Contributions
Abstract: PB1802
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by reciprocal chromosomal translocation t(9;22)(q34;q11), resulting in the formation of the BCR-ABL1 fusion oncogene. One of the most common CML in vitro model is the K562 BCR-ABL1-positive human erythroleukemia cell line derived from a female patient with CML in blastic phase (CML-BP) and representing an important tool for the studies of malignant hematopoiesis in last decades. Although K562 karyotype was described several times, detailed genomic analysis of this cell line is not yet available and to our best knowledge there are no publications yet describing complex genomic landscape of K562 cells.
Aims
The aim of our study was to determine the mutational landscape of K562 cell line using next-generation sequencing (NGS). Additionally classical fluorescence in situ hybridization (FISH) with BCR and ABL1 probes was performed to confirm cytogenetics.
Methods
The K562 cell line was purchased from DSMZ (Braunschweig, Germany). We analyzed almost 1300 genes implicated in human cancer using custom designed capture (SeqCap EZ, NimbleGen, Roche) followed by high-throughput sequencing on Illumina HiSeq 1500. Common variants (>1%) gathered in ESP6500 and 1000 genomes projects and our internal exome database were filtered out and the subsequent analysis was focused on putative protein damaging variants with the frequency in the database from NHLBI GO exome sequencing project less or equal to 0.01. We used different bioinformatic tools for variant effect prediction (eg. PolyPhen-2, SIFT, IntOGen). Mutations were confirmed with Sanger sequencing. FISH was performed using commercially available probes (Vysis, Abbott, USA), that identifies BCR-ABL1/ABL1-BCR fusion genes.
Results
Sequencing and bioinformatical analysis revealed 88 variants with potential biological significance. We detected Q136fs*13 mutation in TP53, which has already been described in K562 cell line previously by ATCC, but we have also identified several new mutations in genes involved in tumorigenesis and drug resistance (Table 1). Moreover, cytogenetic analysis showed both multiplication of the BCR and ABL1 genes and amplification of the BCR-ABL1 fusion gene (Ph chromosome is present in at least four additional copies).
Mutation | NCBI Reference |
TP53 p.Q136fs*13 (known) | NM_001126114.2 |
ASXL1 p.Y591* | NM_015338.5 |
MLH1 p.K175fs | NM_001258271.1 |
BIRC6 p.A3622V | NM_016252.3 |
AKT3 p.G37* | NM_001206729.1 |
BRCA1 p.I540V | NM_007297.3 |
Conclusion
We describe several new mutations in such genes as ASXL1, BRCA1 or MLH1 in one of the most frequently used cell line in leukemia research, K562 erythroleukemia. Our results confirm high level of genomic instability in the blastic phase of CML represented by the K562 cell line and add new, valuable information for researchers who want to employ this cell line. The awareness of the genomic aberrations present in the K562 erythroleukemia cell line is essential for further studies as those aberrations may have a significant impact on the observed results.
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Genomic instability, Cytogenetic abnormalities, Chronic myeloid leukemia, Blast crisis
Abstract: PB1802
Type: Publication Only
Background
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by reciprocal chromosomal translocation t(9;22)(q34;q11), resulting in the formation of the BCR-ABL1 fusion oncogene. One of the most common CML in vitro model is the K562 BCR-ABL1-positive human erythroleukemia cell line derived from a female patient with CML in blastic phase (CML-BP) and representing an important tool for the studies of malignant hematopoiesis in last decades. Although K562 karyotype was described several times, detailed genomic analysis of this cell line is not yet available and to our best knowledge there are no publications yet describing complex genomic landscape of K562 cells.
Aims
The aim of our study was to determine the mutational landscape of K562 cell line using next-generation sequencing (NGS). Additionally classical fluorescence in situ hybridization (FISH) with BCR and ABL1 probes was performed to confirm cytogenetics.
Methods
The K562 cell line was purchased from DSMZ (Braunschweig, Germany). We analyzed almost 1300 genes implicated in human cancer using custom designed capture (SeqCap EZ, NimbleGen, Roche) followed by high-throughput sequencing on Illumina HiSeq 1500. Common variants (>1%) gathered in ESP6500 and 1000 genomes projects and our internal exome database were filtered out and the subsequent analysis was focused on putative protein damaging variants with the frequency in the database from NHLBI GO exome sequencing project less or equal to 0.01. We used different bioinformatic tools for variant effect prediction (eg. PolyPhen-2, SIFT, IntOGen). Mutations were confirmed with Sanger sequencing. FISH was performed using commercially available probes (Vysis, Abbott, USA), that identifies BCR-ABL1/ABL1-BCR fusion genes.
Results
Sequencing and bioinformatical analysis revealed 88 variants with potential biological significance. We detected Q136fs*13 mutation in TP53, which has already been described in K562 cell line previously by ATCC, but we have also identified several new mutations in genes involved in tumorigenesis and drug resistance (Table 1). Moreover, cytogenetic analysis showed both multiplication of the BCR and ABL1 genes and amplification of the BCR-ABL1 fusion gene (Ph chromosome is present in at least four additional copies).
Mutation | NCBI Reference |
TP53 p.Q136fs*13 (known) | NM_001126114.2 |
ASXL1 p.Y591* | NM_015338.5 |
MLH1 p.K175fs | NM_001258271.1 |
BIRC6 p.A3622V | NM_016252.3 |
AKT3 p.G37* | NM_001206729.1 |
BRCA1 p.I540V | NM_007297.3 |
Conclusion
We describe several new mutations in such genes as ASXL1, BRCA1 or MLH1 in one of the most frequently used cell line in leukemia research, K562 erythroleukemia. Our results confirm high level of genomic instability in the blastic phase of CML represented by the K562 cell line and add new, valuable information for researchers who want to employ this cell line. The awareness of the genomic aberrations present in the K562 erythroleukemia cell line is essential for further studies as those aberrations may have a significant impact on the observed results.
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Genomic instability, Cytogenetic abnormalities, Chronic myeloid leukemia, Blast crisis