Identification and characterization of acute myeloid leukemia stem cells

Ossenkoppele, G.J. [Promotor]Schuurhuis, G.J. [Copromotor]Zweegman, S. [Copromotor

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

The new violet laser dye, Krome Orange, allows an optimal polychromatic immunophenotyping based on CD45-KO gating.

Item does not contain fulltextBACKGROUND: Polychromatic immunophenotyping improves characterization of leukocyte subpopulations and their malignant counterparts. However, the lack of various fluorochrome-labeled monoclonal antibodies (MoAbs) hinders the formation of multi-color panels. CD45 appears to be an important MoAb for immunophenotyping of these cells. Plotted against the side scatter, CD45 provides immunological cell differentiation and the ability to recognize various normal and malignant leukocyte subpopulations. CD45 is commonly used and labeled with various fluorochromes and as a result, is incorporated in multi-color panels as a conjugate of less available fluorochromes, such as the violet laser dyes. However, these dyes (e.g. Pacific Orange/PO) often possess low fluorescence intensity, which may be too weak to differentiate between populations. The new organic dye Krome Orange (KO, emission at 528 nm) appears to be a more intense violet laser dye, serving as an alternative to PO. METHODS: Intensities of CD45 conjugated with FITC, PE, ECD, PE-Cy5, PE-Cy7, PO and KO were tested in different cell sources. Various lineage markers were sequentially back gated on CD45-KO to identify subpopulations. A 10-color MoAb panel for determination of aberrancies in small cell samples was composed to test specificity of CD45-KO. CONCLUSIONS: We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants

Aberrant marker expression patterns on the CD34+CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38- population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38- cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38- population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38- cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38- cells in complete remission, indicating the presence of malignant CD34+CD38- cells. Thus, by identifying residual malignant CD34+CD38- cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy