Biologic and clinical features of childhood gamma delta T-ALL: identification of STAG2/LMO2 γδ T-ALL as an extremely high risk leukemia in the very young.

PURPOSE: Gamma delta T-cell receptor-positive acute lymphoblastic leukemia (γδ T-ALL) is a high-risk but poorly characterized disease. METHODS: We studied clinical features of 200 pediatric γδ T-ALL, and compared the prognosis of 93 cases to 1,067 protocol-matched non-γδ T-ALL. Genomic features were defined by transcriptome and genome sequencing. Experimental modeling was used to examine the mechanistic impacts of genomic alterations. Therapeutic vulnerabilities were identified by high throughput drug screening of cell lines and xenografts. RESULTS: γδ T-ALL in children under three was extremely high-risk with 5-year event-free survival (33% v. 70% [age 3-<10] and 73% [age ≥10], P =9.5 x 10 -5 ) and 5-year overall survival (49% v. 78% [age 3-<10] and 81% [age ≥10], P =0.002), differences not observed in non-γδ T-ALL. γδ T-ALL in this age group was enriched for genomic alterations activating LMO2 activation and inactivating STAG2 inactivation ( STAG2/LMO2 ). Mechanistically, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping resulting in deregulation of gene expression associated with T-cell differentiation. Drug screening showed resistance to prednisolone, consistent with clinical slow treatment response, but identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which was efficaciously targeted by Poly(ADP-ribose) polymerase (PARP) inhibition, with synergism with HDAC inhibitors. Ex-vivo drug screening on PDX cells validated the efficacy of PARP inhibitors as well as other potential targets including nelarabine. CONCLUSION: γδ T-ALL in children under the age of three is extremely high-risk and enriched for STAG2/LMO2 ALL. STAG2 loss perturbs chromatin conformation and differentiation, and STAG2/LMO2 ALL is sensitive to PARP inhibition. These data provide a diagnostic and therapeutic framework for pediatric γδ T-ALL. SUPPORT: The authors are supported by the American and Lebanese Syrian Associated Charities of St Jude Children's Research Hospital, NCI grants R35 CA197695, P50 CA021765 (C.G.M.), the Henry Schueler 41&9 Foundation (C.G.M.), and a St. Baldrick's Foundation Robert J. Arceci Innovation Award (C.G.M.), Gabriella Miller Kids First X01HD100702 (D.T.T and C.G.M.) and R03CA256550 (D.T.T. and C.G.M.), F32 5F32CA254140 (L.M.), and a Garwood Postdoctoral Fellowship of the Hematological Malignancies Program of the St Jude Children's Research Hospital Comprehensive Cancer Center (S.K.). This project was supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: U10CA180820, UG1CA189859, U24CA114766, U10CA180899, U10CA180866 and U24CA196173. DISCLAIMER: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding agencies were not directly involved in the design of the study, gathering, analysis and interpretation of the data, writing of the manuscript, or decision to submit the manuscript for publication.

Laboratory Aspects of Minimal / Measurable Residual Disease Testing in B-Lymphoblastic Leukemia.

Minimal residual disease detection provides critical prognostic predictor of treatment outcome and is the standard of care for B lymphoblastic leukemia. Flow cytometry-based minimal residual disease detection is the most common test modality and has high sensitivity (0.01%) and a rapid turnaround time (24 hours). This article details the leukemia associated immunophenotype analysis approach for flow cytometry-based minimal residual disease detection used at St. Jude Children's Research Hospital and importance of using guide gates and back-gating.

Laboratory Aspects of Minimal / Measurable Residual Disease Testing in B-Lymphoblastic Leukemia.

Minimal residual disease detection provides critical prognostic predictor of treatment outcome and is the standard of care for B lymphoblastic leukemia. Flow cytometry-based minimal residual disease detection is the most common test modality and has high sensitivity (0.01%) and a rapid turnaround time (24 hours). This article details the leukemia associated immunophenotype analysis approach for flow cytometry-based minimal residual disease detection used at St. Jude Children's Research Hospital and importance of using guide gates and back-gating.

Clinicopathologic and prognostic features of TdT-negative pediatric B-lymphoblastic leukemia.

Little is known about B-lymphoblastic leukemia (B-ALL) that lacks expression of terminal deoxynucleotidyl transferase (TdT). To address this, we performed the largest study to date of TdT-negative B-ALL using data from St. Jude Total XV and XVI clinical trials. Compared to TdT-positive B-ALL (n = 896), TdT-negative B-ALL (n = 21) was associated with younger age (median, 1.4 versus 6.8 years, P < 0.001), higher white blood cell count (median, 52.8 versus 9.9 × 109/L, P < 0.001), absence of hyperdiploidy (0 versus 27.8%, P = 0.002), KMT2A rearrangement (100 versus 1.9%, P < 0.001), and inferior 5-year event-free survival (EFS) (76.2 versus 90.3%, P = 0.047). In the context of KMT2A-rearranged B-ALL (n = 38), TdT-negativity was significantly associated with the MLLT1 rearrangement partner (P = 0.026) but was not independently predictive of survival, suggesting that the high-risk features of TdT-negative B-ALL are secondary to underlying KMT2A rearrangements. Finally, we compared the sensitivity of TdT-negativity to neuron-glial antigen 2 (NG.2) expression for the detection of KMT2A rearrangements and found that 63% of KMT2A-rearranged B-ALL cases not identified by NG.2 were TdT-negative. The results of this study expand the spectrum of immunophenotypic features that are specific for high-risk KMT2A rearrangements in pediatric B-ALL and can be readily implemented using existing standard acute leukemia flow cytometry panels.

Genome-wide discovery of somatic coding and noncoding mutations in pediatric endemic and sporadic Burkitt lymphoma.

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

The Comparative Sensitivity of Immunohistochemical Markers of Megakaryocytic Differentiation in Acute Megakaryoblastic Leukemia.

OBJECTIVES: Immunohistochemistry (IHC) staining of core biopsy sections often plays an essential role in the diagnosis of acute megakaryoblastic leukemia (AMKL). The goal of this study was to define the relative sensitivities of commonly used stains for markers of megakaryocytic differentiation. METHODS: The sensitivities of IHC stains for CD42b, CD61, and von Willebrand factor (vWF) were compared in 32 cases of pediatric AMKL. RESULTS: The sensitivities of CD42b, CD61, and vWF were 90.6%, 78.1% and 62.5%, respectively. When CD42b and CD61 were used together, the combined sensitivity increased to 93.6%. There were no cases in which vWF was positive when both CD42b and CD61 were negative. CONCLUSIONS: CD42b can reliably be used as a solitary first-line marker for blasts of megakaryocytic lineage, whereas CD61 may be reserved for infrequent cases that are CD42b negative. There is no role for the routine use of vWF when CD42b and CD61 are available.

CD30 Expression in Pediatric Neoplasms, Study of 585 Cases.

CD30 is a member of the tumor necrosis factor receptor superfamily, member 8 (TNFRSF8), and its normal expression is restricted to activated T and B cells. In tumor cells, CD30 expression is most commonly associated with lymphoid malignancies (Hodgkin and non-Hodgkin lymphomas) and is a therapeutic target using anti-CD30 antibody. CD30 expression has been reported also in mostly adult non-lymphoid malignancies, raising the possibility of CD30-targeted therapy for additional tumors. In this study, we examined the incidence of CD30 expression in 251 hematopoietic and 334 non-hematopoietic cases of pediatric tumors. As expected, strong and membranous CD30 staining was seen in anaplastic large cell lymphoma, classical Hodgkin lymphoma, and embryonal carcinoma while variable staining was seen in diffuse large B cell lymphoma. In addition, positive CD30 staining was also seen in cases of neuroblastoma (33 of 56), neoplasm with chondroid differentiation (8 of 25), myeloid neoplasms (11 of 120), hemangioma (2 of 12), and mature teratoma (1 of 11). In neuroblastoma, the CD30 expression was generally restricted to cells with ganglion differentiation; staining of ganglion cells was also seen in the one positive case of mature teratoma. In neoplasm with chondroid differentiation, the positive cases were chondrosarcoma (3 of 5), chondroblastic osteosarcoma (2 of 10), and chondroblastoma (3 of 7). In acute myeloid leukemia, the CD30 positive cases were more common in AML with monocytic differentiation but did not correlate with any specific molecular change. We conclude that CD30 expression in pediatric tumors is more general than anticipated and future studies are warranted to understand the biologic and therapeutic significances.

Prognostic Significance of Major Histocompatibility Complex Class II Expression in Pediatric Adrenocortical Tumors: A St. Jude and Children's Oncology Group Study.

PURPOSE: Histologic markers that differentiate benign and malignant pediatric adrenocortical tumors are lacking. Previous studies have implicated an association of MHC class II expression with adrenocortical tumor prognosis. Here, we determined the expression of MHC class II as well as the cell of origin of these immunologic markers in pediatric adrenocortical tumor. The impact of MHC class II gene expression on outcome was determined in a cohort of uniformly treated children with adrenocortical carcinomas. EXPERIMENTAL DESIGN: We analyzed the expression of MHC class II and a selected cluster of differentiation genes in 63 pediatric adrenocortical tumors by Affymetrix Human U133 Plus 2.0 or HT HG-U133+PM gene chip analyses. Cells expressing MHC class II were identified by morphologic and immunohistochemical assays. RESULTS: MHC class II expression was significantly greater in adrenocortical adenomas than in carcinomas (P = 4.8 ×10-6) and was associated with a higher progression-free survival (PFS) estimate (P = 0.003). Specifically, HLA-DPA1 expression was most significantly associated with PFS after adjustment for tumor weight and stage. HLA-DPA1 was predominantly expressed by hematopoietic infiltrating cells and undetectable in tumor cells in 23 of 26 cases (88%). CONCLUSIONS: MHC class II expression, which is produced by tumor-infiltrating immune cells, is an indicator of disease aggressiveness in pediatric adrenocortical tumor. Our results suggest that immune responses modulate adrenocortical tumorigenesis and may allow the refinement of risk stratification and treatment for this disease. Clin Cancer Res; 22(24); 6247-55. ©2016 AACR.

Pediatric MLBL: challenges remain.

In this issue of Blood, Gerrard et al report the outcome and histologic classification of children and adolescents with mediastinal large B-cell lymphoma (MLBL), and highlight the need for new treatment strategies.

Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells.

Patients with Down syndrome (trisomy 21, T21) have hematologic abnormalities throughout life. Newborns frequently exhibit abnormal blood counts and a clonal preleukemia. Human T21 fetal livers contain expanded erythro-megakaryocytic precursors with enhanced proliferative capacity. The impact of T21 on the earliest stages of embryonic hematopoiesis is unknown and nearly impossible to examine in human subjects. We modeled T21 yolk sac hematopoiesis using human induced pluripotent stem cells (iPSCs). Blood progenitor populations generated from T21 iPSCs were present at normal frequency and proliferated normally. However, their developmental potential was altered with enhanced erythropoiesis and reduced myelopoiesis, but normal megakaryocyte production. These abnormalities overlap with those of T21 fetal livers, but also reflect important differences. Our studies show that T21 confers distinct developmental stage- and species-specific hematopoietic defects. More generally, we illustrate how iPSCs can provide insight into early stages of normal and pathological human development.

α-Hemoglobin-stabilizing protein is a sensitive and specific marker of erythroid precursors.

α-Hemoglobin-stabilizing protein (AHSP) is an abundant erythroid-specific chaperone protein that facilitates incorporation of nascent α-globin into hemoglobin A. We characterized AHSP expression by immunohistochemistry in a panel of 100 neoplastic and reactive bone marrow biopsies and splenic tissue with extramedullary hematopoiesis and compared it with established erythroid markers CD71 and CD235a. In all cases, AHSP expression was limited to physiological nucleated erythroid precursors (EPs) and blasts in erythroid leukemias. Although CD71 also stained EPs, it additionally stained nonerythroid malignant cells to varying extents in acute leukemia, diffuse large B-cell lymphoma, metastatic carcinomas, and small round blue cell tumors. In contrast, CD235a staining was erythroid-specific but stained non-nucleated red blood cells in all specimens, limiting its utility. We conclude that AHSP is superior to CD71 and CD235a for detecting normal and neoplastic nucleated EPs.

Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.

The use of human pluripotent stem cells for laboratory studies and cell-based therapies is hampered by their tumor-forming potential and limited ability to generate pure populations of differentiated cell types in vitro. To address these issues, we established endodermal progenitor (EP) cell lines from human embryonic and induced pluripotent stem cells. Optimized growth conditions were established that allow near unlimited (>10(16)) EP cell self-renewal in which they display a morphology and gene expression pattern characteristic of definitive endoderm. Upon manipulation of their culture conditions in vitro or transplantation into mice, clonally derived EP cells differentiate into numerous endodermal lineages, including monohormonal glucose-responsive pancreatic ß-cells, hepatocytes, and intestinal epithelia. Importantly, EP cells are nontumorigenic in vivo. Thus, EP cells represent a powerful tool to study endoderm specification and offer a potentially safe source of endodermal-derived tissues for transplantation therapies.

Early molecular detection of central nervous system relapse in a child with systemic anaplastic large cell lymphoma: case report and review of the literature.

We report a case of anaplastic large cell lymphoma (ALCL) with central nervous system relapse in an 11-year-old boy. The relapse was suspected on morphologic examination of the cytospin preparations of the cerebrospinal fluid (CSF) with a WBC of 10 cells/microl. CSF relapse was confirmed using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) for abnormal ALK expression or gene structure. The patient developed large intracranial metastases, despite systemic, and intrathecal chemotherapy. This case demonstrates the feasibility of detecting ALCL in paucicellular CSF specimens and suggests that even low CSF involvement can herald massive parenchymal disease.

Cell division rates of primary human precursor B cells in culture reflect in vivo rates.

Bone marrow stroma-based cultures provide a powerful model for studying cell division and apoptosis of primary human precursor B cells. Studies using this model are elucidating the mechanisms by which stromal cells inhibit apoptosis of cultured normal precursor B cells and have demonstrated that the apoptotic rate of cultured leukemic precursor B cells can predict clinical outcome in acute lymphoblastic leukemia. In contrast to apoptosis, cell division in this model has not been well characterized. In this study, we quantified the rates of cell division in cultured primary human normal and leukemic precursor B cells by labeling precursor B cells with the fluorescent dye carboxyfluorescein diacetate, succinimyl ester. Based on the rate of decreasing fluorescent signal over 3 weeks, normal CD19(+), CD10(+) precursor B cells divided once every 90.5 hours, a number that correlates well with the known in vivo rate of 65.5 hours. The division rates were similar among different cultures and constant throughout the 3 weeks of culture, suggesting that the variable expansions of precursor B cells seen among different samples and culture durations are not secondary to different cell division rates. Unlike normal cells, cultured leukemic B cells had a heterogeneous division rate that ranged from once every 26-240 hours. These rates correlated well with their respective in vivo proliferation index. These findings indicate that the stroma-based cultures faithfully replicate in vivo cell division rates and can be used to elucidate the pathways that regulate cell division of primary human precursor B cells.

Identification of cyclin D3 as a direct target of E2A using DamID.

The transcription factor E2A can promote precursor B cell expansion, promote G(1) cell cycle progression, and induce the expressions of multiple G(1)-phase cyclins. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated the 1-kb promoter of cyclin D3, which contains two E boxes. However, deletion of the E boxes did not disrupt the transactivation by E2A, raising the possibility of indirect activation via another transcription factor or binding of E2A to non-E-box DNA elements. To distinguish between these two possibilities, promoter occupancy was examined using the DamID approach. A fusion construct composed of E2A and the Escherichia coli DNA adenosine methyltransferase (E47Dam) was subcloned in lentivirus vectors and used to transduce precursor B-cell and myeloid progenitor cell lines. In both cell types, specific adenosine methylation was identified at the cyclin D3 promoter. Chromatin immunoprecipitation analysis confirmed the DamID findings and localized the binding to within 1 kb of the two E boxes. The methylation by E47Dam was not disrupted by mutations in the E2A portion that block DNA binding. We conclude that E2A can be recruited to the cyclin D3 promoter independently of E boxes or E2A DNA binding activity.