Brooks, Christopher

Abstract Background: CD123 is the α-subunit of the high-affinity receptor for interleukin-3; its expression in normal bone marrow (BM) is highly restricted, however CD123 is highly expressed in certain hematolymphoid malignancies. In a pilot study of 11 systemic mastocytosis (SM) patients (Lasho et al., Leukemia, 2015), we made the following novel observations (i) CD123 was aberrantly expressed on neoplastic mast cells (MC); and (ii) there was focal proliferation of plasmacytoid dendritic cells (PDC) around MC aggregates, suggesting a tumor promoting role for PDC. Objectives: To study MC CD123 expression by IHC in a larger cohort of SM patients to ascertain: (i) whether there is a significant difference in CD123 expression between SM subtypes; (ii) whether focal PDC proliferation around MC aggregates is correlated with CD123 expression; and (iii) whether CD123 expression has prognostic value for overall survival (OS). Methods: The current study was approved by the Mayo Clinic institutional review board. SM was classified as per standard criteria. Archived BM slides were stained for tryptase and CD123 as previously described (Lasho et al., Leukemia, 2015). Slides were reviewed independently by 2 hematopathologists who were blinded with regards to SM subgroups and scored on the following pre-defined parameters: (i) CD123 expression yes/no; (ii) CD123 expression strong/weak; (iii) focal PDC proliferation yes/no; and (iv) BM MC%. Results: We studied 58 SM patients and 3 controls. Of the former, 23 had indolent SM (ISM), 10 aggressive SM (ASM), 23 SM with associated hematological disease (SM-AHD), and 2 had mast cell leukemia (MCL). Of the SM-AHD patients, 10 (43%) had CMML, 5 (22%) myeloproliferative neoplasm-unclassified (MPN-u), 3 (13%) PV, 2 (9%) acute myeloid leukemia, and 1 each had lymphoma, myeloma and myelodysplastic syndrome. The median follow up from diagnosis was 32 months (range 0.1 to 162). Over this period, 3 patients (5%) had leukemic transformation and 30 patients (52%) died. Concordance rates between hematopathologists for CD123 positive/negative, CD123 strong/weak, focal PDC proliferation yes/no and MC% were 97%, 94%, 98% and 100%, respectively. MC CD123 expression was absent in control cases but demonstrable in 37 (64%) SM patients; expression rates were 100%, 61%, 57%, and 0% in ASM, ISM, SM-AHD and MCL, respectively (p=0.02). Similar to MCL, both cases with SM-AML showed absent MC CD123 expression. Strong MC CD123 expression (comparable to background interstitial PDC) was observed in 23 patients (62%), including 85% with SM-AHD, 60% with ASM, and 43% with ISM (p=0.08). Focal PDC proliferation around MC aggregates, regardless of CD123 expression status, was noted in 44 patients (76%), including 90% with ASM, 87% with ISM, 65% with SM-AHD and 0% with MCL (p=0.02). Focal PDC proliferation was significantly higher in CD123-positive versus -negative patients (87% versus 50%, p=0.005). OS was predicted by SM subtype; the median OS for ISM, ASM, SM-AHD and MCL patients was not reached, 30 months, 25 months and 3 months, respectively (p=0.005). CD123 expression status did not have prognostic value for OS overall, however, in SM-AHD patients, CD123 expression was associated with significantly inferior survival (p=0.04), independent of the associated disease (ie, CMML vs AML vs MPN vs other). Survival outcome of SM-CMML patients expressing MC CD123 (n=3, median OS=11 months) was significantly inferior as compared to patients who did not (n=7, median OS=44 months), independent of age and absolute monocyte count (p=0.01). CD123 staining intensity had borderline significance for OS in the overall cohort (p=0.08). When restricted to non-indolent SM cases, strong CD123 expression was associated with significantly worse overall survival (15 versus 88 months, p=0.04). Conclusions: In this cohort, aberrant MC CD123 expression was observed in the majority of SM patients with highest prevalence in ASM patients and uniformly absent expression in leukemic phase SM (MCL or SM-AML). Focal PDC proliferation around MC aggregates was significantly higher in CD123-positive cases, indicating a potential functional interaction. CD123 expression identified SM-AHD patients with inferior OS, especially those with SM-CMML. Strong CD123 staining intensity may be associated with inferior OS in non-indolent SM patients. CD123 expression on MC and PDC constitutes a novel therapeutic target in non-leukemic SM. Disclosures Pardanani: Stemline: Research Funding. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Abstract Background: CD123 (alpha chain of the interleukin-3 receptor [IL-3RA]) heterodimerizes with CD131 (βc, common beta chain) to constitute the high-affinity receptor for IL-3. CD123 is not expressed on hematopoietic stem/progenitor cells from normal bone marrow (BM). In contrast, CD123 is highly expressed in CD34+/CD38- cells from acute myeloid leukemia (AML) patients, which recapitulate the leukemic phenotype in NOD/SCID mice; CD123, therefore, is a marker for leukemic stem cells in AML (Jordan CT et al., Leukemia 2000). CD123 is also highly expressed on CD4+/56+ leukemic cells in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematodermic neoplasm. The validity of CD123 as a rational therapeutic target was illustrated by clinical efficacy data for SL-401, a biologic target therapy directed to IL-3R, in patients with BPDCN and AML, respectively (Frankel AE et al., Blood 2014 andFrankel AE et al., J Clin Oncol 31, 2013 (suppl; abstr 7029)). Objectives: To study CD123 expression as a potential therapeutic target in myeloproliferative neoplasms (MPN). Methods: The study was approved by our institutional review board and patients provided written consent for sample collection. MPN diagnosis was based on WHO 2008 criteria. Surface antigen expression on hematopoietic cells of various lineages was interrogated using the 4-color multiparametric flow cytometer, FACSCaliber TM (BD Biosciences, San Jose, CA). Data analysis was performed using CellQuest Pro Software (BD Biosciences). Results: We studied a total of 21 MPN patients and 3 normal controls. Of the former, 14 patients had systemic mastocytosis (SM) (indolent SM=9, SM with associated myeloid neoplasm=4 and aggressive SM=1), 6 had primary myelofibrosis (PMF) and 1 had eosinophilic leukemia transformed to AML. Normal controls: Three normal BM samples were studied; the number of CD123 positive cells in the total population was <1%. Rare CD34+/CD38- cells were uniformly CD123-. Of the CD123+ cells, only a minor subset (<10%) coexpressed myeloid/granulocyte lineage (CD13+, CD15+ or CD16+) or monocyte/macrophage lineage (CD14+ or CD11b+) markers. Systemic mastocytosis: The data were informative for 6 patients (3 with peripheral blood [PB], 1 with BM, and 2 with paired PB and BM samples) for whom a sufficient number of mast cells (MC) were identified for immunophenotyping. Neoplastic MC were defined as CD117 hi/SSC hi/CD45 hi/CD34-/CD25+/FcεRI+. CD123 expression was seen in the majority of MC for 4 patients (#1-4, CD123 percentage positive, PB/BM): 91%/n.a. (#1), 75%/81% (#2), 84%/n.a. (#3), and 88%/n.a. (#4). In contrast, one patient (#5) had a minority of CD123+ MC (33%/28%). Clonal eosinophilia: PB was studied; the WBC differential count showed 41% leukemic blasts and 57% eosinophils (both confirmed to be clonally related based on FISH studies). Both cell populations were predominantly CD123+ (≥90%). Primary myelofibrosis: We studied PB samples from 6 PMF patients. Approximately 1-2% of circulating cells marked as CD123+; of these, expression was notable on CD34-/CD38+ cells (median 54%; range 41-74% positive). We found a larger proportion (30-50%) of CD123+ cells that coexpressed CD13+, CD16+ or CD11b+ representing monocytes, immature myeloid cells and granulocytes, as compared to normal BM controls. Studies on BM samples from PMF patients are ongoing. Conclusions: CD123 (IL-3RA) is expressed on relevant primary cells of interest in select MPNs, namely neoplastic mast cells in SM patients and eosinophils in clonal eosinophilia patients. In PMF, a minor population of circulating cells was CD123+ with a biased distribution on myeloid lineage cells as compared to normal BM samples. We are currently studying CD123 expression in additional MPN patients, and also analyzing CD123 expression on BM trephine biopsies by immunohistochemistry. In addition, the cytotoxicity assessment of SL-401 against MPN cell lines and primary cells is ongoing. Overall, the aforementioned data support the clinical development of SL-401 in patients with MPNs and clinical trials are currently being planned. Disclosures Brooks: Stemline Therapeutics: Employment, Equity Ownership. Pardanani:Stemline Therapeutics Inc.: Research Funding.