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.

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.

Haematology laboratory parameters to assess efficacy of CD19-, CD22-, CD33-, and CD123-directed chimeric antigen receptor T-cell therapy in haematological malignancies.

INTRODUCTION: Chimeric antigen receptor (CAR) T cell products are available to treat relapsed/refractory B-lymphoblastic leukaemia/lymphoma (B-ALL), diffuse large B-cell lymphoma, mantle-cell lymphoma, and myeloma. CAR products vary by their target epitope and constituent molecules. Hence, there are no common laboratory assays to assess CAR T cell expansion in the clinical setting. We investigated the utility of common haematology laboratory parameters to measure CAR T cell expansion and response. METHODS: Archived CellaVision images, absolute lymphocyte counts, and Sysmex CPD parameters spanning 1 month after CD19-CAR, UCAR19, CD22-CAR, CD33-CAR, and UCAR123 therapy were compared against donor lymphocyte infused control patients. Additionally, CellaVision images gathered during acute EBV infection were analysed. RESULTS: CellaVision images revealed a distinct sequence of three lymphocyte morphologies, common among CD19-CAR, CD22-CAR and UCAR19. This lymphocyte sequence was notably absent in CAR T cell non-responders and stem-cell transplantation controls, but shared some features seen during acute EBV infection. CD19-CAR engraftment kinetics monitored by quantitative PCR show an expansion and persistence phase and mirror CD19-CAR ALC kinetics. We show other novel CAR T cell therapies (UCAR19, CD22-CAR, CD33-CAR and UCAR123) display similar ALC expansion in responders and diminished ALC expansion in non-responders. Furthermore, the CPD parameter LY_WY fluorescence increased within the first week after CD19-CAR infusion, preceding the peak absolute lymphocyte count (ALC) by 3.7 days. CONCLUSION: Autologous and allogeneic CAR T cell therapy produce unique changes in common haematology laboratory parameters and could be a useful surrogate to follow CAR T-cell expansion after infusion.

Transcriptome and unique cytokine microenvironment of Castleman disease.

Castleman disease (CD) represents a group of rare, heterogeneous and poorly understood disorders that share characteristic histopathological features. Unicentric CD (UCD) typically involves a single enlarged lymph node whereas multicentric CD (MCD) involves multiple lymph node stations. To understand the cellular basis of CD, we undertook a multi-platform analysis using targeted RNA sequencing, RNA in-situ hybridization (ISH), and adaptive immune receptor rearrangements (AIRR) profiling of archived tissue from 26 UCD, 14 MCD, and 31 non-CD reactive controls. UCD showed differential expression and upregulation of follicular dendritic cell markers (CXCL13, clusterin), angiogenesis factors (LPL, DLL4), extracellular matrix remodeling factors (TGFß, SKIL, LOXL1, IL-1ß, ADAM33, CLEC4A), complement components (C3, CR2) and germinal center activation markers (ZDHHC2 and BLK) compared to controls. MCD showed upregulation of IL-6 (IL-6ST, OSMR and LIFR), IL-2, plasma cell differentiation (XBP1), FDC marker (CXCL13, clusterin), fibroblastic reticular cell cytokine (CCL21), angiogenesis factor (VEGF), and mTORC1 pathway genes compared to UCD and controls. ISH studies demonstrated that VEGF was increased in the follicular dendritic cell-predominant atretic follicles and the interfollicular macrophages of MCD compared to UCD and controls. IL-6 expression was higher along interfollicular vasculature-associated cells of MCD. Immune repertoire analysis revealed oligoclonal expansions of T-cell populations in MCD cases (2/6) and UCD cases (1/9) that are consistent with antigen-driven T cell activation. The findings highlight the unique genes, pathways and cell types involved in UCD and MCD. We identify potential novel targets in CD that may be harnessed for therapeutics.

Hematopathologic Correlates of CAR T-Cell Therapy.

CD19-targeting chimeric antigen rector (CAR) T-cell products are used for the treatment of relapsed/refractory B-acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and mantle cell lymphoma. The success of CD19-CAR-T cells has led to the investigation of CAR T-cell products targeting different antigens in other hematological malignancies and solid tumors. Clinical laboratories play an important role in the manufacture, distribution, and monitoring of CAR T-cell therapy. Hence, it is important for laboratory professionals to be cognizant of clinicopathologic aspects of CAR T-cell therapy.

Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse.

CD19-directed chimeric antigen receptor (CAR) T cells show characteristic proliferation kinetics after infusion that correlate with response. Clearance of circulating disease, B-cell aplasia (BCA), and cytokine release syndrome (CRS) are used to observe CAR T-cell function, given the lack of commercial CAR T-cell measurement assays. We investigated the utility of common hematology laboratory parameters in 166 patients with B-cell acute lymphoblastic leukemia (B-ALL) who were treated with CAR T-cell therapy targeting CD19. CAR T-cell infusion was followed by disappearance of circulating blasts in 86% of patients at a median of 6 days. After a lag phase, there was a rapid expansion in absolute lymphocyte count (ALC) in the second week that coincided with the appearance of atypical lymphocytes. The expansion phase was followed by a contraction phase with a concomitant decrease in atypical lymphocytes. In vitro CAR T-cell studies showed similar kinetics and morphological changes. Peak ALC and overall expansion was greater in sustained responders compared with that in nonresponders. Patients with early loss of BCA and those with eventual CD19+ minimal residual disease/relapse showed lower overall lymphocyte expansion compared with the controls. Pleomorphic lymphocytosis was noted in the cerebrospinal fluid at post-CAR time points. We conclude that lymphocyte counts and differential can also be used to evaluate CAR T-cell expansion after infusion, along with BCA and CRS. This is the first report to characterize the morphology of CAR T cells and determine the utility of lymphocyte kinetics.

Diagnostic Challenges in Pediatric Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune dysregulation that encompasses a broad range of underlying genetic diseases and infectious triggers. Monogenic conditions, autoimmune diseases, and infections can all drive the phenotype of HLH and associated immune hyperactivation with hypercytokinemia. A diagnosis of HLH usually requires a combination of clinical and laboratory findings; there is no single sensitive and specific diagnostic test, which often leads to "diagnostic dilemmas" and delays in treatment initiation. Ferritin levels, one of the most commonly used screening tests, were collected across a large tertiary care pediatric hospital to identify the positive predictive value for HLH. Herein, we present several cases that illustrate the clinical challenges of confirming an HLH diagnosis. Additionally, we report on the utility of establishing a formal multi-disciplinary group to aid the prompt diagnosis and treatment of patients presenting with HLH-like pathophysiologies.

Characterization of Plasmacytoid Dendritic Cells, Microbial Sequences, and Identification of a Candidate Public T-Cell Clone in Kikuchi-Fujimoto Disease.

OBJECTIVES: Kikuchi-Fujimoto disease (KFD) is a self-limited lymphadenitis of unclear etiology. We aimed to further characterize this disease in pediatric patients, including evaluation of the CD123 immunohistochemical (IHC) staining and investigation of potential immunologic and infectious causes. METHODS: Seventeen KFD cases and 12 controls were retrospectively identified, and the histologic and clinical features were evaluated. CD123 IHC staining was quantified by digital image analysis. Next generation sequencing was employed for comparative microbial analysis via RNAseq (5 KFD cases) and to evaluate the immune repertoire (9 KFD cases). RESULTS: In cases of lymphadenitis with necrosis, >0.85% CD123+ cells by IHC was found to be six times more likely in cases with a final diagnosis of KFD (sensitivity 75%, specificity 87.5%). RNAseq based comparative microbial analysis did not detect novel or known pathogen sequences in KFD. A shared complementarity determining region 3 (CDR3) sequence and use of the same T-cell receptor beta variable region family was identified in KFD LNs but not controls, and was not identified in available databases. CONCLUSIONS: Digital quantification of CD123 IHC can distinguish KFD from other necrotizing lymphadenitides. The presence of a unique shared CDR3 sequence suggests that a shared antigen underlies KFD pathogenesis.

Evidence of thrombotic microangiopathy in children with SARS-CoV-2 across the spectrum of clinical presentations.

Most children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have mild or minimal disease, with a small proportion developing severe disease or multisystem inflammatory syndrome in children (MIS-C). Complement-mediated thrombotic microangiopathy (TMA) has been associated with SARS-CoV-2 infection in adults but has not been studied in the pediatric population. We hypothesized that complement activation plays an important role in SARS-CoV-2 infection in children and sought to understand if TMA was present in these patients. We enrolled 50 hospitalized pediatric patients with acute SARS-CoV-2 infection (n = 21, minimal coronavirus disease 2019 [COVID-19]; n = 11, severe COVID-19) or MIS-C (n = 18). As a biomarker of complement activation and TMA, soluble C5b9 (sC5b9, normal 247 ng/mL) was measured in plasma, and elevations were found in patients with minimal disease (median, 392 ng/mL; interquartile range [IQR], 244-622 ng/mL), severe disease (median, 646 ng/mL; IQR, 203-728 ng/mL), and MIS-C (median, 630 ng/mL; IQR, 359-932 ng/mL) compared with 26 healthy control subjects (median, 57 ng/mL; IQR, 9-163 ng/mL; P < .001). Higher sC5b9 levels were associated with higher serum creatinine (P = .01) but not age. Of the 19 patients for whom complete clinical criteria were available, 17 (89%) met criteria for TMA. A high proportion of tested children with SARS-CoV-2 infection had evidence of complement activation and met clinical and diagnostic criteria for TMA. Future studies are needed to determine if hospitalized children with SARS-CoV-2 should be screened for TMA, if TMA-directed management is helpful, and if there are any short- or long-term clinical consequences of complement activation and endothelial damage in children with COVID-19 or MIS-C.

Diagnostic biomarkers to differentiate sepsis from cytokine release syndrome in critically ill children.

Chimeric antigen receptor (CAR) T-cells directed against CD19 have drastically altered outcomes for children with relapsed and refractory acute lymphoblastic leukemia (r/r ALL). Pediatric patients with r/r ALL treated with CAR-T are at increased risk of both cytokine release syndrome (CRS) and sepsis. We sought to investigate the biologic differences between CRS and sepsis and to develop predictive models which could accurately differentiate CRS from sepsis at the time of critical illness. We identified 23 different cytokines that were significantly different between patients with sepsis and CRS. Using elastic net prediction modeling and tree classification, we identified cytokines that were able to classify subjects as having CRS or sepsis accurately. A markedly elevated interferon γ (IFNγ) or a mildly elevated IFNγ in combination with a low IL1ß were associated with CRS. A normal to mildly elevated IFNγ in combination with an elevated IL1ß was associated with sepsis. This combination of IFNγ and IL1ß was able to categorize subjects as having CRS or sepsis with 97% accuracy. As CAR-T therapies become more common, these data provide important novel information to better manage potential associated toxicities.

Multisystem inflammatory syndrome in children and COVID-19 are distinct presentations of SARS-CoV-2.

BACKGROUNDInitial reports from the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic described children as being less susceptible to coronavirus disease 2019 (COVID-19) than adults. Subsequently, a severe and novel pediatric disorder termed multisystem inflammatory syndrome in children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology.METHODSWe prospectively enrolled hospitalized patients with evidence of SARS-CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data.RESULTSTwenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8, and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. The presence of burr cells on blood smears, as well as Cts, differentiated between patients with severe COVID-19 and those with MIS-C.CONCLUSIONPediatric patients with SARS-CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and those with severe COVID-19.FUNDINGFinancial support for this project was provided by CHOP Frontiers Program Immune Dysregulation Team; National Institute of Allergy and Infectious Diseases; National Cancer Institute; the Leukemia and Lymphoma Society; Cookies for Kids Cancer; Alex's Lemonade Stand Foundation for Childhood Cancer; Children's Oncology Group; Stand UP 2 Cancer; Team Connor; the Kate Amato Foundations; Burroughs Wellcome Fund CAMS; the Clinical Immunology Society; the American Academy of Allergy, Asthma, and Immunology; and the Institute for Translational Medicine and Therapeutics.

CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy.

Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19- subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)- deep remission, whereas 67 patients had a recurrence after achieving a MRD- deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19- leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19- leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19- events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD- remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

Rhabdomyosarcoma and Wilms tumors contain a subpopulation of noggin producing, myogenic cells immunoreactive for lens beaded filament proteins.

Myo/Nog cells are identified by their expression of the skeletal muscle specific transcription factor MyoD and the bone morphogenetic protein inhibitor noggin, and binding of the G8 monoclonal antibody. Their release of noggin is critical for morphogenesis and skeletal myogenesis. In the adult, Myo/Nog cells are present in normal tissues, wounds and skin tumors. Myo/Nog cells in the lens give rise to myofibroblasts that synthesize skeletal muscle proteins. The purpose of this study was to screen human lens tissue, rhabdomyosarcoma cell lines, and tissue sections from rhabdomyosarcoma, Wilms and tumors lacking features of skeletal muscle for co-localization of antibodies to Myo/Nog cell markers and the lens beaded filament proteins filensin and CP49. Immunofluorescence localization experiments revealed that Myo/Nog cells of the lens bind antibodies to beaded filament proteins. Co-localization of antibodies to G8, noggin, filensin and CP49 was observed in most RC13 and a subpopulation of RD human rhabdomyosarcoma cell lines. Western blotting with beaded filament antibodies revealed bands of similar molecular weights in RC13 and murine lens cells. Human alveolar, embryonal, pleomorphic and spindle cell rhabdomyosarcomas and Wilms tumors contained a subpopulation of cells immunoreactive for G8, noggin, MyoD and beaded filaments. G8 was also co-localized with filensin mRNA. Staining for beaded filament proteins was not detected in G8 positive cells in leiomyosarcomas, squamous and basal cell carcinomas, syringocarciomas and malignant melanomas. Lens beaded filament proteins were thought to be present only in the lens. Myo/Nog-like cells immunoreactive for beaded filaments may be diagnostic of tumors related to the skeletal muscle lineage.

Patients with common variable immunodeficiency with autoimmune cytopenias exhibit hyperplastic yet inefficient germinal center responses.

BACKGROUND: The lack of pathogen-protective, isotype-switched antibodies in patients with common variable immunodeficiency (CVID) suggests germinal center (GC) hypoplasia, yet a subset of patients with CVID is paradoxically affected by autoantibody-mediated autoimmune cytopenias (AICs) and lymphadenopathy. OBJECTIVE: We sought to compare the physical characteristics and immunologic output of GC responses in patients with CVID with AIC (CVID+AIC) and without AIC (CVID-AIC). METHODS: We analyzed GC size and shape in excisional lymph node biopsy specimens from 14 patients with CVID+AIC and 4 patients with CVID-AIC. Using paired peripheral blood samples, we determined how AICs specifically affected B-and T-cell compartments and antibody responses in patients with CVID. RESULTS: We found that patients with CVID+AIC displayed irregularly shaped hyperplastic GCs, whereas GCs were scarce and small in patients with CVID-AIC. GC hyperplasia was also evidenced by an increase in numbers of circulating follicular helper T cells, which correlated with decreased regulatory T-cell frequencies and function. In addition, patients with CVID+AIC had serum endotoxemia associated with a dearth of isotype-switched memory B cells that displayed significantly lower somatic hypermutation frequencies than their counterparts with CVID-AIC. Moreover, IgG+ B cells from patients with CVID+AIC expressed VH4-34-encoded antibodies with unmutated Ala-Val-Tyr and Asn-His-Ser motifs, which recognize both erythrocyte I/i self-antigens and commensal bacteria. CONCLUSIONS: Patients with CVID+AIC do not contain mucosal microbiota and exhibit hyperplastic yet inefficient GC responses that favor the production of untolerized IgG+ B-cell clones that recognize both commensal bacteria and hematopoietic I/i self-antigens.

Utility of the immature platelet fraction in pediatric immune thrombocytopenia: Differentiating from bone marrow failure and predicting bleeding risk.

BACKGROUND: Differentiating childhood immune thrombocytopenia (ITP) from other cause of thrombocytopenia remains a diagnosis of exclusion. Additionally factors that predict bleeding risk for those patients with ITP are currently not well understood. Previous small studies have suggested that immature platelet fraction (IPF) may differentiate ITP from other causes of thrombocytopenia and in combination with other factors may predict bleeding risk. METHODS: We performed a retrospective chart review of thrombocytopenic patients with an IPF measured between November 1, 2013 and July 1, 2015. Patients were between 2 months and 21 years of age with a platelet count <50 × 109 /l. Each patient chart was reviewed for final diagnosis and bleeding symptoms. A bleeding severity score was retrospectively assigned. RESULTS: Two hundred seventy two patients met inclusion criteria, 97 with ITP, 11 with bone marrow failure (BMF), 126 with malignancy, and 38 with other causes of thrombocytopenia. An IPF > 5.2% differentiated ITP from BMF with 93% sensitivity and 91% specificity. Absolute immature platelet number (AIPN) was significantly lower in ITP patients with severe to life-threatening hemorrhage than those without, despite similar platelet counts. On multivariate analysis, an IPF < 10.4% was confirmed as an independent predictor of bleeding risk at platelet counts <10 × 109 /l in patients with ITP. CONCLUSIONS: IPF measurement alone has utility in both the diagnosis of ITP and identifying patients at increased risk of hemorrhage. Further study is required to understand the pathophysiological differences of ITP patients with lower IPF/AIPN.

Flow Cytometry in Pediatric Hematopoietic Malignancies.

Utility of flow cytometry in the evaluation of pediatric hematopoietic neoplasms and the differences from adult hematopoietic neoplasms are discussed in this review. Distinction of hematogones from B-lymphoblasts, detection of residual/relapsed disease after novel targeted therapies, and evaluation of pediatric myeloid neoplasms are discussed.

Cytochemical Staining.

Historically, the diagnosis and classification of acute leukemia involved morphologic review of blasts in the peripheral blood and bone marrow smears and cytochemical staining. Cytochemical stains, which are enzymatic colorimetric reactions that occur in the cells of interest, were necessary to assign and confirm myeloid and lymphoid lineage. In the current WHO 2008 Classification of leukemia, immunophenotyping and cytogenetic analysis have largely replaced cytochemical staining in the characterization of acute leukemias. Nonetheless, cytochemical testing remains a useful adjunct assay for the proper classification of acute leukemia in a number of diagnostic settings. This chapter reviews the principles of the most common cytochemical stains, their procedures and guides to interpretation, and results in acute myeloid leukemia.