SBDS Mutations Research Articles

ERCC6L2 Disease and Shwachman-Diamond Syndrome Exhibit Alterations in Metabolomes in Patient Fibroblasts

Introduction Metabolomics is an emerging tool to study metabolic alterations in cancers. It has also the potential to identify clinically relevant biomarkers for diagnosis, risk appraisal, and targeted drug development. In hematology, metabolic reprogramming is essential for normal regulation of hematopoiesis. Association of discrete metabolic alterations with explicit somatic mutations have also been reported in hematological malignancies. We have previously observed glutaminase (GLS) overexpression and improved oxygen consumption rate with excess glutamine in low glucose conditions in patient-derived fibroblasts in ERCC6L2 disease (unpublished). This prompted us to study metabolomic alterations in ERCC6L2 disease and Shwachman-Diamond syndrome (SDS) induced strictly by the germline ERCC6L2 and SBDS mutations. Both inherited bone marrow failure (BMF) syndromes carry considerable tendency of somatic TP53 mutagenesis predisposing patients to a high risk myeloid malignancy. Our aim here was to identify potential drug targets by targeted metabolomics. Methods We examined patient-derived skin fibroblasts (ERCC6L2 disease, n=4; SDS, n=4) and healthy controls (n=4) and performed targeted metabolomics with U-13C-D-glucose and U-13C-L-glutamine labeling. Fibroblasts were seeded on six-well plates and on the following day exposed to 13C-glucose (25mM) or 13C-glutamine (2mM) label containing medium for 30 or 120 minutes. Next, metabolites were extracted and quantified with liquid chromatography mass spectrometer. The results were normalized to total metabolite intensity per sample. Results In 13C-glucose labeled samples, we detected statistically significantly lower intensity of pyruvate M+3 in both ERCC6L2 and SDS fibroblasts compared to control at 30 minutes, which persisted at 120 minutes in SDS cells. In the TCA cycle, malate M+3 labeling indicates pyruvate carboxylase (PC) activity and M+2 labeling pyruvate dehydrogenase activity (PDH) (Figure 1). We found a significantly higher portion of malate M+3 than M+2 in SDS cells at all time points, suggesting lower PDH and higher PC activity in SDS cells. Additionally, we detected higher levels of alanine M+3 in SDS samples. As pyruvate is a source for alanine synthesis, we suspect an alteration in pyruvate metabolism; potentially to overcome challenges in pyruvate oxidation due to either decrease in the respiratory chain function or PDH activity. In 13C-glutamine labeled samples, we detected statistically significantly higher intensity of glutamine M+5 and M+4 in ERCC6L2 samples at 30 minutes compared to control and SDS cells. Glutamine is first converted to glutamate by glutaminase (GLS), after which it enters the TCA cycle as ketoglutarate (KG) (Figure 1). In addition to glutamine, we detected an increase, although not statistically significant, in glutamate M+5 and M+4, as well as KG M+5, in ERCC6L2 cells at 30 minutes. A similar trend was observed in the subsequent TCA cycle intermediates succinic acid M+4, fumarate M+4, and malate M+4. At 120 minutes, there was no difference in metabolite intensities between the groups, suggesting a potentially faster glutamine uptake by ERCC6L2 cells. Conclusions Our analysis using non-hematopoietic tissue provides understanding of metabolic weak links not confounded by somatic events occurring early in the disease course in ERCC6L2 and SDS patients. Our findings imply increased glutamine utilization in ERCC6L2 cells and altered pyruvate metabolism in SDS cells. Therefore, these metabolic pathways are attractive drug targets (summarized in Figure 1). Further studies are needed to determine their potential in supporting the compromised hematopoiesis to reduce the stress for somatic mutagenesis.

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.

Lymphoid Malignancies in Shwachman-Diamond Syndrome: Incidence, Clinical Features, and Outcomes from the North American SDS Registry

Introduction: Many marrow failure conditions are associated with myeloid malignancy predisposition and a subset carry an increased risk of both myeloid and lymphoid malignancies. The increased risk of myeloid malignancy is known in Shwachman-Diamond syndrome (SDS), however, the risk of lymphoid malignancy in SDS has not been previously reported. Through the North American SDS Registry (SDSR), we identified patients with SDS diagnosed with lymphoma. We report the clinical features and outcomes in these patients as well as the incidence rates of lymphoma, MDS, and AML in this cohort. Methods: Patients with biallelic SBDS mutations enrolled on the SDSR were included for analysis. Age- and sex-specific incidence rates from the SEER Program (2016-2020) were multiplied by person-years of observation to obtain expected numbers of events. The crude rate per 100,000 (100,000*observed number of malignancies/person-years of follow-up) and the standardized incidence ratio (SIR) were calculated. Results: Out of a total of 217 patients with SDS with biallelic SBDS mutations in the SDSR, we identified 3 subjects who developed lymphoma. Case 1: A male patient with biallelic SBDS mutations (c.258 + 2T>C; c.653G>A, p.Arg218Gln) presented with dyspnea and was diagnosed with primary mediastinal B-cell lymphoma at age 16 years. Bone marrow exam was negative for lymphoma but revealed a new del20q clone. He received 6 cycles of R-CHOP. His course was complicated by febrile neutropenia, pleural effusion, deep vein thrombosis, and decreased cardiac ejection fraction requiring subsequent omission of doxorubicin and carvedilol initiation. He is in remission >3 years from diagnosis. Case 2: A male patient with biallelic SBDS mutations (c.258 + 2T>C; c.183_184TA>CT, p.Lys62*) was diagnosed at age 23 years with diffuse large B-cell lymphoma (DLBCL) of the distal stomach and duodenal bulb. Bone marrow exam was negative for malignancy but demonstrated an abnormal karyotype: 46,XY,t(11;16)(q23;q24), del(20)(q11.2q13.3)[9]/46,XY[11]. He received 5 cycles of R-CHOP therapy, which was complicated by febrile neutropenia and S. aureus sepsis. Four years later, he was diagnosed with relapsed DLBCL of the distal ileum and concurrent AML with 5q deletion. He achieved complete remission of both malignancies after 2 cycles of cyclophosphamide, vincristine, prednisone, obinutuzumab, azacitidine, and venetoclax. Therapy was complicated by B. cereus bacteremia with brain abscesses and endocarditis and C. difficile infection. He underwent a matched unrelated donor hematopoietic stem cell transplant and is in remission >180 days from transplant. Case 3: A male patient with biallelic SBDS mutations (c.258 + 2T>C; c.183_184TA>CT, p.Lys62*) was diagnosed with Stage IVB classical Hodgkin lymphoma at age 12 years after presenting with mediastinal syndrome. Bone marrow biopsy was negative for findings suggestive of MDS or malignancy, however, the initial PET-CT was consistent with medullary involvement. He initiated therapy with steroids, doxorubicin, vinblastine, dacarbazine, and brentuximab vedotin and received focal radiation. His course was complicated by recurrent febrile neutropenia, C. difficile colitis, and cellulitis. Due to infectious complications and delayed count recovery after 2 cycles, despite chemotherapy dose reductions, he was transitioned to brentuximab and nivolumab. He demonstrated complete response by Deauville criteria prior to cycle 5 of brentuximab and nivolumab. Among 217 patients with biallelic SBDS mutations in the SDSR, the median age was 12.8 years (range: 0.3 - 52.8). The observed number, rates, and SIR for lymphoma, MDS, and AML are given in Table 1. A high SIR indicates a higher incidence in the SDSR relative to the SEER population in 2016-2020. The observed rate of lymphoma was 28.5 times higher in SDS than the expected rate in the general population. Conclusions: The incidence of lymphoma among patients with SDS is increased relative to the general population but is lower than the incidence of MDS or AML. Increased toxicity with standard therapies was observed in these patients. Given the increased risk of MDS/AML in SDS, which may be further increased by antecedent chemotherapy, non-genotoxic therapies should be investigated for lymphoma in SDS. Our results show that SDS should be added to the list of marrow failure conditions predisposing to both myeloid and lymphoid malignancies.

Routine Whole Genome Sequencing for All Children with Hematological Malignancies Defines a New Standard of Care - Data of the First 152 Cases from the NHS England Genomic Medicine Service

Background There is emerging evidence that whole genome sequencing (WGS) can benefit children with hematological malignancies, even when compared to extensive molecular assays. However, studies have been restricted to selected patient groups, and so there is a need to evaluate the feasibility and impact of WGS delivered as part of children's routine care. In England, following the 100k genome project, since early 2021 paired germline and tumor WGS has been made available to all children with a suspected malignancy via the nationalized Genomic Medicine Service. We set out to assess the real world impact of routine WGS, where sequential unselected children were sequenced, and results were used in clinical practice. Methods Clinical and diagnostic information was collected from all patients presenting to Great Ormond Street Hospital (GOSH) hematology department over a 2-year period. WGS findings were superimposed onto the center's standard of care (SOC) and WGS impact was evaluated. Results Between 1 January 2021 and 31 December 2022, 152 children with new diagnoses or relapsed disease had paired WGS, with an uptake rate of 83% (76/92) in the first year and (79/83) 95% in the second. Barriers to recruitment were clinicians not offering WGS (n=16) in year 1, and early death (n=2) or parents declining consent (n=2) in year 2. WGS was performed for all 20 diseases that were treated (Figure 1). Importantly, WGS matched all SOC findings by other genetic tests, including targeted DNA high throughput sequencing (HTS) and RNA-fusion panels. WGS revealed additional oncogenic drivers in 79/152 (52%) of patients, compared to all other genomic tests (including HTS and RNA-fusion panels). WGS aided diagnosis in 11% (17/152) of cases through picking up a novel lesion that defined the child's malignancy, and for 7% (10/152) of patients, it was able to place them into a new formal cytogenetic risk category. In 25/152 (17%) cases care was changed solely because of WGS (Figure 2), with a similar rate in year 1 (14%) and 2 (17%). In 3 cases of B-acute lymphoblastic leukemia (B-ALL) diagnostic testing failed to reveal a risk-defining lesion but WGS identified good risk variants (ETV6-RUNX1 and High Hyperdiploidy) allowing de-escalation of chemotherapy. IGH-DUX4 re-arrangements, solely picked up by WGS in 2 cases of B-ALL, led to 1 child not receiving escalated chemotherapy. In a child presenting with mixed phenotype leukemia, WGS alone revealed a KMT2A-USP2 fusion, which directed therapy, as well as providing an MRD marker. In 12 patients presenting with marrow failure (subsequently diagnosed with aplastic anemia or MDS), WGS was prioritized with a median turnaround time of 2 weeks. WGS was able to give an accelerated diagnosis, facilitating timely therapy. 9 children were found to have pathogenic germline variants. This included 2 cases of an accelerated diagnosis of expected mutations (e.g. an SBDS mutation). Other unexpected germline mutations (e.g. a TP53 mutation presenting with leukemia, and an incidental PMS2 mutation in the context of aplastic anemia), led to cascade testing for patients and their families, and tailored surveillance programs. WGS made 9 children eligible for novel therapeutics, including patients found to have a RAS pathway mutation solely by WGS, and patients who were hypermutated. Conclusion WGS can successfully be delivered as a nationalized genomics medicine service, for all children with suspected hematological malignancies. WGS can match conventional and advanced genomic diagnostics in a single assay, with obvious economic benefits of test consolidation. Beyond this, it delivers additional germline and somatic findings that aid diagnosis, offer therapeutic opportunities, and tangibly change the care patients and families receive. WGS has the additional benefit of future proofing diagnostic and research capabilities, as over time analysis pipelines can be modified and new variants added. For all children presenting with marrow failure, WGS was practice changing, providing all necessary read-outs, and facilitating timely therapy. In the case of the most common childhood malignancy, ALL, one test can replace all diagnostics, including IG/TCR MRD marker screening and pharmacogenomics for TPMT/NUDT15. With continued improvements in turnaround time, and reducing costs, we propose that WGS could feasibly replace multiple molecular diagnostic tests for pediatric hematological malignancies.

Targeting Nucleotide Biosynthesis Ameliorates Neutropenia in a Zebrafish Model of Dnajc21-Mutant Shwachman-Diamond Syndrome

Germline mutations in the ribosome maturation factors, SBDS, DNAJC21 and EFL1, lead to Shwachman-Diamond syndrome (SDS), a type of inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency and growth restriction. Further, SDS patients have a 36% increased cumulative risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) by 30 years of age. Given the rarity of SDS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Mouse and zebrafish models of SBDS mutations exhibit early lethality. In contrast, no in vivo models of DNAJC21 mutations have been published to date. We generated the first in vivo model of dnajc21 loss using zebrafish ( Danio rerio), given their highly conserved hematopoietic program and ease of genetic manipulation. Introduction of a homozygous deletion mutation in the zebrafish dnajc21 gene using CRISPR-Cas9 genomic editing resulted in stunted body growth and neutropenia, recapitulating hallmark SDS patient phenotypes. Whole-mount in situ hybridization for key hematopoietic lineage markers revealed reduced l-plastin +mature leukocytes and hbbe3 + mature erythrocytes in dnajc21 mutant embryos at 48 hours post-fertilization (hpf). A concomitant accumulation of myb + hematopoietic stem and progenitor cells (HSPCs) both in the dorsal aorta (equivalent to aorta-gonad-mesonephros) and the caudal hematopoietic tissue (fetal liver equivalent) was observed. Treatment of embryos with zebrafish granulocyte colony stimulating factor (gcsf) mRNA resulted in a modest expansion of neutrophils in dnajc21 mutant zebrafish compared to wildtype. Further, we observed reduced cell proliferation marked by phosphohistone-H3 (pH3) as well as low ATP:AMP levels measured by liquid chromatography/mass-spectrometry (LC/MS) in the dnajc21 mutants, suggesting a poor bioenergetic profile. At 4 months of age, flow cytometry and histopathological analysis identified reduced mature myelomonocytes and mild erythroid dysplasia in the whole kidney marrow (human bone marrow equivalent) of mutant fish. The link between TP53 somatic mutations and MDS/AML has been established in SBDS-mutant SDS, but has not been explored for DNAJC21-mutant SDS. We hence crossed our dnajc21 mutant zebrafish with a zebrafish line carrying a gain-of-function mutation (R217H) in the tp53 gene. Compound mutant fish had improved neutrophil counts but importantly, this was accompanied by an expansion of myeloid progenitors and significant erythroid dysplasia. To understand the disease mechanisms underlying the neutropenia and hypoproliferation in SDS, we performed bulk RNA sequencing of whole zebrafish larvae at 48 hpf. Biological processes such as oxidative stress response, drug catabolism and pre-mRNA spliceosome assembly were downregulated; whereas, acyl-CoA and nucleotide metabolism were upregulated in the mutants. In parallel, we performed untargeted metabolomics by LC/MS to determine the metabolic changes induced by dnajc21 loss. Upregulated pathways included urea cycle, amino acid and phenylacetate metabolism. Pyrimidine biosynthesis, including both the de novo and salvage pathways were downregulated in the mutants. Remarkably, exogenous supplementation with uridine or thymidine significantly improved neutrophil counts in the mutants. Our findings suggest that in dnajc21-mutant SDS1) neutropenia is caused by a combination of impaired granulocyte differentiation and cell proliferation; 2) the kidney marrow features myelodysplasia-like changes and 3) nucleotide biosynthesis may be a previously unrecognized therapeutic strategy in SDS.

Outcomes of Hematologic Complications in Shwachman Diamond Syndrome: High Risk Features and Implications for Surveillance

Background Shwachman Diamond syndrome (SDS) is characterized by genetic predisposition to bone marrow failure and TP53-mutated myeloid malignancies. Data are scarce to guide surveillance strategies. Objectives This study aims to characterize the presentation and outcomes of hematologic complications in 218 patients with SDS with the goal of informing surveillance. Design/Method Data was obtained from medical records of 218 study subjects with biallelic mutations in SBDS/DNAJC21/ EFL1, or heterozygous mutations in SRP54 consented to the SDS Registry or the Bone Marrow Failure/MDS study which are longitudinal prospective cohort studies. Results The median age of the study cohort was 12.2 years (yr) (range 0.1-52.8 yr). Of 204 patients with SBDS mutations, 12 developed severe BMF requiring transplant (median age 2.9 yr, range 0.4-39.5), 16 developed MDS (median age 15.9 yr, range 0.5-45), 11 developed AML (median age 27.7 yr, range 9.4-47.3) and 2 developed lymphoma (ages 17.0 and 23.8 yr). Of the 48 subjects with SBDS mutations who were adults >18 years (median 25.5, range 18.5-52.8), 16 (33%) developed a myeloid malignancy. Of 20 patients with an SRP54 mutation, 1 developed AML at age 15.2. One patient with biallelic EFL1 mutations died of BMF at age 0.7 yr. Further analysis focused on patients with SBDS mutations. Five patients with MDS and 1 patient with AML were on G-CSF at the time of malignancy diagnosis. Only 2 patients who developed AML had had surveillance, but neither included somatic mutation analysis. The diagnosis of SDS was unrecognized prior to malignancy diagnosis for 5 with MDS (age range 13.8 to 21.5 yr) and 2 with AML (age 37.8 and 47.3 yr). Three patients were diagnosed with MDS on surveillance marrow exam. All patients with AML presented with symptoms which led to diagnosis. Of the 16 patients who initially presented with MDS, 5 progressed to AML. Median survival for those who presented with MDS was 1.3 yr (range 0.2-14.9). Ten were deceased, 4 were alive with median follow up of 3.4 yr (range 1.0-14.9). Two additional patients were <100 days out from HSCT. Of 9 patients with MDS who proceeded to HSCT without prior cytoreductive therapy, 4 were alive with a median follow up of 3.1 yr (range 0.2-14.4) post-HSCT, 5 were deceased from treatment related mortality (TRM) (n=2), relapsed/refractory disease (n=2) or osteosarcoma at 10 years post-HSCT (n=1). Of 6 patients who received cytoreductive therapy, 3 died prior to HSCT with active disease (n=2) or TRM (n=1). Of the 3 who proceeded to HSCT post-cytoreductive therapy, 2 are alive at 0.1 and 0.7 yr post HSCT, and 1 died of TRM. One patient who received no therapy died 7.7 years post-diagnosis. Median survival following AML diagnosis was 1 year (range 0.2-5.6). Of the 11 patients with AML, 8 died with relapsed/refractory disease, 1 died of TRM, and 2 survive in remission at 2 and 5.6 years post-AML diagnosis. Of the two patients who proceeded directly to HSCT, one is alive 5.6 years post-HSCT. Nine patients received a variety of cytoreductive regimens for AML, including AraC/Idarubicin, hypomethylating agents +/- venetoclax, of whom 6 were able to proceed to HSCT. None of the 9 adult patients >18 years with SBDS mutations and AML survived. Eight patients with SDS undergoing surveillance marrow exams developed high risk features (HRF), including progressive dysplasias, dropping blood counts with rising marrow cellularity, high risk clonal cytogenetic abnormalities, and large or rapidly growing TP53 clones. Median age of patients with HRF was 19.7 yrs (range 1.3-40.9). All 6 patients transplanted for HRF are alive and well with a median follow up of 1.8 yr (0.1-4.5) post-BMT (Figure 1); 2 patients are currently undergoing evaluation for HSCT. None of the patients transplanted for HRF developed grade 3 or 4 GVHD. One patient with a large TP53 clone prior to transplant is alive without a detectable TP53 clone 0.9 years post-BMT. Conclusions AML carries a poor prognosis for patients with SBDS mutations, so early referral for novel therapies should be considered. Marrow surveillance including morphology, cytogenetics, FISH, and somatic mutation analysis identified patients who developed HRF. Survival was excellent for patients transplanted for HRF. Although SDS is rare and patient numbers are small, these data show that regular comprehensive marrow surveillance can identify HRF to inform the need for closer monitoring or transplant prior to progression to malignancy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phenotypic Variation in Two Siblings Affected with Shwachman-Diamond Syndrome: The Use of Expert Variant Interpreter (eVai) Suggests Clinical Relevance of a Variant in the KMT2A Gene.

Introduction. Shwachman-Diamond Syndrome (SDS) is an autosomal-recessive disorder characterized by neutropenia, pancreatic exocrine insufficiency, skeletal dysplasia, and an increased risk for leukemic transformation. Biallelic mutations in the SBDS gene have been found in about 90% of patients. The clinical spectrum of SDS in patients is wide, and variability has been noticed between different patients, siblings, and even within the same patient over time. Herein, we present two SDS siblings (UPN42 and UPN43) carrying the same SBDS mutations and showing relevant differences in their phenotypic presentation. Study aim. We attempted to understand whether other germline variants, in addition to SBDS, could explain some of the clinical variability noticed between the siblings. Methods. Whole-exome sequencing (WES) was performed. Human Phenotype Ontology (HPO) terms were defined for each patient, and the WES data were analyzed using the eVai and DIVAs platforms. Results. In UPN43, we found and confirmed, using Sanger sequencing, a novel de novo variant (c.10663G > A, p.Gly3555Ser) in the KMT2A gene that is associated with autosomal-dominant Wiedemann–Steiner Syndrome. The variant is classified as pathogenic according to different in silico prediction tools. Interestingly, it was found to be related to some of the HPO terms that describe UPN43. Conclusions. We postulate that the KMT2A variant found in UPN43 has a concomitant and co-occurring clinical effect, in addition to SBDS mutation. This dual molecular effect, supported by in silico prediction, could help to understand some of the clinical variations found among the siblings. In the future, these new data are likely to be useful for personalized medicine and therapy for selected cases.

P835: GENETIC BACKGROUND AND CLINICAL CHARACTERISTICS OF CONGENITAL NEUTROPENIAS IN ISRAEL

Background: Congenital neutropenias comprise a heterogenous group of disorders causing low neutrophil counts with a variable clinical presentation. Patients may suffer from severe infections and have a high risk of myeloid transformation. To date, over 30 genes are known to cause congenital neutropenias, with a diverse relative frequency in different ethnicities. Understanding the spectrum of genetic causes of neutropenia in ethnically diverse populations and the related clinical presentation may direct the molecular work-up and clinical care. Israel is enriched with multiple ethnicities and high consanguinity. Aims: To evaluate the clinical and genetic spectrum of patients with congenital neutropenias in Israel. Methods: All patients diagnosed with congenital neutropenia from the Israeli Inherited Bone Marrow Failure registry were included. Data were extracted from the registry and patients’ charts. Sanger sequencing was first performed for ELANE or G6PC3, followed by less commonly mutated genes. In recent years, patients with wild-type ELANE/G6PC3 were referred for targeted next generation sequencing, followed by whole exome sequencing. Results: The cohort encompasses 66 patients, from 50 families, including 7 with cyclic neutropenia. Fifty patients (76%) were genetically diagnosed; 22 (33% of the cohort) had mutations in ELANE, 9 (13.6%) had homozygous G6PC3 mutations, 7 (10.6%) SBDS mutations, 6 (9%) patients had SRP54 mutations, two each had heterozygous GFI1 and homozygous JAGN1 mutations and one each had TAZ and SLC37A1 mutations. All patients with G6PC3 and GFI1 mutations were of consanguineous Muslim Arab origin. Extra-hematopoietic manifestations were common among patients with mutations in G6PC3. Most patients (82%) presented with severe infections and this was also the most common cause of death. Eight patients (12%) developed myeloid transformation; in 6 of those a somatic molecular work-up was performed, and truncating mutations in the granulocyte-colony stimulating factor were detected in 4. Eighteen (27%) patients underwent hematopoietic stem cell transplantation, most often due to insufficient response to treatment with granulocyte-colony stimulating factor. Summary/Conclusion: The prevalence of genes causing congenital neutropenias in Israel is unique, as it includes a relatively high frequency of patients with mutations in G6PC3 and an absence of patients with HAX1 mutations, thus directing the genetic work-up and the clinical care. Similar to other registries, a molecular diagnosis was not achieved for 24% of the patients, suggesting yet unknown genetic causes.

Abstract 5777: Germline variants of cancer predisposition genes in a large cohort of Chinese sarcoma patients

Abstract Background: Sarcomas represent a rare and heterogeneous group of soft tissue and bone cancers, including more than 100 histological subtypes. Although genomic profiling has identified oncogenic fusion genes as the major molecular drivers of many sarcoma subtypes, direct targeting of these fusion genes is clinically challenging and novel treatment approaches are needed. Methods: To explore new therapeutic targets in sarcoma, we performed germline testing of 148-cancer predisposition genes (OncoPanscan࣪, Genetronhealth) on genomic DNA from a cohort of 822 unselected Chinese sarcoma patients. Results: The median age of this cohort was 45 (1-88) years, and 84% were soft tissue sarcoma (STS), 16% were osteosarcoma. 9.3% (n =82) of patients harbored at least one predicted pathogenic germline variant in 37 cancer disposition genes including APC, BRCA1, BRCA2, CDKN2A, CHEK2, DICER1, MSH6, PMS2, SBDS, SMARCA4, and TP53. Twenty-four (2.9%) patients had a variant that mapped to a known sarcoma-associated cancer predisposition syndrome gene (CHEK2, DICER1, NF1, RECQL4, SMARCA4, TP53, WRN). The most frequently mutated genes are involved in the DNA damage repair pathway, with a prevalence of 6.3% (n =52). Twelve patients carried pathogenic mutations in TP53, which was associated with cancer-predisposing Li-Fraumeni syndrome (LFS). Interestingly, we also observed 12 patients harboring the same SBDS c.258+2T&amp;gt;C variant. Biallelic SBDS mutations were linked to Shwachman-Diamond Syndrome (SDS) associated with leukemia predisposition. Additionally, four patients carried pathogenic mutations in SMARCA4 (n =2) and SMARCE1 (n =2), two genes encoding key components of the BAF chromatin-remodeling complex. Patients with pathogenic SMARCA4 germline variants could participate in clinical trials of EZH2 inhibitor tazemetostat. Four patients harbored pathogenic germline variants in core homologous recombination genes (BRCA1/2 and PALB2), which may be targeted with platinum-based chemotherapy or PARP inhibitor olaparib. Conclusions: In summary, our genomic profiling approach revealed novel predisposition variants associated with sarcoma, and some of them can serve as therapeutic targets for future clinical investigation. Our work provided proof of principle that sarcoma patients may benefit from genetic counseling and participate in genomically-matched clinical trials. Citation Format: Jingnan Shen, Xiaomo Li, Xianbiao Xie, Si Liu, Tonghui Ma. Germline variants of cancer predisposition genes in a large cohort of Chinese sarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5777.

Hematologic complications with age in Shwachman-Diamond syndrome

AbstractShwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with leukemia predisposition. An understanding of the hematologic complications of SDS with age could guide clinical management, but data are limited for this rare disease. We conducted a cohort study of 153 subjects from 143 families with confirmed biallelic SBDS mutations enrolled on the North American Shwachman Diamond Registry or Bone Marrow Failure Registry. The SBDS c.258 + 2T>C variant was present in all but 1 patient. To evaluate the association between blood counts and age, 2146 blood counts were analyzed for 119 subjects. Absolute neutrophil counts were positively associated with age (P < .0001). Hemoglobin was also positively associated with age up to 18 years (P < .0001), but the association was negative thereafter (P = .0079). Platelet counts and marrow cellularity were negatively associated with age (P < .0001). Marrow cellularity did not correlate with blood counts. Severe marrow failure necessitating transplant developed in 8 subjects at a median age of 1.7 years (range, 0.4-39.5), with 7 of 8 requiring transplant prior to age 8 years. Twenty-six subjects (17%) developed a myeloid malignancy (16 myelodysplasia and 10 acute myeloid leukemia) at a median age of 12.3 years (range, 0.5-45.0) and 28.4 years (range, 14.4-47.3), respectively. A lymphoid malignancy developed in 1 patient at the age of 16.9 years. Hematologic complications were the major cause of mortality (17/20 deaths; 85%). These data inform surveillance of hematologic complications in SDS.

Impact and Dynamics of TP53 Mutated Clones in Shwachman Diamond Syndrome in a Series of 80 Patients

Introduction : Hematological complications (HC) as Aplastic anemia (AA) and myelodysplasia and acute leukemia (MDS/AL) are frequent and life threatening in patients with Shwachman Diamond Syndrome (SDS) with SBDS mutations. The therapy of such events is based on Hematopoetic stem cell transplantation (HSCT), which results remain quite poor, especially in case of malignancy. So far, it is difficult to anticipate to such HC and a lot is expected from the study of clonal evolution prior HC. Methods: A Targeted panel of 43 genes involved in MDS/AL (sensibility 1%) has been evaluated in 80 patients with SBDS mutation, representative of a nation based cohort of 154 patients. This cross sectional study has been completed by a prospective study for 40 patients evaluated at several time points. Results: The evaluation was performed in various situations: steady state i.e. no haematological complication, in MDS/AL and AA and lastly after HSCT. At the first evaluation, somatic mutation was found in 21 patients (30%) among the 70 in steady state and in 7 of the 8 cases with HC (6/6 cases with MDS/AL, in 1 among the 2 cases with AA) while the 1 of the 2 patients long term survivors after HSCT have no mutation and the other one kept a TP53 clone with a normal blood count and a low (1.5%) variant allele frequency (VAF). Among the 40 patients with several time points, 17 have a mutation at the first time points, but 10 others had additional mutation later. Globally, the most frequent gene involved was TP53 (82%) while mutations in other genes have been observed rarely. VAF in patients with vs without HC is lower (median VAF 0% vs 22.8% respectively p &amp;lt; 0.001) . Complex caryotype, monosomy 7, Iso7q were associated with P53 clone while in Del20q, 8 patients out 14 have a P53 mutations. The comparison between blood and bone marrow results allow the possibility to monitor such mutations in blood. Clonal evolution in one patient who presents a MDS in the course of the follow up had shown a competition between clones. Conclusion: Acquired TP53 is extremely frequent in patients with SBDS mutations, even in the absence of HC, but the prevalence as well as the VAF increased in case of HC. When sequential evaluation could be performed, competition between clones is frequent and a clinical decision remains therefore difficult, just on the evaluation of a time point. Acknowledgments: The French SCN registry is supported by grants from Amgen, Chugai, Prolong Pharma, X4 Pharma, Inserm, the Association 111 les Arts, the Association RMHE, the Association Sportive de Saint Quentin Fallavier. The authors thank the association IRIS and Mrs Grosjean and Mr Gonnot(ASSQF), the association Barth France for their support. Disclosures Sicre de Fontbrune: Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Renard:Jazz Pharmaceuticals: Research Funding. Tournilhac:ABBVIE: Consultancy, Honoraria, Other: Travle grant; INNATE Pharma: Consultancy, Honoraria; GILEAD: Consultancy, Honoraria, Other: Travel Grant; Takeda: Consultancy, Honoraria, Other: Travel grant; Janssen: Consultancy, Honoraria, Other: Travel grant.

Distinct Genetic Pathways Define Leukemia Predisposition Versus Adaptive Clonal Hematopoiesis in Shwachman-Diamond Syndrome

Background: Shwachman-Diamond Syndrome (SDS) is a bone marrow failure disorder caused by impaired removal of EIF6 from the nascent 60S ribosome subunit, resulting in defective ribosome assembly. SDS patients have a high risk of myeloid neoplasms (MN) and the prognosis of those that develop MN is poor. Knowledge of the kinetics and functional consequences of somatic mutation acquisition in SDS may offer insight into mechanism of transformation and the potential for therapuetic intervention. Methods: We performed whole exome sequencing of 45 samples from 30 patients, and validated recurrent somatically mutated genes using targeted sequencing with error suppression in prospectively collected samples from 110 patients in the North American SDS Registry. We correlated mutation status with clinical outcome and performed functional studies to understand the consequence of somatic mutations in SDS. Results: We detected somatic mutations in 74 of 98 (76%) patients with germline biallelic SBDS mutations (median 2 mutations/patient, range 0-21). We found no mutations in patients with SDS-like disease; those who have clinical features of SDS without disease defining mutations. Of the 83 patients with SDS without a MN diagnosis, 60 (72%) had detectable clonal hematopoiesis (CH), 40 of whom had more than one mutation (median 3, range 1-21). The most frequently mutated genes were EIF6 (60/98, 61%),TP53 (44/98, 45%), PRPF8 (12/98, 12%), and CSNK1A1 (6/98, 6%). Among SDS patients with TP53 mutated CH, 90.9% (30 of 33) had concurrent EIF6 mutations. To determine whether EIF6 and TP53 mutations occur in the same or different clones, we performed single cell DNA sequencing. Among the 47 clones identified with either EIF6 or TP53 mutations, 24 had a sole EIF6 mutation, and 21 had a sole TP53 mutation, showing that these mutations arise in separate clones. To study the functional consequences of EIF6 missense mutations, we cloned 7 patient-derived mutations and generated cell lines expressing wild-type or mutant EIF6 cDNA. We found six mutants (I13N, R67W, G69S, P73R, A194T, G196R) reduced levels of EIF6 protein compared with wild type EIF6, despite comparable abundance of mRNA. The most common recurrent mutation, N106S, was found in 20% of patients and, by contrast to others listed above, did not change protein expression. This mutation is located at the EIF6/60S protein interface and disrupted the interaction of N106S-EIF6 with the 60S subunit as measured by polysome profiling followed by western blotting. To compare the effects of EIF6 versus TP53 somatic mutations in context of SDS deficient translation, we measured ribosome maturation and translation in SDS cells containing shRNAs targeting EIF6 or TP53. EIF6 knockdown ameliorated the SDS defect, reflected by improved ribosome joining (normalization of the 80:60s ratio) and enhanced protein translation (increased O-propargyl-puromycin incorporation), whereas TP53 knockdown had no effect. Knockdown of EIF6 in SDS deficient cells decreased p53 pathway activation as demonstrated by decreased CDKN1A expression. TP53 mutations were significantly associated with MN diagnosis (p=0.023), but were also common in SDS CH and typically stable over time. To identify the characteristics associated with transformation, we analyzed exomes from 7 patients with TP53 mutated myeloid malignancy for allelic imbalances at the TP53 locus and found that all 7 had biallelic alteration of TP53. Using single cell DNA sequencing from serial samples, we observed that TP53 LOH can precede transformation by several years and can distinguish pre-leukemic clones from indolent clones with monoallelic TP53 alterations. Somatic EIF6 mutations were not found in the leukemic clones. These results suggest early detection of TP53 LOH may distinguish clones with leukemic potential. Conclusions: In SDS, impairment of ribosome maturation drives selection of clones with somatic EIF6 or TP53 mutations. EIF6 mutations promote competitive fitness by rescuing the SDS ribosome defect and decreasing p53 pathway activation, and do not contribute to malignant transformation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. These results provide genetic evidence that germline SBDS deficiency causes a global, disease-specific HSC fitness constraint that drives parallel development of somatic CH and provides a mechanistic rationale for clinical surveillance. Disclosures Dale: Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Research Funding; X4 Pharmaceuticals: Honoraria. Gansner:Alnylam Pharmaceuticals: Current Employment, Current equity holder in private company. Edwards:Jazz Pharmaceuticals: Consultancy, Honoraria. Fleming:DISC Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Bluebird Bio: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding.

Синдром Швахмана — Даймонда: современные генетические аспекты заболевания из группы рибосомопатий

Objective of the Review: To analyse new DNAJC21, EFL1, SRP54 mutations causing ribosome biogenesis defects and presenting with clinical symptoms similar to the symptoms of Shwachman – Diamond syndrome (SDS). Key Points. SDS is a ribosomapathy and is characterised by pancreatic exocrine insufficiency, defective hematopoiesis, musculoskeletal anomalies, and a high risk of myelodysplastic syndrome and acute myeloid leukemia. About 90% of SDS patients have biallelic SBDS mutations. However, 10–20% of patients with a set of symptoms typical of SDS did not have any pathovars in SBDS gene; therefore, we searched for other candidate genes. In addition to SDS, genetic disorders associated with defected maturation, deficient structure or function of ribosomes and ribonucleoprotein complexes include Diamond – Blackfan anemia, cartilage and hair hypoplasy (McKusick type metaphyseal chondrodysplasia), congenital diskeratosis, 5q-syndrome, and others. These syndromes are similar to SDS. All these conditions are associated with medullary deficiency at least in one hematopoiesis chain. All five conditions are associated with a high risk of cancer. Conclusion. SDS is a genetically determined condition belonging to ribosomapathies. Ribosomapathies are caused by mutations in genes that participate in the synthesis of ribosomal proteins and factors, functioning at various stages of their assembly, and give origin to a number of clinical phenotypes, including haematological malignancies and cancer. In clinical practice, SDS is diagnosed on the basis of typical clinical symptoms and if pathogenic SBDS mutations are present. The issue whether SDS is a genetic heterogenetic ribosomapathy or a mutation of other genes causing defective ribosome synthesis and SDS-like symptoms, is disputable and requires further research. Keywords: Shwachman – Diamond syndrome, genes, SBDS, DNAJC21, EFL1, SRP54, ribosomapathy.

Response to High Dose G-CSF Treatment (20µg/kg/d or Higher) of Patients with Congenital Neutropenia: An Analysis By the Scnir in Europe

Background: Congenital neutropenia (CN) is a group of rare inborn genetic disorders of hematopoiesis including a variety of different gene mutations and different patterns of inheritance. Independent of the underlying genetic subtypes, CN patients benefit from G-CSF maintenance treatment, which improved the life expectancy and quality of life significantly. The majority of CN patients documented by the SCNIR Europe respond well to G-CSF treatment and achieve and maintain neutrophil response with an anticipated ANC of greater 1000/µL with a median G-CSF dose of 4.77 µg/kg/day. However, the G-CSF dose used is very heterogeneous and dose increase is dependent on the decision of each treating physician. Here we report on the incidence, clinical characteristics and outcome of patients receiving G-CSF doses of 20µg/kg/d or higher. Methods: In the European database of the SCNIR we have identified 420 CN patients with different genetic subtypes (including SDS and GSD1b) who are treated with G-CSF. Non-response is defined as median neutrophil counts below 500/µL upon G-CSF doses of 20µg/kg/day or higher. Partial responders have median ANCs of 500 to 999/µL with these G-CSF doses or severe infections despite ANCs of greater 1000/µL. Eighty of the 420 pts received G-CSF doses of 20µg/kg/d or higher. Results: According to the above described response criteria we identified 26 non- responders (6.2%), 18 partial responders (4.3%) and 36 patients reaching ANCs above 1000 with G-CSF doses of 20µg/kg/d or higher (32 patients receiving 20 to 39µg/kg/d; 4 patients receiving more than 50µg/kg/d) out of the 420 CN patients treated with G-CSF. Although patients with different genotypes receive long-term G-CSF treatment, only ELANE or JAGN1 mutations were identified in both non- and partial responders. Analysing ELANE patients separately, 9.3% were non-responders and 5.3 % partial responders to G-CSF. While gender ratio male/female is almost even in the CN cohort (1.08 including 6 males with x-linked TAZ mutations) as well as in the ELANE subgroup (0.87), the gender ratio for ELANE non-responders is 0.2 and 0.4 in ELANE partial responders. In 50% of the identified non- and partial responders the genotype is still unclassified. In 3 patients who were not included in the analysis G-CSF non-response was due to a homozygous CSF3R mutation. These patients respond well to GM-CSF but not to G-CSF. Conclusions: Non-response or partial response to high doses of G-CSF treatment is correlated with distinct genotypes (ELANE, JAGN1, SRP54 and so far unclassified). However, requirement of high G-CSF doses (20µg/kg/d or higher) does not per se indicate non-response, since a substantial number of patients reach sufficient ANCs with these G-CSF doses, especially in patients with ELANE and HAX1 genotype. Treating physicians often do not increase G-CSF doses to &gt;20µg/kg/d to rule out response to high doses. Patients with CSF3R homozygous mutations cannot respond to G-CSF, but respond well to GM-CSF, while ELANE or JAGN1 patients do not respond to GM-CSF. While patients with HAX1 mutations may require high G-CSF doses, but then respond well, patients with SBDS or other mutations (e.g. G6PC3, CXCR4, GFI-1, TAZ etc.) did not require high G-CSF doses for a longer period or were non-responsive to G-CSF treatment. Our data support the dosing recommendation of the SCNIR not to stop G-CSF at 20µg/kg/day, but to increase further, since a substantial number of patients are still responsive. Disclosures No relevant conflicts of interest to declare.

The Shwachman-Diamond Syndrome Registry: Hematologic Complications

▪The Shwachman-Diamond Syndrome Registry (SDSR) was established in December 2008 with the goal of understanding the natural history and biology of SDS to improve the lives of people with SDS. The SDSR has enrolled 220 patients with biallelic SBDS mutations or SDS-Like features. Longitudinal data has been collected for 176 patients with a median duration of follow-up of 10.7 (0.3-52.8) years. Biallelic SBDS mutations were noted in 117 patients, and biallelic DNAJC21 mutations in one patient. Sequencing of genetically undefined cases did not identify EFL1 or SRP54 mutations. Ongoing characterization of SBDS mutation-negative individuals has identified subgroups of clinically defined SDS individuals, as well as a more heterogenous subgroup with features of SDS but who do not meet classic diagnostic criteria. Clinical characteristics and outcomes of patients with biallelic SBDS mutations were examined. AML developed in 5 patients and MDS in 11 patients at a median age 37.9 years (range, 19.5-47.3) and 10.7 years (range, 1-45) respectively. No solid tumors were diagnosed. 21 individuals have undergone stem cell transplantation. Overall survival was 91% in the 10 years since registry establishment, with deaths mainly caused by myelodysplasia (MDS) (n=3) and acute myeloid leukemia (AML) (n=4). One individual with MDS died of hepatic failure unrelated to MDS.Hematologic parameters, trends over time, and clinical correlations were examined.. Cytopenias were almost universal (98.8%) and often intermittent, with neutropenia the most frequent in 98.8%, anemia in 16.5% and thrombocytopenia in 45.3%. Bone marrow evaluations were available in 98, with 88.2% demonstrating marrow hypoplasia. Notable, marrow hypoplasia was progressive, with average marrow cellularity of 74% in patients <1 year of age (n=13), 45% in marrows of children 1-19 years of age (n=277), and 38% in marrows of adults 19-43 years of age (n=38). Cases with discordance between marrow cellularity and cytopenias were noted, so a systematic analysis of the patterns of marrow cellularity and blood counts is ongoing. Marrow dysmorphologies were common and present in in 40.9%, 49.5%, and 28% of the erythroid, myeloid and megakaryocyte lineages respectively, highlighting the need for review from pathologists experienced in the baseline dysmorphologies common in SDS. Marrow cytogenetic data were available for 89 patients of whom 30 (33%) developed clonal abnormalities diagnosed at median age of 8.67 years (range, 0.3-38.9). The most common clonal abnormalities were iso(7q) (n=4) and del(20q) (n=13). Blood count patterns associated with these clonal abnormalities were variable. Notably, iso7q clones were transient while the majority of del20q clones were persistent. One patient with a history of an iso(7q) clone and one with a del(20q) clone progressed to MDS.There is a paucity of evidence to guide optimal surveillance strategies for leukemia predisposition syndromes. The SDSR revealed wide variation in clinical practice amongst local hematologists including: 1) no surveillance for well-appearing patients, 2) intermittent blood counts, or 3) bone marrow exams of variable frequency with variable testing of cytogenetics and FISH. Some patients were not followed by hematology because they looked well until they presented with AML. In one illustrative case, a teenage patient with a transient iso7q clone had improving hemoglobin, platelet counts, absolute neutrophil counts, and reduction in red cell macrocytosis over the year prior to diagnosis of MDS. Additional clinical and laboratory studies are underway to provide evidence-based guidelines and to develop more sensitive tests for optimal surveillance of patients with predisposition to myeloid malignancies. DisclosuresMyers:Novartis: Membership on an entity's Board of Directors or advisory committees; Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Frequency and Evolution of TP53 Mutant Clones in Shwachman Diamond Syndrome. a Cohort Study from the French Severe Chronic Neutropenia (SCN) Registry

Context: Shwachman Diamond disease (SDS) is caused by an SBDS mutation, is typically associated with neutropenia and exocrine pancreas deficiency. Pancytopenia, myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), are life-threatening complications of SDS. To date, the sole risk factors identified for SDS are early symptoms (before age of 3 months) and mild chronic anemia or thrombocytopenia(1).Methods: To determine which mutations underlie clonal development and leukemic changes in SDS, we screened a series of patients with congenital neutropenias at various time points of follow-up. We used a consensus NGS panel of 41 genes involved in the development of myeloid malignancies (Haloplex® Agilent) (2, 3). Patients with SBDS mutations included in the French Severe Congenital Neutropenia registry or followed in the Leuwen University were screened when bone marrow samples were available as well as other subtypes of congenital neutropenia.Results: Among the 139 SDS patients, bone marrow samples of 23 patients were available for screening at various time points. We found isolated somatic TP53 mutations in 10 cases. In addition one patient had concomitant FLT3 TKD and TP53 mutations, and another patient had the recurrent IDH1 p.Arg132Cys variant. Strikingly, no TP53 mutations were observed when the screening was extended to 70 non-SDS neutropenia patients. None of the 11 SDS patients without any detectable mutations (with a threshold of detection of 0.5%) had any severe hematological expression nor presented any major hematological complications at time of sampling. By contrast, among the 12 SDS patients with somatic mutations, AML or MDS were observed in 3 cases, 1 with the IDH1 mutation (Variant allelic frequency (VAF): 42%), 1 with the recurrent TP53 p.Gly245Ser mutation (VAF:53%), 1 with two TP53 mutations (VAFs : 19% and 37%). Severe cytopenias without MDS or AML were found in 2 other cases, one with an isolated TP53 mutation at 24%, and one with both TP53 and FLT3 mutations around 45%. In the 7 remaining patients, allele frequencies of TP53 variants were found below 2% in four cases, and at 3%, 14%, and 37% in the 3 other patients. By sequential analysis in one patient we found a p.Val272Met variant (1.1% at 9 years) which was no more observed but was replaced by the recurrent p.Arg175His mutation (4% at 12 years and 14% at 15 years). Figure 1 depicts the allele frequencies of the variants with time among the 23 patients. The three patients with MDS / AML died despite hematopoietic stem cell transplantation (HSCT) in two of them. In contrast, the two patients with severe cytopenias and TP53 mutations who were transplanted are doing well three years after HSCT, and no TP53 mutation was detected one year after transplant.Conclusion: TP53 mutations are associated with hematological complications and specifically acquired in SDS when compared to other congenital neutropenias. This is in line with the frequency of complex karyotype MDS/AML in SDS(1). Routine evaluation of TP53 load in SDS patients may offer a powerful tool to screen SDS who may be susceptible to have severe hematological complications in a preemptive transplantation strategy setting.

Genetic Alterations Predict Outcomes in Patients with Myelodysplastic Syndrome Receiving Allogeneic Hematopoietic Stem Cell Transplantation

▪Background: Allogeneic hematopoietic stem cell transplantation (HCT) is the only potentially curative therapy for myelodysplastic syndrome (MDS). However, mortality after HCT for MDS is high, with deaths attributable to both relapsed disease and transplant-related complications. The genetic lesions that drive MDS pathogenesis have the potential to predict the outcome of patients following HCT.Methods: We performed targeted sequencing of 127 genes on pre-HCT blood samples from 1514 patients who received allogeneic HCT for MDS between 2005 and 2014. All MDS patients enrolled in the Center for International Blood and Marrow Transplant Research (CIBMTR) Repository were considered for inclusion. Patients were excluded only if blasts were >/= 20% or if they had diagnosis of CMML or MDS/MPN overlap. The median age of patients was 59 years (range 0.4 - 77 years), including 241 (16%) < 40 years of age. Donors were 8/8 HLA-matched unrelated donors (n = 862, 57%), HLA-identical siblings (n = 181, 12%), < 8/8 HLA-matched unrelated donors (n = 296, 20%), or umbilical cord blood (n = 174, 11%).Results:TP53 mutations were independently associated with poor overall survival (HR 1.71, 95% CI 1.45-2.02, p < 0.0001) and a shorter time to relapse (HR 2.34, 1.85-3.00, p < 0.0001). In a multivariable model the adverse prognostic significance of TP53 mutations was independent of recipient age, performance status, hematologic status at time of HCT, bone marrow blast count, and karyotype. RAS pathway mutations were independently associated with a shorter time to relapse (HR 1.73, 1.34-2.23, p < 0.0001) and JAK2 V617F mutations were associated with higher transplant-related mortality (TRM) (HR 2.13, 1.38-3.27, p = 0.0006).Patients with therapy-related MDS (t-MDS) comprised 21% of the cohort (n = 311) and had significantly worse survival than patients with primary MDS (HR 1.4, 1.2-1.6, p = 0.0002). Only TP53 (OR 3.8, p <0.0001) and the p53 regulator PPM1D (OR 4.8, p < 0.0001) were selectively mutated in t-MDS. Overall survival was inferior in t-MDS patients with TP53 mutations (HR 1.7, 1.3-2.3, p = 0.0004), but similar in t-MDS patients without TP53 mutations compared to patients with primary MDS (HR 1.1, 0.9-1.4, p = 0.37).In univariate analysis, young recipient age was powerfully associated with good post-transplant survival. However, we identified a group of adult patients under 40 years old with compound heterozygous mutations in the Shwachman-Diamond Syndrome (SDS)-associated SBDS gene that had remarkably poor prognosis and no long-term survivors (HR 6.0, 1.6-23.0, p = 0.009). All patients with biallelic SBDS mutations had somatic TP53 mutations, suggesting a biological synergy between SBDS and TP53 mutations in clonal MDS transformation of genetically-defined SDS.Finally, we used recursive partitioning to identify 6 distinct MDS subgroups based on the association between clinical and genetic variables and post-HCT survival, then evaluated each subgroup to identify links to relapse or TRM (Figure 1). Consistent with our multivariable model, the presence of TP53 mutations was the most important prognostic variable, with mortality driven by relapse and not influenced by conditioning intensity. In patients age 40 and older without TP53 mutations, two subgroups had poor outcomes: those with RAS pathway mutations, with median OS of 11 months and high risk of relapse (p = 0.001), and those with JAK2 mutations, with median OS of 6 months and high risk of TRM (p < 0.001). Among patients under 40 years old without TP53 mutation, we identified two subgroups based on evaluation of three high-risk clinical features (t-MDS, platelets < 30 x 109/L at HCT, or bone marrow blasts at diagnosis >/= 15%). Patients < 40 years old with at least one high-risk feature had 3 year OS of 49.0%, associated with high TRM (p < 0.001), while those with no high-risk features had 3 year OS of 81.4%.Conclusions: We found that TP53 mutations are the most important predictor of prognosis in allo-HCT for MDS, independent of other clinical and genetic variables, and that the adverse outcome in TP53 mutated MDS is not influenced by conditioning intensity, donor type, or graft source. TP53 mutations account for the characteristically poor outcomes of clinically distinct subgroups, including t-MDS and genetically-defined SDS. We present a multivariable model that identifies 6 distinct MDS subgroups with differential post-HCT survival and differences in relapse and TRM. [Display omitted] DisclosuresLindsley:Takeda Pharmaceuticals: Consultancy; MedImmune: Research Funding. Mar:H3 Biomedicine: Other: Spouse's employment.

Germline and Somatic Genetic Characterization of Shwachman-Diamond Syndrome

The North American Shwachman-Diamond Syndrome Registry (SDSR) opened in 2008 to improve our understanding of the natural history of Shwachman-Diamond Syndrome (SDS), improve medical outcomes, and facilitate research. The diagnosis of SDS was defined by either biallelic SBDS gene mutations or by the clinical combination of exocrine pancreatic dysfunction with bone marrow failure. Median age of study subjects is 11.2 years (range, 0.6-52.8). SBDS genetic reports were available for 168 subjects. Eighty-one had biallelic SBDS mutations, while 48 individuals lacked SBDS mutations. Ongoing characterization of SBDS mutation-negative individuals has identified subgroups of SDS individuals meeting clinical diagnostic criteria as outlined above, as well as a more heterogenous subgroup with features of SDS but in whom a clinical diagnosis could not be confirmed by classic diagnostic criteria. Amongst those with biallelic SBDS mutations, cytopenias were noted in all but 1 subject. Intermittent neutropenia was noted in 97% (n=70/72) and was the most frequent hematologic abnormality. Anemia was noted in 53% (n=39/73) and thrombocytopenia in 64% (n=48/75). Congenital anomalies were seen in 50% (n=39/78).The mutational spectrum of SBDS was explored. In 80 of 81 patients harboring biallelic SBDS mutations, the c.258+2 T>C intron 2 splice donor mutation was found in at least one of the two mutant SBDS alleles. In these cases, the mutation spectrum of the second mutant SBDS allele included missense mutations, splice site mutations, and truncating mutations. The one patient lacking the intron 2 splice donor mutation had a c.183_184delTAinsCT mutation together with a c.523 C>T mutation in SBDS confirmed to be in trans. This results in the combination of a truncating p.Lys62X mutation with a p.R175W missense mutation at a conserved residue predicted to be deleterious. This patient presented with neonatal severe aplastic anemia requiring platelet and red cell transfusions with neutrophil counts of 0-200 unresponsive to G-CSF. The marrow showed irregular islands of cartilage surrounded by osteoid consistent with a disorder of bone formation. A low level of SBDS protein is expressed by the c.258+2T>C variant, so the absence of this hypomorphic allele may have contributed to this exceptionally severe phenotype.Bone marrow reports were available for 67 subjects with biallelic SBDSmutations. Marrow hypocellularity was noted in 79% (n=49/62). Mild morphologic marrow dysplasia was observed in 58% (n=35/60). Clonal abnormalities developed in 36%. The most common clonal abnormality was del20q in 16% (n=10/64). Isochromosome 7 was noted in 2% (n=1/64). Three individuals developed AML at ages 19.5, 38, and 39 years. Eleven (14%) have undergone hematopoietic stem cell transplantation (HSCT), 10 for MDS or AML and 1 for severe aplastic anemia.Given the frequency of del20q clones in SDS, we studied clinical features of the 10 SDSR subjects with del20q clones. Median age of this group was 17 years (range, 10.8-29.3). Congenital anomalies were noted in 80% (n=8/10). Frequency of cytopenias was similar to that of non-del20q subjects, with neutropenia, anemia and thrombocytopenia seen in 90%, 50%, and 80% respectively. Bone marrow pathology reports were available for 9 subjects. All were noted to have hypocellular marrows as well as mild marrow dysplasia. In many patients, the clone was persistent or grew over time, with longest duration of 14.6 years. Progression to MDS was reported in 3 subjects who initially had an isolated del20q clone. Two developed an additional loss of chromosome 7. Progressive marrow dysplasia and falling blood counts were seen in two subjects. All three were treated with HSCT at 5.4, 5.7 and 7 years. Deletion of 20q in SDS has been hypothesized to result in a milder hematologic phenotype due to compensatory deletion of eIF6. eIF6 binds to the nascent 60S ribosomal subunit and sterically inhibits joining of the 60S to the 40S ribosomal subunits. SBDS functions to facilitate release of eIF6 thus promoting assembly of the mature 80S ribosome. These data from the SDSR suggest that SDS patients with del20q clones remain at risk for clonal evolution. Higher patient numbers are needed to quantitate risk of MDS in SDS patients with del20q.The SDSR continues to expand and mature as a resource for biological and clinical studies in this rare disorder advancing our understanding of marrow failure and clonal evolution. DisclosuresDavies:Novartis: Honoraria. Dale:Amgen: Consultancy, Honoraria, Research Funding.

Modeling Bone Marrow Failure and MDS in Shwachman Diamond Syndrome Using Induced Pluripotent Stem Cells

Myelodysplastic syndrome (MDS) caused by monosomy 7 or del(7q) is a frequent clonal abnormality that arises in the context of inherited bone marrow failure syndromes, such as Shwachman Diamond Syndrome (SDS). Monosomy 7/del(7q) also develops in a subset of patients with acquired aplastic anemia or de novo MDS in the general population. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation, most frequently to acute myelogenous leukemia (AML). Bone marrow failure and clonal evolution to MDS and AML remain major causes of morbidity and mortality for individuals with SDS. Currently, the only curative therapy for these hematological complications is a hematopoietic stem cell transplant. Prognosis is extremely poor once SDS patients develop leukemia. The basis for this propensity to develop monosomy 7 clones remains unclear. The longterm aim of this study is to understand the molecular mechanisms underlying leukemia predisposition and develop more effective treatments.Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in bone marrow failure remains a critical question. The lack of synteny between murine versus human chromosome 7 has posed a major barrier to the development of mouse models of monosomy 7/del(7q). To study the biological and molecular consequences of monosomy 7/del(7q) in SDS, induced pluripotent stem cells (iPSCs) were generated from bone marrow mononuclear cells of two patients with SDS. Each patient harbored homozygous c.258+2 T>C mutations in the canonical splice donor site of intron 2 in the SBDS gene. The SDS-iPSCs retained the pathogenic homozygous IVS2+2 T>C SBDS mutations, expressed stem cell markers, formed teratomas, and expressed reduced levels of SBDS protein similar to levels noted in the primary patient samples. Proliferation of 4 distinct SDS-iPSC clones derived from two different patients was reduced relative to wild type controls without an increase in cell death. SDS-iPSC formed smaller embryoid bodies with reduced production of CD34+ hematopoietic stem/progenitor cells. Hematopoietic differentiation from CD34+ to CD45+ cells was also impaired. Preliminary data suggest that SDS-iPSCs retain the capacity to give rise to hematopoietic stem/progenitor cells and early myeloid progenitor cells in vitro. These populations were also observed in primary SDS patient-derived bone marrow samples. Because the number of CD34+ cells derived from SDS-iPSCs are limiting, a previously reported 5 transcrition factor re-specification system was used to expand multilineage hematopietic progenitors for further characterization. SDS iPSCs were able to differentiate into an expandable CD34+ population in vitro. Further studies to characterize the hematopoietic impairment in SDS iPSC and primary marrow samples are ongoing.To model del(7q) in SDS iPSCs, a deletion of the MDS-associated long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q at locus (11.2) was confirmed by karyotyping and by qPCR across chromosome 7. The SDS (del7q) iPSCs retained the SBDS pathogenic mutations, expressed stem cell markers, and formed teratomas. Proliferation of the SDS del(7q) iPSC was markedly impaired compared to isogenic SDS iPSCs. No increase in cell death was observed in the SDS del7q iPSCs. Studies are in progress to determine the effects of del7q on hematopoiesis. Investigation is ongoing to determine the molecular consequences of deleting 7q. These isogenic SDS+/- del(7q) iPS models provide a platform to study the role of 7q loss in clonal evolution from bone marrow failure and to screen for novel therapeutic compounds or pathways to treat bone marrow failure and MDS. DisclosuresNo relevant conflicts of interest to declare.

The North American Shwachman-Diamond Syndrome Registry: Genetically Undefined Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an inherited marrow failure syndrome associated with exocrine pancreatic dysfunction and an increased risk of myelodysplasia and leukemia. The majority of individuals with SDS carry biallelic SBDS gene mutations, however a subset of patients remain genetically undefined. The objective of this study was to compare the clinical characteristics of patients with and without SBDS mutations. To address these questions, we conducted a retrospective study of patients enrolled on the North American Shwachman-Diamond Syndrome Registry (SDSR). Clinical data from the SDSR were available for 55 individuals with biallelic SBDS mutations and 16 individuals who fulfilled clinical diagnostic criteria for SDS but lacked biallelic mutations in the SBDS gene. Study subject ages for SBDS mutation positive and negative cohorts span 2-52.4 and 2.8-21.4 years with median ages of 12.4 and 10.9 years respectively. Cytopenias were present for both SBDS mutation positive and negative cohorts, with neutropenia the most common event in 94% and 81% respectively. Bone marrow hypocellularity was reported in 91% of those with SBDS mutations and 69% of those without. Marrow dysplasia was reported in 65% of those with SBDS mutations and none of those without. Clonal abnormalities were present in 44% and 25% of those with and without SBDS mutations with median age of initial appearance at 9 years (0.8-45.1) and 7 years (1.2-14) respectively. Abnormalities included del7q and del20q in both groups as well as iso7q, trisomy 8 and others in the SBDS mutation positive group. Clonal abnormalities were all transient in the SBDS mutation negative cohort. One SBDS mutation positive individual developed AML. None of the SBDS mutation negative individuals developed malignancy or progressed to require HSCT thus far. Pancreatic dysfunction determined by low serum trypsinogen or pancreatic isoamylase was similar in both cohorts 79% vs 80%. However, only 27% (15/55) of SBDS mutation positive individuals reported requiring enzyme therapy with 33% (18/55) documenting failure to thrive, in contrast to 75% (12/16) of SBDS mutation negative individuals with 73% (11/15) having failure to thrive. A broad spectrum of congenital anomalies were reported in 55% and 56% of SBDS mutation positive and negative individuals respectively, with skeletal anomalies being the most common in both groups. Medical comorbidities commonly reported in both groups included eczema and endocrinopathies. Elevated liver transaminases were seen in 27% of SBDS mutation positive individuals but this was not seen in the SBDS mutation negative cohort.Conclusion: Patients with genetically undefined (SBDS mutation negative) SDS share clinical characteristics with SBDS mutation positive patients; however, the risk of leukemia in the genetically undefined patients remains unclear due to low patient numbers with short follow-up. Further studies of this young cohort are required to inform medical management and to advance our understanding of genetic etiology, mechanism, disease pathophysiology and treatment of these marrow failure disorders. DisclosuresDale:Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau.