Healthy controls were provided by the Institute for Medical Informatics, Biometry and Epidemiology (IMIBE), University Hospital Essen, Essen, Germany, as part of the Heinz-Nixdorf Recall MultiGeneration (HNRM) study. This study served to extend the Heinz-Nixdorf Recall Study (HNRS), whose objectives and study design were published previously (26). Both studies were approved by the responsible institutional ethics committees and followed strict internal and external quality assurance protocols. Written informed consent was obtained from all participants. In this manuscript, n = 11 of the overall analyzed n = 111 blood samples were included to match the presented aCML case in age and gender (n = 6) or age only (n = 5) (Table 1). Blood of the indicated patient suffering from atypical chronic myeloid leukemia (aCML) was drawn within the out- and in-patients units of the Department of Hematology (University Hospital, Essen, Germany) after written informed consent was obtained. All blood samples were obtained in EDTA-supplemented tubes and transported for 30 minutes up to 1 hour in a VACUETTE® transport container (VTC) (Greiner Bio-One, Kremsmünster, Austria) according to the UN 3373 regulation.
To detect somatic, mutational events, a molecular screen was set up analyzing 65 candidate genes in unseparated patient leukocytes derived from peripheral blood samples taken soon after 1st diagnosis, at follow-up-1 after ruxolitinib (sample taken 7 months after start of treatment) and follow-up-2 (sample taken 12 months after start of treatment). DNA of unenriched leukocytes from these consecutive samples was analyzed by next generation gene capture based deep sequencing (NGS) using a custom myeloid gene panel (Agilent SureSelect QXT, target enrichment protocol for loci ABL1 (E4-11), ARID1A, ASXL1 (E12), ATRX (E8_10, 17_35), BCOR, BCORL1, BRAF (E15), CALR (E9), CBL (E8,9), CLBB (E9,10), CBLC (E7), CEBPA, CSF3R (E13-17), CSMD1, CSNK1A1 (E3,4), CUX1, DNMT3A, EED, ETNK1, ETV6, EZH2, FLT3 (E14-15,20) , GATA1, GATA2, GNAS (E7-9), HRAS, IDH1 (E4), IDH2 (E4), IKZF1, JAK2 (E12-16), JAK3, KIT (E2,8-17), KDM6A (syn. UTX), KMT2A (syn. MLL), KRAS, MPL (E4-12), NPM1 (E12), NRAS, PDGFRA (E12,14,18), PHF6, PIGA, PRPF40B, PTEN (E5,7), PTPN11 (E3,13), RAD21, RUNX1, SETBP1 (E4), SF1, SF3A1, SF3B1 (E13-16), SH2B3 (E2), SMC1A (E2,3,10-12,16-18), SMC3, SRSF2 (E1), STAG1, STAG2, STAT3 (E3,21), SUZ12 (E10-16), TET2, THPO, TP53, U2AF1 (E2,6), U2AF2, WT1 (E7,9), ZRSR2, coding exons +/-20 bp, „E“ denotes exon) on an Illumina MiSeq platform. The sequencing runs yielded 2.4 to 3.5 million reads for the samples with totals of 4.7 to 10.6 gigabases in the untrimmed raw data of the sequencing runs, whereof 91.9 %, 95.6 %, and 94.7 % had quality scores exceeding Q30, resulting in average coverages of 857, 823, and 1042 reads per base, respectively. The LOD for somatic mutations varies depending on mutation type, percentage of neoplastic cells in the sample and copy number of individual loci. Mostly, mutations with VAF > 4 % can be detected with our bioinformatics pipeline: Bioinformatics and evaluation of sequence data after cutadapt Version: 1.9.1, bwa Version: 0.7.5a-r405, SAMtools Version: 1.2 (using htslib 1.2.1). Software: Seqnext (JSI) Version 4.3.1; if required for confirmative Sanger: Seqpilot (JSI) Version 4.4.0 Analyzed NGS data after trimming were 100 % above QS-cutoff > 30 (mostly ≥ 38). The ROI were 100 % over minimal sequencing deepness of 100. Mutation nomenclature according to HGVS. Reference sequences of genes in which mutations were detected are given in italics: ASXL1_NM_015338_c.1934dup, p.Gly646Trpfs*12; CSF3R_NM_000760_c.1853C>T p.Thr618Ile plus presumably germline variant CSF3R_ NM_000760_c.1795C>A, p.His599Asn; TET2_NM_001127208_c.3320C>G p.Ser1107Ter; TET2_ NM_001127208_c.4222G>T p.Gly1408Ter; CEBPA_NM_04364_c.1004T>A p.Leu335Gln; EZH2_NM_004456_c.2069G>A p.Arg690His; NRAS_NM_002524_c.35G>A p.Gly12Asp; STAG2_NM_001042749_c.1178T>A p.Leu393Ter; U2AF1_NM_006758_c.460T>A p.Cys154Ser.
Neutrophil isolation and migration assay conditions
For all healthy controls, neutrophils were isolated from 3 ml EDTA-supplemented blood via density centrifugation using PolymorphprepTM (Cat. No.: 1114683, AXIS-SHIELD, Oslo, Norway) as previously described (13). In short, PolymorphprepTM was overlaid with blood at a 1:1 ratio and centrifuged at 450 rcf for 30 minutes without brake. Polymorphonuclear cells (PMN) were collected and washed with sterile PBS (Cat. No.: P04-36500, PAN-Biotech, Aidenbach, Germany). Erythrocytes were lysed for 10 minutes at room temperature (RT) in lysis buffer, containing 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in distilled H2O. After another washing step in sterile PBS, cells were resuspended in sterile hematopoietic progenitor growth medium (HPGM, Cat. No.: PT-3926, Lonza, Basel, Switzerland) and automatically counted using a Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA, USA). Since PolymorphprepTM isolation did not reliably separate neutrophils from the aCML patient, isolations of aCML neutrophils after day 14 were carried out using magnetic negative isolation with the MACSxpress® Neutrophil Isolation Kit (Cat. No.: 130-104-434, Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s instructions. Residual erythrocytes were also magnetically depleted using MACSxpress® Erythrocyte Depletion Kit (Cat. No.: 130-098-196, Miltenyi Biotec) according to manufacturer’s instructions. Afterwards, purified neutrophils were washed in sterile PBS, resuspended in sterile HPGM and automatically counted. Comparability of the procedures was ensured by side-by-side measurements of the same sample on day 14 (Supplemental Figure 2A) and as previously detailed (13). The neutrophil migration assay was performed as previously described (13). Briefly, purified neutrophils were seeded in a 96 Well µ-Plate (Cat. No.: 89621, ibidi, Martinsried, Germany) at a density of 8,250 cells per well (growth area: 0.56 cm2) in 198 µl HPGM supplemented with serum replacement 3 (SR3, final concentration: 0.3x, Cat. No.: S2640, Sigma-Aldrich, Munich, Germany). Neutrophils were stimulated with 2 µl fMLP (final concentration: 10 nM; Cat. No.: F3506, Sigma-Aldrich, Munich, Germany), 2 µl human recombinant CXCL1 (final concentration: 100 ng/ml; Cat. No.: 275-GR-010/CF, R&D Systems, Minneapolis, MN, USA), or 2 µl human recombinant CXCL8 (final concentration: 100 ng/ml; Cat. No.: 208-IL-010/CF, R&D Systems). As all stimuli were reconstituted in sterile PBS, the addition of 2 µl PBS alone served as a vehicle control. The plates were centrifuged and incubated at 37 °C, 5 % CO2 for 20 minutes before microscopy.
Time-lapse microscopy and auto-tracking
All samples were imaged in a Leica DMI6000 B (Leica Microsystems, Wetzlar, Germany) coupled to a workstation running Leica Application Suite X (LASX, Leica Microsystems) with a motorized stage with a HC PL FLUOTAR L 20x/0.40 DRY objective (Cat. No.: 11506243, Leica Microsystems) at an imaging rate of one frame every 8 seconds for one hour at 37 °C, without CO2. The generated movies were exported as *.mov files. These files were analyzed with the Automated Cellular Analysis System (ACAS, Metavi-Harmony software, MetaVi Labs, Austin, TX, USA; [email protected]). The evaluation interval was set to 30 s, the minimum track duration to 60 s, the movement threshold to 8 µm and the microscopy resolution to 0.458716 pixel/µm.
100,000 purified neutrophils were stained with the following antibodies: CD15 VioBlue (dilution: 1:100, clone: VIMC6, Cat. No.: 130-113-488, Miltenyi Biotec), CD16 FITC (dilution: 1:100, clone: REA423, Cat. No.: 130-113-392, Miltenyi Biotec), fMLP receptor Alexa Fluor 647 (final dilution: 1:100, clone: 5F1, Cat. No.: 565623, BD Biosciences, San Jose, CA), CXCR1 PE (dilution: 1:100, clone: 8F1, Cat. No.: 130-105-352, Miltenyi Biotec), and CXCR2 PE-Vio770 (dilution: 1:20, clone: REA208, Cat. No.: 130-100-930, Miltenyi Biotec). After an incubation step of 15 minutes in the dark at 4 °C, the suspensions were diluted 1:1 with PBS and analyzed on a MACSQuant VYB (Miltenyi Biotec).
Manual cell size analysis
To quantify the changes in cellular morphology of aCML neutrophils and neutrophils from healthy donors, the size of cells in the respective video were manually analyzed using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2017.). For that, the first image of every video was exported as *.tif from the LASX software and imported to ImageJ. Subsequently, the outer cell margins were manually marked as regions of interest (ROI) and the occupied area was computed by ImageJ’s ROI manager. Results were given in µm². Additionally, five age- and gender-matched probands were quantified as controls.
All statistical analyses were performed using GraphPad PrismTM (Version 6.07, GraphPad Software, San Diego, CA, USA). Experimental data were plotted as bar graphs or scatter dot plots. Statistical computation, such as computation of p-values and others, was performed as described in the respective figure legends.