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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 (TGFβ, SKIL, LOXL1, IL-1β, 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.

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.

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

Pediatric leukemia remains a significant contributor to childhood lethality rates. However, recent development of new technologies including next-generation sequencing has increased our understanding of the biological and genetic underpinnings of leukemia, resulting in novel diagnostic and treatment paradigms. The most prevalent pediatric leukemias include B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). These leukemias are highly heterogeneous, both clinically and genetically. There are multiple genetic subgroups defined by the World Health Organization (WHO), each with distinct clinical management. Clinical laboratories have started adopting genomic testing strategies to include high throughput sequencing assays which, together with conventional cytogenetic techniques, enable optimal patient care. This review summarizes genetic and genomic techniques used in clinical laboratories to support management of pediatric leukemia, highlighting technical, biological and clinical advances. We illustrate clinical utilities of comprehensive genomic evaluation of leukemia genomes through clinical case examples, which includes the interrogations of hundreds of genes, and multiple mutation mechanisms using next generation sequencing technologies. Finally, we provide a future perspective on clinical genomics and precision medicine.

We describe the case of a 16 year-old female with mixed phenotype acute leukemia B/myeloid, NOS (formerly biphenotypic leukemia) with masked hypodiploidy and somatic TP53 and CDKN2A/B deletions. She achieved morphologic remission with lymphoid-directed multi-agent chemotherapy, but experienced an early medullary relapse 11 months from initial diagnosis. Her case details the unusual finding of hypodiploidy in a patient with ambiguous lineage leukemia and highlights the complexity of therapy selection for these high-risk patients.

Background Chronic myeloid leukemia (CML) comprises ~3 % of pediatric leukemia. Although therapy with tyrosine kinase inhibitors (TKIs) is highly effective for CML, multiple factors have been identified as predictive of treatment failure. Chromosomal abnormalities involving the MECOM locus at 3q26 portend therapy resistant disease in adults, yet have never been described in pediatric patients and have not been associated with T lymphoblastic progression. Case presentation We present a case of an 11-year-old boy with CML possessing the unique combination of T lymphoblastic transformation and a subclone harboring inv(3)(q21q26.2) at diagnosis. This is the first reported case of pediatric CML with inv(3)(q21q26.2) and the first case of T lymphoblastic progression associated with this karyotype. The patient was treated with single agent TKI therapy with robust initial response. Marrow histology at one month showed restoration of trilineage hematopoiesis and BCR-ABL RT-PCR at three months showed a 1.4 log reduction in transcript levels. Conclusions The karyotypic abnormality of inv(3)(q21q26.2) in CML is not restricted to adult patients. Moreover, while chromosome 3 abnormalities are markers of TKI resistance in adults, our patient showed a robust early response to single agent TKI therapy. This finding suggests pediatric CML with inv(3)(q21q26.2) may have distinct features and more favorable treatment responses than those described in adults.