Immunoprofiling of leukemic stem cells CD34+/CD38−/CD123+ delineate FLT3/ITD-positive clones

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous clonal disorder presenting with accumulation of proliferating undifferentiated blasts. Xenograft transplantation studies have demonstrated a rare population of leukemia-initiating cells called leukemic stem cells (LSCs) capable of propagating leukemia that are enriched in the CD34+/CD38− fraction. LSCs are quiescent, resistant to chemotherapy and likely responsible for relapse and therefore represent an ideal target for effective therapy. LSCs are reported to overexpress the alpha subunit of the IL-3 receptor (CD123) compared to normal CD34+/CD38− hematopoietic stem cells. It has not been demonstrated whether CD123-positive (CD34+/CD38−) subpopulation is enriched for any clonal markers of AML or any LSC properties. The aims of this study were to investigate whether FMS-like tyrosine kinase (FLT3)/internal tandem duplication (ITD) mutations are present at LSC level and whether FLT3/ITD mutation is confined to LSC as defined by CD34+/CD38−/CD123+ and not CD34+/CD38−/CD123−. Methods Thirty-four AML cases were analyzed by five-color flow cytometry and sequential gating strategy to characterize of CD34+/CD38−/CD123+ cells. These cells were sorted, analyzed by PCR, and sequenced for FLT3/ITD. Results In this study, we confirm significant expression of CD123 in 32/34 cases in the total blast population (median expression = 86 %). CD123 was also expressed in the CD34+/CD38− cells (96 ± 2 % positive) from 28/32 for CD123+ AML. CD123 was not expressed/low in normal bone marrow CD34+/CD38− cells (median expression = 0 %, range (0–.004 %). AML samples were tested for FLT3/ITD (10 positive/25). FLT3/ITD+ AML cases were sorted into two putative LSC populations according to the expression of CD123 and analyzed for FLT3/ITD again in the stem cell fractions CD34+/CD38−/CD123+ and CD34+/CD38−/CD123−. Interestingly, FLT3/ITD was only detected in CD34+/CD38−/CD123+ (7/7) and not in CD34+/CD38−/CD123− subpopulation (6/7). Conclusions This finding shows that FLT3/ITD are present at LSC level and may be a primary and not secondary event in leukemogenesis, and the oncogenic events of FLT3/ITD happen at a cell stage possessing CD123. It shows that CD123 immunoprofiling provides further delineation of FLT3+ LSC clone. This novel finding provides a rationale for treatment involving CD123-targeting antibodies with intracellular FLT3 inhibitors directed against CD34+/CD38−/CD123+. This may result in more effective anti-LSC eradication

Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)

Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations

Fludarabine, Cytarabine, Granulocyte Colony-Stimulating Factor, and Idarubicin With Gemtuzumab Ozogamicin Improves Event-Free Survival in Younger Patients With Newly Diagnosed AML and Overall Survival in Patients With NPM1 and FLT3 Mutations.

PURPOSE: To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. PATIENTS AND METHODS: One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). RESULTS: There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. CONCLUSION: Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit

Biomarkers in nasopharyngeal carcinoma and ionizing radiation

© Springer Science+Business Media Dordrecht 2015.Cancer of the nasopharynx is an epithelial cancer developed in the retro-nasal area in the nasal cavity. Nasopharyngeal carcinoma (NPC) is prevalent in the endemic regions including Southern China and Southeast Asia. The primary tumor of NPC is small and usually causes no symptoms. Patients often present with bilateral glands enlargement and local/regional lymph node metastasis leading to poor prognosis and local control rate. Therefore, there is a desperate need to develop early diagnosis method to improve the treatment outcome. Detection of EBV-derived biomarkers in tissues and EBV DNA in the peripheral blood of NPC patients opens up the possibility to monitor the disease using molecular markers with high sensitivity. Among the biomarkers in tissue, in situ hybridization for EBER remains to be the most efficient and reliable way because EBER is the most abundantly expressed viral transcript. In addition to diagnosis value, EBER, EBNA1, and LMP1 levels in tissue after radiation therapy could serve as biomarkers to evaluate response to radiotherapy. In light of the detection of EBV DNA in plasma, serum, nasal brush, and saliva, EBV DNA is particularly useful for periodic monitoring of NPC patients. A systematic review has revealed that circulating EBV DNA could be applied as noninvasive diagnostic biomarker for NPC. Moreover, the close association of circulating EBV DNA with the clinical outcomes of treatment provided new tool to predict the treatment outcome. Nevertheless, circulating EBV DNA was not detectable in all the undifferentiated NPC patients, and EBV latent infection was not associated with WHO-1 NPC, which limits the utility of EBV DNA. Further studies are warranted to identify complementary biomarkers originating from the human cancer cells to overcome the limitation of EBV-based biomarkers in NPC screening and monitoring treatment outcome.Link_to_subscribed_fulltex