biomarkers. Additional evidence is required to determine the association of these biomarkers with prognosis and cancer risk, and their potential as druggable targets for FA therapy.

Systematic search for the UBA1 mutation in men after a first episode of venous thromboembolism: A monocentric study

Systematic search for the UBA1 mutation in men after a first episode of venous thromboembolism: A monocentric study

“REAL-WORLD” RESULTS OF HYPOMETHYLANT THERAPY IN MYELODYSPLASTIC SYNDROME, CHRONIC MYELOMONOCYTIC LEUKEMIA AND OLIGOBLASTIC ACUTE MYELOID LEUKEMIA

Myelodysplastic Syndrome (MDS) represents a heterogeneous group of myeloid neoplasms, resulting in progressive bone marrow failure and cytopenias. Although the allogeneic hematopoietic stem cell transplantation (HSCT) is the only potential curative treatment, only few patients are candidates. In this regard, hypomethylating agents (HMA)- Azacitidine (AZA) and decitabine (DEC) - appears as a standard treatment for higher-risk MDS, in addition to being considered in oligoblastic acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Although AZA improved the overall survival (OS) in clinical trials, results in real-world are discordant. This study aims to evaluate the clinical characteristics and outcomes of MDS patients treated with HMA in an academic hospital. Retrospective cohort, over 18 years of age, diagnosed with MDS, oligoblastic AML and CMML, de novo or secondary, treated with HMA and followed up at the cytopenias outpatient clinic at HCFMUSP from 2010 to June 2021. Data were collected from medical records and stored in REDcap. IWG 2006 criteria were used for response assessment. Kaplan-Meier curves were used for survival graphs, and Cox model regression for uni and multivariate analysis. 70 patients, 60 (85%) treated with AZA and 10 (15%) with DEC. Median age at diagnosis was 60.9 years (IQR 48.7 – 68.3), 61.4% male. In relation to diagnoses, 28 had de novo MDS (32.9% with excess of blasts; 4.3% with multilineage dysplasia; 2.9% with ring sideroblasts); 7 had AML (10%); 24 secondary myeloid neoplasm (21.4% therapy related; 7.1% with germline/ Fanconi predisposition; 5.7% after acquired aplastic anemia). The median score of IPSS and IPSS-R was respectively 1.5 and 6.0 (high risk). Before treatment 35.4% of patients had poor or very-poor karyotype. Response evaluation was assessed in 51 patients (72.8%) at the end of fourth cycle. The overall response rate (ORR) was 66.6%, including complete, partial and marrow responses, besides hematological improvement. Twelve patients (17%) undergone HSTC. The median OS, since starting HMA, was 11.5 months (IQR 8.1 – 13.8 95% CI), with median follow-up of 15.9. In univariate analysis, variables associated with worse OS were late initiation of HMA (HR 2.39 95% CI 1.18 – 4.84, p =0.015), age (HR 1.03 95% CI 1.01 – 1.05, p =0.007), poor-risk karyotype (HR 4.8 95% CI 2.29 – 10, p <0.001), hemoglobin < 8.5 (HR 3.44 95% CI 1.56 – 7.88, p =0.002) and platelets < 80.000 (HR 3.5 95% CI 1.49 – 8.23, p =0.002). In multivariate analysis, parameters associated with worse OS were transfusion dependency (HR 2.26 95% CI 1.27 – 4.04, p = 0.006) and poor karyotype (HR 2.53 95% CI 1.41 – 4.57, p =0.002). Thirty-five patients (50%) had febrile neutropenia, and 30% delayed one or more cycles. The results reinforce the poor outcomes of higher-risk MDS despite the use of HMA. Some real-world studies previously published had already showed a suboptimal drug benefit, probably by the underutilization and low persistence of the treatment, in a subgroup of critically ill patients. This retrospective analysis confirms the poor prognosis of higher-risk MDS treated with HMA. Poor-risk karyotype and transfusion dependency are the main factors that impact the outcomes.

Heterozygous Variants in the DNA-binding Domain of c-Myb May Affect Normal B/T Cell Development.

Heterozygous Variants in the DNA-binding Domain of c-Myb May Affect Normal B/T Cell Development.

MDS-420 Low Risk Myelodysplastic Syndromes in Morocco

Myelodysplastic syndromes (MDS) are a diverse group of clonal hematopoietic malignancies, characterized by ineffective hematopoiesis, progressive bone marrow failure, cytogenetic and molecular abnormalities, and variable risk of progression to acute myeloid leukemia (AML). Clinically, MDS patients often present with symptoms related to peripheral cytopenia (tiredness, pallor, infections, bruising, bleeding), although they may also be asymptomatic with abnormal blood counts found on routine evaluation. The most commonly used scores are the WHO Prognostic Scoring System (WPSS), International Prognostic Scoring System (IPSS), and more recently, the Revised International Prognostic Scoring System (RIPSS). In addition to the factors that make up these scores, other independent prognostic factors have been identified such as age, the Eastern Cooperative Oncology Group (ECOG) score, ferritin level, beta 2 microglobulin and lactic dehydrogenase (LDH). Our study includes all patients diagnosed with MDS between January 2004 and December 2018, over an age range of 5 months to 95 years, encompassing 272 patients. Data were collected from the patients' files. According to the prognostic score, we found 73 patients with a low risk, 165 patients with intermediate I risk; based on this score, 88% of the myelodysplasias in our series were of low risk. According to the IPSS-R, we found that 67 patients had a very low risk, 126 patients were at low risk, 36 patients were at intermediate risk. According to the IPSS and IPSS-R, low risk patients are children and adolescents, with a stable evolution. Abstinence from therapy was recommended as the treatment choice in 20% of patients with low-risk MDS. In our series, 74% of cases received symptomatic treatment including: 67% of cases were transfused with packed red blood cells, 22% were transfused with platelet units, 6% received iron chelators, 4% were treated with erythropoietin, 24% were treated with antibiotic therapy. Our results are consistent with published studies regarding the epidemiology, the clinic, and prognosis. Our study confirms the prognostic value of bone marrow karyotype and validates the WPSS IPSS and RIPSS scores in predicting overall survival of Moroccan patients with myelodysplastic syndromes.

A Narrative Review on Fanconi Anemia: Genetic and Diagnostic Considerations.

Fanconi anemia (FA) is an autosomal recessive disorder, both genetically and phenotypically. It is characterized by chromosomal instability, progressive bone marrow failure, susceptibility to cancer, and various other congenital abnormalities. It involves all the three cell lines of blood. So far, biallelic mutations in 21 genes and one x-linked gene have been detected and found to be associated with FA phenotype. Signs and symptoms start setting in by the age of 4 to 7 years, mainly hematological symptoms. This includes pancytopenia, that is, a reduction in the number of white blood cells (WBCs), red blood cells (RBCs), and platelets. Therefore, the main criteria for diagnosis of FA include skeletal malformations, pancytopenia, hyperpigmentation, short stature, urogenital abnormalities, central nervous system, auditory, renal, ocular, and familial occurrence. Patients showing signs and symptoms of FA should be thoroughly evaluated. A complete blood count will reveal a reduced number of RBC, WBC, and platelets, that is, pancytopenia. Chromosomal breakage study/stress cytogenetics should be done in patients with severe pancytopenia. Momentousness timely diagnosis of current disease, prenatal diagnosis, and genetic counseling should be emphasized.