Hypomethylating agents are associated with high rates of hematologic toxicity in patients with secondary myeloid neoplasms developing after acquired aplastic anemia.

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Reduced toxicity matched sibling bone marrow transplant results in excellent outcomes for severe congenital neutropenia.

Severe congenital neutropenia (SCN) is caused by germline mutations, most commonly in ELANE, impacting neutrophil maturation and leading to high risk of life-threatening infections. Most patients with ELANE-mutant SCN can achieve safe neutrophil counts with chronic Granulocyte-Colony Stimulating Factor (G-CSF). However, up to 10% of patients have neutropenia refractory to G-CSF and require allogeneic stem cell transplant. Traditional conditioning for these patients includes busulfan and cyclophosphamide which is associated with significant toxicities. We present five patients with SCN without myeloid malignancy transplanted using a reduced toxicity regimen of busulfan, fludarabine and thymoglobulin. 5 pediatric patients with SCN underwent matched sibling donor bone marrow transplant (MSD-BMT) between 2014-2022 on or per CHP14BT057 (NCT02928991), a prospective, single center trial testing elimination of cyclophosphamide from conditioning in pediatric patients with single lineage inherited BMF syndromes. All patients had MSDs and no evidence of MDS. Conditioning consisted of PK-adjusted busulfan, fludarabine, and thymoglobulin, with calcineurin inhibitor and mycophenolate mofetil GVHD prophylaxis. With median follow-up of 48.4 months, overall and event-free survival were 100%. There was no acute GVHD and one instance of chronic limited GVHD. Patients exhibited >95% donor myeloid chimerism at 5 years post-BMT. Two patients experienced CMV reactivation without end-organ disease, and no other viral reactivation or significant infections occurred. MSD-BMT with reduced toxicity myeloablation for SCN provides excellent outcomes while minimizing toxicity. These data suggest that busulfan, fludarabine, and ATG can be considered an efficacious, low-toxicity standard of care regimen for patients with SCN undergoing MSD-BMT.

Disparities in Cytomegalovirus Infection Rates by Race and Ethnicity among Pediatric Allogeneic Hematopoietic Cell Transplantation Recipients at a Single Center.

Previous literature has reported cytomegalovirus (CMV) infection rate disparities among racial/ethnic groups of hematopoietic cell transplantation (HCT) recipients. Because race and ethnicity categorizations are social constructs unlikely to affect biological systems, it is likely there are covariates on the pathway to CMV detection, known as mediators, that can explain the observed disparity. Recent developments in mediation analysis methods enable the analysis of time-to-event outcomes, allowing an investigation of these disparities to also consider the timing of CMV infection detection relative to HCT. This study aimed to explore whether racial and ethnic CMV infection disparities existed within a population of HCT recipients at our center, and whether clinical covariates explained any observed association. The study cohort included all recipients of allogeneic HCT performed at the Children's Hospital of Philadelphia between January 2004 and April 2017 who were CMV PCR-negative pretransplantation, had known donor/recipient CMV serology, and were under blood CMV PCR surveillance. Subjects were followed for 100 days post-HCT. Accelerated failure time models using subject's reported race/ethnicity, dichotomized into non-Hispanic White (NHW) and non-NHW, and exposure and time to CMV detection as outcomes examined whether selected clinical factors-donor/recipient CMV serostatus, recipient age, indication for HCT, hematopoietic cell source, match quality-mediated any identified exposure-outcome association. The analysis included 348 HCTs performed in 335 subjects, with 86 episodes (24.7%) in which CMV was detected via PCR analysis. The accelerated failure time model without mediators estimated that non-NHW subjects had fewer CMV-free survival days (time ratio, .21; 95% confidence interval, .10 to .44). Any hypothesized mediator mediated at most 5% of the total association between race/ethnicity and time to CMV detection. Non-NHW HCT recipients had fewer CMV-free survival days than NHW recipients; none of the clinical factors hypothesized to mediate this association accounted for a significant component of total association. Further research should focus on nonclinical factors influenced by systemic racism to better understand their effect on CMV infection among HCT recipients.

Uncovering the Genetic Etiology of Inherited Bone Marrow Failure Syndromes Using a Custom-Designed Next-Generation Sequencing Panel.

Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for ∼30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, ≥99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 single-nucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing-based panel testing should be a part of routine diagnostics in patients with suspected IBMFS.

Management of Fanconi anemia beyond childhood.

Fanconi anemia (FA) has long been considered a severe inherited bone marrow failure (BMF) disorder of early childhood. Thus, management of this multisystem disorder has previously been unfamiliar to many hematologists specializing in the care of adolescents and young adults (AYA). The increased diagnosis of FA in AYA patients, facilitated by widely available germline genomic testing, improved long-term survival of children with FA following matched sibling and alternative donor hematopoietic stem cell transplantation (HSCT) performed for BMF, and expanding need in the near future for long-term monitoring in patients achieving hematologic stabilization following ex vivo gene therapy are all reasons why management of FA in AYA populations deserves specific consideration. In this review, we address the unique challenges and evidence-based practice recommendations for the management of AYA patients with FA. Specific topics addressed include hematologic monitoring in AYA patients yet to undergo HSCT, management of myeloid malignancies occurring in FA, diagnosis and management of nonhematologic malignances and organ dysfunction in AYA patients with FA, and evolving considerations for the long-term monitoring of patients with FA undergoing gene therapy.

How I treat cytopenias after CAR T-cell therapy.

Increasing use of chimeric antigen receptor T-cell therapy (CAR-T) has unveiled diverse toxicities warranting specific recognition and management. Cytopenias occurring after CAR-T infusion invariably manifest early (<30 days), commonly are prolonged (30-90 days), and sometimes persist or occur late (>90 days). Variable etiologies of these cytopenias, some of which remain incompletely understood, create clinical conundrums and uncertainties about optimal management strategies. These cytopenias may cause additional sequelae, decreased quality of life, and increased resource use. Early cytopenias are typically attributed to lymphodepletion chemotherapy, however, infections and hyperinflammatory response such as immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome may occur. Early and prolonged cytopenias often correlate with severity of cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. Bone marrow biopsy in patients with prolonged or late cytopenias is important to evaluate for primary disease and secondary marrow neoplasm in both pediatric and adult patients. Commonly, cytopenias resolve over time and evidence for effective interventions is often anecdotal. Treatment strategies, which are limited and require tailoring based upon likely underlying etiology, include growth factors, thrombopoietin-receptor agonist, stem cell boost, transfusion support, and abrogation of infection risk. Here we provide our approach, including workup and management strategies, for cytopenias after CAR-T.

Automation of Hematopoietic Cell Transplantation Outcomes Reporting Leads to Dramatic Reduction in Errors Reported to Real-World Data Registry.

Institutions that perform hematopoietic cell transplantation (HCT) are required by law to report standardized, structured data on transplantation outcomes. A key post-transplantation outcome is engraftment, the time between HCT infusion and reemergence of circulating neutrophils and platelets. At our center, we found that manual chart abstraction for engraftment data was highly error-prone. We developed a custom R/Shiny application that automatically calculates engraftment dates and displays them in an intuitive format to augment the manual chart review. Our hypothesis was that use of the application to assist with calculating and reporting engraftment dates would be associated with a decreased error rate. The study was conducted at a single tertiary care institution. The application was developed in a collaborative, multidisciplinary fashion by members of an embedded cellular therapy informatics team. Retrospective validation of the application's accuracy was conducted on all malignant HCTs from February 2016 to December 2020 (n = 198). Real-world use of the application was evaluated prospectively from April 2021 through April 2022 (n = 53). The Welch 2-sample t test was used to compare error rates preimplementation and postimplementation. Data were visualized using p charts, and standard special cause variation rules were applied. The accuracy of reported data postdeployment increased dramatically; the engraftment error rate decreased from 15% to 3.8% for neutrophils (P = .003) and from 28% to 1.9% for platelets (P < .001). This study demonstrates the effective deployment of a custom R/Shiny application that was associated with significantly reduced error rates in HCT engraftment reporting for operational, research, and regulatory purposes. Users reported subjective satisfaction with the application and that it addressed difficulties with the legacy manual process. Identifying and correcting erroneous data in engraftment reporting could lead to a more efficient and accurate nationwide assessment of transplantation success. Furthermore, we show that it is possible and practical for academic medical centers to create and support embedded informatics teams that can quickly build applications for clinical operations in a manner compliant with regulatory requirements.

Inherited bone marrow failure with macrothrombocytopenia due to germline tubulin beta class I (TUBB) variant.

Germline mutations in tubulin beta class I (TUBB), which encodes one of the ß-tubulin isoforms, were previously associated with neurological and cutaneous abnormalities. Here, we describe the first case of inherited bone marrow (BM) failure, including marked thrombocytopenia, morphological abnormalities, and cortical dysplasia, associated with a de novo p.D249V variant in TUBB. Mutant TUBB had abnormal cellular localisation in transfected cells. Following interferon/ribavirin therapy administered for transfusion-acquired hepatitis C, severe pancytopenia and BM aplasia ensued, which was unresponsive to immunosuppression. Acquired chromosome arm 6p loss of heterozygosity was identified, leading to somatic loss of the mutant TUBB allele.

Pathogenicity and impact of HLA class I alleles in aplastic anemia patients of different ethnicities.

Acquired aplastic anemia (AA) is caused by autoreactive T cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some patients with AA. However, pathogenicity, structural characteristics, and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 patients with AA from 2 multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a potentially novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or transplant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult patients with AA. Our study provides insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved understanding of clonal evolution in AA.

Does immune destruction drive all forms of bone marrow failure?

Current paradigms of bone marrow failure (BMF) pathophysiology suggest that immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs) drives acquired aplastic anemia. In contrast, loss of HSPCs due to senescence and/or apoptosis causes BMF in inherited BMF syndromes. In this issue of the JCI, Casado and colleagues challenge this dichotomous conception by demonstrating that NK cell-dependent, immune-mediated hematopoietic suppression and HSPC clearance drive BMF in Fanconi anemia (FA). They show that genotoxic stress upregulates natural killer group 2 member D ligands (NKG2D-L) on FA HSPCs leading to NK cell cytotoxicity through NKG2D receptor activation. Inhibition of NKG2D-NKG2D-L interactions enhanced FA HSPC clonogenic potential and improved cytopenias in vivo. These results provide alternative targets for the development of immunosuppressive therapies to reduce HSPC loss and mitigate the risk of hematologic malignancies in FA.

Non-myeloablative conditioning is sufficient to achieve complete donor myeloid chimerism following matched sibling donor bone marrow transplant for myeloproliferative leukemia virus oncogene (MPL) mutation-driven congenital amegakaryocytic thrombocytopenia: Case report.

Background: Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare platelet production disorder caused mainly by loss of function biallelic mutations in myeloproliferative leukemia virus oncogene (MPL), the gene encoding the thrombopoietin receptor (TPOR). Patients with MPL-mutant CAMT are not only at risk for life-threatening bleeding events, but many affected individuals will also ultimately develop bone marrow aplasia owing to the absence of thrombopoietin/TPOR signaling required for maintenance of hematopoietic stem cells. Curative allogeneic stem cell transplant for patients with CAMT has historically used myeloablative conditioning; however, given the inherent stem cell defect in MPL-mutant CAMT, a less intensive regimen may prove equally effective with reduced morbidity, particularly in patients with evolving aplasia. Methods: We report the case of a 2-year-old boy with MPL-mutant CAMT and bone marrow hypocellularity who underwent matched sibling donor bone marrow transplant (MSD-BMT) using a non-myeloablative regimen consisting of fludarabine, cyclophosphamide, and antithymocyte globulin (ATG). Results: The patient achieved rapid trilinear engraftment and resolution of thrombocytopenia. While initial myeloid donor chimerism was mixed (88% donor), due to the competitive advantage of donor hematopoietic cells, myeloid chimerism increased to 100% by 4 months post-transplant. Donor chimerism and blood counts remained stable through 1-year post-transplant. Conclusion: This experience suggests that non-myeloablative conditioning is a suitable approach for patients with MPL-mutant CAMT undergoing MSD-BMT and is associated with reduced risks of conditioning-related toxicity compared to traditional myeloablative regimens.

Hematologic abnormalities in Aicardi Goutières Syndrome.

BACKGROUND: Because of the broad clinical spectrum, heritable autoinflammatory diseases present a management and therapeutic challenge. The most common genetic interferonopathy, Aicardi Goutières Syndrome (AGS), is associated with early onset neurologic disability and systemic inflammation. The chronic inflammation of AGS is the result of dysregulation of interferon (IFN) expression by one of nine genes within converging pathways. While each AGS subtype shares common features, distinct patterns of severity and potential for systemic complications amongst the genotypes are emerging. Multilineage cytopenias are a potentially serious, but poorly understood, complication of AGS. As immunomodulatory treatment options are developed, it is important to characterize the role of the disease versus treatment in hematologic abnormalities. This will allow for better understanding and management of cytopenia. METHODS: In total, 142 individuals with molecularly-confirmed AGS were included. Information on genotype, demographics, and all available hematologic laboratory values were collected from existing medical records. As part of a clinical trial, a subset of this cohort (n = 52) were treated with a janus kinase inhibitor (baricitinib), and both pre- and post-treatment values were included. Abnormal values were graded based on Common Terminology Criteria for Adverse Events (CTCAE v5.0), supplemented with grading definitions for thrombocytosis, and were compared across genotypes and baricitinib exposure. RESULTS: In total, 11,184 laboratory values were collected over a median of 2.54 years per subject (range 0-22.68 years). To reduce bias from repeated sampling within a limited timeframe, laboratory results were restricted to the most abnormal value within a month (n = 8485). The most common abnormalities were anemia (noted in 24% of subjects prior to baricitinib exposure), thrombocytopenia (9%), and neutropenia (30%). Neutropenia was most common in the SAMHD1 cohort and increased with baricitinib exposure (38/69 measurements on baricitinib versus 14/121 while not on baricitinib). Having an abnormality prior to treatment was associated with having an abnormality on treatment for neutropenia and thrombocytopenia. CONCLUSION: By collecting available laboratory data throughout the lifespan, we were able to identify novel patterns of hematologic abnormalities in AGS. We found that AGS results in multilineage cytopenias not limited to the neonatal period. Neutropenia, anemia, and thrombocytopenia were common. Moderate-severe graded events of neutropenia, anemia, and leukopenia were more common on baricitinib, but rarely of clinical consequence. Based on these results, we would recommend careful monitoring of hematologic parameters of children affected by AGS throughout the lifespan, especially while on therapy, and consideration of AGS as a potential differential diagnosis in children with neurologic impairment of unclear etiology with hematologic abnormalities. Trial registration ClinicalTrials.gov Identifier: NCT01724580 ClinicalTrials.gov Identifier: NCT03921554.

Unrelated donor α/ß T cell- and B cell-depleted HSCT for the treatment of pediatric acute leukemia.

Unrelated donor (URD) hematopoietic stem cell transplant (HSCT) is associated with an increased risk of severe graft-versus-host disease (GVHD). TCRαß/CD19 depletion may reduce this risk, whereas maintaining graft-versus-leukemia. Outcome data with TCRαß/CD19 depletion generally describe haploidentical donors, with relatively few URDs. We hypothesized that TCRαß/CD19-depletion would attenuate the risks of GVHD and relapse for URD HSCT. Sixty pediatric and young adult (YA) patients with hematologic malignancies who lacked a matched-related donor were enrolled at 2 large pediatric transplantation centers between October 2014 and September 2019. All patients with acute leukemia had minimal residual disease testing, and DP typing was available for 77%. All patients received myeloablative total body irradiation- or busulfan-based conditioning with no posttransplant immune suppression. Engraftment occurred in 98%. Four-year overall survival was 69% (95% confidence interval [CI], 52%-81%), and leukemia-free survival was 64% (95% CI, 48%-76%), with no difference between lymphoid and myeloid malignancies (P = .6297 and P = .5441, respectively). One patient (1.7%) experienced primary graft failure. Relapse occurred in 11 patients (3-year cumulative incidence, 21%; 95% CI, 11-34), and 8 patients (cumulative incidence, 15%; 95% CI, 6.7-26) experienced nonrelapse mortality. Grade III to IV acute GVHD was seen in 8 patients (13%), and 14 patients (26%) developed chronic GVHD, of which 6 (11%) had extensive disease. Nonpermissive DP mismatch was associated with higher likelihood of acute GVHD (odds ratio, 16.50; 95% CI, 1.67-163.42; P = .0166) but not with the development of chronic GVHD. URD TCRαß/CD19-depleted peripheral HSCT is a safe and effective approach to transplantation for children/YAs with leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02323867.

Betibeglogene Autotemcel Gene Therapy for Non-ß0/ß0 Genotype ß-Thalassemia.

BACKGROUND: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent ß-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the ß-globin (ßA-T87Q) gene. METHODS: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent ß-thalassemia and a non-ß0/ß0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months). RESULTS: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed. CONCLUSIONS: Treatment with beti-cel resulted in a sustained HbAT87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-ß0/ß0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.).

LNK (SH2B3) inhibition expands healthy and Fanconi anemia human hematopoietic stem and progenitor cells.

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to alleviate hematologic symptoms in monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi anemia (FA). However, for FA and other BMF syndromes, insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein LNK (SH2B3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs results in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhances cytokine-mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment of FANCD2-depleted FA-like HSCs was markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.

Comparison of hematopoietic cell transplant conditioning regimens for hemophagocytic lymphohistiocytosis disorders.

BACKGROUND: Allogeneic hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis (HLH) disorders is associated with substantial morbidity and mortality. OBJECTIVE: The effect of conditioning regimen groups of varying intensity on outcomes after transplantation was examined to identify an optimal regimen or regimens for HLH disorders. METHODS: We studied 261 patients with HLH disorders transplanted between 2005 and 2018. Risk factors for transplantation outcomes by conditioning regimen groups were studied by Cox regression models. RESULTS: Four regimen groups were studied: (1) fludarabine (Flu) and melphalan (Mel) in 123 subjects; (2) Flu, Mel, and thiotepa (TT) in 28 subjects; (3) Flu and busulfan (Bu) in 14 subjects; and (4) Bu and cyclophosphamide (Cy) in 96 subjects. The day 100 incidence of veno-occlusive disease was lower with Flu/Mel (4%) and Flu/Mel/TT (0%) compared to Flu/Bu (14%) and Bu/Cy (22%) (P < .001). The 6-month incidence of viral infections was highest after Flu/Mel (72%) and Flu/Mel/TT (64%) compared to Flu/Bu (39%) and Bu/Cy (38%) (P < .001). Five-year event-free survival (alive and engrafted without additional cell product administration) was lower with Flu/Mel (44%) compared to Flu/Mel/TT (70%), Flu/Bu (79%), and Bu/Cy (61%) (P = .002). The corresponding 5-year overall survival values were 68%, 75%, 86%, and 64%, and did not differ by conditioning regimen (P = .19). Low event-free survival with Flu/Mel is attributed to high graft failure (42%) compared to Flu/Mel/TT (15%), Flu/Bu (7%), and Bu/Cy (18%) (P < .001). CONCLUSIONS: Given the high rate of graft failure with Flu/Mel and the high rate of veno-occlusive disease with Bu/Cy and Flu/Bu, Flu/Mel/TT may be preferred for HLH disorders. Prospective studies are warranted.

Inducible Sbds deletion impairs bone marrow niche capacity to engraft donor bone marrow after transplantation.

Bone marrow (BM) niche-derived signals are critical for facilitating engraftment after hematopoietic stem cell (HSC) transplantation (HSCT). HSCT is required for restoration of hematopoiesis in patients with inherited BM failure syndromes (iBMFSs). Shwachman-Diamond syndrome (SDS) is a rare iBMFS associated with mutations in SBDS. Previous studies have demonstrated that SBDS deficiency in osteolineage niche cells causes BM dysfunction that promotes leukemia development. However, it is unknown whether BM niche defects caused by SBDS deficiency also impair efficient engraftment of healthy donor HSC after HSCT, a hypothesis that could explain morbidity noted after clinical HSCT for patients with SDS. Here, we report a mouse model with inducible Sbds deletion in hematopoietic and osteolineage cells. Primary and secondary BM transplantation (BMT) studies demonstrated that SBDS deficiency within BM niches caused poor donor hematopoietic recovery and specifically poor HSC engraftment after myeloablative BMT. We have also identified multiple molecular and cellular defects within niche populations that are driven by SBDS deficiency and are accentuated by or develop specifically after myeloablative conditioning. These abnormalities include altered frequencies of multiple niche cell subsets, including mesenchymal lineage cells, macrophages, and endothelial cells; disruption of growth factor signaling, chemokine pathway activation, and adhesion molecule expression; and p53 pathway activation and signals involved in cell cycle arrest. Taken together, this study demonstrates that SBDS deficiency profoundly impacts recipient hematopoietic niche function in the setting of HSCT, suggesting that novel therapeutic strategies targeting host niches could improve clinical HSCT outcomes for patients with SDS.

The predictive value of PNH clones, 6p CN-LOH, and clonal TCR gene rearrangement for aplastic anemia diagnosis.

Acquired aplastic anemia (AA) is a life-threatening bone marrow aplasia caused by the autoimmune destruction of hematopoietic stem and progenitor cells. There are no existing diagnostic tests that definitively establish AA, and diagnosis is currently made via systematic exclusion of various alternative etiologies, including inherited bone marrow failure syndromes (IBMFSs). The exclusion of IBMFSs, which requires syndrome-specific functional and genetic testing, can substantially delay treatment. AA and IBMFSs can have mimicking clinical presentations, and their distinction has significant implications for treatment and family planning, making accurate and prompt diagnosis imperative to optimal patient outcomes. We hypothesized that AA could be distinguished from IBMFSs using 3 laboratory findings specific to the autoimmune pathogenesis of AA: paroxysmal nocturnal hemoglobinuria (PNH) clones, copy-number-neutral loss of heterozygosity in chromosome arm 6p (6p CN-LOH), and clonal T-cell receptor (TCR) γ gene (TRG) rearrangement. To test our hypothesis, we determined the prevalence of PNH, acquired 6p CN-LOH, and clonal TRG rearrangement in 454 consecutive pediatric and adult patients diagnosed with AA, IBMFSs, and other hematologic diseases. Our results indicated that PNH and acquired 6p CN-LOH clones encompassing HLA genes have ∽100% positive predictive value for AA, and they can facilitate diagnosis in approximately one-half of AA patients. In contrast, clonal TRG rearrangement is not specific for AA. Our analysis demonstrates that PNH and 6p CN-LOH clones effectively distinguish AA from IBMFSs, and both measures should be incorporated early in the diagnostic evaluation of suspected AA using the included Bayesian nomogram to inform clinical application.

Case Report: Immune Dysregulation Due to Toxoplasma gondii Reactivation After Allogeneic Hematopoietic Cell Transplant.

Disseminated toxoplasmosis is an uncommon but highly lethal cause of hyperferritinemic sepsis after hematopoietic cell transplantation (HCT). We report two cases of disseminated toxoplasmosis from two centers in critically ill adolescents after HCT: a 19-year-old who developed fever and altered mental status on day +19 after HCT and a 20-year-old who developed fever and diarrhea on day +52 after HCT. Both patients developed hyperferritinemia with multiple organ dysfunction syndrome and profound immune dysregulation, which progressed to death despite maximal medical therapies. Because disseminated toxoplasmosis is both treatable and challenging to diagnose, it is imperative that intensivists maintain a high index of suspicion for Toxoplasma gondii infection when managing immunocompromised children, particularly in those with known positive T. gondii serologies.