Contributions
Abstract: EP890
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
Molecular studies and conventional cytogenetics are essential for patients with myelodysplastic syndromes (MDS) to establish a correct diagnosis and to optimize their prognosis and management. Routinely, these analyses are performed in bone marrow (BM) samples, especially cytogenetics as it is difficult to obtain metaphases in peripheral blood (PB) samples. Analysis of cell-free DNA (cfDNA), also known as liquid biopsy, has been reported as a reliable approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alteration analysis in cfDNA from MDS patients.
Aims
To analyze the molecular and cytogenetic abnormalities of cfDNA using next generation sequencing (NGS) in patients with MDS.
Methods
BM aspirates and PB samples were collected from 57 newly diagnosed or treatment-naïve patients with MDS: 1 MDS-SLD, 33 MDS-MLD, 3 MDS-RS-SLD, 10 MDS-RS-MLD, 2 MDS-del(5q), 5 MDS-BE-1, 1 MDS-BE-2 and 2 MDS-U. PB samples from 33 healthy controls were also studied. Molecular characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patients. Libraries were prepared using a custom panel including 48 myeloid-associated genes and genomic regions localized at the most frequently altered chromosomes in MDS (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina technology with a 3000x minimum coverage. 14/57 (24.6%) presented cytogenetic/FISH alterations at the time of diagnosis, 2 of them with alterations not covered by the panel (+14, del(9q)). Copy number variant (CNV) analysis was performed by NGS to detect cytogenetic alterations in both cfDNA and BM, and confirmed by chromosomal microarrays (CMA) in BM DNA (CytoScan/OncoScan, ThermoFisher).
Results
We obtained a median amount of total cfDNA of 60.1 ng/ml in MDS patients that was significantly higher than that obtained from healthy controls (median 5.16 ng/ml) (p<0.001, Mann-Whitney). Sequencing of BM DNA and cfDNA showed a comparable mutational profile (153/161 mutations, 95.0% concordance). The most frequently mutated genes were TET2 (47.4%), SF3B1 (29.8%), ASXL1 (21.1%), SRSF2 (19.3%), DNMT3A (15.8%), ZRSR2 (14.0%) and U2AF1 (12.3%). A strong correlation was observed between the VAF of BM and cfDNA (r=0.787, p<0.001, Spearman). There were 7 discordant mutations: 3 were only detected in cfDNA and 4 were only detected in BM. These 7 discordant mutations presented a lower variant allele frequency (VAF) (median 7.41%, range 2.86–25.53%) when compared to the VAF observed in cfDNA in the whole cohort (median 31.44%, range 0.74–98.28%).
CNV analysis by NGS showed cytogenetic alterations in 9/57 MDS patients in both BM DNA and cfDNA. 8/12 (73%) cases with altered karyotype/FISH were detected by NGS, and the remaining were identified in few metaphases (2 cases presented +8 and 1 case +4) and in one case a del(5q) only detected by FISH. Interestingly, in a patient without analyzable metaphases in the karyotype, del(20q) was found by NGS and was confirmed by CMA. Overall, CMA and NGS were highly concordant to detect chromosomal aberrations although they do not reach the detection achieved by karyotype/FISH. However, all the cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA (100% concordance).
Conclusion
The analysis of cfDNA allows the characterization of the molecular abnormalities in patients with MDS. Cytogenetic alterations were detectable in most cases by NGS in both BM DNA and cfDNA although with a lower detection rate than karyotype/FISH.
Keyword(s): Diagnosis, Genomics, Molecular cytogenetics, Myelodysplasia
Abstract: EP890
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
Molecular studies and conventional cytogenetics are essential for patients with myelodysplastic syndromes (MDS) to establish a correct diagnosis and to optimize their prognosis and management. Routinely, these analyses are performed in bone marrow (BM) samples, especially cytogenetics as it is difficult to obtain metaphases in peripheral blood (PB) samples. Analysis of cell-free DNA (cfDNA), also known as liquid biopsy, has been reported as a reliable approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alteration analysis in cfDNA from MDS patients.
Aims
To analyze the molecular and cytogenetic abnormalities of cfDNA using next generation sequencing (NGS) in patients with MDS.
Methods
BM aspirates and PB samples were collected from 57 newly diagnosed or treatment-naïve patients with MDS: 1 MDS-SLD, 33 MDS-MLD, 3 MDS-RS-SLD, 10 MDS-RS-MLD, 2 MDS-del(5q), 5 MDS-BE-1, 1 MDS-BE-2 and 2 MDS-U. PB samples from 33 healthy controls were also studied. Molecular characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patients. Libraries were prepared using a custom panel including 48 myeloid-associated genes and genomic regions localized at the most frequently altered chromosomes in MDS (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina technology with a 3000x minimum coverage. 14/57 (24.6%) presented cytogenetic/FISH alterations at the time of diagnosis, 2 of them with alterations not covered by the panel (+14, del(9q)). Copy number variant (CNV) analysis was performed by NGS to detect cytogenetic alterations in both cfDNA and BM, and confirmed by chromosomal microarrays (CMA) in BM DNA (CytoScan/OncoScan, ThermoFisher).
Results
We obtained a median amount of total cfDNA of 60.1 ng/ml in MDS patients that was significantly higher than that obtained from healthy controls (median 5.16 ng/ml) (p<0.001, Mann-Whitney). Sequencing of BM DNA and cfDNA showed a comparable mutational profile (153/161 mutations, 95.0% concordance). The most frequently mutated genes were TET2 (47.4%), SF3B1 (29.8%), ASXL1 (21.1%), SRSF2 (19.3%), DNMT3A (15.8%), ZRSR2 (14.0%) and U2AF1 (12.3%). A strong correlation was observed between the VAF of BM and cfDNA (r=0.787, p<0.001, Spearman). There were 7 discordant mutations: 3 were only detected in cfDNA and 4 were only detected in BM. These 7 discordant mutations presented a lower variant allele frequency (VAF) (median 7.41%, range 2.86–25.53%) when compared to the VAF observed in cfDNA in the whole cohort (median 31.44%, range 0.74–98.28%).
CNV analysis by NGS showed cytogenetic alterations in 9/57 MDS patients in both BM DNA and cfDNA. 8/12 (73%) cases with altered karyotype/FISH were detected by NGS, and the remaining were identified in few metaphases (2 cases presented +8 and 1 case +4) and in one case a del(5q) only detected by FISH. Interestingly, in a patient without analyzable metaphases in the karyotype, del(20q) was found by NGS and was confirmed by CMA. Overall, CMA and NGS were highly concordant to detect chromosomal aberrations although they do not reach the detection achieved by karyotype/FISH. However, all the cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA (100% concordance).
Conclusion
The analysis of cfDNA allows the characterization of the molecular abnormalities in patients with MDS. Cytogenetic alterations were detectable in most cases by NGS in both BM DNA and cfDNA although with a lower detection rate than karyotype/FISH.
Keyword(s): Diagnosis, Genomics, Molecular cytogenetics, Myelodysplasia