Impact of scFv on functionality and safety of third generation CD123 CAR T cells.

Chimeric antigen receptor (CAR) T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen. scFv selection should involve safety profiling with evaluation of the efficacy/toxicity balance, especially when the target antigen also is expressed on healthy cells. Here, to assess differences in terms of efficacy and on-target/off-tumor effects, we five different CARs targeting CD123 by substituting only the scFv. In in vitro models, T cells engineered to express three of these five CD123 CARs were effectively cytotoxic on leukemic cells without increasing lysis of monocytes or endothelial cells. Using the IncuCyte® system, we confirmed the low cytotoxicity of CD123 CAR T cells on endothelial cells. Hematotoxicity evaluation using progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR T cells were less cytotoxic on hematopoietic stem cells. Using a humanized mouse model, we confirmed that CD123- cells were not eliminated by the CD123 CAR T cells. Two CD123 CAR T cells reduced tumor infiltration and increased overall survival of mice in three in vivo models of blastic plasmacytoid dendritic cell neoplasm. In an aggressive version of this model, bulk RNA sequencing analysis showed that these CD123 CAR T cells upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Together, these results emphasize the importance of screening different scFvs for the development of CAR constructs to support selection of cells with the optimal risk-benefit ratio for clinical development.

Genetics and Epigenetics in Neoplasms with Plasmacytoid Dendritic Cells.

Plasmacytoid Dendritic Cells (pDC) are type I interferon (IFN)-producing cells that play a key role in immune responses. Two major types of neoplastic counterparts for pDC are now discriminated: Blastic pDC Neoplasm (BPDCN) and Mature pDC Proliferation (MPDCP), associated with myeloid neoplasm. Two types of MPDCP are now better described: Chronic MyeloMonocytic Leukemia with pDC expansion (pDC-CMML) and Acute Myeloid Leukemia with pDC expansion (pDC-AML). Differential diagnosis between pDC-AML and BPDCN is particularly challenging, and genomic features can help for diagnosis. Here, we systematically review the cytogenetic, molecular, and transcriptional characteristics of BPDCN and pDC-AML. BPDCN are characterized by frequent complex karyotypes with recurrent MYB/MYC rearrangements as well as recurrent deletions involving ETV6, IKZF1, RB1, and TP53 loci. Epigenetic and splicing pathways are also particularly mutated, while original processes are dysregulated, such as NF-kB, TCF4, BCL2, and IFN pathways; neutrophil-specific receptors; and cholinergic signaling. In contrast, cytogenetic abnormalities are limited in pDC-AML and are quite similar to other AML. Interestingly, RUNX1 is the most frequently mutated gene (70% of cases). These typical genomic features are of potential interest for diagnosis, and also from a prognostic or therapeutic perspective.

Plasmacytoid Dendritic Cells, a Novel Target in Myeloid Neoplasms.

Plasmacytoid dendritic cells (pDC) are the main type I interferon producing cells in humans and are able to modulate innate and adaptive immune responses. Tumor infiltration by plasmacytoid dendritic cells is already well described and is associated with poor outcomes in cancers due to the tolerogenic activity of pDC. In hematological diseases, Blastic Plasmacytoid Dendritic Cells Neoplasm (BPDCN), aggressive leukemia derived from pDCs, is well described, but little is known about tumor infiltration by mature pDC described in Myeloid Neoplasms (MN). Recently, mature pDC proliferation (MPDCP) has been described as a differential diagnosis of BPDCN associated with acute myeloid leukemia (pDC-AML), myelodysplastic syndrome (pDC-MDS) and chronic myelomonocytic leukemia (pDC-CMML). Tumor cells are myeloid blasts and/or mature myeloid cells from related myeloid disorders and pDC derived from a clonal proliferation. The poor prognosis associated with MPDCP requires a better understanding of pDC biology, MN oncogenesis and immune response. This review provides a comprehensive overview about the biological aspects of pDCs, the description of pDC proliferation in MN, and an insight into putative therapies in pDC-AML regarding personalized medicine.

Transcriptomic and genomic heterogeneity in blastic plasmacytoid dendritic cell neoplasms: from ontogeny to oncogenesis.

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.

BPDCN: When polychemotherapy does not compromise allogeneic CD123 CAR-T cell cytotoxicity.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy with poor prognosis and no treatment consensus. Combining chemotherapy and immunotherapy is a promising strategy to enhance therapeutic effect. Before combining these therapies, the influence of one on the other has to be explored. We set up a model to test the combination of polychemotherapy - named methotrexate, idarubicine, dexamethasone, and L-asparaginase (MIDA) - and CD123 CAR-T cell therapy. We showed that CD123 CAR-T cells exert the same effect on BPDCN models alone, or after MIDA regimen. These data support a preclinical rationale to use immunotherapy after a treatment with polychemotherapy for BPDCN patients.

Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs 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 pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs 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 pre-pDC stage. In all cases, pDCs, 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 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.

CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is associated with a remarkably poor prognosis and with no treatment consensus. The identification of relevant therapeutic targets is challenging. Here, we investigated the immune functions, antileukemia efficacy and safety of CD28/4-1BB CAR T cells targeting CD123 the interleukin (IL)-3 receptor alpha chain which is overexpressed on BPDCN. We demonstrated that both retroviral and lentiviral engineering CD28/4-1BB CD123 CAR T cells exhibit effector functions against BPDCN cells through CD123 antigen recognition and that they efficiently kill BPDCN cell lines and BPDCN-derived PDX cells. In vivo, CD28/4-1BB CD123 CAR T-cell therapy displayed strong efficacy by promoting a decrease of BPDCN blast burden. Furthermore we showed that T cells from BPDCN patient transduced with CD28/4-1BB CD123 CAR successfully eliminate autologous BPDCN blasts in vitro. Finally, we demonstrated in humanized mouse models that these effector CAR T cells exert low or no cytotoxicity against various subsets of normal cells with low CD123 expression, indicating a potentially low on-target/off-tumor toxicity effect. Collectively, our data support the further evaluation for clinical assessment of CD28/4-1BB CD123 CAR T cells in BPDCN neoplasm.

Post-Essential Thrombocythemia Myelofibrosis and Multiple Isodicentric Y Chromosomes: A Unique Case among a Rare Association.

Multiple isodicentric Y chromosomes [idic(Y)] is a rare cytogenetic abnormality, most exclusively described in constitutional karyotypes. Only recently has this entity been reported in hematologic neoplasms such as myeloid disorders, albeit these cases remain very scarce. The possible involvement of increasing copies of potential proto-oncogenes located on the multiple idic(Y) led to consider one of them, CRLF2, as a target for kinase inhibitors. We report here, to our knowledge, the first case of multiple idic(Y) in a patient with myelofibrosis secondary to essential thrombocythemia. The patient received ruxolitinib therapy with initial good clinical response.

How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients?

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.

Early diagnosis of celiac disease in IgA deficient children: contribution of a point-of-care test.

BACKGROUND: The serological diagnosis of celiac disease (CD) often relies on the presence of anti-tissue transglutaminase (tTG) IgA autoantibodies. Patients suffering from selective IgA deficiency (IgAD) are often not aware of their IgA deficiency and are tested as CD negative, delaying considerably the diagnosis. The detection of IgG against deamidated gliadin peptides (DGP) has high specificity and better sensitivity than IgG anti-tTG. A multi-analytic lateral-flow immunochromatographic assay (CD-LFIA) based on the detection of IgA and IgG anti-DGP and total IgA was shown to have a good diagnostic accuracy for CD. The aim of this study was to evaluate the clinical accuracy of its use in children suffering from IgAD. METHODS: 45 IgAD children ranging from 1.1 to 17.4 years and suspected of CD or having high CD risk factors were referred from outpatient clinics located in the area of Rhone-Alpes (France) to the Hospices Civils de Lyon, Paediatric Hospital-Gastroenterology-Hepatology- Nutrition Department for further CD investigations. The CD investigations, including the sample collection, were performed within the Paediatric Hospital-Gastroenterology-Hepatology- Nutrition Department, and the serological testing was performed at the Lyon-Sud Hospital-Immunology Laboratory. The diagnosis of CD was based on IgG anti-tTG serology, biopsy results and patient follow-up. The serum samples were retrospectively tested on the CD-LFIA test. RESULTS: A total of eight (8) patients were diagnosed as new CD. All were correctly identified by the CD-LFIA. The test yielded four (4) false positive results. Two patients with positive IgG anti-tTG were negative on CD-LFIA, but were classified as CD negative based on biopsy results and patient follow-up. The remaining 33 patients were found negative by both methods. The specificity and sensitivity of CD-LFIA was of 89.2% [74.6-97.0] and of 100% [63.1-100] respectively. The negative predictive value (NPV) was of 100% [89.4-100], and the Likelihood Ratio for Negative Test (LR-) was of 0 [0.0-0.91]. CONCLUSIONS: CD-LFIA is a useful, non-invasive and rapid tool to rule out CD in primary care paediatric patients having CD-related symptoms and IgAD. Patients having a positive CD-LFIA result could be then readily directed to secondary care setting for further evaluation by standard serology and biopsy.