Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Patient-Derived Xenografts Are Faithful Genomic and Phenotypic Models of Primary Leukemia and Respond to the IL3 Receptor Targeting Agent SL-401 In Vivo

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive acute leukemia/lymphoma recently classified as a malignant transformation of plasmacytoid dendritic cells (pDCs) and a subtype of acute myeloid leukemia (AML). BPDCN has no standard treatment and a poor prognosis, with median survival <1 year. A significant roadblock to better understanding BPDCN is a lack of adequate model systems. We generated patient-derived xenografts (PDX) of BPDCN in NOD/Scid/IL2rgnull (NSG) mice. Bone marrow or peripheral blood cells involved by BPDCN blasts (CD45 low, CD123 high, HLA-DR high, CD3 neg) were transplanted into irradiated NSG recipients. Nine of 16 BPDCNs caused lethal leukemia involving blood, spleen, and bone marrow 2-6 months after transplantation. All nine BPDCN PDXs were serially transplantable. Flow characterization of each patient's BPDCN and corresponding xenograft revealed no major differences in BDCA2, BDCA4, FCeR1, ILT7, or cytoplasmic TCL1 staining. All samples maintained high expression of the human interleukin-3 (IL3) receptor (IL3Ralpha/CD123), a hallmark feature of BPDCN. To further characterize BPDCN pathogenesis we performed whole transcriptome sequencing (RNA-seq) on sorted blasts from 11 patients and on normal pDCs isolated from 4 healthy donors. These were compared to RNA-seq in six PDXs. The spectrum of mutations in BPDCN transcriptomes overlapped with that seen in other hematologic malignancies, particularly myeloid disorders, and was similar to reported DNA mutations in BPDCN, including in ASXL1, CTCF, IDH2, NRAS, RUNX1, STAG2, TET2, and TP53. Particularly striking was the presence of a canonical mutation in an RNA splicing factor in 7 of 11 cases (SRSF2 P95H/L/R in four, ZRSR2 R295* and gene locus deletion in two, and SF3B1 K666N in one). Known oncogenic mutations in the original disease were retained in the PDXs, including all splicing factor mutations, with the exception of an IDH2 R140Q that was lost in one PDX. BPDCN PDXs grouped together in unsupervised clustering of expression profiles, distinct from AML and ALL PDXs in an analysis of 134 models from the DFCI Public Repository of Xenografts (http://PRoXe.org). Gene set enrichment analysis (GSEA) of KEGG and REACTOME pathways associated with differentially expressed genes between primary BPDCNs and non-malignant pDCs revealed signatures related to dendritic cell activation, cell cycle, and apoptosis. In addition, 3 of the top 11 sets were genes involved in mRNA processing, mRNA splicing, and processing capped intron-containing pre-mRNAs (all FDR<1e-6). To test the efficacy of BPDCN-targeted therapy using primary human leukemias in vivo, we performed a pre-clinical trial in NSG mice using SL-401, a recombinant biologic consisting of a fusion protein of IL3 and diphtheria toxin. Three independent BPDCN xenografts were injected into 20 NSG mice each, and followed by weekly peripheral blood monitoring for human CD45 and CD123. When leukemia burden reached >0.5% in at least half of the mice in the cohort, animals were randomized to receive SL-401 at 100 ug/kg or vehicle intraperitoneally daily for 5 days. Two mice in each group were sacrificed at day 7 for response assessment, and peripheral blood was followed weekly in the remaining mice for evidence of progression (>5% human CD45/CD123-positive cells). 7 days after treatment, mice receiving SL-401 had dramatic reductions in BPDCN in the peripheral blood, spleen, and bone marrow (0.31% vs 37.6% in marrow of SL-401 vs vehicle). SL-401 prolonged progression-free survival in all BPDCNs tested (12 vs 48 days, P<0.0001 by log-rank test). At the time of progression after SL-401, relapsing mice were re-randomized to receive a 2nd and in some cases 3rd cycle of SL-401 or vehicle. Repeated treatment in mice that progressed after SL-401 resulted in second and third peripheral blood remissions. All PDXs responded to SL-401 including those with and without splicing factor and TP53 mutations. CD123 expression was maintained at high levels on all SL-401 treated BPDCNs even after repeated cycles. Primary xenografts of BPDCN are faithful models of the human disease, maintain genetic and transcriptomic characteristics of the original tumor, and respond to multiple courses of IL3-DT in vivo, suggesting that they provide a valuable resource to study disease biology and response/resistance to targeted therapy. Disclosures Chen: Stemline Therapeutics, Inc.: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lane:Stemline Therapeutics, Inc.: Research Funding.

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Features of non-activation dendritic state and immune deficiency in blastic plasmacytoid dendritic cell neoplasm (BPDCN)

Blood Cancer Journal ◽

10.1038/s41408-019-0262-0 ◽

2019 ◽

Vol 9 (12) ◽

Cited By ~ 3

Author(s):

Hannah C. Beird ◽

Maliha Khan ◽

Feng Wang ◽

Mansour Alfayez ◽

Tianyu Cai ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cell ◽

Peripheral Blood ◽

Profile Analysis ◽

Hematologic Malignancy ◽

Plasmacytoid Dendritic Cell ◽

Serum Samples ◽

Molecular Features ◽

Cell Neoplasm ◽

Poor Outcomes

AbstractBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, male-predominant hematologic malignancy with poor outcomes and with just one recently approved agent (tagraxofusp). It is characterized by the abnormal proliferation of precursor plasmacytoid dendritic cells (pDCs) with morphologic and molecular similarities to acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) in its presentation within the bone marrow and peripheral blood. To identify disease-specific molecular features of BPDCN, we profiled the bone marrow, peripheral blood, and serum samples from primary patient samples using an in-house hematologic malignancy panel (“T300” panel), transcriptome microarray, and serum multiplex immunoassays. TET2 mutations (5/8, 63%) were the most prevalent in our cohort. Using the transcriptome microarray, genes specific to pDCs (LAMP5, CCDC50) were more highly expressed in BPDCN than in AML specimens. Finally, the serum cytokine profile analysis showed significantly elevated levels of eosinophil chemoattractants eotaxin and RANTES in BPDCN as compared with AML. Along with the high levels of PTPRS and dendritic nature of the tumor cells, these findings suggest a possible pre-inflammatory context of this disease, in which BPDCN features nonactivated pDCs.

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Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Harbors Frequent Splicesosome Mutations That Cause Aberrant RNA Splicing Affecting Genes Critical in pDC Differentiation and Function

Blood ◽

10.1182/blood.v128.22.738.738 ◽

2016 ◽

Vol 128 (22) ◽

pp. 738-738 ◽

Cited By ~ 4

Author(s):

Katsuhiro Togami ◽

Vikas Madan ◽

Jia Li ◽

Alexandra-Chloe Villani ◽

Siranush Sarkizova ◽

...

Keyword(s):

Dendritic Cell ◽

Rna Splicing ◽

Intron Retention ◽

Plasmacytoid Dendritic Cell ◽

Splicing Factor ◽

Splicing Factors ◽

Rna Seq ◽

Aberrant Splicing ◽

Recurrent Mutations ◽

Cell Neoplasm

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy thought to result from transformation of plasmacytoid dendritic cells (pDCs). Clinical outcomes are poor and pathogenesis is unclear. To better understand BPDCN genomics and disease mechanisms, we performed whole exome- (12 BPDCNs), targeted DNA- (additional 12 BPDCNs), bulk whole transcriptome RNA- (12 BPDCNs and 6 BPDCN patient-derived xenografts [PDXs]), and single cell RNA-sequencing (scRNA-seq) compared to normal DCs. We observed RNA splicing factor mutations in 16/24 cases (7 ZRSR2, 6 SRSF2, 1 each SF3B1, U2AF1, SF3A2, SF3B4). Additional recurrent alterations were in genes known to be mutated in other blood cancers: TET2, ASXL1, TP53, GNB1, NRAS, IDH2, ETV6, DNMT3A, and RUNX1. From exome sequencing we also discovered recurrent mutations in CRIPAK (6/12 cases), NEFH (4/12), HNF1A (2/12), PAX3 (2/12), and SSC5D (2/12) that may be unique to BPDCN. ZRSR2 is notable among the recurrently mutated splicing factors in hematologic malignancies in that all mutations are loss-of-function (e.g., nonsense, frameshift). Of note, BPDCN is very male predominant, ZRSR2 is located on chrX and all mutations are in males. ZRSR2 plays a critical role in "minor" or U12-type intron splicing (only 0.3% of all introns). Thus, we hypothesized that mis-splicing, possibly of U12 genes, contributes to BPDCN pathogenesis. Using RNA-seq, we measured aberrant splicing in BPDCN. Intron retention was the most frequent abnormality in ZRSR2 mutant BPDCNs and PDXs compared to non-mutant cases. ZRSR2 mutant intron retention predominantly affected U12 introns (patients: 29.4% of retained introns, P<0.0001; PDX: 94%, P<0.0001). To test if ZRSR2 loss directly causes U12 intron retention in otherwise isogenic cells, we performed ZRSR2 knockdown using doxycycline-inducible shRNAs in the BPDCN cell line, CAL1, which has no known splicing factor mutation. RNA-seq was performed 0, 2, and 7 days after addition of doxycycline in 3 independent clones each of control or ZRSR2 knockdown. Consistent with what we observed in primary BPDCN, intron retention events were higher in ZRSR2 compared to control shRNA cells after 7 days of doxycycline (mean 885.7 vs 122.7 events, P=0.041). Aberrant intron retention after ZRSR2 knockdown largely involved U12 introns (30/732 U12 vs 37/207,344 U2 introns, P<0.0001). SRSF2 and SF3B1 mutations in BPDCN were at hotspots seen in other cancers: SRSF2 P95H/L/R and SF3B1 K666N, mutants that induce specific types of aberrant splicing (Kim, Ca Cell 2015; Darman, Cell Rep 2015). Mutant BPDCNs demonstrated the same aberrations: SRSF2, exon inclusion/exclusion based on CCNG/GGNG exonic splicing enhancer motifs; SF3B1, aberrant 3' splice site recognition. We hypothesized that aberrant splicing may affect RNAs important for pDC development or function. To further define genes uniquely important in BPDCN, we performed scRNA-seq on 4 BPDCNs and on DCs from healthy donors. By principal component analysis, BPDCNs were more similar to pDCs than to conventional DCs (cDCs) or other HLA-DR+ cells. However, several critical genes for pDC function had markedly lower expression in BPDCN including the transcription factors IRF4 and IRF7. Next we determined which genes were commonly mis-spliced in splicing factor mutant BPDCNs. Strikingly, this list included genes already known to be important in driving DC biology or identified in our scRNA-seq as being differentially expressed between BPDCN and healthy pDCs, including IRF7, IRF8, IKZF1, FLT3, and DERL3. To determine if splicing factor mutations affect DC function, we stimulated ZRSR2 knockdown or control CAL1 cells with Toll-like receptor (TLR) 7, 8, and 9 agonists (R848 or CpG oligo). ZRSR2 knockdown inhibited upregulation of the CD80 costimulatory molecule and aggregation of CAL1 cells, suggesting impairment in activation. Using mouse conditional knock-in bone marrow in ex vivo multipotent progenitor assays, DC differentiation induced by FLT3 ligand was biased toward pDCs and away from cDCs in SRSF2 P95H mutant compared to wild-type cells. However, cDC and monocyte differentiation in the presence of GM-CSF was not affected. In conclusion, splicing factors are frequently mutated in BPDCN and lead to specific splicing defects. Splicing factor mutations may promote BPDCN by affecting pathways important in DC maturation or activation, which could contribute to transformation. Disclosures Seiler: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy.

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Pre-Clinical Studies of Anti-CD123 CAR-T Cells for the Treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Blood ◽

10.1182/blood.v128.22.4039.4039 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4039-4039 ◽

Cited By ~ 12

Author(s):

Tianyu Cai ◽

Roman Galetto ◽

Agnès Gouble ◽

Julianne Smith ◽

Antonio Cavazos ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Dendritic Cell ◽

Research Funding ◽

Plasmacytoid Dendritic Cell ◽

Newly Diagnosed ◽

Car T Cells ◽

Car T ◽

Cell Neoplasm

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy which originates from the precursors of plasmacytoid dendritic cells. Data on the biology of BPDCN are limited, patient outcomes have historically been poor and there remains no established standard of care. CD123/IL3Rα is overexpressed in nearly 100% of patients with BPDCN. Considering the urgent unmet medical need for patients with BPDCN, targeting CD123 emerged as an attractive therapeutic target given its accessibility (cell surface) and differential expression (markedly over-expressed on BDPCN blasts as compared to normal hematopoietic stem cell compartment). UCART123 (CD123CAR/RQR8+_TCRαβ-_T-cells) are genetically modified allogeneic T-cells (obtained from healthy volunteer donors by leukapheresis) containing (i) an anti-CD123 CAR (CD123 scFv-41BB-CD3z) and (ii) an RQR8 depletion ligand that confers susceptibility to rituximab. The cell surface expression of the T cell receptor (TCR) is depleted through the inactivation of the TCRa constant (TRAC) gene using Cellectis' TALEN® technology. In this study, we examined efficacy of this first allogenic anti-CD123 CAR-T cells in pre-clinical models of BPDCN. We first analysed the level of expression of CD123 in the bone marrow of 8 patients with newly diagnosed BPDCN, and compared with CD123 expression on blasts of 28 newly diagnosed AML patients. CD123 expression levels were significantly higher in BPDCN (mean fluorescence intensity (MFI) range 3,484-17,937) compared to AML (MFI range 360-5,073) (p<0.01). In vitro cytotoxic activity of UCART123 cells was evaluated by co-culturing UCART123 cells with primary human BPDCN, at a 10:1 effector to target ratio, using flow cytometry. The results show significant cytotoxic activity of UCART123 cells against BPDCN samples compared to cells co-cultured with non-transduced TCRab- T-cells (NTD) (Fig. 1A). We next evaluated antigen-specific UCART123 cell degranulation by staining of CD107α, a lysosomal associated membrane protein residing in cytolytic granule membranes. The results indicated that specific degranulation of UCART123 cells is observed upon co-culture with CD123 (+) target cells. The capacity of UCART123 cells to secrete cytokines, in particular IFNγ, in response to specific stimulation with CD123 (+) BPDCN cells was examined using a BioLegend's LEGENDplexTM assay. UCART123 cells stimulated by CD123 (+) BPDCN cells, but not NTD cells, secreted high levels of IFNγ in the culture supernatants (Fig. 1B). To evaluate in vivo anti-tumor activity of UCART123 cells, we established patient-derived xenograft (PDX) from a patient with relapsed BPDCN in NSG-SGM3 mice. Upon engraftment, mice were randomized into 4 groups (9 mice/group). Mice received either a single tail vein injection of vehicle, 10×106 NTD T-cells, and 3×106 or 10×106 UCART123 cells. All mice in vehicle-treated group and in the group that received 10×106 NTD cells died by day 53, with high tumor burden of BPDCN detected in peripheral blood, spleen and bone marrow. Both cell doses of UCART123 significantly extended mice survival (Fig. 1C) and reduced or eliminated circulating BPDCN cells. 57 days after UCART123 cells injection, one mouse from UCART123 10×106 group was sacrificed. UCART123 cells were detected in spleen (16.4% CAR+ cells) and bone marrow (1.1% CAR+ cells) using human CD5 antibody or CD123-Fc protein. In summary, UCART123 causes specific killing of BPDCN cells, associated with antigen-specific T-cell degranulation and robust levels of IFNg production. Our preliminary data indicate persistence of UCART123 cells in vivo in an NSG-S model of primary BPDCN. Most importantly, UCART123 therapy results in BPDCN eradication and long-term disease-free survival in primary BPDCN PDX mice. Additional PDX studies are ongoing and will be presented. These results demonstrate pre-clinical proof-of principle of high anti-BPDCN activity of UCART123 allogeneic CAR T-cells, and warrant further clinical testing of this approach in human clinical trials in BPDCN. Disclosures Galetto: Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy. Guzman:Cellectis: Research Funding. Konopleva:Cellectis: Research Funding; Calithera: Research Funding.

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In Utero Cltc-ALK Fusion In Hematopoietic Progenitor Cells As a First Step Of Leukemogenesis In Blastic Plasmacytoid Dendritic Cell Neoplasm

Blood ◽

10.1182/blood.v122.21.2587.2587 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2587-2587

Author(s):

Kiriko Tokuda ◽

Minenori Eguchi-Ishimae ◽

Mariko Eguchi ◽

Eiichi Ishii

Keyword(s):

Dendritic Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Fusion Gene ◽

Plasmacytoid Dendritic Cell ◽

In Utero ◽

Guthrie Card ◽

Myeloid Neoplasm ◽

Cell Neoplasm ◽

Blast Cells

Abstract Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a subtype of myeloid leukemia mainly affecting the elderly and often accompanied by cutaneous legions. It is a rare disease, and neither the genetic nor clonal origin of the disease is known. We report the first case of BPDCN with clathrin heavy chain (CLTC)-anaplastic lymphoma kinase (ALK) fusion gene. We performed a detailed analysis to understand the origin of the tumor cells and the leukemic process involved. Samples and Results Samples were collected from a female infant who was admitted under the diagnosis of hemophagocytic lymphohistiocytosis (HLH) at 1 month of age. One month later, leukemic blasts appeared in the peripheral blood showing karyotypic abnormality 46,XX,t(2;17;8)(p23;q23;p23). Fluorescence in situ hybridization with break apart probes covering the ALK gene revealed translocation of the 3’-ALK signal to der(17) and loss of the 5’ ALK signal on der(2). CLTC-ALK fusion was identified by direct sequencing of the RT-PCR product obtained from the peripheral blood specimen. Although HLH symptoms improved after one course of chemotherapy, blast cells re-appeared in the peripheral blood and bone marrow after 3 courses of chemotherapy, with a karyotype of 45, XX, t(2;17;8)(p23;q23;p23), -7. Multicolor flow cytometry showed the blast cells were weakly positive for CD4 and negative for CD3, and expression of CLTC-ALKwas confirmed in these cells. Some of the blasts were highly positive for CD123 and CD303, indicating the plasmacytoid dendritic cell phenotype and leading to the diagnosis of BPDCN. The rest of the blasts were positive for CD56 and weakly positive for CD123. Nearly half of this CD4+CD56+ population was also positive for monocytic marker, CD14. The possibility of in utero origin of the leukemic cells was tested by analyzing the presence of CLTC-ALK fusion in the Guthrie card. The genomic breakpoint of the CLTC-ALKfusion was determined by inverse PCR, and then 24 pieces of the Guthrie card containing the neonatal blood were tested for the existence of the cells carrying the same fusion breakpoint. The testing revealed the prenatal origin of the fusion gene. To explore the origin of leukemic transformation in the patient, the presence of the CLTC-ALK fusion gene was assessed in genomic DNA extracted from subpopulations sorted from the patient’s peripheral blood. As well as leukemic CD4+CD3- cells, most of the monocytes possessed the CLTC-ALK fusion gene, and a small portion of T cells, B cells and neutrophils were also positive for genomic CLTC-ALK fusion. Immature cells with high CD34 expression but without lineage markers separated from the peripheral blood were also positive for CLTC-ALKfusion. Conclusions The CLTC-ALK fusion gene was identified for the first time as the leukemia-promoting abnormality in an infant case of myeloid neoplasm BPDCN, indicating the tumorigenic potential of CLTC-ALK in myeloid progenitor cells. In addition, activated monocytes with the CLTC-ALK fusion might be responsible for the occurrence of HLH in the patient. Formation of the CLTC-ALK fusion was suggested to have occurred in a hematopoietic progenitor cells in utero, and the subsequent acquisition of monosomy 7, one of the myeloid lineage-oriented abnormalities, might have determined the cell fate to a myeloid neoplasm in this patient. Disclosures: No relevant conflicts of interest to declare.

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Efficient Engraftment and Disease Replication of Myelodysplastic Syndromes Using a Novel Humanized Mice Model

Blood ◽

10.1182/blood.v126.23.4100.4100 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4100-4100

Author(s):

Yuanbin Song ◽

Ashley Taylor ◽

Anthony Rongvaux ◽

Tingting Jiang ◽

Nikolai A. Podoltsev ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Epigenetic Alterations ◽

Histologic Analysis ◽

Nsg Mice ◽

Xenotransplantation Model ◽

Human Cytokines

Abstract Myelodysplastic Syndromes (MDS) are a heterogeneous disorder of the hematopoietic stem cell caused by a large number of genetic and epigenetic alterations. With the development of novel therapeutics a reliable model to test the drugs' efficacy in correlation with genetic and epigenetic alterations and disease phenotype is essential. Recent advances in the field of MDS xenotransplantation have been achieved by transgenic expression of human cytokines in the murine host as well as by co-transplantation of primary patient derived mesenchymal stromal cells (MSCs) concurrent with MDS stem cells. However, neither model to date affords efficient transplantation of MDS at a scale that allows in vivo mechanistic studies or provides a platform to develop and test novel therapeutics. We sought to establish a MDS xenotransplantation model in humanized immunodeficient mice amenable to mechanistic in vivo studies and therapeutic testing. Several murine cytokines essential for hematopoiesis are non-crossreactive with their human counterpart. "MISTRG" mice express several human, non-crossreactive cytokines, namely M -CSF, I L-3, GM-CSF, and T hrombopoietin from the respective murine loci, as well as human macrophage receptor signal regulatory protein-alpha (S IRPα) to prevent murine macrophage-mediated immune rejection in the Rag2-/- IL2rγ-/- background (Rongvaux et al. Nature Biotech 32(4): 364 - 372, 2014). To establish a reliable, efficient MDS xenotransplantation model we optimized the host irradiation dose, transplantation route, CD34+ cell number and cell preparation. Mice were allowed to engraft for >10 weeks. Peripheral blood (PB), bone marrow (BM), and spleen were analyzed for engraftment by flow cytometry. BM, spleen, and liver were also fixed and sectioned for histologic analysis. Human CD45+ cells were sorted from engrafted MISTRG bone marrow and genomic testing was performed by cytogenetics, FISH, and/or targeted exome sequencing. MISTRG mice consistently supported higher engraftment in peripheral blood and bone marrow than NSG mice for the majority of MDS samples assessed. Out of 25 different patient's BM samples, including 6 RCMD, 5 RAEB I, 12 RAEB II, and 2 CMML patient samples, 23 samples engrafted in MISTRG mice, while 19 of the samples were transplanted concurrently into NSG mice (6 RCMD, 3 RAEB I, and 11 RAEB II patient samples) out of which 12 samples engrafted. Mice were classified as engrafted when huCD45+ cells accounted for over 1% of all nucleated cells in BM. On average, huCD45 engraftment was 7.3-fold higher in MISTRG than in NSG mice (17.78% vs. 2.45%). 56.1% of all MISTRG mice compared with 26.7% of NSG mice transplanted with MDS were engrafted. The number of engrafted MISTRG mice per sample ranged from 2-10 mice could be further improved with optimal bone marrow sample collection. We verified engraftment of the MDS clone via cytogenetics, FISH, and/or targeted exome sequencing, also revealing preserved clonal distribution and mutant allele frequencies in engrafted mice. Flow cytometric analysis of lineage differentiation revealed robust myeloid engraftment in MISTRG mice as opposed to NSG mice. In addition terminal differentiation of myeloid cells was markedly improved in MISTRG over NSG mice, with immunophenotypic concordance between engrafted MISTRG mice and the patient's primary bone marrow. Histologic analysis showed striking similarities between engrafted MISTRG bone marrow and the concurrent patient's bone marrow, such as marked dysplasia and clustering of human CD61 positive megakarocytes with resultant myelofibrosis as evident by reticulin staining. MISTRG mice lack the DNA repair defect inherent to NSG mice and are thus tolerant of chemotherapeutic agents. Studies testing hypomethylating drugs and targeted agents are now underway to establish MISTRG as promising "co-clinical" model for MDS. In conclusion, physiologic expression of essential non-crossreactive human cytokines greatly facilitates long-term engraftment of MDS patient derived CD34+ HSPCs in the murine immunodeficient host. MISTRG mice engraft lower and higher grade MDS with replication of the disease genotypes and phenotypes, supporting higher engraftment in bone marrow and blood than NSG mice for all MDS samples assessed. MISTRG mice may provide a xenotransplantation model for MDS allowing us to study the biology of the disease and to test therapeutics in vivo. Disclosures No relevant conflicts of interest to declare.

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Clinical and Pathological Features and Differential Diagnosis of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): a Series of Cases

Blood ◽

10.1182/blood.v128.22.5183.5183 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5183-5183 ◽

Cited By ~ 1

Author(s):

Gabriela Cesarman-Maus ◽

Carmen Lome ◽

Karla Adriana Espinosa ◽

Carmen Marcela Quezada-Fiallos ◽

Silvia Rivas ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Differential Diagnosis ◽

Dendritic Cell ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Plasmacytoid Dendritic Cell ◽

Cell Neoplasm ◽

Skin Infiltration ◽

Acute Myeloid

Abstract Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is a recently recognized highly aggressive malignant proliferation of plasmacytoid dendritic-cell (PDC) precursors which consistently express CD4, CD56 and CD123. Mortality is high despite transplant and the response to treatment is poor, except for preliminary results with conjugated anti-CD123. Clinically, cutaneous involvement is the most common feature with or without the presence of initial bone marrow infiltration, however patients may present with bone marrow-only disease. BPDCN is underdiagnosed, and can be confused with several entities, including acute myeloid leukemia. We describe our experience with 7 cases of BPDCN, their clinical and pathological presentation, and describe possible misdiagnosis and the antibodies that may help in corroborating BPDCN. See table for clinical characteristics at diagnosis. Diagnosis of BPDCN must take into account clinical, morphological and immunohistochemical analysis (IHC), since these tumors variably express markers that may be shared with other neoplasias including CD56 and TCL-1. When IHC is not categorical for BPDCN, the WHO recommends reporting the cases as AML of ambiguous lineage. The typical IHC includes CD4, CD43, CD45RA, CD56, CD123, TCL1, CLA and CD68. The absence of CD56 does not exclude the diagnosis. Markers shared with other hematological tumors include: CD7, CD33, CD2,CD36 and CD38 and TdT. Thus differential diagnosis should be done primarily with A) Skin infiltration by acute myeloid leukemia (myeloperoxidase +, 7- lysozyme +, CD34 +, CD117 +/-) B) Skin Infiltration by T / NK extra nodal lymphoma ( CD8 , cytotoxic cytoplasmic granules [CCG] , granzyme B, perforin, TIA1 and EBER) C) cutaneous peripheral T lymphoma ( +/- CD8, CD2 +/-, +/- CD5, CD7 +/- and variably positive CCG´s). D) Other histiocytic and dendritic cell-neoplasms may also be considered in the differential diagnosis however the histological appearance is usually characteristic. BPDCN is a poorly known entity that should be suspected by both clinician and pathologist in order to make a correct diagnosis. Table Table. Disclosures No relevant conflicts of interest to declare.

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In vivo and in vitro sensitivity of blastic plasmacytoid dendritic cell neoplasm to SL-401, an interleukin-3 receptor targeted biologic agent

Haematologica ◽

10.3324/haematol.2014.111740 ◽

2014 ◽

Vol 100 (2) ◽

pp. 223-230 ◽

Cited By ~ 36

Author(s):

F. Angelot-Delettre ◽

A. Roggy ◽

A. E. Frankel ◽

B. Lamarthee ◽

E. Seilles ◽

...

Keyword(s):

Dendritic Cell ◽

Plasmacytoid Dendritic Cell ◽

Biologic Agent ◽

Interleukin 3 ◽

In Vitro Sensitivity ◽

Cell Neoplasm

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A Novel Xenograft Model of Cytogenetically Normal Acute Myeloid Leukemia Harboring RUNX1 and ASXL1 Mutations Demonstrates Residual Disease after Anthracycline/Cytarabine-Based Chemotherapy

Blood ◽

10.1182/blood.v124.21.9.9 ◽

2014 ◽

Vol 124 (21) ◽

pp. 9-9

Author(s):

Umayal Sivagnanalingam ◽

Marlene Balys ◽

Allison Eberhardt ◽

Nancy Wang ◽

John M. Ashton ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Residual Disease ◽

Chemotherapy Resistance ◽

Driver Mutations ◽

Therapeutic Approaches ◽

Tail Vein ◽

Nsg Mice ◽

Novel Therapeutic

Abstract Introduction: Cytogenetically normal acute myeloid leukemia (CN-AML) patients harboring RUNX1 mutations have a poor prognosis with standard, anthracyline/cytarabine-based chemotherapy and novel therapeutic approaches are urgently needed. Development of novel therapeutic approaches in RUNX1-mutated, CN-AML is hindered by a lack of adequate in vivo models. Munker et al. have generated a RUNX1-mutated, CN-AML cell line (termed CG-SH) from an AML patient and characterized its properties in vitro [Leukemia Research 33 (2009) 1405-1408; PMID: 19414191]; however, its potential to model the disease in vivo has not previously been explored. Moreover, CG-SH has not been comprehensively examined for additional AML driver mutations that might contribute to its biological properties. Our hypothesis was that CG-SH cells would efficiently engraft immune-deficient mice and demonstrate residual disease after anthracycline/cytarabine-based chemotherapy, rendering it a robust, in vivo platform to explore novel therapeutic approaches targeting RUNX1-mutated CN-AML. Methods: CG-SH cells were generously provided to us by Dr. Reinhold Munker of LSU-Shreveport and cultured in RPMI containing 12% FCS + P/S. For xenograft transplantation studies, CG-SH were injected via tail vein into 8-10 week old NOD/SCID/IL2Rg null (NSG) mice. Engraftment of CG-SH in NSG mice was determined by quantifying the percentage of human CD45+ (hCD45+) cells in bone marrow, spleen, and peripheral blood by flow cytometry. Whole exome sequencing and identification of sequence variations in genes known to be recurrently mutated in AML was done in conjunction with the Genomics Research Center at the University of Rochester Medical Center. Results: In initial experiments, CG-SH were confirmed to harbor the previously reported mutation in exon 8 of RUNX1 (c.1213_1214insCCCC) by Sanger Sequencing and to be cytogenetically normal by conventional karyotyping. Since our goal was to assess response of CG-SH-engrafted NSG mice to AML-like chemotherapy, and mice pre-conditioned with irradiation do not tolerate this therapy, we first tested the ability of CG-SH to engraft non-irradiated NSG mice. We found that tail vein injection of 1e6 CG-SH cells directly from culture into non-irradiated NSG mice resulted in high-level engraftment; at 6 weeks, mean engraftment in bone marrow, spleen, and peripheral blood was 71 +/- 9%, 49 +/- 12%, and 35 +/- 16%, respectively. Since CG-SH cells grow slowly in culture, we wanted to know the fewest number of cells necessary to establish leukemia in non-irradiated NSG mice. Limiting dilution analysis demonstrated that as few as 1000 CG-SH cells were sufficient to establish leukemia in non-irradiated NSG mice. At an equivalent cell dose, CG-SH engraftment levels were approximately 100-fold greater than those of M9-ENL cells, a different leukemia line commonly used for engraftment studies in NSG mice. Given the chemotherapy resistance demonstrated by patients with RUNX1-mutated, CN-AML, we hypothesized that mice xenografted with CG-SH cells would demonstrate residual disease after anthracycline/cytarabine-based chemotherapy. Mice xenografted with CG-SH cells were allowed to develop leukemia and then treated with a 5-day regimen of doxorubicin and cytarabine adapted for NSG mice [Wunderlich et al. Blood 2013; 121(12):e90-e97; PMID: 23349390]. Disease response was assessed 4 days after completion of chemotherapy (Figure 1A). Chemotherapy treatment resulted in eradication of CG-SH cells from the spleen and peripheral blood (data not shown), but persistence of a low-level of disease in the bone marrow (Figure 1B). To better understand the spectrum of AML driver mutations in CG-SH, we conducted whole exome sequencing. In addition to the RUNX1 and NRAS mutations previously reported, we identified mutations in ASXL1, which frequently co-occur with RUNX1 mutations in CN-AML, and CEBPA. Conclusions: Tail vein injection of CG-SH cells directly from culture into non-irradiated NSG mice efficiently generates leukemia in recipient animals that is not eradicated with AML-like chemotherapy. As such, these xenografts are a robust, in vivo platform to explore mechanisms of chemotherapy resistance and novel therapeutic approaches in CN-AML with RUNX1 and ASXL1 mutations. Figure 1A Figure 1A. Figure 1B Figure 1B. Disclosures No relevant conflicts of interest to declare.

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Blastic plasmacytoid dendritic cell neoplasm with skin and bone marrow involvement: Report of three cases

World Journal of Clinical Cases ◽

10.12998/wjcc.v9.i33.10293 ◽

2021 ◽

Vol 9 (33) ◽

pp. 10293-10299

Author(s):

Jiang-Hong Guo ◽

Hong-Wei Zhang ◽

Li Wang ◽

Wei Bai ◽

Jin-Fen Wang

Keyword(s):

Bone Marrow ◽

Dendritic Cell ◽

Plasmacytoid Dendritic Cell ◽

Bone Marrow Involvement ◽

Cell Neoplasm

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The diagnostics of blastic plasmocytoid dendritic cell neoplasm: report of five cases

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2021-20-3-60-67 ◽

2021 ◽

Vol 20 (3) ◽

pp. 60-67

Author(s):

I. A. Demina ◽

S. A. Kashpor ◽

O. I. Illarionova ◽

M. E. Dubrovina ◽

A. A. Dudorova ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Dendritic Cell ◽

Plasmacytoid Dendritic Cell ◽

Skin Lesions ◽

Flow Cytometry Data ◽

Hematological Disorders ◽

Pediatric Hematology ◽

National Medical ◽

Cell Neoplasm

The diagnosis of rare hematological disorders requires a comprehensive clinical and laboratory investigation with careful interpretation of all test results. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is one of such rare entities. We have performed a retrospective analysis of the results of immunophenotyping, cytomorphology and cytogenetics of bone marrow tumor cells from 5 patients with BPDCN aged from 8 to 51 years. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. No specific characteristics of blasts were found. No correlation with the treatment and outcomes was noted as well: 3 patients died of progression or relapse (2 and 1, respectively). Bone marrow immunophenotyping is probably the most valuable laboratory test which allows physicians to establish the proper diagnosis in the absence of skin lesions. Flow cytometry immunophenotyping is the only technique used to determine the antigen profile that enables us to distinguish normal plasmacytoid dendritic cells from tumor ones by the presence (or absence) of the expression of CD2, CD7, CD38, CD56, CD303 etc. In the present paper, we provide a detailed description of five cases of BPDCN and main methods for flow cytometry data analysis. The parents of the patients agreed to use the information, including photos of children, in scientific research and publications.

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