A major challenge in the health care management of HL is finding optimal balance between treatment efficacy and risk of toxicity. Nowadays there is no reliable and precise tool for evaluation of treatment response and MRD monitoring due to scarce presence and difficult availability of neoplastic cells. The cell-free DNA (cfDNA) in HL reflects its mutational profile and could be source for genotyping assays. We developed a specific NGS panel for analysis of 13 genes involved in pathogenesis of HL. Selected variants detected by NGS were subsequently used for MRD monitoring by droplet PCR (dPCR). Our cohort consisted of 48 pts: 20 females/28 males; median age of dg 39.5 years. Histological subtypes: 28 pts (58%) NSCHL; 14 pts (29%) MCCHL; 1 pt (2%) NLPHL; 1 pt (2%) LRCHL (4 pts not determined). Forty four samples were obtained at dg/ 4 in relapse. CfDNA was extracted from peripheral blood plasma using QiaAmp Circulating nucleic acid kit (Qiagen). Specific NGS panel covering coding sequences (including UTRs) of 13 selected genes was designed. For library preparation we used SureSelect XT HS2 technology (Agilent Technologies) based on "target enrichment" with molecular barcodes. Sequencing was performed on a NovaSeq6000 (Illumina). Data were analyzed with the SureCall software (Agilent Technologies) with sensitivity of 1,0 % VAF. The detected variants were annotated using COSMIC, dbSNP, Ensembl and ClinVar. Selected variants were further monitored by dPCR (QIAcuity Digital PCR System; Qiagen) with sensitivity of 0,1 % VAF. Mutations were detected in 22/48 (46%) pts. The most frequently mutated genes were STAT6 (12/48 pts), TNFAIP3 (10/48 pts), XPO1 (7/48 pts), SOCS1 (7/26 pts). Frameshift deletions prevailed in TNFAIP3 and SOCS1 genes. Most mutations in the STAT6 (p.N417Y/D) and XPO1 (p.E571K) genes were hotspots. We monitored levels of these variants by dPCR during the course of disease and correlate results with clinical and PET-CT data. Fast, sensitive and noninvasive detection of mutations means an important improvement in diagnostics, prognostics, and monitoring of HL. NGS/dPCR approach would refine the evaluation of treatment response fundamentally. Correlation of mutational load with continuous PET examination would reduce the amount of false-positive results and enable us to use more precise and safe therapy de-escalation. DPCR proved to be sensitive, fast and afordable technology for MRD testing.
Supported: IGA_LF_2022_001 and MZCR-RVO(FNOL,00098892)