Treatment of patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN): focus on the use of tagraxofusp and clinical considerations.

BPDCN is an aggressive myeloid malignancy with a poor prognosis. It derives from the precursors of plasmacytoid dendritic cells and is characterized by CD123 overexpression, which is seen in all patients with BPDCN. The CD123-directed therapy tagraxofusp is the only approved treatment for BPDCN; it was approved in the US as monotherapy for the treatment of patients aged ≥2 years with treatment-naive or relapsed/refractory BPDCN. Herein, we review the available data supporting the utility of tagraxofusp in treating patients with BPDCN. In addition, we present best practices and real-world insights from clinicians in academic and community settings in the US on how they use tagraxofusp to treat BPDCN. Several case studies illustrate the efficacy of tagraxofusp and discuss its safety profile, as well as the prevention, mitigation, and management of anticipated adverse events.

Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity.

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

Developing a mechanistic translational PK/PD model for a trifunctional NK cell engager to predict the first-in-human dose for acute myeloid leukemia.

Natural killer cell engagers (NKCEs), a treatment that stimulates innate immunity, have lately gained attention owing to their favorable safety profile, and their efficacy. Natural killer (NK) cell activation is driven by immune synapse formation between drugs, NK cells, and tumor cells. However, no clear translational modeling approach has been reported for first-in-human (FIH) dose estimation of humanized NKCEs. We developed the first translational mechanistic synapse-driven pharmacokinetic/pharmacodynamic (PK/PD) model for a trifunctional NKp46/CD16a-CD123 (CD123-NKCE) by integrating (i) in vitro target cell cytotoxicity in MOLM-13 tumor cell lines at varying effector-to-tumor cell ratios and incubation intervals; (ii) nonhuman primate PK and profiles of CD123+ cells and NKP46+ NK cells; and (iii) healthy human or patients with acute myeloid leukemia system-specific parameters. To depict direct tumor cell killing by the innate immunity, no transit compartment was included in PK/PD model structures. Model predictions suggested an intrapatient dose escalation of 10/30/100 µg/kg twice weekly to be selected as the starting dose in the FIH trial. However, sensitivity analyses revealed that CD123+ cell growth rate constant and maximal tumor killing rate constant were the key uncertainties to the recommended active dose. This novel translational model structure can be used as the basis to predict clinical PK/PD data for CD123-NKCE, and the translational strategy may serve as a foundation for future advancements of NKCEs.

Tagraxofusp in myeloid malignancies.

Tagraxofusp (or SL-401) is a recombinant molecule composed of human interleukin-3 that binds CD123 on neoplastic cells fused to a truncated diphtheria toxin (DT). Tagraxofusp's most significant success has come from studies involving patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive disease that is usually refractory to conventional chemotherapy. Tagraxofusp had an acceptable safety profile and high efficacy in early phase I/II studies on patients with BPDCN. Another phase II study confirmed the good response rates, resulting in Food and Drugs Administration and European Medicine Agency approval of tagraxofusp for the treatment of BPDCN. Considering its high efficacy and its manageable safety profile, tagraxofusp has been suddenly explored in other myeloid malignancies with high expression of cell surface CD123, both in monotherapy or combination strategies. The triplet tagraxofusp-azacytidine-venetoclax appears to be of particular interest among these combinations. Furthermore, combination strategies may be used to overcome tagraxofusp resistance. The downregulation of DPH1 (diphthamide biosynthesis 1), the enzyme responsible for the conversion of histidine 715 on eEF2 to diphthamide, which is then the direct target of ADP ribosylation DT, is typically associated with this resistance phenomenon. It has been discovered that azacitidine can reverse DHP1 expression and restore sensitivity to tagraxofusp. In conclusion, the success of tagraxofusp in BPDCN paved the way for its application even in other CD123-positive malignancies. Nowadays, several ongoing trials are exploring the use of tagraxofusp in different myeloid neoplasms. This review aims to summarize the actual role of tagraxofusp in BPDCN and other CD123-positive myeloid malignancies.

Tagraxofusp for blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy that presents with characteristic dark purple skin papules, plaques, and tumors, but may also involve the bone marrow, blood, lymph nodes, and central nervous system. The disease, which commonly affects older men but can also present in children, is associated with a distinct immunophenotype including universal expression of CD123, the α chain of the interleukin 3 receptor. Recently, tagraxofusp, a CD123-targeting drug consisting of the ligand for CD123, interleukin 3, conjugated to a truncated diphtheria toxin payload was approved for treatment of BPDCN. This was the first agent specifically approved for BPDCN and the first CD123 targeted agent in oncology. Here, we review the development of tagraxofusp, and the key preclinical insights and clinical data that led to approval. Tagraxofusp treatment is associated with a unique toxicity, capillary leak syndrome (CLS), which can be severe but is manageable with proper patient selection and monitoring, early recognition, and directed intervention. We outline our approach to the use of tagraxofusp and discuss open questions in the treatment of BPDCN. Overall, tagraxofusp represents a unique targeted therapy and a step forward in meeting an unmet need for patients with this rare disease.

Tandem bispecific CD123/CLL-1 CAR-T cells exhibit specific cytolytic effector functions against human acute myeloid leukaemia.

OBJECTIVES: The treatment of refractory and recurrent acute myeloid leukaemia (AML) is still a challenge with poor response rates and short survival times. In an attempt to solve this problem, we constructed a tandem bispecific chimeric antigen receptor (CAR) targeting CD123 and C-type lectin-like molecule 1 (CLL-1), two different AML antigens, and verified its cytotoxic effects in vitro. METHODS: We established and cultured K562 cell lines expressing both CD123 and CLL1 antigens. Single-target CAR-T cells specific to CD123 and CLL1 were engineered, alongside tandem CD123/CLL1 bispecific CAR-T cells. Flow cytometry was used to determine cell phenotypes, transfection efficiencies, cytokine release, and CAR-T-cell proliferation, and an lactate dehydrogenase assay was used to detect the cytotoxicity of CD123/CLL-1 bispecific tandem CAR-T cells in vitro. RESULTS: Two types of tandem CAR-T cells exhibited significant killing effects on CLL-1 + CD123+ leukaemia cell lines and primary AML tumour cells. The killing efficiency of tandem CAR-T cells in the case of single antigen expression is comparable to that of single target CAR-T cells. When faced with dual target tumour cells, dual target CAR-T cells significantly surpass single target CAR-T cells. CD123/CLL-1 CAR-T cells in tandem targeted and killed CD123- and CLL-1-positive leukaemia cell lines and released a large number of cytokines. CONCLUSIONS: CD123/CLL-1 CAR-T cells in tandem can simultaneously target CD123 and CLL-1 on AML cells, demonstrating a significant ability to kill single antigens and multi-target tumour cells. This suggests that CD123/CLL-1 CAR-T cells exhibit significant advantages in the expression of multiple antigens in a wide range of target cells, which may help overcome the challenges posed by tumour heterogeneity and evasion mechanisms.

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy.

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD).

Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with an aggressive clinical course and poor prognosis. BPDCN is most often characterized by its presentation with distinct cutaneous lesions. Bone marrow involvement, lymphadenopathy, splenomegaly, and/or cytopenias are also seen to varying degrees. BPDCN presents with diffuse, monomorphous blasts with irregular nuclei, fine chromatin, and scant, agranular cytoplasm. Expression of CD4, CD56, and CD123 is the hallmark of BPDCN. The presence of ≥4 of CD4, CD56, CD123, TCL1, TCF4, and CD303 is necessary for the diagnosis of BPDCN. Prior to December 2018, management of BPDCN revolved around intensive chemotherapy using acute myeloid leukemia or acute lymphoblastic leukemia regimens. However, responses were transient with poor overall survival (OS). Allogeneic stem cell transplantation (alloSCT) is the only potentially curative treatment for BPDCN. Even so, only a minority of patients are candidates for alloSCT given the preponderance of disease in older individuals. For the few fit patients who are candidates for alloSCT, the aim is to achieve complete remission prior to alloSCT. Tagraxofusp (SL-401), a recombinant fusion protein containing interleukin-3 fused to truncated diphtheria toxin, was the first approved CD123-targeted therapy for BPDCN based on a phase I/II clinical trial showing a 90% overall response rate. It was approved by the FDA on December 21, 2018. Capillary leak syndrome is an important adverse effect of tagraxofusp that requires close monitoring. Several clinical trials are underway to study other regimens for the treatment of BPDCN, including IMGN632 (pivekimab sunirine), venetoclax (alone and in combination with hypomethylating agents), CAR-T cells, and bispecific monoclonal antibodies.

IRF8 may be a useful marker for blastic plasmacytoid dendritic cell neoplasm, especially with weak CD123 expression.

We highlight the utility of interferon regulatory factor 8 (IRF8), a novel marker of monocytic and dendritic cell lineages, in the diagnosis of a case of blastic plasmacytoid dendritic cell neoplasm (BPDCN) presenting initially in the skin. A 60-year-old male with a previous history of myelodysplastic syndrome presented with cutaneous nodules on chest and scalp. A punch biopsy specimen of a skin nodule showed a diffuse dermal infiltrate of atypical mononuclear cells. The neoplastic cells expressed CD4, CD56, CD43, and TdT but showed minimal reaction for TCL-1 and CD123, and were negative for CD34, CD117, and MPO, confounding the diagnosis. IRF8 performed in retrospect was strongly positive. A new punch biopsy specimen of a chest nodule showed the blastoid tumor cells were positive for TCL-1, CD4, and CD56, but dim CD123. Subsequent bone marrow involvement showed blastoid tumor cells with intense positivity for CD123, CD4, and CD56, which was supportive of the BPDCN diagnosis. BPDCN cases with weak or variable CD123 and TCL-1 expression represent a potential diagnostic pitfall. In a recent study, 15 cases of BPDCN showed uniformly strong staining for IRF8, while CD123 was dim or negative in 4 of these 15 cases. We suggest IRF8 may be a useful marker for BPDCN, especially in cases with weak or variable expression of CD123 and TCL1.

ENPP4 and HOXA3 as potential leukaemia stem cell markers in acute myeloid leukaemia.

INTRODUCTION: Acute myeloid leukaemia (AML) is a heterogeneous malignant disease with a high degree of treatment failure using chemotherapy. Leukaemia stem cells (LSCs) are CD34+CD38- early progenitors associated with poor prognosis in AML. A unique LSC phenotype that excludes rare normal haematopoietic stem cells (HSC) is still elusive. This study aimed to determine expression of selected potential LSC markers in normal and leukaemic myeloid cells and correlate prognosis in AML patients. MATERIALS AND METHODS: Flow cytometry and RT-qPCR measured expressions of ALDH, IL3RA/CD123, CLEC12A/CLL-1/CD371, HOXA3 and ENPP4. Normal cord blood (n=3) and blood monocytes (n=5) represented HSC and mature cells, respectively. Myeloid leukaemia cell lines (THP-1, KG-1a, K562 and HL-60) represented progenitor cells at various stages of maturation. AML samples included chemo-resistant (n=8), early relapse (n=2) and late relapse (n=18). RESULTS: Combining protein/gene expressions, CD34+CD38- was a feature of immature cells seen in cord blood, KG-1a, and K562 but not more mature cells (blood monocytes and HL-60). Normal cells expressed CD371 while mature cells (blood monocytes and HL-60) lacked CD123. ENPP4 was not expressed on normal cells while HOXA3 was expressed only on cord blood and THP-1. In AML, CD123, HOXA3, ENPP4 (but not CD371) were significantly increased in the CD34+CD38- fraction of chemo-resistant patients while ALDH was associated with chemo-resistance. CONCLUSION: CD34+CD38- presented an immature phenotype and with ALDH were associated with poor prognosis. CD123, HOXA3 and ENPP4 further enriched the LSC population. ENPP4 has not been reported and has the advantage of not being expressed on HSC and normal monocytes.

Expanding the Immunophenotypic Spectrum of Neoplastic and Reactive Plasmacytoid Dendritic Cells.

OBJECTIVES: Targeted therapies for blastic plasmacytoid dendritic cell neoplasm (BPDCN) have presented a diagnostic dilemma for differentiating residual BPDCN from reactive plasmacytoid dendritic cells (pDCs) because these conditions have a similar immunoprofile, necessitating discovery of additional diagnostic markers. METHODS: Fifty cases of BPDCN involving bone marrow (26/50) and skin (24/50) as well as other hematologic malignancies (67) and nonneoplastic samples (37) were included. Slides were stained using a double-staining protocol for the following immunohistochemical marker combinations: TCF4/CD123, TCF4/CD56, SOX4/CD123, and IRF8/CD123. RESULTS: The nuclear marker SOX4 is expressed in neoplastic pDCs; in our cohort, SOX4/CD123 showed 100% sensitivity and 98% specificity in distinguishing BPDCN from reactive pDCs and other neoplasms. TCF4/CD56 had a 96% sensitivity and 100% specificity for BPDCN. IRF8 is a nonspecific marker that is positive in BPDCN and pDCs as well as other myeloid malignancies. CONCLUSIONS: The novel immunohistochemical combination SOX4/CD123 distinguishes BPDCN, including CD56-negative BPDCN, from both reactive pDCs and other neoplasms. Because of their high diagnostic sensitivity and specificity, the double-staining marker combinations TCF4/CD123, TCF4/CD56, and SOX4/CD123 can be used to confirm lineage in BPDCN cases and detect minimal/measurable residual disease in tissue specimens.

Improving the anti-acute myeloid leukemia activity of CD123-specific engager T cells by MyD88 and CD40 costimulation.

The outcome of patients with acute myeloid leukemia remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors or bispecific T-cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T-cell engagers (CD123.ENG T cells) have anti-acute myeloid leukemia activity. However, like chimeric antigen receptor T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigen-positive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible co-stimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization. CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of a chemical inducer of dimerization, as judged by cytokine production (interferon-γ, interleukin-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved antitumor activity in an acute myeloid leukemia xenograft model. This translated into a significant survival advantage in comparison to that of mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor pathway activation via MyD88 and provision of co-stimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.

The Importance of Toll-like Receptor 9 Expression on Monocytes and Dendritic Cells in the Context of Epstein-Barr Virus Infection in the Immunopathogenesis of Primary Glomerulonephritis.

Toll-like receptor 9 (TLR9) is activated by unmethylated cytosine-phosphate-guanosine (CpG) dinucleotides found in the genomes of pathogens such as Epstein-Barr virus (EBV). The aim of this study was to determine the role of TLR9 in the immunopathogenesis of IgA nephropathy (IgAN) and membranoproliferative glomerulonephritis (MPGN) in the context of Epstein-Barr virus (EBV) infection. For this purpose, the frequency of TLR9-positive monocytes and dendritic cells (DCs, i.e., BDCA-1; myeloid dendritic cells, and BDCA-2; plasmocytoid dendritic cells) was studied, and a quantitative analysis of the concentration of TLR9 in the serum of patients diagnosed with IgAN and MPGN was undertaken. Higher frequencies of TLR9-positive DCs and monocytes in IgAN and MPGN patients were observed as compared with the control group. Patients diagnosed with GN exhibited a higher percentage of BDCA-1+CD19- and BDCA-2+CD123+ DCs than patients in the control group. Moreover, serum TLR9 concentration was shown to be significantly correlated with EBV DNA copy number/µg DNA, IgG, IgM, serum albumin, total protein in 24-h urine collection test and the frequency of BDCA-2+CD123+ DCs in peripheral blood. Our findings confirm that TLR9 may be involved in the development of IgAN and MPGN.

Generation and proof-of-concept for allogeneic CD123 CAR-Delta One T (DOT) cells in acute myeloid leukemia.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory hematological tumors, showing impressive complete remission rates. However, around 50% of the patients relapse before 1-year post-treatment. T-cell 'fitness' is critical to prolong CAR-T persistence and activity. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells; in this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent Vδ1+γδ T cells, represent a promising allogeneic platform. METHODS: Here we generated and preclinically validated, for the first time, 4-1BB-based CAR-DOTs directed against the interleukin-3α chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts. RESULTS: CD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo, even on tumor rechallenge. CONCLUSIONS: Our results provide the proof-of-concept for a DOT-based next-generation allogeneic CAR-T therapy for AML.

CD123 Antagonistic Peptides Assembled with Nanomicelles Act as Monotherapeutics to Combat Refractory Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Due to the development of drug resistance to traditional chemotherapies and high relapse rate, AML still has a low survival rate and there is in an urgent need for better treatment strategies. CD123 is widely expressed by AML cells, also associated with the poor prognosis of AML. In this study, we fabricated nanomicelles loaded with a lab-designed CD123 antagonistic peptide, which were referred to as mPO-6. The antagonistic and therapeutic effects were investigated with CD123+ AML cell lines and a refractory AML mouse (AE and CKITD816V) model. Results show that mPO-6 can specifically bind to the CD123+ AML cells and inhibit the cell viability effectively. Intravenous administration of mPO-6 significantly reduces the percentage of AML cells' infiltration and prolongs the median survival of AML mice. Further, the efficiency of mPO-6 is demonstrated to interfere with the axis of CD123/IL-3 via regulating the activation of STAT5, PI3K/AKT, and NF-κB signaling pathways related to cell proliferation or apoptosis at the level of mRNA and protein in vivo and in vitro. In conclusion, the novel CD123 antagonistic peptide micelle formulation mPO-6 can significantly enhance apoptosis and prolong the survival of AML mice by effectively interfering with the axis of CD123/IL-3 and therefore is a promising therapeutic candidate for the treatment of refractory AML.

Long-Term Benefits of Tagraxofusp for Patients With Blastic Plasmacytoid Dendritic Cell Neoplasm.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive myeloid malignancy. We report long-term results, including data from the continued access phase, of the largest prospective BPDCN trial evaluating the CD123-targeted therapy tagraxofusp (TAG) in adults with treatment-naive and relapsed/refractory BPDCN. The primary outcome was complete response (CR) + clinical CR (CRc: CR with residual skin abnormality not indicative of active disease). Eighty-four (65 treatment-naive and 19 relapsed/refractory) of 89 patients received TAG 12 µg/kg once daily; the median follow-up was 34.0 months. For treatment-naive patients, the overall response rate was 75%; 57% achieved CR + CRc. The median time to remission was 39 (range, 14-131) days, and the median CR + CRc duration was 24.9 (95% CI, 3.8 to not reached) months. Nineteen patients (51%) with CR + CRc were bridged to stem-cell transplant, with a median CR + CRc duration of 22.2 (range, 1.5-57.4) months. Most common adverse events were increased alanine (64%) or aspartate (60%) aminotransferase and hypoalbuminemia (51%); most occurred in cycle 1 and were transient. Capillary leak syndrome occurred in 21% of patients (grade ≥ 3: 7%). In first-line patients with BPDCN, TAG monotherapy resulted in high and durable responses, allowing many to bridge to stem-cell transplant. TAG was generally well-tolerated with a predictable and manageable safety profile.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN): A promising future in the era of targeted therapeutics.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy arising from precursor dendritic cells. BPDCN cells characteristically express several markers on their cell surfaces including CD123, CD4, and CD56. Because of its rarity and challenging clinical presentation, there was no standard of care in managing BPDCN for decades and its prognosis overall was poor. However, as understanding of this rare neoplasm has increased, so have treatment options. The conventional cytotoxic chemotherapy regimens once used in the treatment of BPDCN were modest in their impact on disease relapse until paired with hematopoietic stem cell transplant. Although recent data suggest that there still remains a role for chemotherapeutic agents, targeted modalities have expanded the overall BPDCN treatment landscape. The CD123-targeted agent, tagraxofusp, was the first Food and Drug Administration-approved monotherapy in the treatment of BPDCN. Since its inception, several CD123-targeted and other cell-surface agents have been investigated, with many agents still in the preclinical stages. Although relapsed/refractory disease and central nervous system disease both remain formidable areas of research, there are several promising therapeutic approaches that could have a significant impact on the trajectory of treatment. This review will provide detailed insight on the novel drugs currently in use and those being explored in the management of BPDCN.

Prognostic Impact and Phenotype of Residual Acute Myeloid Leukemia Stem Cells.

BACKGROUND: Leukemia stem cells (LSCs) have been demonstrated to be more therapy-resistant than leukemic blast cells reflecting measurable residual disease (MRD). CD34+CD38- cell frequency is an independent factor for relapse prediction and could therefore be used in the future to improve MRD assessment in acute myeloid leukemia (AML). This protocol is designed to enable accurate and reproducible immunophenotypic detection of measurable residual stem cell disease necessary for proper therapeutic decision and report their prognostic value in AML patients. METHODS: Fifty-four Novo AML adult patients diagnosed in the onco-hematology service of the "20 August 1953" Hospital in Casablanca. We analyzed phenotype and frequency of CD45dim CD34+CD38- cells in bone marrow samples from patients with AML and non-myeloid malignancies using six-color flow cytometry and a simple one-tube essay. RESULTS: For evaluation of leukemic stem cells, our gate strategy was based on the selection of CD34+CD38 - stem cells and leukemia associated immunophenotype approach. Positivity of CD123 or/and aberrant expression of primitive markers CD117 and HLA DR on stem cells discriminate leukemia stem cells from normal hematopoietic stem cells. We reported a statistically significant difference between expressions of primitive markers (CD117 and HLA DR) on leukemic stem cells. In addition, the frequency of LSCs after complete remission in post-induction was persistent in 50% of AML patients. CONCLUSIONS: Overall, we show that CD34+CD38-CD123+ as a basic phenotype, with aberrant phenotype detection of HLA DR and CD117 markers on stem cells, contributes to detecting LSCs which indicates the poor prognosis.

The Expressions of CD30 and CD123 of Mastocytosis in Taiwan.

Mastocytosis is a rare disease with a low incidence in Asia-Pacific populations. CD30 and CD123 may have potential prognostic and therapeutic value, but the results are inconsistent. Because racial disparities may exist, we aim to evaluate the expressions of CD30 and CD123 in a series of mastocytosis cases in Taiwan. Twelve patients with systemic and 7 with cutaneous forms of mastocytosis were studied. The expressions of CD30 and CD123 were correlated with the clinical features of the patients. Eighty-three percent (10/12) of patients with systemic mastocytosis (SM) had an associated hematological neoplasm. Four of the SM patients had both "B" and "C" findings, and they had a median survival time of 0.9 months. CD30 expression was positive in 50% (6/12) of SM cases and 100% (6/6) of cutaneous mastocytosis cases. CD123 was expressed focally or weakly in only 2 SM-associated hematological neoplasm cases. The distribution of mastocytosis subtypes and the expression of CD30 and CD123 in Taiwan differed from those reported in North America and Europe. However, mastocytosis, especially indolent forms, is easily overlooked as its heterogeneous and nonspecific clinical manifestations. A high index of suspicion and improved diagnostic methods can be helpful.

Targeting CD123 in blastic plasmacytoid dendritic cell neoplasm using allogeneic anti-CD123 CAR T cells.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.