Abstract: The exact disease state of chronic myeloid leukemia (CML) patients in deep molecular remission is unknown, since even the most sensitive quantitative polymerase chain reaction (qPCR) methods cannot identify the patients who are prone to relapse after treatment withdrawal. Consequently, new strategies are needed to unravel the true disease state in these patients. Through evaluation of FACS sorted stem- and progenitor cells with standard qPCR and interphase fluorescence in situ hybridization (iFISH) assays, we here demonstrate that residual disease can be assessed at the stem cell level in patients with 4-log reduction in BCR-ABL expression (MR4). Fresh bone marrow aspirates were acquired from 17 CML patients treated with tyrosine kinase inhibitors (TKIs) for a period of 12-119 months. Samples were enriched for CD34+ cells, stained with fluorescent monoclonal antibodies and FACS sorted. In the sort setup we included the human myeloid inhibitory C-type lectin-like receptor (hMICL) antigen (van Rhenen et al Blood 2007, Larsen et al Cytometry 2012), which identifies granulocyte-macrophage committed progenitors. Sorted subsets were: Hematopoietic stem cells (HSCs, CD34+CD38-), hMICL+ progenitors (MpP; CD34+CD38+hMICL+), and hMICL- progenitors (MnP; CD34+CD38+hMICL-). Sorted cells were evaluated for their content of Philadelphia positive (Ph+) cells using both iFISH and a sensitive qPCR assay optimized to small cell samples. Eleven of the 17 studied patients had attained MR4 for a median of 30 months (range: 11 – 88), with the remainder having achieved major molecular response (MMR). In the sorted CD34+ subsets (fig 1A) our sensitive qPCR assay only detected BCR-ABL expression in 10/48 (21%) subsets (fig 1B), despite a median input of 85,000 cells (range: 600 – 370,000). Considering these data, we were surprised to find that iFISH detected Ph+ cells in 14/34 (41%) subsets in frequencies ranging from 2% to 14%, and most frequently in the HSC fraction (fig 1C). Taken together, iFISH proved significantly better than qPCR in detecting residual Ph+ cells in the sorted fractions (p=0.038), especially within the HSC fraction (p=0.005) (fig 1D). Moreover, in subsets where both methods were applied, all qPCR positive subsets were also iFISH positive (4/33) while 9/33 samples were qPCR-/iFISH+, including all positive samples from MR4 patients (fig 1E).Figure 1Detection of residual Ph+ cells in FACS sorted CD34+ cell subpopulations in CML patients in MMR and MR4. (A) FACS sorting of HSCs, MpPs, and MnPs. (B) Expression of BCR-ABL mRNA (on the International Scale (IS)) in the sorted CD34+ subsets. Error bars denote the median upper boundary of BCR-ABL expression (± 95% confidence interval). (C) Residual Ph+ cells as detected by iFISH in the sorted CD34+ subsets. (D) Detection of residual Ph+ cells in sorted subsets by iFISH and mRNA-based qPCR. Error bars denote SEM. (E) Combined qPCR and iFISH status of samples analyzed by both modalities.Figure 1. Detection of residual Ph+ cells in FACS sorted CD34+ cell subpopulations in CML patients in MMR and MR4. (A) FACS sorting of HSCs, MpPs, and MnPs. (B) Expression of BCR-ABL mRNA (on the International Scale (IS)) in the sorted CD34+ subsets. Error bars denote the median upper boundary of BCR-ABL expression (± 95% confidence interval). (C) Residual Ph+ cells as detected by iFISH in the sorted CD34+ subsets. (D) Detection of residual Ph+ cells in sorted subsets by iFISH and mRNA-based qPCR. Error bars denote SEM. (E) Combined qPCR and iFISH status of samples analyzed by both modalities. Combining the data from all sorted subsets within the individual patients (fig 2), iFISH detected Ph+ cells in at least one CD34+ fraction in 9/10 patients, while qPCR only found 5/15 positive (p=0.013). Moreover, qPCR positivity was significantly less frequent in MR4 patients (1/10) compared to MMR patients (4/5, p=0.017), while iFISH positivity was equally frequent (p=1.0), thus indicating that low BCR-ABL producing Ph+ cells may be even more predominant in the MR4 patients.Figure 2Detection of residual disease in the CD34- and in at least one of the analyzed CD34+ fractions of the individual patients as assessed by both iFISH and mRNA-based qPCR.Figure 2. Detection of residual disease in the CD34- and in at least one of the analyzed CD34+ fractions of the individual patients as assessed by both iFISH and mRNA-based qPCR. To our knowledge, we are the first to directly demonstrate the discrepancy between DNA- and mRNA-based methods when assessing persisting Ph+ stem- and progenitor cells in patients with MR4. Pre-selection of CD34+ stem- and progenitor cells unmasked residual disease, and enabled detection of Ph+ cells by iFISH in almost all analyzed patients, including 5/6 of patients with MR4. Our results show that the residual Ph+ cells in CML patients with deep molecular response are low BCR-ABL producers, and that DNA-based methods are required to define the next level of response beyond the achievement of undetectable BCR-ABL mRNA. This approach demonstrates that residual disease can be assessed at the stem cell level in CML patients with MR4, and may provide us with a tool for early and safe identification of candidates for TKI withdrawal. Disclosures: Hokland: Novartis: Research Funding.

Abstract: Although chronic myeloid leukemia (CML) originates from a primitive hematopoietic stem cell (HSC), it is the more differentiated progenitor cells that drive the expansion of the malignant clone. In addition, previous studies of chronic phase CML have shown that, despite the marked leukocytosis observed here, megakaryocyte-erythroid progenitors dominate the progenitor fraction. We sought to elucidate this by employing the new marker for leukemic stem cells, the human myeloid inhibitory C-type lectin-like receptor (hMICL), in the study of progenitor cell expansion in CML. Bone marrow or peripheral blood stem cells were acquired from 11 normal donors and 31 CML patients at diagnosis in chronic phase and/or after 3-119 months of tyrosine kinase inhibitor (TKI) treatment. Cells were stained with fluorescent monoclonal antibodies and FACS sorted into HSCs (CD34+CD38-), hMICL+ progenitors (MpP; CD34+CD38+hMICL+), and hMICL- progenitors (MnP; CD34+CD38+hMICL-). Sorted cell subsets were subjected to growth in a 14-day methylcellulose assay and analyzed quantitatively for expression of the BCR-ABL fusion transcript. In normal donors, hMICL expression reproducibly identified a well-defined subpopulation of the CD34+CD38+ cells (fig 1A). The MpPs were highly enriched for cells of granulocyte-macrophage progenitors (GMP) phenotype compared to the MnPs (p=0.012) (fig 1B). Sorted MpPs produced almost exclusively granulocyte and/or macrophage (CFU-GM) colonies (median: 92% of colonies), while colonies from MnPs were dominated by BFU-Es (91%) and, as opposed to the MpPs, also contained CFU-GEMM colonies (0.64%) (fig 1C). Thus, hMICL seems to be a useful marker for the GMP population.Figure 1Immunological and functional properties of MpPs in normal donors. (A) Identification of MpPs, MnPs, and HSCs within the CD34+ compartment. (B) Cells with GMP phenotype (CD34+CD38+CD123lowCD45RA+) in MpP and MnP subsets. (C) Colony growth of bone marrow mononuclear cells (MNC) and sorted MpPs and MnPs in a 14-day methylcellulose assay. Error bars denote SDs.Figure 1. Immunological and functional properties of MpPs in normal donors. (A) Identification of MpPs, MnPs, and HSCs within the CD34+ compartment. (B) Cells with GMP phenotype (CD34+CD38+CD123lowCD45RA+) in MpP and MnP subsets. (C) Colony growth of bone marrow mononuclear cells (MNC) and sorted MpPs and MnPs in a 14-day methylcellulose assay. Error bars denote SDs. In CML at diagnosis we found decreased numbers of MpPs (mean 23% of CD34+CD38+ cells (range: 6.3-48%)), compared to the normal donors (33% (18-48%), p=0.030) (fig 2A). Extraordinarily, the MpP fraction varied considerably in size among CML patients, and 12/23 patients had MpP fractions within the 90% reference range (RR) of normal donors (MpPHIGH patients) and thus distinctly higher than the remaining patients (MpPLOW patients) (fig 2B-C). High MpP fractions significantly correlated with high WBC (Spearman's r = 0.47, p=0.049) (fig 2D), high neutrophil counts (r = 0.55, p=0.043), large spleen size (r = 0.66, p=0.0069), and low hemoglobin at the time of diagnosis (r = -0.58, p=0.014). Within the progenitor compartment, high ratio of BCR-ABL in the MpP to BCR-ABL in the MnP significantly correlated with large MpP fractions (r = 0.54, p=0.021).Figure 2Human MICL expression in chronic phase CML patients. (A) Fraction of MpPs in normal donors and CML patients at diagnosis. (B) Typical immunological profiles of MpPLOW patients and (C) MpPHIGH patients. (D) Correlation between MpP fraction size and total white blood cell count at the time of diagnosis. (E) Development of MpP fraction size in individual patients after 3-6 months (solid lines) and after 12-119 months (dotted lines) of TKI treatment in MpPLOW patients and (F) MpPHIGH patients.Figure 2. Human MICL expression in chronic phase CML patients. (A) Fraction of MpPs in normal donors and CML patients at diagnosis. (B) Typical immunological profiles of MpPLOW patients and (C) MpPHIGH patients. (D) Correlation between MpP fraction size and total white blood cell count at the time of diagnosis. (E) Development of MpP fraction size in individual patients after 3-6 months (solid lines) and after 12-119 months (dotted lines) of TKI treatment in MpPLOW patients and (F) MpPHIGH patients. During the first 6 months of TKI treatment differing developments in MpP fraction size were observed for MpPLOW and MpPHIGH patients. While MpPLOW patients showed increasing MpP fractions during the first 6 months of treatment (fig 2E), 4/4 and 2/4 MpPHIGH patients displayed a decrease in MpPs at 3 and 6 months, respectively (fig 2F). Thus, in these patients, the majority of the Ph+ progenitor cells being cleared seemed to be GMPs. In conclusion, our data demonstrate that hMICL is an early marker of granulocyte-macrophage differentiation, and provides a readily accessible approach to assessing the GMP population during TKI therapy in CML. Using the present approach we have uncovered a higher degree of variability in the composition of the progenitor compartment at diagnosis than previously reported, and shown that a significant proportion of the patients have expanded GMP populations. Ongoing studies are aimed at determining whether these patients may represent patients with a more advanced form of disease at the time of diagnosis. Disclosures: Stentoft: Novartis: Consultancy, Financial support for relevant congress participation Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers-Squibb: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Danish Regions: Membership on an entity’s Board of Directors or advisory committees. Hokland:Novartis: Research Funding.

Abstract: Abstract 4427 Very rarely, Philadelphia positive (Ph+) chronic myeloid leukemia (CML) presents with isolated thrombocythemia (CML-T) with no or only slight elevation of neutrophil counts. Due to their rarity, such patients have not been studied in detail, especially whether they constitute a separate disease entity, where the initiating leukemic hit originates from a more lineage-committed progenitor cell type than the one affected in classical CML. To evaluate this, we studied a 68-year-old woman who during a follow-up for follicular lymphoma presented with a sudden thrombocythemia of 2062×109/L, a basophil count of 0.94×109/L but no increase in neutrophil count (3.9×109/L). A bone marrow (BM) biopsy revealed slight to moderate hyperplasia with an increased number of small, hypolobulated megakaryocytes, but no increase in the myelopoietic compartment. Apart from the Ph1 positivity, no molecular aberrations were seen by cytogenetic analysis, array CGH, or extensive molecular testing; noteworthy, the JAK2 V617F mutation was absent. The patient was treated with three different Tyrosine Kinase Inhibitors (TKI's), all of which had to be terminated after a total of 26 weeks of therapy due to severe side effects (dyspnea, acrocyanosis of the fingers and fatigue). At present, she is on pegylated interferon alpha-2a, which is well tolerated. In a longitudinal study of the stem cell biology in this patient we centered on three BM aspirates obtained at diagnosis as well as 3 and 9 months following diagnosis. Subsets of progenitor- and mature lineage committed cells were FACS-purified and analyzed for the presence of leukemic cells by FISH and qPCR technique and additionally subjected to growth in a 14-day methylcellulose assay. At diagnosis, the CD34+ compartment consisted of 10% immature (CD34+CD38-) and 90% mature (CD34+CD38+) progenitors, both of which were enriched for Ph+ cells (74% and 78%, respectively) (Figure 1A-B). Moreover, the colony-forming cells (CFC's) from sorted CD34+ cells were BCR-ABL+ with the erythroid lineage (BFU-E; 94%) dominating and only 4% myeloid lineage (CFU-G, CFU-M, and CFU-GM) and 2% multi-lineage (CFU-GEMM). By qPCR analysis of picked colonies we, moreover, observed that all types of colonies were BCR-ABL+ (Figure 1C). After three months of TKI therapy the patient responded with a 2.6 log decrease in BCR-ABL expression in BM mononuclear cells, the BCR-ABL+ CFC's disappeared and the distribution of erythroid and myeloid colonies normalized. However, specific analysis of the progenitor compartment showed reductions of only 0.5 log in CD34+CD38- cells and 1.6 log in CD34+CD38+ cells. At 9 months after the diagnosis, after pausing TKI treatment for 3 months, BCR-ABL expression in peripheral blood resurged and further analysis identified Ph+ cells by FISH in CD66+ neutrophils (10%) and CD19+CD10+ B-cell precursors (2%), and by expression of BCR-ABL in CD19+ B-cells (positive in one of three triplicate wells) and CD3+ T-cells (0.000068 K562 equivalents) (Figure 1D). These data show, despite the highly unusual presentation with isolated thrombocythemia, that the origin of this form of CML is very similar to classical CML with pan-lineage involvement and a resistance of progenitor cells to TKI therapy. The selective expansion of megakaryocytic lineage cells in this form of CML, however, remains enigmatic and will require further insight into the mechanics of differentiation in the malignant clone at the level of common myeloid progenitors or earlier. Figure 1. Delineation of stem cell biology in a CML patient presenting with isolated thrombocythemia. (A) FACS sorting of CD34+CD38− and CD34+CD38+ cells at diagnosis and 3 months after. (B) Initial treatment response in whole BM and FACS sorted CD34+CD38+ and CD34+CD38− cells. (C) Lineage involvement of the CML clone 9 months after diagnosis (after 3 months of pausing TKI treatment). (D) Growth of FACS sorted CD34+ cells in CFC-assay at diagnosis and 3 and 9 months after. : BCR-ABL expression measured by qPCR, and normalized to K562 equivalents (K562eq). : Fraction of Ph+ cells evaluated by FISH. Figure 1. Delineation of stem cell biology in a CML patient presenting with isolated thrombocythemia. (A) FACS sorting of CD34+CD38− and CD34+CD38+ cells at diagnosis and 3 months after. (B) Initial treatment response in whole BM and FACS sorted CD34+CD38+ and CD34+CD38− cells. (C) Lineage involvement of the CML clone 9 months after diagnosis (after 3 months of pausing TKI treatment). (D) Growth of FACS sorted CD34+ cells in CFC-assay at diagnosis and 3 and 9 months after. : BCR-ABL expression measured by qPCR, and normalized to K562 equivalents (K562eq). : Fraction of Ph+ cells evaluated by FISH. Disclosures: No relevant conflicts of interest to declare.

Abstract: In flow cytometry, a compensation matrix is commonly reused over time, especially in clinical laboratories, to save time and reagents. However, generating a same‐day compensation matrix is considered the best practice by many experts. BD Biosciences developed Cytometer Setup and Tracking software to deliver proper instrument characterization, performance tracking, and stability. BD's “Application Settings” enable daily cytometer adjustments of photomultiplier tube (PMT) settings to correct for day‐to‐day variations in instrument performance. Here, we investigated if using Application Settings would improve data stability over time, including the impact on data stability when reusing a compensation matrix. We consecutively analyzed peripheral blood mononuclear cell (PBMC) aliquots from a single healthy donor together with 8‐peak Rainbow beads and daily compensation controls (22 runs in total over 6.5 months). We found larger variation within both PBMC subset quantifications and median fluorescence intensity (MFI) levels when using Application Settings (i.e., daily adjusted PMTs) compared to fixed PMT voltages (both with the same Day 0 compensation matrix applied). This larger variation was partly due to errors in compensation, but was also seen for Rainbow beads MFI data (not impacted by compensation), and thus likely produced by imprecise adjustments of PMTs by Applications Settings. Notably, the larger variation observed with Application Settings was most pronounced on a few days of the experiment with very large deviations, whereas on most days Application Settings and Fixed PMTs performed similar. The present results call for caution in using Application Settings in longitudinal studies, especially if also reusing a compensation matrix. In contrast, reusing a compensation matrix over time with fixed PMT voltages yielded stable results comparable with running same‐day compensation controls. © 2020 International Society for Advancement of Cytometry

Abstract: Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.