Detection of clinically relevant variants in the TP53 gene below 10% allelic frequency: A multicenter study by ERIC, the European Research Initiative on CLL

Abstract

In chronic lymphocytic leukemia, the reliability of next-generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low-VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low-VAF variants on time-to-second-treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low-VAF TP53 variants had shorter TTST and OS versus wt-TP53 patients, and the relative risk of second-line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low-VAF TP53 variants and wt-TP53 patients, while patients with high-VAF TP53 variants had inferior OS compared to wild type-TP53 cases. Altogether, NGS-based approaches are technically capable of detecting low-VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set-up. Finally, whereas low-VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment.

Figure 1

Results of interlaboratory comparison of NGS methods. (A) Results provided by 41 laboratories testing 44 methods. Black and orange dots represent variants present in reference samples with VAF values measured by ddPCR; the color distinguishes variants reported (black) and not reported (orange) by the laboratory. Methods (X‐axis) are ordered by the lowest variant allele frequency (VAF) that was detected without missing any variant present in reference samples and without false positive results (red dots; VAF value reported by the laboratory). (B) Correlation of median VAF values reported by participants and VAF values obtained from droplet digital PCR (ddPCR) analysis for individual variants. Spearman correlation coefficient R = 0.9849. (C). Overall performance of tested methods. Blue area shows the cumulative proportion of the methods (Y‐axis) in relation to the lowest VAF (values measured by ddPCR; X‐axis) that was reliably detected, that is, all variants present in reference samples were reported without false positivity. Red line shows the cumulative proportion of methods reporting false positive results (VAF value reported by the laboratory).

Figure 2

Survival in patients analyzed in 12 participating centers stratified by TP53 mutation status. (A, B) Time to second treatment (TTST) in patients not receiving targeted treatment frontline stratified by variant allele frequency (VAF) (A) and by VAF and del(17p) presence (B). (C) TTST in patients treated with frontline targeted agents stratified by VAF. Event = 2nd treatment or death, censored = untreated and alive at last follow‐up. (D) Overall survival (OS) from therapy initiation. Low‐VAF = 1%–10%; high‐VAF = >10% VAF. Targeted treatment = BcR or BCL‐2 inhibitor; for details on treatment see Supporting Information S1: Table 6. Group comparison–LogRank test with Benjamini‐Hochberg correction of p‐values. HR, hazard ratio: numbers in brackets show a 95% confidence interval.

Figure 3

Multivariate Cox model for TTST and OS. Wild‐type TP53, mutated IGHV, and FCR‐like therapy were used as reference; 1058 patients with data available for all parameters were included. Frontline CIT–other, chemoimmunotherapy other than FCR‐like; Frontline other–other therapies. CI, confidence interval; HR, hazard ratio.

Figure 4

Relative risk for TTST (A) and OS (B) in relation to TP53 mutation VAF in patients not receiving targeted treatment frontline. The increasing relative risks of the event (TTST: 2nd treatment or death, OS: death) were obtained from the Cox model with penalized spline term for continuous predictor “VAF highest” centered at 0, that is, TP53 wildtype. VAF highest – % VAF of a single variant in the sample or the highest VAF if multiple variants were present.