Impact of CD123 expression, analyzed by immunohistochemistry, on clinical outcomes in patients with acute myeloid leukemia.

Aberrant expression of the interleukin-3 receptor alpha chain (IL3RA or CD123) is frequently observed in patients with a subset of leukemic disorders, including acute myeloid leukemia (AML), particularly in leukemia stem cells. We analyzed the relationships between immunohistochemical (IHC) expression, including that of CD123, and clinical outcomes. This study involved a retrospective analysis of 48 patients diagnosed with de novo AML (M0-M5, n = 48) at our hospital between February 2008 and September 2015. Among patients with de novo AML, CD123 expression was associated with a failure to achieve complete response (CR) to initial induction chemotherapy (P = 0.044) and poor overall survival (OS) (P = 0.036). This is the first study using IHC to demonstrate that CD123 expression is associated with a poor CR rate and poor OS in de novo AML patients. These results support previous reports using flow cytometry (FCM). CD123 expression may thus be useful for assessing AML patients' prognoses. At the time of diagnosis, CD123 expression analysis using IHC may represent a clinically useful assessment for de novo AML patients.

CD123 expression patterns and selective targeting with a CD123-targeted antibody-drug conjugate (IMGN632) in acute lymphoblastic leukemia.

The potential of CD123-targeted therapies in acute lymphoblastic leukemia/lymphoma remains largely unexplored. We examined CD123 expression levels in a large cohort of patients with acute lymphoblastic leukemia/lymphoma and assessed the in vitro impact of IMGN632, a conjugate of CD123-binding antibody with a novel DNA-alkylating payload. CD123 expression on leukemic blasts was surveyed using multicolor/multiparameter flow cytometry. The in vitro effect of IMGN632 was evaluated on B acute lymphoblastic leukemia/lymphoma cell lines and primary B acute lymphoblastic leukemia/lymphoma blasts. The study cohort (n=213) included 183 patients with B acute lymphoblastic leukemia/lymphoma and 30 with T acute lymphoblastic leukemia/lymphoma. CD123 expression was more prevalent in B acute lymphoblastic leukemia/lymphoma than in T acute lymphoblastic leukemia/lymphoma (164/183, 89.6% versus 13/30, 43.3%; P<0.0001), and within B acute lymphoblastic leukemia/lymphoma CD123 expression was more prevalent in Philadelphia chromosome-positive patients than in Philadelphia chromosome-negative patients (96.6% versus 86.3%; P=0.033). In T acute lymphoblastic leukemia/lymphoma, 12/13 (92.3%) patients with CD123-positive blasts had either early T precursor (ETP) or early non-ETP immunophenotype. IMGN632 was highly cytotoxic to B acute lymphoblastic leukemia/lymphoma cell lines, with half maximal inhibitory concentrations (IC50) between 0.6 and 20 pM. In five of eight patients' samples, low picomolar concentrations of IMGN632 eliminated more than 90% of the B acute lymphoblastic leukemia/lymphoma blast population, sparing normal lymphocytes. In conclusion, CD123 expression is prevalent across acute lymphoblastic leukemia/lymphoma subtypes, and the CD123-targeted antibody-drug conjugate IMGN632 demonstrates promising selective activity in preclinical models of B acute lymphoblastic leukemia/lymphoma.

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping.

BACKGROUND: While it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies. METHODS: CD123 expression was evaluated using standardized EuroFlow immunophenotyping in 139 pediatric AML, 316 adult AML, 193 pediatric BCP-ALL, 69 adult BCP-ALL, 101 pediatric T-ALL, and 28 adult T-ALL patients. Paired diagnosis-relapse samples were available for 57 AML and 19 BCP-ALL patients. Leukemic stem cell (LSC) data was available for 32 pediatric AML patients. CD123 expression was evaluated based on mean fluorescence intensity, median fluorescence intensity, and percentage CD123 positive cells. RESULTS: EuroFlow panels were stable over time and between laboratories. CD123 was expressed in the majority of AML and BCP-ALL patients, but absent in most T-ALL patients. Within AML, CD123 expression was lower in erythroid/megakaryocytic leukemia, higher in NPM1 mutated and FLT3-ITD mutated leukemia, and comparable between LSC and leukemic blasts. Within BCP-ALL, CD123 expression was higher in patients with (high) hyperdiploid karyotypes and the BCR-ABL fusion gene. Interestingly, CD123 expression was increased in BCP-ALL relapses while highly variable in AML relapses (compared to CD123 expression at diagnosis). CONCLUSIONS: Authors evaluated CD123 expression in a large cohort of acute leukemia patients, based on standardized and reproducible methodology. Our results may facilitate stratification of patients most likely to respond to CD123 targeted therapies and serve as reference for CD123 expression (in health and disease). © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and B regulatory cells in patients with Inflammatory Bowel Disease.

The aim of the study was to characterize and to quantify peripheral and tissue. IL-35- and IL-37-producing cells in Inflammatory Bowel Disease (IBD) patients. We studied a total of 38 active UC, 31 inactive UC, 17 active CD, and 13 inactive CD and 50 non-inflamed tissues as control group. Gene expression was measured by real time polymerase chain reaction (RT-PCR) and protein expression was evaluated in tissue by immunohistochemistry and in peripheral blood mononuclear cells by flow cytometry. Higher levels of IL-35 was produced by intestinal regulatory B cells and circulating regulatory CD4(+) and CD8(+) T cells in active vs. inactive disease or healthy donors (P<0.05). The IL-37 was conspicuously synthesized by circulating B cells, active natural killer cells and monocytes. These results suggest that down-regulation of inflammation in active IBD patients might be based on the increased expression of IL-35 and IL-37.

Perspectives for therapeutic targeting of gene mutations in acute myeloid leukaemia with normal cytogenetics.

The acute myeloid leukaemia (AML) genome contains more than 20 driver recurrent mutations. Here, we review the potential for therapeutic targeting of the most common mutations associated with normal cytogenetics AML, focusing on those affecting the FLT3, NPM1 and epigenetic modifier genes (DNMT3A, IDH1/2, TET2). As compared to early compounds, second generation FLT3 inhibitors are more specific and have better pharmacokinetics. They also show higher anti-leukaemic activity, leading to about 50% of composite complete remissions in refractory/relapsed FLT3-internal tandem duplication-mutated AML. However, rapid relapses invariably occur due to various mechanisms of resistance to FLT3 inhibitors. This issue and the best way for using FLT3 inhibitors in combination with other therapeutic modalities are discussed. Potential approaches for therapeutic targeting of NPM1-mutated AML include: (i) reverting the aberrant nuclear export of NPM1 mutant using exportin-1 inhibitors; (ii) disruption of the nucleolus with drugs blocking the oligomerization of wild-type nucleophosmin or inducing nucleolar stress; and (iii) immunotherapeutic targeting of highly expressed CD33 and IL3RA (CD123) antigens. Finally, we discuss the role of demethylating agents (decitabine and azacitidine) and IDH1/2 inhibitors in the treatment of AML patients carrying mutations of genes (DNMT3A, IDH1/2 and TET2) involved in the epigenetic regulation of transcription.

Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia.

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

Expression of CD90, CD96, CD117, and CD123 on different hematopoietic cell populations from pediatric patients with acute myeloid leukemia.

BACKGROUND AND AIMS: In trying to contribute to our knowledge on the biology of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) from pediatric acute myeloid leukemia (AML), in the present study we analyzed the expression of four cell surface antigens relevant to human hematopoiesis-CD90, CD96, CD117, and CD123-in bone marrow from pediatric AML patients and normal control subjects. METHODS: CD34(+) CD38(-) cells (enriched for HSC) and CD34(+) CD38(+) cells (enriched for HPC) were resolved on the basis of CD34 and CD38 expression. Concomitantly, expression of CD90 and CD96 or CD117 and CD123 was assessed by multicolor flow cytometry in each cell population. RESULTS: CD90 and CD117 were expressed in a low proportion of CD34(+) CD38(-) and CD34(+) CD38(+) cells and no significant differences were observed between normal marrow and AML at diagnosis. In contrast, CD96(+) cells and CD123(+) cells were found at significantly higher levels in both cell populations from AML at diagnosis, as compared to normal marrow. Levels of both cell surface markers after treatment remained higher than in normal marrow. DISCUSSION: These results show an increased frequency of CD96(+) and CD123(+) cells within the CD34(+) cell population from pediatric AML; this is consistent with the findings reported previously for adult AML. Our study supports the notion that expression of such antigens should be explored for their use as markers for diagnosis and prognosis.

IL-3 promotes osteoblast differentiation and bone formation in human mesenchymal stem cells.

IL-3 is an important cytokine that regulates hematopoiesis. We have previously demonstrated that IL-3 is a potent inhibitor of osteoclastogenesis and bone resorption. In the present study, we have investigated the role of IL-3 on human osteoblast differentiation and bone formation. We found that IL-3 in a dose-dependent manner increases osteoblast differentiation and matrix mineralization in human mesenchymal stem cells (MSCs). IL-3 significantly enhances the expression of osteoblast specific genes such as alkaline phosphatase, collagen type-I, osteocalcin and osteopontin; and Runx-2 and osterix transcription factors. Moreover, IL-3 induces the expression of bone morphogenetic protein-2 (BMP-2), and activates smad1/5/8. IL-3 enhances osteoblast differentiation and BMP-2 secretion through JAK/STAT pathway. Interestingly, IL-3 promotes in vivo bone regeneration ability of MSCs. Thus, we reveal for the first time that IL-3 enhances human osteoblast differentiation and bone formation in both in vitro and in vivo conditions, and suggest its therapeutic potential for bone formation in important bone diseases.

Human basophils express amphiregulin in response to T cell-derived IL-3.

BACKGROUND: Amphiregulin, a member of the epidermal growth factor family, is expressed by activated mouse T(H)2 cells. Amphiregulin produced by mouse hematopoietic cells contributes to the elimination of a nematode infection by a type 2 effector response. OBJECTIVE: To identify the human peripheral blood cell population expressing amphiregulin. METHODS: Amphiregulin-expressing cells were identified by flow cytometry of cell surface markers and histologic staining. Histamine and amphiregulin in supernatants were measured by enzyme immunoassay. Quantitative real-time PCR was used to measure mRNA expression. RESULTS: Stimulation of human PBMCs by anti-CD3 + anti-CD28 antibodies induced expression of amphiregulin mRNA and protein by a non-T-cell population. The amphiregulin-producing cells were basophils, as judged by morphology and expression of CD203c and CD123 (IL-3 receptor α chain). Activated mouse basophils also produced amphiregulin. Amphiregulin expression by basophils in response to anti-TCR stimulation required IL-3 produced by T cells, and IL-3 alone induced high levels of amphiregulin expression by purified basophils. Amphiregulin was expressed at much higher levels when human basophils were stimulated by IL-3 than by IgE cross-linking, whereas the opposite was true for IL-4 expression and histamine release. Heparin-binding epidermal growth factor-like growth factor was also expressed by IL-3-stimulated human basophils. PBMCs from human subjects with asthma contained significantly higher numbers of basophils able to produce amphiregulin compared with controls with or without allergy. CONCLUSION: IL-3 can induce basophils to express high levels of amphiregulin, which may contribute to tissue remodeling during type 2 immune responses such as asthma.

Overexpression of CD123 correlates with the hyperdiploid genotype in acute lymphoblastic leukemia.

We evaluated CD123 expression in 95 pediatric and 24 adult ALL patients and compared the results with the CD123 expression in normal B-cell precursors. Early B-cell precursors were negative while intermediate precursors and mature B cells showed weak CD123 expression. Leukemic blasts in 31% of precursor-B ALL samples exhibited strong expression of CD123, 61% had moderate CD123 expression and 8% were negative; 81.5% of ALL with hyperdiploid karyotype (>/= 52 chromosomes) showed strong CD123 overexpression. In contrast, cases with ETV6/RUNX1 rearrangement had weak CD123 expression. Our study suggests that overexpression of CD123 is an aberrant phenotype present in a subset of precursor-B ALL with hyperdiploid genotype, and represents an additional marker of good prognosis in pediatric precursor-B ALL. Moreover, aberrant CD123 expression in ALL is a good marker for monitoring of minimal residual disease.

Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia.

In acute myeloid leukemia (AML), apart from the CD34(+)CD38(-) compartment, the side population (SP) compartment contains leukemic stem cells (LSCs). We have previously shown that CD34(+)CD38(-) LSCs can be identified using stem cell-associated cell surface markers, including C-type lectin-like molecule-1 (CLL-1), and lineage markers, such as CD7, CD19, and CD56. A similar study was performed for AML SP to further characterize the SP cells with the aim of narrowing down the putatively very low stem cell fraction. Fluorescence-activated cell sorting (FACS) analysis of 48 bone marrow and peripheral blood samples at diagnosis showed SP cells in 41 of 48 cases that were partly or completely positive for the markers, including CD123. SP cells in normal bone marrow (NBM) were completely negative for markers, except CD123. Further analysis revealed that the SP fraction contains different subpopulations: (a) three small lymphoid subpopulations (with T-, B-, or natural killer-cell markers); (b) a differentiated myeloid population with high forward scatter (FSC(high)) and high sideward scatter (SSC(high)), high CD38 expression, and usually with aberrant marker expression; (c) a more primitive FSC(low)/SSC(low), CD38(low), marker-negative myeloid fraction; and (d) a more primitive FSC(low)/SSC(low), CD38(low), marker-positive myeloid fraction. NBM contained the first three populations, although the aberrant markers were absent in the second population. Suspension culture assay showed that FSC(low)/SSC(low) SP cells were highly enriched for primitive cells. Fluorescence in situ hybridization (FISH) analyses showed that cytogenetically abnormal colonies originated from sorted marker positive cells, whereas the cytogenetically normal colonies originated from sorted marker-negative cells. In conclusion, AML SP cells could be discriminated from normal SP cells at diagnosis on the basis of expression of CLL-1 and lineage markers. This reveals the presence of a low-frequency (median, 0.0016%) SP subfraction as a likely candidate to be enriched for leukemia stem cells.

Identification of genes with abnormal expression changes in acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the most common and deadly forms of hematopoietic malignancies. We hypothesized that microarray studies could identify previously unrecognized expression changes that occur only in AML blasts. We were particularly interested in those genes with increased expression in AML, believing that these genes may be potential therapeutic targets. To test this hypothesis, we compared gene expression profiles between normal hematopoietic cells from 38 healthy donors and leukemic blasts from 26 AML patients. Normal hematopoietic samples included CD34+ selected cells (N = 18), unselected bone marrows (N = 10), and unselected peripheral bloods (N = 10). Twenty genes displayed AML-specific expression changes that were not found in the normal hematopoietic cells. Subsequent analyses using microarray data from 285 additional AML patients confirmed expression changes for 13 of the 20 genes. Seven genes (BIK, CCNA1, FUT4, IL3RA, HOMER3, JAG1, WT1) displayed increased expression in AML, while 6 genes (ALDHA1A, PELO, PLXNC1, PRUNE, SERPINB9, TRIB2) displayed decreased expression. Quantitative RT/PCR studies for the 7 over-expressed genes were performed in an independent set of 9 normal and 21 pediatric AML samples. All 7 over-expressed genes displayed an increased expression in the AML samples compared to normals. Three of the 7 over-expressed genes (WT1, CCNA1, and IL3RA) have already been linked to leukemogenesis and/or AML prognosis, while little is known about the role of the other 4 over-expressed genes in AML. Future studies will determine their potential role in leukemogenesis and their clinical significance.

Distribution of dendritic cell subtypes in primary oral squamous cell carcinoma is inconsistent with a functional response.

Dendritic cell (DC) function will likely be important for immunological cancer therapies, but our knowledge of the roles of DCs in tumors is limited, in part because most studies were performed before DC subtypes were defined. We studied the distributions of immature epidermal, immature dermal, mature, and plasmacytoid DCs in 63 primary tumors and eight lymph node metastases from oral squamous cell carcinoma patients without evidence of distant metastasis. Immature CD207/Langerin+ and CD209/DC-SIGN+ DCs were present in the primary tumor region, but CD209/DC-SIGN+ cells rarely infiltrated the tumor. Mature DCs were rare, and presence of CD123+ plasmacytoid DCs was associated with poorer outcome. Unexpectedly, migration and maturation of DCs was associated with a worse outcome. Overall, the distribution of DC subtypes indicated that ineffective DC response to tumor is a failure of DC function rather than recruitment, suggesting that a strategy of shifting the balance of secreted factors in the tumor milieu may be more effective in restoring anti-tumor immune function than current methods restoring only one population of DCs to the immunosuppressive tumor region.