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C. Brieghel et al.
However, Sanger sequencing and fluorescence in situ hybridization (FISH) fail to detect 4-5% of newly diag- nosed and untreated patients with CLL harboring low bur- den TP53muts (Sanger negative) without concomitant del(17p).13,14 Deep-targeted next-generation sequencing (NGS) of TP53 has shown that low burden TP53muts with a variant allele frequency (VAF) as low as 0.3% have similar outcome to patients with high burden TP53muts (Sanger positive).13,14 However, recent data from the UK CLL4 trial indicated that low burden TP53muts impacted neither OS nor PFS for patients treated with chemothera- py.15 For newly diagnosed patients harboring only one TP53ab, better OS is demonstrated compared to patients with both del(17p) and TP53mut. Similarly, patients with del(17p) and additional low burden TP53mut show better OS compared to patients with additional high burden TP53mut.16,17 Thus, the impact of additional TP53ab war- rants further investigation.
Upon therapy, the prevalence and size of TP53 clones increase due to clonal evolution and acquisition of new TP53muts.18,19 Targeted therapy is established as the stan- dard of care for patients with TP53 aberrated CLL.20,21 Whether patients with low burden TP53ab may benefit more from targeted therapy compared to standard CIT still remains open for investigation, as the evidence avail- able so far does not allow definitive guidelines to be for- mulated.12 Thus, studies to elucidate a technical and a clin- ically significant limit of detection (LOD) for TP53mut are warranted to guide clinical decisions for these patients.
We here describe a robust NGS assay for detection of TP53mut as low as 0.2% VAF. In order to investigate a clinically relevant LOD for low burden TP53mut, we assessed the impact of TP53mut at diagnosis and at time of treatment in a single center cohort of patients with CLL.
Methods
Patients and materials
All consecutive patients diagnosed with CLL from a single cen- ter sampled between January 2007 and October 2014 were includ- ed in the study (Online Supplementary Figure S1). Samples from patients obtained within 200 days of the diagnostic flow cytome- try were considered newly diagnosed.21 To assess the clinical impact of TP53ab at time of treatment, samples obtained up to 200 days before treatment were included for a separate analysis. All available samples considered newly diagnosed and/or sampled at time of treatment were sequenced. Due to the retrospective nature of the study, TP53 analysis was performed on peripheral blood mononuclear cells (PBMCs) and not on purified CLL cells. For 244 patients (81% of the newly diagnosed cohort) with avail- able flow cytometry data at time of sampling, 197 patients (81%) had CLL populations more than 70% of the PBMCs (see Online Supplementary Methods), thus we report VAFs based on PBMCs.
Patients’ characteristics and clinical data were obtained from medical records and registries; CLL-International Prognostic Index (CLL-IPI) factors in terms of age (≤65 vs. >65 years), Binet stage (A vs. B or C), beta-2-microglobulin (β2M) (<4.0 mg/L vs. ≥4.0 mg/L), IGHV mutational status (germline identity <98% vs. ≥98%), and TP53ab only by FISH [no del(17p) vs. del(17p)] were included.3,22 Del(17p) was considered positive if present in at least 10% of 200 interphases. The study was approved by the Danish National Committee on Health Research Ethics, the Data Protection Agency and the Health Authorities involved.
TP53 mutational analysis by deep-targeted sequencing A high sensitivity TP53 assay was established based on serial 10-fold dilutions of DNA from patient samples with donor DNA. By including a dilution step for each sequenced sample, back- ground noise was filtered and an LOD was established at 0.2% VAF (Online Supplementary Methods, Online Suppplementary Table S1 and Online Suppplementary Figure S2). For each patient, DNA extracted from PBMCs was analyzed undiluted and diluted 20% (dilution factor 5) in DNA derived from the SU-DHL4 cell line. A known TP53mut (p.Arg273Cys) harbored in the cell line DNA acted as internal control of dilution grade. Using 100 ng gDNA per reaction, TP53 exons 2-10 incl. 2 bp intronic overlap for splice sites were amplified with 30 cycles of PCR using Phusion® HSII High- Fidelity DNA polymerase (Life Technologies, Waltham, MA, USA). A list of the primers used is provided in Online Supplementary Table S2. In brief, library preparation was performed following manufacturer protocol KAPA DNA Library Preparation (Nimblegen). Using SeqCap Adapter Kit A and B (Roche NimbleGen) or NEXTflexTM DNA Barcodes 96 (Bioo Scientific, Austin, TX, USA), libraries were pooled (24 or 96 samples per lane) and sequenced as paired-end on a HiSeq2500 using HiSeq® SBS Kit v.4 (2x125 base PE, Illumina) to obtain a minimum target
read depth of 20,000x.
Bioinformatic workflow
A workflow for detection of low burden variants was devel- oped in CLC Biomedical Genomics Workbench 3.0 (CLC BGW, Qiagen, Hilden, Germany) as described in the Online Supplementary Methods. Achieving a median coverage of 144,158 reads (99% of region > 26,217x), we applied both a dilution match algorithm (DMA) and a stereotypic error model (SEM) described in detail in the Online Supplementary Methods. In brief, only variants that diluted correctly were called TP53mut by DMA (Online Supplementary Figure S3), while SEM identified outliers from the position-specific and nucleotide-specific background noise as true TP53mut based on the distribution of stereotypic errors (Online Supplementary Figures S4 and S5).13 Results from both DMA and SEM were compared using contingency tables, and only true pos- itive variants were considered true mutations and used in subse- quent analyses (Online Supplementary Table S3 and Online Supplementary Figure S6).
Validation by droplet digital PCR and Capture based targeted next-generation sequencing
Droplet digital PCR (ddPCR) was used to validate initial low burden variants. Allele specific Prime AssayTM probes (Bio-Rad, Hercules, CA, USA) were applied for triplicate analyses using QX200TM Droplet DigitalTM PCR System and QuantaSoftTM 1.7 (Bio-Rad) according to instructions from the manufacturer. A cus- tom made SeqCap EZ Choice gene panel (Roche Nimblegen) con- taining TP53 exons 2-10 was used to validate mutations with a VAF of 1% or over, as described in the Online Supplementary Methods.
Statistical analysis
Time to event was calculated from date of diagnosis for treat- ment-free survival (TFS), and from date of diagnosis or first date of treatment for OS. Patients were followed until initiation of CLL- specific treatment or death or end of follow up, which ever came first, defined as TFS, and until death or end of follow up, whichev- er came first, defined as overall survival (OS). Analyses were per- formed using the Kaplan-Meier method, and log-rank test was used to compare outcome. TP53mut were stratified into high and low burden mutations (VAF>10% and VAF ≤10%, respectively)
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haematologica | 2019; 104(4)