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The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis. Significance: Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains. This article is highlighted in the In This Issue feature, p. 171

R code of our analysis clock like mutational signatures in Hodgkin lymphoma WGS and WES.

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis.Significance:Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains.This article is highlighted in the In This Issue feature, p. 171

All supplementary figures S1-S17

R code of our analysis for driver mutation discovery in Hodgkin lymphoma WGS and WES.

Supplementary Tables S1-S20

Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. Objective.— To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. Design.— We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription–polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. Results.— Concurrently collected saliva and mid-turbinate swabs had greater than 95% positive agreement with NP swabs when obtained within 10 days of symptom onset. Positive agreement of saliva and mid-turbinate samples collected from children with symptom onset >10 days prior, or without symptoms, was 82% compared to NP swab samples. Cycle threshold (Ct) values for mid-turbinate nasal samples more closely correlated with Ct values from NP samples than from saliva samples. Conclusions.— These findings suggest that all 3 sample types from children are useful for SARS-CoV-2 diagnostic testing by RT-PCR, and that concordance is greatest when the child has had symptoms of COVID-19 within the past 10 days. This study provides scientific justification for using sample types other than the NP swab for SARS-CoV-2 testing in pediatric populations.

The correlation between cardiovascular disease and iron deficiency anemia (IDA) is well documented but poorly understood. Using a multi-disciplinary approach, we explore the hypothesis that the biophysical alterations of red blood cells (RBCs) in IDA, such as variable degrees of microcytosis and decreased deformability may directly induce endothelial dysfunction via mechanobiological mechanisms. Using a combination of atomic force microscopy and microfluidics, we observed that subpopulations of IDA RBCs (idRBCs) are significantly stiffer and smaller than both healthy RBCs and the remaining idRBC population. Furthermore, computational simulations demonstrated that the smaller and stiffer idRBC subpopulations marginate toward the vessel wall causing aberrant shear stresses. This leads to increased vascular inflammation as confirmed with perfusion of idRBCs into our "endothelialized" microfluidic systems. Overall, our multifaceted approach demonstrates that the altered biophysical properties of idRBCs directly lead to vasculopathy, suggesting that the IDA and cardiovascular disease association extends beyond correlation and into causation.

Relapse- and continuous complete remission (CCR)-associated pediatric acute myeloid leukemia (AML) patient bone marrows collected at the time of diagnosis (Dx), end of induction (EOI) and relapse were analyzed by single cell RNA sequencing. A novel AML blasts-associated 7-genes signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH) displayed a strong correlation with blast percentages and overall survival in the TARGET AML dataset (HR = 2.3; P-value = .007). Distinct clusters of AML-blasts at Dx were observed for relapse- and CCR-associated samples with differential expression of genes associated with survival. Relapse-associated samples demonstrated enrichment of exhausted T cells and M2 macrophages as opposed to inflammatory M1 macrophages in CCR-associated samples at Dx. EOI treatment resistant blast cells overexpressed fatty acid oxidation, tumor growth and stemness genes. Also, a relapse-associated EOI samples T cells subset showed downregulation of MHC Class I and regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse-/CCR-associated tumor microenvironment transcriptome landscape.

TO THE EDITOR: Mixed phenotype acute leukemia (MPAL) represents a heterogeneous group of leukemias accounting for 2% to 5% of all newly diagnosed acute leukemias.[1][1][⇓][2][⇓][3][⇓][4]-[5][5] Although most cases of acute leukemia display the differentiation pattern of a single lineage

Background: Iron deficiency anemia (IDA), which affects individuals of all ages worldwide, is an often overlooked and undertreated component of chronic disease, despite data correlating its association with adverse outcomes in patients with cardiovascular disease (von Haehling, Nat Rev Cardiol, 2015). While red blood cells (RBCs) in IDA are known to be smaller and contain less hemoglobin than healthy RBCs, how RBC deformability is altered in IDA remains poorly understood; some ektacytometry studies have observed impaired deformability in iron deficient RBCs (idRBCs), while others described either unchanged or increased deformability (Brandão, Clin Hemorheol Microcirc, 2009). Here we ask: can single cell biophysical techniques definitively determine whether idRBCs are less deformable than healthy RBCs and how heterogenous that phenomena may be? Recent investigations into IDA's role in cardiovascular disease have generally focused on the myocardium and coronary vasculature, yet much regarding other physiologic implications remains unknown, including whether idRBCs cause microvascular obstruction or vasculopathy. To address such questions, we leveraged a suite of microvascular models we developed. Methods: We first coupled our microfluidic capillary model with μEXACT, our customized automated particle tracking program for hematologic cell-based assays, to collect high-throughput velocity tracking of single RBCs from a healthy control and 2 IDA patients (anemic for age, ferritin Results: sDI distribution histograms were obtained for healthy and IDA patient RBCs. The mean sDIs for IDA patient RBCs were decreased in comparison to the healthy RBCs. Additionally, both IDA patient's RBCs lacked a subpopulation of highly deformable RBCs, likely reticulocytes, seen in the healthy RBCs (Fig 1C). There was no evidence of microchannel occlusion for the healthy control or IDA patient whole blood samples in either the straight 100μm microchannels or branching 30μm microfluidic devices (Fig 2D). Finally, in our endothelialized microfluidic model, endothelium exposed to IDA patient RBCs exhibited increased VCAM-1 and E-selectin expression over endothelium exposed to healthy RBCs (Fig 3B). Conclusions: By utilizing an array of microfluidic models we can develop a more comprehensive understanding of the role idRBCs play systemically on microvasculature. Our combined microfluidic and image analysis system demonstrated decreased deformability in idRBCs and can offer detection of subpopulation differences that cannot be fully characterized with bulk techniques such as ektacytometry. So far, our data demonstrates that while no microvascular occlusion occurs, idRBCs contribute to endothelial inflammation. Additionally, the observation that physical interactions between endothelial cells and idRBCs are sufficient to cause endothelial inflammation warrants further investigation, as generally idRBCs had not been viewed as pro-inflammatory. Ongoing studies will couple unique sDI distribution curves with the degree of endothelial inflammation, as well as elucidate how these changes are associated with the degree of IDA or clinical events such as the initiation of iron supplementation. Utilizing atomic force microscopy to better understand how the idRBC membrane impacts deformability and developing biophysical computer simulations to determine if increased idRBC-endothelium interactions are observed in silico are also planned. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

Introduction Despite recent improvement in outcomes for de novo disease, pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains challenging to treat at relapse. Investigation into genomic markers of treatment response and therapy resistance offers an opportunity to further enhance outcomes for these patients. We previously identified a T-ALL blast-associated gene signature at diagnosis (Dx) and characterized the immune microenvironment in Dx T-ALL marrow samples using single cell transcriptome analysis (Bhasin et al. Blood 2020(ASH)). This approach allowed us to generate a granular expression map of both the T-ALL landscape and the Dx bone marrow (BM) immune microenvironment. Here we expand this work by evaluating samples collected from the same patients Dx and End of Induction (EOI) BM samples from pediatric T-ALL patients. The use of paired samples provides insight into treatment-induced changes in the microenvironment. Further, the inclusion of both minimal residual disease (MRD) positive and MRD negative samples allowed us to compare differences between these groups. Methods Using the 10X genomics platform, we profiled the single cell transcriptome of ~18,000 BM and immune microenvironment cells from viably frozen samples collected from T-ALL patients at Dx or EOI. Five paired Dx and EOI samples and one EOI sample from a patient with relapsed T-ALL were evaluated, for a total of 11 samples. Three paired samples were MRD positive at EOI and two were MRD negative; the relapsed sample was MRD negative. Cell clustering was performed using the Seurat package and differential expression analysis was performed using R/Bioconductor packages (Hao et al. Cell 2021). Cell communication analysis was conducted using the CellChat R tool (v 1.0.0) to infer cell-cell communication within the EOI MRD positive and MRD negative subsets and compare their communication networks (Jin et al. Nature Comm 2021). Results Using our previously described blast-associated gene signature (Bhasin et al. ASH 2020) we were able to identify residual blast populations at EOI in MRD-positive samples. Comparative analysis of gene profiles at Dx and EOI showed significant changes in the microenvironment cell populations with highest increase in erythroid cell populations after induction therapy. The gene expression profiles were significantly different for immune cells at Dx and EOI and the relapsed sample had greater similarity to the Dx samples indicating a persistent immunosuppressive environment. Clustering analysis of the EOI samples (3 MRD positive and 2 MRD negative) demonstrated the presence of patient specific blast cells in MRD positive samples that retained patient-specific transcriptomeheterogeneity at EOI (Fig.1A). Analysis of communication networks between different cell types based on receptor and ligand expression levels between different cell types identified a CD34 + cluster of stem cells that had different interactions with other immune populations in the MRD positive and negative subsets. Differential expression analysis between the MRD positive and MRD negative cells in this CD34 + stem cell cluster identified higher expression of myeloid associated genes such as CEBPB, CEBPD, AZU1 in the MRD negative group relative to the MRD positive cells, which showed higher expression of B-cell related genes such as IGHM, VPREB1, CD79A/ B along with upregulation of P13K signaling in B-lymphocytes, B-cell receptor signaling and autophagy pathways. Analysis of upstream regulators based on the differential gene signature between the MRD positive and MRD negative group demonstrated upregulation of MYC and TCF3 activity and inhibition of TGFB1, CSF3 and CEBPA in MRD positive compared to MRD negative samples (Fig.1B). Conclusions: Leukemic blasts exhibit patient-specific gene expression signatures that are present at EOI in MRD positive samples. Exploration of the impact of minimal residual disease at EOI revealed differential gene expression patterns in stem cells from MRD positive samples, characterized by activation of B cell related signaling pathways and regulators such as MYC and TCF3. In contrast, a more myeloid-like expression signature was observed in stem cells from MRD negative samples. These findings open the avenues for exploration of therapeutic targets of T-ALL progression. Figure 1 Figure 1. Disclosures DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes. Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro. Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies. We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels. Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Figure 1 Figure 1. Disclosures Abukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a small fraction of Hodgkin and Reed-Sternberg (HRS) tumor cells (~1%) which are surrounded by an extensive immune infiltrate. The rare nature of HRS cells limits the ability to study the genomics of cHL using standard platforms. To circumvent this, our group has optimized fluorescence-activated cell sorting to isolate HRS cells and intratumor B- and T- cells and to perform whole exome sequencing (WES; Reichel, Blood 2015). To date, however, there have been no reports on whole genome sequencing (WGS) of cHL. Methods: We performed flow-sorting of HRS cells and WGS to define the genomic landscape of cHL including: i) mutational processes involved in pathogenesis, ii) large and focal copy number variants, iii) structural variants including complex events, iv) the sequence and evolution of molecular events in cHL. We interrogated WGS from 25 cases of cHL: 10 pediatric patients (age40). Intra-tumoral T-cells were used as germline control. An additional 36 cHL cases were evaluated by WES. Results: The average depth of coverage among the 25 WGS cases was 27.5x. After having identified and removed amplification-based palindromic sequencing artifacts, we observed a median of 5006 single base substitutions (SBS; range 1763-18436). Pediatric and AYA patients had a higher SBS burden compared to older adults (median 5279 vs. 2945, p=0.009). Five main SBS signatures were identified: SBS1 and SBS5 (aging), SBS2 and SBS13 (APOBEC), and SBS25 (chemotherapy, in a relapsed case). A dNdScv driver discovery analysis performed on the combined WES and WGS cases identified 24 driver genes including BCL7A and CISH which had not been previously reported as drivers in cHL. An investigation of copy number alterations (CNAs) confirmed high ploidy in cHL (median 2.95, range 1.66-5.33). Whole genome duplication was identified in 64% cases. We also observed clear evidence of complex events such as chromothripsis (n=4), double minutes (dm, n=2), breakage-fusion-bridge (bfb; n=4). Some of these events were responsible for the acquisition of distinct drivers. For example, we observed one dm and one bfb responsible for CD274 and REL gains, respectively (>10 copies). Leveraging the high prevalence of large chromosomal gains, we performed an investigation of the relative timing of acquisition of driver mutations. Clonal mutations within chromosomal gains can be defined as duplicated (VAF~66%; acquired before the gain) or non-duplicated (VAF~33%; acquired before or after the gain). Sixty-one percent (152/249) of driver genes were duplicated suggesting that they were acquired prior to large chromosomal gains. Next, we used the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains to estimate the molecular time of each duplicated segment (Rustad, Nat Comm 2020). In 11/22 genomes the final CNA profile was acquired through at least two temporally distinct events. To convert these relative estimations into absolute timing (i.e., the age at which events occurred), we leveraged the clock-like mutation signatures (SBS1, SBS5). We first confirmed that the SBS1 and SBS5 mutation rate were constant over time (R 2=0.84; p Conclusion: Here we report the first WGS in cHL. We identify novel drivers and genomic mechanisms involved in cHL pathogenesis. We found that mutations in driver genes are often acquired earlier then chromosomal gains, potentially preceding the cHL diagnosis by several years. In addition, we observed key differences in biology of cHL across age groups including accelerated mutagenesis and increased mutational burden among younger patients. Disclosures Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Oberley: Caris LIfe Science: Current Employment. Lim: EUSA Pharma: Honoraria. Landgren: Janssen: Other: IDMC; Celgene: Research Funding; Janssen: Honoraria; Amgen: Honoraria; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Moskowitz: Merck & Co., Inc.: Research Funding. Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Elemento: Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; Champions Oncology: Consultancy; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Janssen: Research Funding. Roth: Janssen: Consultancy; Merck: Consultancy.

Hemophagocytic Lymphohistiocytosis (HLH) is a hyperinflammatory disorder that may be encountered as a primary or secondary phenomenon. HLH secondary to lymphoma has been described, more frequently in adults than in children. T-cell/Histiocyte-rich B-cell lymphoma (THRLBCL) is a large B-cell lymphoma that resides in a microenvironment of robust host immune response and has previously been associated with HLH in adults. Here, we describe the first case of HLH secondary to THRLBCL in an adolescent patient.

Introduction: While advances in front-line conventional chemotherapy have increased the likelihood of attaining remission in pediatric AML, relapse rates remain high (25-35%), and novel therapies are needed (Zhang, Savage et al. 2019). The clinical and molecular heterogeneity of AML makes it complex to study and creates challenges for the development of novel therapies (Bolouri, Farrar et al. 2018). It is important to identify cells and pathways underlying relapse to facilitate development of novel therapies. Single-cell RNA Sequencing (scRNA-Seq) allows in-depth analysis of the heterogeneous AML landscape to provide a detailed view of the tumor microenvironment, revealing populations of blasts and immune cells which may be relevant to relapse or complete remission. Methods: We analyzed ~36,500 cells from 14 pediatric AML bone marrow samples in our institutional biorepository, spanning different AML subtypes and 3 healthy children to generate a comprehensive scRNA-Seq landscape of immature AML-associated blasts and microenvironment cells. Samples collected at the time of diagnosis (Dx), end of induction (EOI), and relapse (Rel) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy, Gartung et al. 2019). After normalization of scRNA-Seq data, the cell clusters were identified using principal component analysis and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al, 2018). Differential expression, pathways and systems biology analysis between relapsed and remission patients reveal differences for specific cell clusters (Panigrahy, Gartung et al. 2019). To determine the clinical outcome association of our AML blast specific markers, survival analysis was performed on AML TARGET data (https://ocg.cancer.gov/programs/target) using cox proportional hazard survival approach. To characterize AML blast cells with high accuracy, we used support vector machine (SVM), an Artificial Intelligence based feature extraction and model development approach (Bhasin, Ndebele et al. 2016). Results:ScRNA-Seq analysis of paired Dx and EOI samples using UMAP identified three blast cell clusters with significant gene expression differences among different patients, indicating heterogeneity of AML blast cells (Fig 1a, b). Comparative analysis of the three Dx enriched blast cell clusters with other cells identified a "core blast cell signature" with overexpression of genes like AZU1, CLEC11A, FLT3, and NREP (Fig 1c). These core AML-blast genes were linked to significant activation of the Wnt/Ca2+, Phospholipase C, and integrin signaling pathways (Z score >2 and P-value The scRNA-Seq of AML specific blast cells from relapsed and remission samples exhibited a different clustering pattern indicating different transcriptome landscapes. Relapse-associated AML cell clusters expressed high levels of AZU1, S100A4, LGALS1, and GRK2 genes (Fig 2a). Analysis of the non-AML tumor microenvironment demonstrated enrichment of T/NK in relapsed samples, with differential expression of T cell regulatory/activation genes (Fig 2b, c). ScRNA-Seq showed enrichment of monocyte/macrophage cell clusters in remission samples with distinct relapse- and remission-specific clusters. Remission associated macrophage/monocyte clusters showed overexpression of S100A10, FTH1, CST3 and IFITM2 genes (Fig 2d). Similarly, enrichment of T cell and monocyte/macrophage clusters was observed in relapse and remission samples respectively during EOI. Conclusions: Using single cell transcriptomics we developed a novel potential gene signature to characterize heterogenous AML blast populations with high sensitivity. These genes and the pathways they regulate implicate potential therapeutic targets in pediatric AML. Single cell transcriptome analysis also enabled identification of cell clusters with modulated gene expression at both Dx and EOI that may be useful in predicting relapse/remission. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is characterized by proliferation of immature T-cells and accounts for ~15% of pediatric ALL. T-ALL blasts are phenotypically diverse and are sub-classified into pro-, pre-, cortical and mature T-ALL based on the stage of differentiation of the leukemic clone. Early T precursor ALL (ETP-ALL) is a T-ALL subtype associated with higher risk of relapse (Raetz and Teachey, 2016). Bulk sequencing approaches have revealed valuable information about modulated genes in T-ALL; however, little is understood about the interplay between tumor cells and the immune microenvironment. We present a comprehensive single cell RNA sequencing (scRNASeq) analysis of T-ALL samples with the purpose of characterizing the heterogenous tumor and microenvironment cells in order to identify dysregulated genes in leukemia cells and investigate oncogenic signaling pathways. Methods: We profiled 16,280 cells from 5 diagnostic pediatric T-ALL bone marrow samples using the Chromium single cell transcriptomics platform (10x Genomics, Pleasanton, CA). To compare T-ALL versus healthy bone marrow profiles and identify T-ALL-specific malignant blast cell populations, we included data from 3 healthy pediatric bone marrow samples from a recent study (Caron et al, 2020). Dimension reduction using the Uniform Manifold Approximation and Projection (UMAP) approach was used to identify unique cell type clusters (Becht et al, 2018). Using the TARGET dataset (https://ocg.cancer.gov/programs/target), we further evaluated the prognostic significance of identified malignant blast-specific genes by performing Kaplan-Meier survival analysis and compared gene expression patterns and tumor microenvironment makeup between ETP-ALL and non-ETP T-ALL patient samples. Results: We successfully characterized leukemic blasts (CD7+, CD99+ and CD3D+) and other major immune cell types (T cells, B cells, monocytes, erythroid precursors) using the expression of established marker genes (Fig 1A, C). Clustering analysis revealed patient-specific leukemia blast cell clusters (Fig 1B). Differential expression analysis between CD3D+ patient-specific leukemia clusters and CD3D+ clusters comprised of normal T-cells identified a set of 385 promiscuous genes that are significantly differentially expressed between malignant and normal T-cells (p-value 1.3, p-value Conclusions: We identified a gene signature characterizing heterogenous T-ALL blast populations. External validation using the TARGET dataset identified genes within the signature that are associated with poor outcomes in T-ALL. Differences in the composition of the immune microenvironment in ETP-ALL versus non-ETP T-ALL samples provide a promising area for future study. Further studies will be carried out with relapsed and non-relapsed T-ALL samples to validate this leukemia signature. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Lineage switch in acute leukemias is a well-reported occurrence; however, most of these cases involve a switch from either lymphoid to myeloid or myeloid to lymphoid lineage. Here, we report a case of a 14-year-old male with B-cell acute lymphoblastic leukemia (B-ALL) who initially responded well to standard chemotherapy but then later developed mixed phenotype acute leukemia (MPAL) at relapse, likely reflecting a clonal evolution of the original leukemia with a partial phenotypic shift. The patient had a del(9)(p13p21) in his leukemia blasts at diagnosis, and the deletion persisted at relapse along with multiple additional cytogenetic aberrations. Interestingly, the patient presented with an isolated testicular lesion at relapse, which on further analysis revealed both a lymphoid and myeloid component. Unfortunately, the patient did not respond well to treatment at relapse and eventually succumbed to his disease. To our knowledge, an isolated extramedullary MPAL at relapse in a patient with previously diagnosed B-ALL has not been reported in the literature before.

Atypical marginal zone hyperplasia (AMZH) of mucosa-associated lymphoid tissue (MALT) closely resembles lymphoma in that it shows expansion of the marginal zones with prominent intraepithelial B lymphocytes, is immunoglobulin light-chain restricted, and may show aberrant CD43 expression. However, unlike lymphoma, it does not show rearrangement of the immunoglobulin heavy chain gene (immunoglobulin H [IgH]) by polymerase chain reaction (PCR), and it behaves in a benign fashion. We identified AMZH in 2 pediatric solid organ transplant recipients who presented with adenotonsillar hypertrophy. To date, the patients have experienced a self-limited course in the absence of treatment or reduction of immunosuppression. Atypical marginal zone hyperplasia is a pitfall for posttransplant lymphoproliferative disorder and MALT lymphoma in the pediatric solid organ transplant population. In transplant patients with a lambda-restricted B-cell clone and marginal zone hyperplasia in native MALT sites, PCR for IgH and IgK gene rearrangement is essential to prevent misdiagnosis.

We report a 1-year-old female child presenting with hypereosinophilia who was found to have concurrent myeloid sarcoma and a central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT). She was later found to have a germline mutation in SMARCB1. Concurrent hematologic malignancy and CNS AT/RT have not previously been described in the context of a SMARCB1 loss-of-function germline mutation.

Anita K. McElroy, Joseph A. Hilinski, Carlos R. Abramowsky, Ronald Jaffe, Sunita I. Park, Bahig M. Shehata, and ToddM. Cooper Divisions of Infectious Diseases and Hematology/Oncology, Department of Pediatrics, and Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia;, and Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania

Introduction: Burkitt Lymphoma (BL) is a highly aggressive mature B cell neoplasm comprising 30% of Non-Hodgkin lymphoma in children. BL can be difficult to discriminate from other mature B cell lymphomas, and distinguishing BL from Diffuse Large B Cell Lymphoma (DLBCL) is a commonly encountered diagnostic dilemma. While c-myc translocation as detected by fluorescence in situ hybridization is more often associated with BL, DLBCLs may also have the translocation, and therapy is often started prior to obtaining this result. Early and rapidly obtained diagnostic clues would therefore be helpful to establish the appropriate therapeutic regimen. CD58 (aka LFA-3) is a cell adhesion molecule typically found on antigen presenting cells and is highly overexpressed in B-lymphoblastic leukemia (B-LL)(Chen et al, Blood 2001). In contrast, CD58 expression decreases as physiologic B cells mature in the bone marrow (Lee et al, Am J Clin Pathol 2005). Despite findings of abnormal CD58 expression in B-LL, CD58 expression by flow cytometry in BL and other mature B lymphomas, including DLBCL, has not been previously described in children or adults. Thus our objective was to investigate quantitative CD58 expression in BL and other pediatric B cell malignancies by flow cytometry. Methods: CD58 has been included in the flow cytometry diagnostic panel for all hematopoietic malignancies at our institution since 2009. In a single center retrospective study, we reviewed flow cytometry data from clinical specimens obtained at initial diagnosis of BL (n=18) and other mature B lymphomas (n=16) from 2009-2015. The "other mature B lymphomas" group consisted of DLBCL (n=6), post-transplant lymphoproliferative disorder (n=3), primary mediastinal large B cell lymphoma (n=2), pediatric follicular lymphoma (n=2), B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL (n=2), and extranodal marginal zone lymphoma (n=1). We also investigated CD58 expression of consecutive initial diagnostic bone marrow aspirates of B-LL (n=20) and Stage I-III hematogones (n=25) from bone marrow aspirates of patients with benign hematologic conditions (all from 2014). All samples included in this analysis were from patients 18 years of age or younger. CD58 expression was quantified as arbitrary mean fluorescence intensity (MFI) units. Initial comparisons were performed using a Kruskal-Wallis test, with post-hoc pairwise comparisons computed with the Nemenyi test. Results: CD58 expression by BL was found to be low (median MFI 427). In contrast, other mature B lymphomas and B-LL had significantly higher expression of CD58 when compared to BL (median MFI 1480 and 2342 respectively, p Conclusions: Our data suggest that CD58 expression is significantly lower in BL compared to DLBCL, other mature B lymphomas, B-LL, and maturing B cells in pediatric patients. CD58 expression may be a useful diagnostic tool to discriminate BL from other pediatric B cell malignancies. Consistent with our data, low expression of cell adhesion molecules LFA-1 and CD58/LFA-3 in cultured Burkitt cell lines has been demonstrated previously (Billaud et al, Blood 1990), and our findings support the previous data. The authors also hypothesized that decreased expression of CD58 may serve as a mechanism of immune escape in BL. Correlation of CD58 surface proteins with RNA expression may be a first step to elucidate mechanistically why BL cells have decreased CD58, and how that may provide a survival advantage. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Chronic myeloproliferative disorders (CMPDs) are clonal hematopoetic stem cell disorders resulting in the increased production of cells of the myeloid lineage. The recently described G to T point mutation at nucleotide 1849 in the JAK2 gene may be a specific molecular marker to aid in the diagnosis of CMPDs, including polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF). Previously described methods of detection for this point mutation include sequencing, allele-specific PCR, and restriction digestion. We report here the development of a novel method to detect the JAK2 point mutation using high resolution amplicon melting curve analysis. Genomic DNA was obtained from peripheral blood samples from patients with PV, ET, CIMF, and suspected CMPD using the Qiagen DNA Blood kit (Valencia, CA). DNA was quantified using PicoGreen (Invitrogen) fluorescent dye followed by spectrophotometry and samples were normalized to a standard concentration. Real time PCR was performed on the Roche Light Cycler in the presence of the double stranded DNA binding dye LCGreen1 using primers that flank the point mutation. High Resolution Melting curve analysis was then performed on the HR-1 instrument (Idaho Technologies), and mutation status assigned by comparing the melting curve position and shape to controls of known mutation status. For comparison, the samples were also restriction digested using BsaX I and analyzed by agarose gel electrophoresis. Mutation status was confirmed by sequencing of the PCR amplicons (Lark Technologies, Houston, TX) for a subset of specimens. Twenty six patients with known CMPD as defined by the WHO criteria (2001) were included in the study. In addition, seven patients with suspected CMPD were studied. The point mutation in JAK2 was identified in 9 (100%) of 9 PV patients, 7 (58%) of 12 ET patients, 1 (50%) of 2 CIMF patients, 3 (100%) of 3 CMPD NOS patients, and 2 (29%) of 7 patients with suspected CMPD. Patients testing homozygous for the JAK2 mutation included 6 patients with PV, 1 patient with ET, and 1 patient with CMPD, NOS; all other positive patients were heterozygous for the mutation. We observed excellent correlation between the melting curve analysis and restriction digest results. High resolution melting curve analysis is a rapid, sensitive, low reagent cost method to assess for the JAK2 point mutation in cases of suspected or known CMPDs. The advantages of this technique include the ability to discriminate heterozygous from homozygous mutations, increased sensitivity as compared to sequencing, rapid turn around time, no post-amplification modification, and the ability to screen for other mutations or polymorphisms within exon 12 of the JAK2 gene.