Inherited Bone Marrow Failure Disorder Research Articles

Clinical Characteristics of Shwachman-Diamond Syndrome in China - Result from Childhood Bone Marrow Failure Diseases Register of China Alliance for Blood Diseases(cBMFR-CABD)

OBJECTIVE This study aimed to identify the clinical features of 73 children diagnosed with SDS from 22 centres in China. METHODS Childhood Bone Marrow Failure Diseases Register of China Alliance for Blood Diseases (cBMFR-CABD) was a bidirectional multicenter cohort study that collected data from 22 paediatric blood disease treatment centres from all over China. Patients were enrolled in the sub-centres from 1.1.2011 that were aged < 18 years at diagnosis and met the diagnostic criteria for an inherited bone marrow failure disorder. The clinical data for the 73 SDS patients analyzed in this study were collected between 1.1.2011 and 31.12.2022. RESULTS Among the 73 SDS children, 38 were male and 35 were female. Short stature (height<-2SD) was found in 53.4 % (39/73) patients. Chronic diarrhea was observed in 24.7 % (18/73) patients. In children evaluated for intellectual and physical malformations, 7 patients (17.3 %,6/52) had mental retardation and 14 (20.5 %, 15/73) patients had physical deformities. Genetic testing was available in 63 patients. seventeen (27.0 %) patients carry bi-allelic c.258+2T>C and c.183-184TA>CT SBDS mutations. Eleven patients (17.5%) are homozygous for the c.258+2T>C mutation. Four patients (6.3%) were compound heterozygous for c.258+2T>C and exon 2 deletions. One patient carries bi-allelic mutation of DNAJC21. This patient had a history of repeated infections and pancytopenia.Bone marrow smear shows multilineage dysplasia. Bone marrow results were available for evaluation in 54 patients including 67 % (36/54) BMF, 30 %(16/54) MDS, 4 % (2/54) AML.TP53 mutation testing was available in 15 patinets with eight (53.3%) patients carry TP53 mutation.Karyotype analysis was available in 36 patinets with 5 patients were complex karyotype. Among the 56 patients available for follow-up, 17 patients underwent haematopoietic stem cell transplantation, 5 patients died (one infection,three transplant-related death and one relapse). Among the 39 non-HSCT patients, 3 patients died of AML transformation. Three-year overall survival of all patients were 91.7%(95%CI 99.5 %~ 83.9% ), BMF patients were 100.0%(95%CI 100.0%~100.0% ), MDS/AML patients were 67.7%(95%CI 94.0%~ 41.4% ). CONCLUSION Our results provide the clinical features of the largest SDS cohort in China. A high incidence of short stature was observed. Bi-allelic c.258+2T>C with c. 183-184 TA > CT SBDS mutations were the most common mutation type. HSCT is an effective treatment modality in SDS. However, AML transformation is still a major risk factor for children with SDS.

Dyskeratosis Congenita: A Case Report

Dyskeratosis Congenita (DC) is a rare inherited bone marrow failure disorder characterized by lacy reticulated skin pigmentation, nail dystrophy and mucosal leukoplakia. In DC bone marrow failure is the main cause of premature death with an additional predisposition to malignancy. DC results from progressive shortening of telomeres resulting in DNA replication. X-linked recessive is the main mode of inheritance. Most of the patients respond to Androgen therapy. This is a case report of a 5-year-old boy presented with features of bone marrow failure and had abnormality of skin, oral mucosa and nail. His complete blood count with peripheral blood film showed pancytopenia and no blast cell. Hypocellular marrow was present in his Bone marrow examination. Chromosomal analysis- fluorescence in situ hybridization (FISH) revealed very short telomere of chromosome. Microarray genotyping showed DKC1 gene abnormality. BANGLADESH J CHILD HEALTH 2022; VOL 46 (1) : 39-41

UBTF tandem Duplications Account for a Third of Advanced Pediatric MDS without Genetic Predisposition to Myeloid Neoplasia

Introduction: Recurrent tandem duplications (TD) in exon 13 of the upstream binding transcription factor gene (UBTF) have recently been identified in 4% of pediatric AML (Umeda et al. 2022). UBTF-TD AMLs were associated with adolescent age, normal karyotype, trisomy 8, as well as FLT3-ITD and WT1 mutations. Response to chemotherapy was poor and probability of overall survival (OS) at 5 years only 44%. Here, we evaluate the role of UBTF-TD in pediatric MDS. Methods: We studied 104 consecutive patients (pts) diagnosed with primary MDS with excess blasts below the age of 18 years enrolled in the prospective studies 98 or 2006 of the European Working Group of MDS in Childhood (EWOG-MDS) in Germany between July 1, 1998 and June 30, 2021. Pts with MDS secondary to chemo- or radiation therapy, acquired aplastic anemia and pts with classical inherited bone marrow failure disorder had been excluded from the analysis. High molecular weight DNA was extracted from stored bone marrow (BM) samples or archived smears. Screening for UBTF-TD was performed by PCR using 6-FAM-labelled primers covering exon 13 of the UBTF gene followed by high resolution fragment analysis. For samples showing aberrant signals indicating UBTF-TDs, Sanger sequencing confirmed the presence of a mutation. All UBTF mutant samples were also analyzed using targeted next generation sequencing (Archer Myeloid panel) for the presence of co-occurring alterations. Results:UBTF-TDs were detected in 25 of the 104 pts (24%; 16 males/9 females), while 79 pts (76%; 45 males, 34 females) showed no UBTF mutation (UBTF-wt). Variant allelic frequencies of UBTF-TD ranged from 11%-79% (median 41%). Median age at diagnosis in UBTF-TD and UBTF-wt pts was not different (11.0 vs. 9.8 years). UBTF-TD pts had higher absolute neutrophil counts (2.7 vs. 0.9 x109/L, p=0.01) and more often normoblasts on peripheral blood smears (92% vs 43%, p<0.01). Blast percentage in BM was similar with <10% blasts in 40% and 38% of UBTF-TD and wt pts, respectively. BM cellularity was normal or increased in 92% of UBTF mutated pts compared to 72% of UBTF-wt pts (p<0.01). Cytogenetic analyses revealed striking differences according to UBTF mutational status with the following karyotypes (TD vs wt): normal (72% vs 33%), -7 (0% vs 30%), del(7q) (0% vs 2%), +8 (24% vs 9%), +8 plus other (0% vs 4%), random (4% vs 7%) and structural complex (0% vs 7%). Probability of OS at 5 years for pts with UBTF-TD was inferior (0.50, 0.28-0.72) to that of pts with UBTF-wt (0.73, 0.62-0.84; p=0.03). Of the 104 pts, 30 pts had a genetic predisposition to myeloid neoplasia, i.e. GATA2 deficiency (n=15), RUNX1 deficiency (n=7), SAMD9/SAMD9L disease (n=6) or neurofibromatosis type 1 (n=2). Of note, none of the pts with predisposition harbored a UBTF-TD. To study the impact of UBTF-TD on outcome of hematopoietic stem cell transplantation (HSCT), pts with germline disease were excluded; of the 72 pts available for HSCT analysis (25 UBTF-TD and 47 UBTF-wt). Therapy prior to HSCT in UBTF-TD and UBTF-wt pts was comparable and consisted of AML-type therapy in 32% vs 21%, less intensive chemotherapy 8% vs 15% and no therapy in 60% vs 64% pts, respectively. Type of donor, stem cell source and conditioning regimen did not differ between the 2 groups, 74% of pts received a busulfan, cyclophosphamide and melphalan conditioning. Event-free survival (EFS) and relapse incidence (CIR) at 5 years of the total cohort were 0.55 (0.39-0.71) and 0.23 (0.15-0.36), respectively. There was no difference in EFS between the 25 UBTF-TD (0.43, 0.22-0.64) and 47 UBTF-wt pts (0.60, 0.45-0.75). However, in pts with normal karyotype, presence of an UBTF-TD was associated with an inferior EFS (0.35 vs. 0.77, p=0.03) and higher CIR compared to wt pts (0.36 vs. 0.06, p=0.03). Co-occurrence of WT1 mutations among UBTF mutated pts was associated with higher CIR (p=0.03); other mutations were less frequent (Figure). Discussion: We identified UBTF-TD in a third of all pediatric pts with advanced primary MDS and absence of germline predisposition. In addition, we noted UBTF-TD at a high frequency in pediatric pts with MDS-related AML (3/15 pts UBTF-TD). In regards to the recent discovery in AML, we conclude that UBTF-TD mutations define a novel subgroup of myeloid neoplasia presenting frequently as pediatric MDS. The high CIR following HSCT particularly in patients with concurrent WT1 mutations outlines an urgent need for new therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Mutations in Ribosomal Protein Genes L9 and L15 Link to DBA with Specific Clinical Phenotypes

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein (RP) gene mutations. The European (EuroDBA) consortium reports here new mutations identified in RP genes L15 and L9 in patients with DBA. Although point mutations have not been previously reported for RPL15 in DBA, we identified 6 unrelated children of whom 4 carried truncating mutations c.242dupA; p.Tyr81X (N=3) and c.85C>T; p.Gln29X (N=1). Additional 2 patients carried RPL15 missense variants c.29T>C; p.Leu10Pro and c.458A>C; p.Lys153Thr. Notably, of the 4 patients with truncating mutations, 3 manifested prenatally with severe hydrops fetalis and required between 4-9 intrauterine transfusions. Even more remarkable is the observation that 3/4 patients with this mutation type rapidly became treatment-independent and maintained normal blood counts until the last follow-up (age 5-16 years). This finding suggests that a previously unknown correlation between genotype and hematological phenotype may underlie the tendency of some DBA patients to undergo treatment independence. Additionally we report a pathogenic splice donor mutation c. -2+1G>C in RPL9, a gene that has not been previously implicated in DBA pathogenesis. The patient presented with DBA and severe colitis at the age of 6 months, and showed dysmorphic features including microcephaly and bilateral thumb abnormality.Cells carrying either RPL15 or RPL9 mutations revealed pre-rRNA processing defects, reduced 60S ribosomal subunit formation, a reduction of de novo protein synthesis, and stabilization of the TP53 tumor suppressor. Zebrafish models reducing either RP recapitulated a loss of hemoglobin-expressing cells in early development. Red cell culture assays of RPL15 - or RPL9 -mutated primary erythroblast cells revealed reductions of cell proliferation and delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. Colon tissue from patient with RPL9 mutation revealed intense epithelial TP53 staining, CD3+ lymphocytic infiltrates, and insufficient crypt regenerationThis study identifies RPL9 as a new DBA-linked gene, identifies recurrent truncating hotspot mutation in RPL15, reaffirms a role for TP53 stabilization in disease pathophysiology, and suggests that certain DBA genotypes may be used for risk stratification. To our knowledge, this is the first report on multiple DBA patients presenting with hydrops fetalis or colitis due to haploinsufficiency of specific RP genes. DisclosuresNo relevant conflicts of interest to declare.

Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1αshort promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19- deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Clinical features and outcomes of patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia: a multicentre, retrospective, cohort study

Data to inform surveillance and treatment for leukaemia predisposition syndromes are scarce and recommendations are largely based on expert opinion. This study aimed to investigate the clinical features and outcomes of patients with myelodysplastic syndrome or acute myeloid leukaemia and Shwachman-Diamond syndrome, an inherited bone marrow failure disorder with high risk of developing myeloid malignancies. We did a multicentre, retrospective, cohort study in collaboration with the North American Shwachman-Diamond Syndrome Registry. We reviewed patient medical records from 17 centres in the USA and Canada. Patients with a genetic (biallelic mutations in the SBDS gene) or clinical diagnosis (cytopenias and pancreatic dysfunction) of Shwachman-Diamond syndrome who developed myelodysplastic syndrome or acute myeloid leukaemia were eligible without additional restriction. Medical records were reviewed between March 1, 2001, and Oct 5, 2017. Masked central review of bone marrow pathology was done if available to confirm leukaemia or myelodysplastic syndrome diagnosis. We describe the clinical features and overall survival of these patients. We initially identified 37 patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia. 27 patients had samples available for central pathology review and were reclassified accordingly (central diagnosis concurred with local in 15 [56%] cases), 10 had no samples available and were classified based on the local review data, and 1 patient was excluded at this stage as not eligible. 36 patients were included in the analysis, of whom 10 (28%) initially presented with acute myeloid leukaemia and 26 (72%) initially presented with myelodysplastic syndrome. With a median follow-up of 4·9 years (IQR 3·9-8·4), median overall survival for patients with myelodysplastic syndrome was 7·7 years (95% CI 0·8-not reached) and 0·99 years (95% CI 0·2-2·4) for patients with acute myeloid leukaemia. Overall survival at 3 years was 11% (95% CI 1-39) for patients with leukaemia and 51% (29-68) for patients with myelodysplastic syndrome. Management and surveillance were variable. 18 (69%) of 26 patients with myelodysplastic syndrome received upfront therapy (14 haematopoietic stem cell transplantation and 4 chemotherapy), 4 (15%) patients received no treatment, 2 (8%) had unavailable data, and 2 (8%) progressed to acute myeloid leukaemia before receiving treatment. 12 patients received treatment for acute myeloid leukaemia-including the two patients initially diagnosed with myelodysplastic who progressed- two (16%) received HSCT as initial therapy and ten (83%) received chemotherapy with intent to proceed with HSCT. 33 (92%) of 36 patients (eight of ten with leukaemia and 25 of 26 with myelodysplastic syndrome) were known to have Shwachman-Diamond syndrome before development of a myeloid malignancy and could have been monitored with bone marrow surveillance. Bone marrow surveillance before myeloid malignancy diagnosis was done in three (33%) of nine patients with leukaemia for whom surveillance status was confirmed and 11 (46%) of 24 patients with myelodysplastic syndrome. Patients monitored had a 3-year overall survival of 62% (95% CI 32-82; n=14) compared with 28% (95% CI 10-50; n=19; p=0·13) without surveillance. Six (40%) of 15 patients with available longitudinal data developed myelodysplastic syndrome in the setting of stable blood counts. Our results suggest that prognosis is poor for patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia owing to both therapy-resistant disease and treatment-related toxicities. Improved surveillance algorithms and risk stratification tools, studies of clonal evolution, and prospective trials are needed to inform effective prevention and treatment strategies for leukaemia predisposition in patients with Shwachman-Diamond syndrome. National Institute of Health.

IMPLICATIONS OF REVERTANT SOMATIC MOSAICISM IN BONE MARROW FAILURE SYNDROMES

Revertant somatic mosaicism (RM) has been reported in genetic diseases including skin disorders and bone marrow failure (BMF) syndromes. We present examples of RM of haematopoietic cells in the context of inherited BMF syndromes with dramatically different clinical outcomes. Diamond Blackfan Anaemia (DBA) is an inherited BMF disorder caused by mutations/deletions in ribosomal proteins. We have a 9 year old male who required regular transfusions until he became transfusion independent at age 4. We identified a 184kb deletion on chromosome 12 encompassing 11 genes including RPS26 (known DBA gene). Significantly, we observed multiple independent copy neutral loss of heterozygosity (CNLOH) events on chromosome 12, correcting the 184kb deletion in a subset of blood cells, but not in hair. We propose RM as a mechanism for correction of the germline deletion resulting in spontaneous remission in the patient. We also recently identified germline mutations in SAMD9L, in a family with thrombocytopaenia, macrocytosis and ALL. In this family, we see examples for somatic mosaicism leading to a severe blood phenotype as well as CNLOH RM causing milder phenotypes in different family members. RM needs to be considered when selecting tissue source for diagnosis in carriers with mild phenotype/remission, and use of patient-derived cell lines for drug screens with multiple rounds of cell replication. We are currently trying to understand and manipulate revertant clone selection and hope this will open windows for rational correction and selection protocols for effective therapeutic intervention. Experimental manipulations to select clones that improve clinical phenotype have substantive implications on efficiencies of gene manipulations, manufacture and quality control requirements of autologous cellular therapies.

Haplo-Identical Transplant Using Post Transplant Cyclophosphamide in Children – Single Centre Experience from a Developing Country

Introduction The access to hematopoietic stem cell transplant [HSCT] for children lacking a matched sibling donor has vastly improved with the introduction of haplo-identical transplant using post-transplant cyclophosphamide(PTCy). There is limited data on the use of PTCy and we present our experience with the use of HaploHSCT with PTCy in children. Patients and Methods Between 2010 and August 2018, 86 children (≤ 15 years) underwent 95 haplo-identical transplants using PTCy as the major graft versus host disease (GVHD) prophylaxis. Disease status at the time of HSCT was classified into Early, Intermediate and Late disease based on modified CIBMTR criteria to include immune deficiencies and bone marrow failure disorders. Data on HSCT and outcomes were collected from the institutional database and individual medical records. Results The median age of these children was 7 years (range: 9 months – 15) including 51 boys and 35 girls. Indications for HSCT included malignant diseases in 35 (AML, ALL) and non-malignant diseases in 51 (immune deficiency, inherited bone marrow failure disorders, aplastic anemia). Myeloablative conditioning was used in 51 transplants and non-myeloablative conditioning in 45. Disease status included Early disease (ED) in 10, Intermediate disease in 53 and Late disease in 32 transplants. 13 haplotransplants were performed as a second rescue transplant after failure of either a Haplo, MUD or sibling donor HSCT. Engraftment was seen in 82% with graft failure in 8% and early death in 10%. The median time to neutrophil engraftment was 18 days (range: 13 – 25). Acute GVHD (grade 2-4) was seen in 34% of evaluable patients while chronic GVHD was seen in 44% of evaluable patients. Infections were a major problem in the first 100 days of HSCT with viral infections (CMV, BK) occurring in 70% of transplants and bacterial infections in 42%. At a median follow up of 28 months, the overall survival is 52%. 44 children (52%) are alive and well. All children transplanted with early disease are alive while survival is 62% with intermediate disease and 25% with late disease. Conclusion Haplo-identical transplants using PTCy is feasible in children and associated with reasonable outcomes. Infections remain a major hurdle to improving overall survival. Disease status at HSCT has a major impact on outcomes.

Aplastic and Hypoplastic Anemias.

1. Suzie A. Noronha, MD* 1. *Division of Pediatric Hematology/Oncology, University of Rochester, Rochester, NY * Abbreviations: AA: : aplastic anemia AML: : acute myeloid leukemia ANC: : absolute neutrophil count ARC: : absolute reticulocyte count CBC: : complete blood cell DBA: : Diamond-Blackfan anemia DC: : dyskeratosis congenita FA: : Fanconi anemia GPI: : glycosylphosphatidylinositol HSCT: : hematopoietic stem cell transplant MCV: : mean corpuscular volume MSD: : matched sibling donor MUD: : matched unrelated donor OS: : overall survival PNH: : paroxysmal nocturnal hemoglobinuria PRCA: : pure red cell aplasia SDS: : Shwachman-Diamond syndrome TEC: : transient erythroblastopenia of childhood 1. Anemia is a common finding in general pediatrics, but knowing when to refer a patient can be challenging. 2. Pediatricians do not always order reticulocyte counts as part of the evaluation of anemia. The reticulocyte count, interpreted in the context of the mean corpuscular volume, is very useful in the development of differential diagnoses for anemia and determination of urgency of referral. 3. Pediatricians (and subspecialists) may not realize that diagnosis of malignancy, particularly head and neck cancers, at an unexpectedly early age may be associated with an inherited bone marrow failure. After completing this article, readers should be able to: 1. Assess the need for referring a patient with anemia based on whether anemia is isolated or accompanied by other hematologic or physical anomalies. 2. Recognize that anemia in the setting of congenital anomalies may signify an inherited bone marrow failure disorder. 3. Understand that inherited bone marrow failure disorders confer a higher risk of malignancy. Children with anemias often initially present to their pediatricians. Anemia may result from blood loss, a destructive process (ie, hemolysis), nutritional deficiency, or poor production (eg, ineffective erythropoiesis or hypoplastic or aplastic marrow) (Table). Hemolytic and nutritional anemias have been discussed elsewhere and are referenced briefly in this article. (1)(2)(3)(4) This review focuses on the broad differential diagnosis for anemia associated with poor production. These anemias can lead to chronic morbidity and require subspecialty care, whereas others may be managed effectively in primary care. This review offers a framework to guide evaluation and the need for subspecialty investigation. View this table: TABLE . Classification of Anemias by Mechanism and Notable Associated Laboratory Data Aplastic and hypoplastic anemias are characterized …

Non-Genotoxic Conditioning Efficiently Depletes Host Hematopoietic Stem Cells and Facilitates Robust Multi-Lineage Engraftment in a Mouse Model of Fanconi Anemia

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow prior to a bone marrow transplant to facilitate engraftment of donor cells. While effective, some major issues with chemotherapy remain which includes off-target genotoxic effects increasing the risk of secondary malignancies. These complications are compounded in disease settings that arise from a deficit in DNA repair pathways where cytotoxic treatments can result in oncogenic transformation, such as Fanconi anemia (FA). FA is an inherited bone marrow failure disorder resulting from an intrinsic defect in DNA repair affecting approximately 130,000 children each year. Currently mutations in 22 genes have been implicated in the pathogenesis of FA. While novel gene-therapy based protocols are showing early promise for this patient populations, the standard treatment for the hematologic complications for FA is a bone marrow transplant. However, secondarily to an underlying sensitivity to DNA damage, these patients are unable to receive standard myeloablative conditioning, which can reduce the efficiency of reconstitution. Avoiding alkylating agents could improve outcomes and success rates in these patients. Furthermore, this methodology could be translated to the allogeneic bone marrow transplantation setting, decreasing the toxicity of this treatment modality.Our approach for characterizing an alternative conditioning regimen for FA patients utilizes immunotoxin conjugates specifically targeting hematopoietic stem cell populations. These non-genotoxic antibody-based drug conjugates utilize saporin (SAP), a ribosomal toxin, to eliminate cells in a targeted manner while leaving the remainder of the marrow compartment intact. Antibodies targeting either CD45 or CD117 were used in a mouse model of FA where expression of their FANCA gene, one of the most common mutations in humans, has been knocked out. Mice conditioned with either of the drugs received various doses of whole marrow from healthy heterozygous littermates. On the day of transplant, mice conditioned with either immunotoxin demonstrated significantly reduced LSK stem cell populations in the marrow similar to cyclophosphamide (Cy) controls (Figure 1A).Peripheral engraftment of donor cells was monitored for 6 months, after which mice were sacrificed for complete analysis of engraftment in the bone marrow compartment. No significant difference was observed in engraftment between Cy and immunotoxin conditioned mice (Figure 1B), and all treatment groups exhibited robust multilineage reconstitution in a cell dose dependent manner. Additionally, Cy treated mice demonstrated greater and sustained weight loss and lower gastrointestinal losses compared to immunotoxin treated mice. Ongoing clonal analysis studies of engrafted cells has demonstrated polyclonal reconstitution, indicating a large number of donor stem cells are actively contributing to hematopoiesis.In this study, we demonstrate that non-genotoxic conditioning approaches both facilitate multilineage engraftment of donor marrow and significantly deplete host hematopoietic stem cell populations. These are crucial since persistence of host hematopoiesis could eventually result in clonal evolution and leukemogenesis in post-transplant FA patients. Achieving both of these conditions through targeted elimination with immunotoxin conjugates represents a major advancement in bone marrow transplantation for FA. We now are initiating studies using immunotoxin-based conditioning for the transplantation of gene-modified syngeneic stem cells and allogeneic cells. We think these studies will inform future clinical trials and provide the groundwork for the next-generation of therapy for FA patients. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment. Palchaudhuri:Harvard University: Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy.

Differences in the bone marrow histology between childhood myelodysplastic syndrome with multilineage dysplasia and refractory cytopenia of childhood without multilineage dysplasia.

Refractory cytopenia of childhood (RCC) is subdivided into myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) and RCC without (w/o) multilineage dysplasia (RCC without MLD). Although RCC is a histomorphological distinct entity, the bone marrow (BM) histology of RCC is not yet characterised in relation to multilineage dysplasia. We investigated the BM histological features of RCC to clarify the characteristics of BM histology of MDS-MLD in childhood compared to RCC without MLD. The BM histology and cytology in 60 RCC patients from the nationwide registry of Japanese Childhood AA-MDS Study Group were reviewed retrospectively. Although a thorough genetic assessment, including GATA2 and/or SAMD9, was not performed, inherited BM failure disorders were excluded by a cytogenetic test, a chromosome fragility test and a telomere length measurement along with careful clinical assessments. Among the 60 patients, 20 (33%) of MDS-MLD and 40 (67%) of RCC w/o MLD were classified according to their BM cytology. We then investigated the BM histological features and compared them between the two groups. The BM cellularity, distribution pattern of haematopoiesis, frequency of left-shifted granulopoiesis, numbers of micromegakaryocytes and p53 immunostaining-positive cells were significantly different between the groups. The BM histology of MDS-MLD in childhood showed higher cellularity, the more common occurrence of diffuse distribution pattern, more frequently left-shifted granulopoiesis and more micromegakaryocytes and p53 immunostaining-positive cells than RCC without MLD. Our results showed that MDS-MLD in childhood had a characteristic BM histology compared to RCC without MLD. The clinical relevance of MDS-MLD in childhood needs to be evaluated.

Combination therapy with atorvastatin and celecoxib delays tumor formation in a Fanconi anemia mouse model.

Fanconi anemia is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Currently, no interventions to prevent or delay the formation of solid tumors are available. Two of the most important hallmarks of Fanconi anemia are inflammation and oxidative stress. In this study, we administrated the antioxidant atorvastatin and the anti-inflammatory drug celecoxib to cohorts of Fancd2-/- /Trp53+/- mice, a model of Fanconi anemia. Treatment started at weaning and continued until the mice developed a palpable mass or suffered from>20% weight loss. Tumor samples and selected tissues were subjected to histopathological examination. χ2 test was performed to analyze tumor incidence, and Kaplan-Meier survival curves were evaluated with log-rank test. In addition, a small cohort of mice was monitored for the safety of the drugs. The combined oral administration of both drugs significantly delayed tumor onset in Fancd2-/- /Trp53+/- mice. Specifically, the treatment delayed the onset of ovarian tumors in Fancd2-/- /Trp53+/- mice and increased the mean ovarian tumor-free survival time by 17%, whereas this combinatorial drug regimen did not have a significant effect on other tumor types. In addition, no detrimental effects on hematopoiesis from the drug treatment were observed during a 12-month safety monitoring. The data presented here suggest that a combination of atorvastatin and celecoxib may be a good candidate for chemoprevention in Fanconi anemia.

Allogeneic Stem Cell Transplantation in Fanconi Anemia and other inherited bone marrow failure disorders

Allogeneic Stem Cell Transplantation in Fanconi Anemia and other inherited bone marrow failure disorders

Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

Clinical heterogeneity in a family with DKC1 mutation, dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome in first cousins.

Dyskeratosis congenita (DC) is an inherited bone marrow failure disorder characterized by mucocutaneous features (skin pigmentation, nail dystrophy and oral leukoplakia), pulmonary fibrosis, hematologic and solid malignancies. Its severe form, recognized as Hoyeraal-Hreidarsson syndrome (HHS), also includes cerebellar hypoplasia, microcephaly, developmental delay and prenatal growth retardation. In literature phenotypic variability among DC patients sharing the same mutation is wellknown. To our knowledge this report describes for the first time a family of DC patients, characterized by a member with features of classic DC and another one with some features of HHS, both with the same mutation in DKC1. Our family confirms again that one mutation can be associated with different phenotypes and different hematological manifestations. It’s possible to speculate that there are likely to be patients who do not clinically fit neatly into either classical DC or HHS, but whose clinical features are due to mutations in DKC1 or in genes responsible for autosomal DC/HHS.

Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice

AbstractFanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2−/− mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2−/− mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2−/−Trp53+/− mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient–derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.

Genetics in Inherited Bone Marrow Failure Disorders

The classical Inherited Bone Marrow Failure Syndromes (IBMFS) such as Fanconi anemia, Dyskeratosis Congenita, Shwachman-Diamond syndrome, and Diamond-Blackfan anemia are a heterogeneous group of disorders, all of which are characterized by impaired hematopoiesis, varying degrees of peripheral cytopenias and marrow hypoplasia and dysplasia. Many of these are associated with an increased risk of clonal dominance and evolution to myelodyplastic syndrome (MDS) and acute myeloid leukemia (AML). For the purposes of this talk, the familial MDS and acute leukemia predisposition syndromes are also included in the broad term IBMFS. The genes responsible for a subset of IBMFS have been identified and will be reviewed. However, the causative mutations in many patients presenting with seemingly inherited marrow failure remain unknown. Gene discovery in IBMFS has been difficult in large part due to the phenotypic heterogeneity of these syndromes. Some patients with IBMFS display a distinct clinical phenotype with associated syndromic abnormalities, others are variable and overlap with one another or with acquired MDS or idiopathic acquired aplastic anemia, and additional cases are more obscure and have evaded classification altogether. Accurate diagnosis of IBMFS inform patient care as it allows appropriate screening of siblings to avoid choosing an affected donor if marrow transplant is indicated and the selection of an appropriate transplant conditioning regiment to avoid undue toxicity. Additionally, accurate diagnosis allows appropriate medical monitoring and early intervention to successfully treat disease-specific non-hematologic medical complications. The application of next generation sequencing approaches for comprehensive genetic screening of IBMFS, including these cryptic or atypical presentations will be reviewed. In addition to providing accurate diagnoses in a subset of patients, genetic characterization in small family kindreds or even in single individuals presents unique opportunities to discover new genes and pathways contributing to dysfunctional hematopoiesis and clonal progression. The frequency of inherited mutations in known IBMFS genes among seemingly idiopathic acquired aplastic anemia patients or pediatric and younger adults with MDS referred for hematopoietic stem cell transplant will be reviewed. Future genetic studies are needed to characterize the secondary genetic events that lead to disease progression in IBMFS. Disclosures No relevant conflicts of interest to declare.

Comparison of a restrictive versus liberal red cell transfusion policy for patients with myelodysplasia, aplastic anaemia, and other congenital bone marrow failure disorders.

This is the protocol for a review and there is no abstract. The objectives are as follows: To assess the efficacy and safety of a restrictive versus liberal red cell transfusion strategy for patients with long-term bone marrow failure. These include myelodysplasia, acquired aplastic anaemia, and other inherited bone marrow failure disorders.

RPS19 Deficiency Leads to GATA1 Downregulation through TNF-Mediated p38 MAPK Activation

Diamond-Blackfan Anemia (DBA) is a rare inherited bone marrow failure disorder, characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. While several studies have found that the tumor suppressor protein p53 contributes to DBA pathogenesis, and that certain GATA1 mutations can give rise to DBA, the link between ribosomal protein mutations and erythroid defects is not well understood.To investigate the molecular pathways downstream of RPS19 deficiency, we infected human cord blood CD34+ cells with RPS19 shRNA lentivirus and observed that RPS19 knockdown resulted in decreased GATA1 mRNA and protein expression, resulting in defective erythropoiesis. We also detected increased TNF-α levels in RPS19 knockdown cells, and in zebrafish that have been treated with RPS19 morpholino. TNF-α was primarily upregulated in the non-erythroid population of RPS19 deficient cells, while its receptor, TNFR1, showed increased expression on the surface of erythroid cells.To understand the relationship between GATA1 and TNF-α, we treated primary hematopoietic CD34+ cells with TNF-α or its inhibitor etanercept. Our results demonstrated that TNF-α treatment reduced GATA1 expression, and that this effect could be rescued by the addition of etanercept. Treatment of RPS19 deficient cells with etanercept improved their erythroid differentiation, and increased GATA1 expression. Additionally, etanercept treatment successfully reversed the anemia phenotype observed in RPS19 deficient zebrafish.To study pathways downstream of TNF-α, we examined phosphorylation of signaling pathways such as p38 MAPK, NFκB, ERK, STAT1, and STAT5 in RPS19 deficient erythroid cells using phospho-flow cytometry. Among these pathways, we found a significant increase in phosphorylation of p38 MAPK, but not ERK, NFκB, STAT1, or STAT5, suggesting that p38 MAPK activation by TNF-α contributes to decreased GATA1 expression in RPS19 cells. We suggest a novel mechanism for the erythroid defects observed in DBA, in which RPS19 deficiency leads to increased TNF-α production in non-erythroid cells, and activation of p38 MAPK, followed by decreased GATA1 expression, in erythroid cells. Our data also suggest that TNF-α inhibitors, such as etanercept, may be beneficial in treating patients with DBA. DisclosuresNo relevant conflicts of interest to declare.