Contributions
Abstract: PB1491
Type: Publication Only
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Next-generation sequencing (NGS) technologies have revolutionized approaches in molecular genetic studies making possible to spell out the mutational status, the genetic and epigenetic variability of chronic lymphocytic leukemia (CLL). The driver mutation’s identification allows for a deep understanding the pathogenesis of CLL, stratifying patients (pts) into prognostic groups and select the appropriate treatment matching it with the genetic lesions.
Aims
We aimed to develop the Lymphoid Targeted NGS Panel for assessing the mutations of pts with CLL and to study the possible correlation of the mutational status with the clinical characteristics of the disease.
Methods
In study were enrolled 26 pts with CLL: treatment-naïve (n=18) and relapsed/refractory (n=8). All cases were diagnosed according to iwCLL criteria (iwCLL, Hallek et al., 2018). According to cytogenetic assay, pts were divided into 3 prognostic groups: favorable (n=16), neutral (n=2) and unfavorable (n=8). Although, 20/26 pts were divided into 2 prognostic groups taking into account the data on the mutational status of IGHV: favorable (n=8) and unfavorable (n=12). Six patients have no available data of IGHV mutational status. Treatment regimens: standard rituximab-containing (n=15) and targeted drugs (n=11). The Lymphoid Targeted NGS Panel was designed as a comprehensive gene panel and covered 117 genes, part of which plays a core role in cellular pathways. DNA samples were extracted from peripheral B-cell lymphocytes via the standard phenol-chloroform method. An average reading depth of 1000x is produced on a MiSeq platform (Illumina, USA). The 2% threshold of allele frequency (VAF) was used. The clinical significance of mutations was established using the following databases: COSMIC, ClinVar, gnomAD with application in silico analysis (Cscape, Cancer Genome Interpreter, SNPs&Go).
Results
Sequencing of 15 pts samples was performed. Genetic aberrations were detected in all DNA samples. We identified 722 variants in 83/117 genes. Frequent mutations were FAT1 (13.9%), KMT2C (13.6%) and RYR1 (4.4%). The clinical significance of 82% of variants was identified as likely benign (n=348) and benign (n=244), 16.2% (n=116) of the mutations was uncertain. Regarding associations, pathogenic and likely pathogenic mutations were identified in 80% pts (12/15) with an unfavorable prognosis. The pathogenic mutations 1.7% (n=12): SF3B1 K700E (n=3), SF3B1 G742D, TP53 Lys120Glu, NOTCH1 P2514fs*4, BRAF D594G, BRAF K601N, PTEN (c.1321-2A>T),MGA R1246X, JAK3 V722L, IDH2 T352P. Likely pathogenic variants (n=2) were ATM Y2080C and NF1 M2217V. The detected variant of the mutation Lys120Glu in the TP53 gene was associated with the presence of del17p13, and correlated with the unfavorable clinical course of the disease in patient CLL-024. Three pts with del13q14 had SF3B1 K700E pathogenic mutation, which accelerated the development of CLL by alternative splicing of RNA and activation mTORC1. It should be noted that 2 pts (CLL-023, CLL-024) with unfavorable prognosis had mutations both in BCR and NOTCH2 genes of uncertain significance. The existing approach to the interpretation of the results does not allow making an unambiguous conclusion about the clinical significance of variants in the IDH2 and JAK3 genes, despite its pathogenic effect.
Conclusion
The obtained data demonstrate the possibility of applying NGS in clinical practice and the approbation of The Lymphoid Targeted NGS Panel. Whereas the clinical significance of some mutations is currently uncertain, further research using NGS technology is required.
Keyword(s): Chronic lymphocytic leukemia, Gene array, Molecular markers, Mutation analysis
Abstract: PB1491
Type: Publication Only
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Next-generation sequencing (NGS) technologies have revolutionized approaches in molecular genetic studies making possible to spell out the mutational status, the genetic and epigenetic variability of chronic lymphocytic leukemia (CLL). The driver mutation’s identification allows for a deep understanding the pathogenesis of CLL, stratifying patients (pts) into prognostic groups and select the appropriate treatment matching it with the genetic lesions.
Aims
We aimed to develop the Lymphoid Targeted NGS Panel for assessing the mutations of pts with CLL and to study the possible correlation of the mutational status with the clinical characteristics of the disease.
Methods
In study were enrolled 26 pts with CLL: treatment-naïve (n=18) and relapsed/refractory (n=8). All cases were diagnosed according to iwCLL criteria (iwCLL, Hallek et al., 2018). According to cytogenetic assay, pts were divided into 3 prognostic groups: favorable (n=16), neutral (n=2) and unfavorable (n=8). Although, 20/26 pts were divided into 2 prognostic groups taking into account the data on the mutational status of IGHV: favorable (n=8) and unfavorable (n=12). Six patients have no available data of IGHV mutational status. Treatment regimens: standard rituximab-containing (n=15) and targeted drugs (n=11). The Lymphoid Targeted NGS Panel was designed as a comprehensive gene panel and covered 117 genes, part of which plays a core role in cellular pathways. DNA samples were extracted from peripheral B-cell lymphocytes via the standard phenol-chloroform method. An average reading depth of 1000x is produced on a MiSeq platform (Illumina, USA). The 2% threshold of allele frequency (VAF) was used. The clinical significance of mutations was established using the following databases: COSMIC, ClinVar, gnomAD with application in silico analysis (Cscape, Cancer Genome Interpreter, SNPs&Go).
Results
Sequencing of 15 pts samples was performed. Genetic aberrations were detected in all DNA samples. We identified 722 variants in 83/117 genes. Frequent mutations were FAT1 (13.9%), KMT2C (13.6%) and RYR1 (4.4%). The clinical significance of 82% of variants was identified as likely benign (n=348) and benign (n=244), 16.2% (n=116) of the mutations was uncertain. Regarding associations, pathogenic and likely pathogenic mutations were identified in 80% pts (12/15) with an unfavorable prognosis. The pathogenic mutations 1.7% (n=12): SF3B1 K700E (n=3), SF3B1 G742D, TP53 Lys120Glu, NOTCH1 P2514fs*4, BRAF D594G, BRAF K601N, PTEN (c.1321-2A>T),MGA R1246X, JAK3 V722L, IDH2 T352P. Likely pathogenic variants (n=2) were ATM Y2080C and NF1 M2217V. The detected variant of the mutation Lys120Glu in the TP53 gene was associated with the presence of del17p13, and correlated with the unfavorable clinical course of the disease in patient CLL-024. Three pts with del13q14 had SF3B1 K700E pathogenic mutation, which accelerated the development of CLL by alternative splicing of RNA and activation mTORC1. It should be noted that 2 pts (CLL-023, CLL-024) with unfavorable prognosis had mutations both in BCR and NOTCH2 genes of uncertain significance. The existing approach to the interpretation of the results does not allow making an unambiguous conclusion about the clinical significance of variants in the IDH2 and JAK3 genes, despite its pathogenic effect.
Conclusion
The obtained data demonstrate the possibility of applying NGS in clinical practice and the approbation of The Lymphoid Targeted NGS Panel. Whereas the clinical significance of some mutations is currently uncertain, further research using NGS technology is required.
Keyword(s): Chronic lymphocytic leukemia, Gene array, Molecular markers, Mutation analysis