University of Groningen Genetic defects in myeloid malignancies and preleukemic conditions Berger, Gerbrig IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Berger, G. (2019). Genetic defects in myeloid malignancies and preleukemic conditions. Rijksuniversiteit Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 26-09-2021 3. Ringsideroblasts in acute myeloid leukemia are associated with adverse risk and result from an aberrant heme-metabolism gene program G Berger, M Gerritsen, TN Koorenhof-Scheele, G Yi, LI Kroeze, M Stevens-Kroef, K Yoshida, E van den Berg, H Schepers, G Huls, AB Mulder, S Ogawa, JHA Martens, JH Jansen and E Vellenga (Submitted) Chapter 3 Abstract Ringsideroblasts (RS) emerge from aberrant erythroid differentiation, resulting in excessive mitochondrial iron accumulation. This is a characteristic feature of myelodysplastic syndromes (MDS) with mutations in spliceosome gene SF3B1, but RS is also observed in patients with acute myeloid leukemia (AML). We therefore characterized the presence of RS in a cohort of AML patients. The RS-AML subgroup was enriched with patients in the ELN adverse-risk category (55%). In line with this finding, 35% of all RS-AML cases had complex cytogenetic aberrancies, and TP53 was most recurrently mutated in this cohort (42%), followed by DNMT3A (29%), RUNX1 (21%) and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole-exome sequencing and SNP array analysis on a subset of patients showed that the RS phenotype did not result from a single-gene defect. Shared genetic defects between erythroblasts and total mononuclear cell fraction within the same 3 patient indicate common ancestry for the erythroid lineage and the myeloid blast cells in RS-AML patients. RNA sequencing of CD34+ AML cells revealed differential gene expression between RS-AML and a separate AML cohort, including genes involved in megakaryocytic/erythroid differentiation and mRNA splicing. Furthermore, several heme-metabolism-related genes were found to be upregulated in RS-AML, as was observed in SF3B1mut MDS. These results demonstrate that erythroblasts share ancestry with malignant myeloid blast cells in RS-AML. Although the genetic background of RS- AML differs from that of RS-MDS, downstream effector pathways may be comparable, providing a possible explanation for presence of RS in AML. ingsideroblasts (RS) are erythroid in low-risk MDS, which is characterized precursor cells that accumulate by a stable clinical course and a low risk R excessive mitochondrial iron of leukemic transformation.(8,13) As a and can be observed in bone marrow core component of the U2 small nuclear smears associated with multiple medical ribonucleoprotein particle (snRNP), conditions.(1) Acquired presence of RS is a SF3B1 is essential for pre-RNA splicing. characteristic feature in myelodysplastic (14) The molecular mechanism by which syndrome (MDS) subtypes, including SF3B1 mutations result in RS formation MDS with single lineage dysplasia is not yet fully understood. A proposed (MDS-RS-SLD), multilineage dysplasia mechanism is that specific patterns of (MDS-RS-MLD) and in combination missplicing result in altered expression with the presence of thrombocytosis of genes that are essential for correct (MDS/MPN-RS-T).(2) Non-malignant programming of erythropoiesis.(15,16) causes of RS include several drugs, The relationship between toxins, alcohol, copper deficiency and genetic defects in SF3B1 and the RS- congenital sideroblastic anemia.(3) This phenotype is not one-to-one; in 10-20% latter group comprises conditions of the MDS-RS patients no mutation caused by inborn defects in genes in the SF3B1 gene is detected.(8-12) that operate in several mitochondrial Moreover, RS can also be present in a pathways, including ALAS2, ABCB7, subset of AML patients(17), while SF3B1 SLC25A38 and HSPA9.(4-7) mutations are infrequent findings in In MDS, the RS phenotype is this disease.(10,17,18) Besides SF3B1, strongly correlated with mutations in the only other correlation between splicing factor 3B subunit 1 (SF3B1), a gene defect and the RS phenotype with an incidence higher than 80%.(8-12) was described for PRPF8, for which SF3B1 mutations are usually observed mutations are reported in ~3% of the 66 Ringsideroblasts in acute myeloid leukemia cases.(19) Other spliceosomal genes that lymphoprep (PAA, Cölbe, Germany) are more frequently mutated in myeloid according to standard procedures. malignancies, including SRSF2, U2AF1, To separate cell fractions, following ZRSR2, have not been implicated in the thawing of viably frozen MNCs, cells RS-phenotype.(9) were washed and stained with a panel In the present study we deter- of antibodies and sorted for purification mined the prevalence of ringsideroblasts of different cell fractions (antibodies in various ontogenic AML subtypes and against CD3, CD34, CD71 and the association of the RS phenotype in CD235a surface markers (conjugates AML with adverse risk characteristics. CD3-FITC (cat. 345763), CD34-APC To identify the landscape of genomic (cat. 555824) or CD34-PE Cy7 (cat. defects that underlies the RS phenotype 348811) or CD34-FITC (cat. 345801, in AML, we performed whole exome BioLegend, Uithoorn, the Netherlands), sequencing, targeted sequencing and CD71-BV786 (cat. 563768) or CD71- SNP-array analysis. Finally, to identify APC (cat. 551374) or CD71-PE (cat. 3 differential expression of genes asso- 555537) and CD235a-BV421 (cat. ciated with the RS phenotype in AML, 562938) or CD235a-APC (cat. 551336), we performed RNA sequencing on antibodies were obtained from BD CD34+-selected AML cells. Bioscience (Breda, The Netherlands) unless otherwise indicated)). Analysis and Material & methods sorting of various cell fractions was performed on MoFlo XDP or Astrios Patients and data collection For this (Dako Cytomation, Carpinteria, CA, USA). study, we collected data from 126 AML Single viable cells were selected based and high-risk MDS (≥10% bone marrow on forward and side scatter profiles in blasts) who were diagnosed between combination with negativity for DAPI January 2000 and April 2018 at the or PI (both Sigma-Aldrich, Saint Louis, University Medical Center Groningen. MO, USA). Blast fraction was defined as The inclusion criterion was the reported CD34 positive, T cell fraction as CD3 presence of ringsideroblasts in the positive and erythroblast fraction as diagnostic bone marrow smear. Patients CD71/CD235a positive. Sorting purity with previously reported MDS-RS was defined as ≥95% and confirmed by were excluded. Diagnosis and risk reanalysis. classification was revised based upon DNA isolation and World Health Organization classification amplification. Genomic DNA from (2016)(2) and European Leukemia Net various cell fractions was extracted with (ELN) recommendations (2017)(20). Bone the NucleoSpin Tissue kit (Macherey- marrow (BM) and/or peripheral blood Nagel, Düren, Germany) according to (PB) from patients were biobanked after the manufacturer’s instructions. In informed consent for investigational use. case of insufficient yield, a maximum The study was conducted in accordance of 70ng DNA was amplified using the with the Declaration of Helsinki and Qiagen REPLI-g kit (Qiagen, Venlo, the institutional guidelines and regulations. Netherlands) in one reaction, according Morphologic and cytogenetic analyses to the manufacturer’s protocol. were accomplished following standard Targeted deep sequencing procedures. using a myeloid gene panel. Targeted Sorting of cell fractions. sequencing of DNA derived from BM The mononuclear cell (MNC) fraction or PB samples obtained at diagnosis was from BM and/or PB was obtained by carried out using the myeloid TruSight density gradient centrifugation using sequencing panel (Illumina, San Diego, 67 Chapter 3 CA, USA). Library preparation was per- (Agilent Technologies, Santa Clara, CA, formed according to the manufacturer’s USA), massively parallel sequencing protocol (Illumina). Aligning and filter- was performed on enriched exome ing of sequence data was performed fragments using the HiSeq 2500 using NextGENe version 2.3.4.2 (Soft- platform (Illumina, San Diego, CA, USA). Genetics, Pennsylvania, US). Cartagenia Alignment of sequences and calling of Bench Lab NGS (Agilent, Santa Clara, mutations was executed our previously CA, USA) was used for analysis of the described in-house pipelines(9,21), with resulting vcf file. Sequencing artifacts minor modifications. The resultant data were excluded using a threshold of 5%. file was analyzed for the presence of A minimal variant read depth of 20 germline variants