Abstract: Blastic plasmacytoid dendritic cell neoplasm is a clonal disease derived from precursors of plasmacytoid dendritic cells (pDC). It is a rare neoplasm involving the skin which may or may not be associated from the outset with a leukemic component. The disease invariably progresses to aggressive leukemic dissemination, leading to a differential diagnosis with acute leukemia. In 2004, we set up a French network to recruit biological data at diagnosis. Diagnosis was according to recommendations (Swerdlow et al, 2008), with, in addition, a mandatory panel of pDC markers (Garnache-Ottou et al, 2009) detected by flow cytometry or by immunohistochemistry on infiltrated blood, bone marrow or cutaneous lesions. In total, 109 cases of BPDCN were included in 35 hospitals (2000-2013). BPDCN is more prevalent in men (sex ratio 4.4/1) and in elderly subjects (median age: 63 years; 7 patients were 〈 20 yo).S kin lesions are very prevalent (85%) with variable lesion types. Blood cell counts show variable leukocytosis (figure 1A) with presence of blasts in 65% of cases. Anemia and thrombopenia were present in 59% and 76% of cases respectively. Bone marrow aspiration showed blastic infiltration in 94% of cases. Indeed, in 7 cases, there was isolated cutaneous involvement at diagnosis, with neither blood nor bone marrow infiltration. Morphologies of blast cells were heterogeneous. Typical morphologies were the most frequent, including medium-sized cells with a blastic round or irregular nucleus, cytoplasm displayed faint and irregular basophilia and no granulation. In a contingent of the blastic populations, we observed small vacuoles in a peculiar arrangement under the cytoplasmic membrane (42%) or the presence of large pseudopodia (28%) or both (17%) (Figure 1B, C, D). Some cases showed a more immature morphology with larger cells, higher nucleo-cytoplasmic ratio, very visible nucleoli and reinforced basophilia (28%) or a pseudomonoblastic morphology (5%) (Figure 1E). Rare cases presented a pseudolymphocytic form (7%, figure 1F) or large granulations in the cytoplasm (2%). Peroxidase and esterase were negative in all cases. Dysplasia of hematopoietic lineages was observed in 29% (figure 1G). For 8% of patients, myelodysplastic syndrome was diagnosed before the diagnosis of BPDCN. Immunophenotype showed that HLA-DR and CD4 were expressed in all cases, but 4 cases did not express CD56 (confirmed using 3 different antibodies). Expression of markers of others hematopoietic lineages was frequent. Among myeloid markers, the most frequent was CD33 (46%), followed by CD117 (23%), whereas CD13, CD11c, CD15 and CD65 were rarely expressed. Monocyte markers (CD14, CD64, CD11b) and myeloperoxidase were never expressed. For the T lineage, CD2 and CD7 were the most frequent (62% and 58% respectively) whereas CD5 was rare (7%). No cytoplasmic or surface CD3 were detected. For the B lineage, CD22 was expressed in 16%, and low levels of cCD79a in 5%. Both were never expressed together, and no CD19, CD20 and immunoglobulins were found. Generally, we observed one of these antigens (Ags) per case, but in 44% of cases, there was a combination of 2 or 3 Ags from 2 or 3 different lineages. Immature Ags such as CD34 and CD133 were never found, and Tdt was found in 14% of cases. Cytogenetic analysis revealed abnormal caryotype in 65% of the 78 caryotypes evaluated, with 20 cases having a complex caryotype. The frequency of the chromosomal abnormalities involved are shown in Figure 1H. In conclusion, we describe the largest series of BPDCN to date in the literature. Detailed clinical and biological data at presentation allow improved recognition of this rare form of acute and aggressive leukemia, enabling early initiation of appropriate management. Figure 1. A: Blood cell count in 109 BPDCN patients at diagnosis. Bars represent the median. B: Typical BPDCN morphology. C: In this case, the nuclei were peripheral, cytoplasm presented heterogenous basophilia, vacuoles were rare but large pseudopodia are frequent. D: Typical morphology with frequent microvacuoles under the cytoplasmic membrane. E: Immature morphology. F: Pseudolymphocytic morphology. G: Presence of dysplasia in myeloid cells with Auer Rods in the granulocytes. The morphology of the Blastic cells is typical. H. Chromosomal abnormalities in 78 caryotypes evaluated: The histogram represents the number of cases in which each chromosome was involved (deletion, gain, translocations). Figure 1. A: Blood cell count in 109 BPDCN patients at diagnosis. Bars represent the median. B: Typical BPDCN morphology. C: In this case, the nuclei were peripheral, cytoplasm presented heterogenous basophilia, vacuoles were rare but large pseudopodia are frequent. D: Typical morphology with frequent microvacuoles under the cytoplasmic membrane. E: Immature morphology. F: Pseudolymphocytic morphology. G: Presence of dysplasia in myeloid cells with Auer Rods in the granulocytes. The morphology of the Blastic cells is typical. H. Chromosomal abnormalities in 78 caryotypes evaluated: The histogram represents the number of cases in which each chromosome was involved (deletion, gain, translocations). Disclosures Bardet: Celgene: Research Funding. Deconinck:CHUGAI: Other: Travel for international congress; PFIZER: Research Funding; ROCHE: Research Funding; NOVARTIS: Other: Travel for international congress; ALEXION: Other: Travel for international congress; JANSSEN: Other: Travel for international congress; LFB loboratory: Consultancy.

Abstract: LXR activation inhibits BPDCN cell survival through the increase of cholesterol efflux, the inhibition of NF-κB, and IL-3 signaling. Treatment with LXR agonists can be proposed as a new therapeutic approach for BPDCN.

Abstract: Neoplasms involving plasmacytoid dendritic cells (pDC) include blastic pDC neoplasms (BPDCN) and other pDC proliferations, where pDC are associated with myeloid malignancies: most frequently chronic myelomonocytic leukemia (CMML) but also acute myeloid leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDC in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DC (cDC) associated in the same sample, by phenotypic and molecular analyses (targeted next-generation sequencing, 70 genes). We compared 15 pDCAML at diagnosis with 21 BPDCN and 11 normal pDC from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDC (4-36%), monocytes in 14 cases (1-10%) and cDC (two cases, 4.8-19%). pDC in pDC-AML harbor a clearly different phenotype from BPDCN: CD4+ CD56– in 100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a prepDC stage. In all cases, pDC, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite the low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.

Abstract: Oncogenesis and ontogeny of blastic plasmacytoid dendritic cell neoplasm (BPDCN) remain uncertain, between canonical plasmacytoid dendritic cells (pDCs) and AXL+ SIGLEC6+ DCs (AS-DCs). We compared 12 BPDCN to 164 acute leukemia by Affymetrix HG-U133 Plus 2.0 arrays: BPDCN were closer to B-cell acute lymphoblastic leukemia (ALL), with enrichment in pDC, B-cell signatures, vesicular transport, deubiquitination pathways, and AS-DC signatures, but only in some cases. Importantly, 1 T-cell ALL clustered with BPDCN, with compatible morphology, immunophenotype (cCD3+ sCD3− CD123+ cTCL1+ CD304+), and genetics. Many oncogenetic pathways are deregulated in BPDCN compared with normal pDC, such as cell-cycle kinases, and importantly, the transcription factor SOX4, involved in B ontogeny, pDC ontogeny, and cancer cell invasion. High-throughput sequencing (HaloPlex) showed myeloid mutations (TET2, 62%; ASXL1, 46%; ZRSR2, 31%) associated with lymphoid mutations (IKZF1), whereas single-nucleotide polymorphism (SNP) array (Affymetrix SNP array 6.0) revealed frequent losses (mean: 9 per patient) involving key hematological oncogenes (RB1, IKZF1/2/3, ETV6, NR3C1, CDKN2A/B, TP53) and immune response genes (IFNGR, TGFB, CLEC4C, IFNA cluster). Various markers suggest an AS-DC origin, but not in all patients, and some of these abnormalities are related to the leukemogenesis process, such as the 9p deletion, leading to decreased expression of genes encoding type I interferons. In addition, the AS-DC profile is only found in a subgroup of patients. Overall, the cellular ontogenic origin of BPDCN remains to be characterized, and these results highlight the heterogeneity of BPDCN, with a risk of a diagnostic trap.

Abstract: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)–like, acute lymphoid leukemia (ALL)–like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])–like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.

Abstract: Introduction: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy derived from plasmacytoid dendritic cells (pDC). Today, no therapeutic consensus exists and only allogeneic hematopoietic cell transplantation provides a durable remission. BPDCN cells express high levels of the IL-3 receptor alpha chain (CD123) and exhibit an increased survival and growth in response to IL-3 (Chaperot et al. 2001). Recently, aberrant NF-κB activation has been reported in BPDCN (Sapienza et al. 2014), and we identified that liver X receptor (LXR) stimulation inhibits NF-κB activation in non-leukemic pDC. LXR represent nuclear receptors promoting cholesterol efflux via the ATP binding cassette A1 and G1 (ABCA1 and ABCG1, respectively) transporters and lipid acceptors, such as apolipoprotein A1 (ApoA1). This process has been shown to repress IL-3-induced proliferation of murine hematopoietic stem cells (Yvan-Charvet et al. 2010). The aim of this study was to investigate LXR activation in BPDCN, and determine whether LXR agonists possess therapeutic effects. Material and Methods: Cells from 13 BPDCN, 22 undifferentiated AML, 23 ALL patients and 4 normal pDC were evaluated for expression of LXR or cholesterol exchange related genes (lxra, lxrb, abca1, abcg1, apoe, srebp1c, ldlr, vldlr), plus nfkb1 gene by Affymetrix mRNA microarray. BPDCN cells (2 established cell lines [GEN2.2, CAL-1], a primary cell line [BES1] and 5 fresh BPDCN samples) were treated with two LXR agonists (T0901317 [T09] or GW3965 [GW] at 1 µM, 10 µM or 50 µM), NF-κB inhibitors (JSH 23, or Bortezomib [Bort]) and the cholesterol acceptor ApoA1 (10 µg/mL). Gene expression (abca1, abcg1, nfkb1, bcl2, bax and bak1) was assessed by qRT-PCR. Cell death was assessed by Annexin V/7AAD staining (flow cytometry) and caspase activation (Western blot). CAL-1 cell proliferation was assessed by dye dilution (cytometry). Cholesterol efflux was measured directly by H3 cholesterol efflux from cells and indirectly by membrane ABCA1 expression (confocal microscopy). NF-κB and IL-3 signaling pathway activation was explored by protein phosphorylation (phospho-RelA and phospho-STAT5, respectively; [confocal microscopy and Western blot] ). A BPDCN xenograft model was used to evaluate LXR agonist effect in-vivo with blast cell infiltration attested by human CD45/CD123/BDCA4 staining (cytometry). Results: BPDCN cells showed a unique transcriptomic signature with LXR-related gene downregulation. LXR agonist treatment for 24h restored LXR target gene expression, including abca1 and abcg1, and induced a significant concentration-dependent killing (Fig. 1). Moreover, caspase 8 and 9 cleavage associated with bcl2 downregulation and bax and bak1 upregulation (0.78, 8 and 12.2 fold mRNA levels, respectively, for GW 50 µM, 6h, n=3) were observed. At a lower non cytotoxic concentration (10 µM), GW treatment inhibited cell proliferation (-25%, p 〈 0.05, n=7) that was reinforced by addition of ApoA1 (-35%, n=2). This suggests a link with cholesterol efflux supported by induction of ABCA1 expression at BPDCN membrane and increase of H3 cholesterol efflux in response to LXR agonists. LXR agonists inhibited IL-3-induced STAT5 phosphorylation and NF-κB signaling pathway at transcriptional (nfkb1, 0.25 fold mRNA level, p 〈 0.05, n=3 fresh BPDCN samples) and RelA phosphorylation levels. This suggests additional mechanisms by which LXR activation affects BPDCN. NF-kB inhibitor treatment (JSH23, 25 µM, 24h) reduced BPDCN survival (-35%, n=3) and proliferation (-20%, p 〈 0.05, n=3). A synergic effect of NF-κB inhibitors (JSH23 25 µM or Bort 30 nM) and LXR agonists (GW or T09, 10 µM), was observed on cell death and inhibition of proliferation (Fig. 1). The in-vivo study revealed a reduced CAL-1 infiltration in the spleen and bone marrow of T09-treated mice (1.2 mg/2 days for 14 days) compared with control mice (62% vs. 33% and 17% vs. 9%, respectively). Effects of LXR plus NF-κB inhibitor treatment in vivo are under investigation. Conclusion: BPDCN exhibit a specific transcriptomic signature with LXR-related gene downregulation. LXR stimulation of BPDCN restores LXR-related gene expression and inhibits survival and proliferation. This may occur via several mechanisms: inhibition of IL-3 and NF-κB signaling, and/or increased cholesterol efflux. A cytotoxic or cytostatic effect was confirmed in-vivo by reduced BPDCN infiltration. Disclosures No relevant conflicts of interest to declare.

Abstract: Acute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are the basis of refractory/relapsed (R/R) disease in 80% of patients with AML not receiving allogeneic transplantation. Methods In this study, we showed that the interleukin-1 receptor accessory protein (IL-1RAP) protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared with the most common potential AML targets, since it is not expressed by the normal hematopoietic stem cell. After establishing the proof of concept for the efficacy of chimeric antigen receptor (CAR) T-cells targeting IL-1RAP in chronic myeloid leukemia, we hypothesized that third-generation IL-1RAP CAR T-cells could eliminate AML LSCs, where the medical need is not covered. Results We first demonstrated that IL-1RAP CAR T-cells can be produced from AML T-cells at the time of diagnosis and at relapse. In vitro and in vivo, we showed the effectiveness of IL-1RAP CAR T-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous IL-1RAP CAR T-cells against primary cells from patients with AML at diagnosis or at relapse. In patient-derived relapsed AML xenograft models, we confirmed that IL-1RAP CAR T-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen, are cytotoxic against primary AML cells and increased overall survival. Conclusion In conclusion, our preclinical results suggest that IL-1RAP CAR T-based adoptive therapy could be a promising strategy in AML treatment and it warrants the clinical investigation of this CAR T-cell therapy.