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TP53 mutations in CLL
Table 1. Patients’ characteristics of the Danish nationwide cohort and study cohorts for newly diagnosed patients and patients at time of treat- ment.
Variable
Age
≤65 years >65 years
Binet stage
Nationwide N (%)
1017 (28.8) 2513 (71.2)
2804 (79.4) 726 (20.6) 0
2233 (86.0) 365 (14.0) 932
1822 (67.9) 861 (32.1) 847
2832 (93.9)
185 (6.1)
513
Newly diagnosed N (%)
20 (41.4) 170 (58.6)
232 (84.7) 42 (15.3) 16
213 (86.6) 33 (13.4) 44
192 (68.1) 90 (31.9) 8*
283 (97.6)
7 (2.4)
0
Time of treatment N (%)
35 (57.4) 26 (42.6)
20 (32.8) 41 (67.2) 0
27 (73.0) 10 (27.0) 24
17 (30.4) 39 (69.6) 5*
55 (90.2) 6 (9.8) 0
A
B/C Unknown
β2M
≤4.0 mg/L >4.0 mg/L Unknown
IGHV
Mutated Unmutated Unknown
FISH
No del(17p) Del(17p) Unknown
*Indicates inconclusive IGHV analysis. β2M: beta-2-microglobulin; FISH: fluorescence in situ hybridization.
including minor TP53muts (VAF<1%).12,18 Since allogeneic stem cell transplantation is considered the only curative treatment in CLL, follow up was censored upon allogeneic stem cell transplan- tation for the cohort analyzed at time of treatment. FISH was not repeated at time of treatment in 5 patients for whom the baseline FISH were extrapolated. All analyses downstream of CLC BGW were performed with R version 3.4.1.23
Results
Patient cohorts and impact of baseline characteristics
A total of 446 patients were included in our study. We excluded 44 patients with unavailable material and 92 patients who were neither newly diagnosed nor sampled at time of treatment. The two final cohorts included 290 newly diagnosed patients and 61 patients sampled at time of treatment, including 50 patients at time of first-line treatment and 11 at time of later lines of treatment (Online Supplementary Figure S1). Median time from date of diag- nosis to sample collection was two days [interquartile range (IQR): 1.25-2.00). During a median follow up of 6.0 years (IQR: 3.9-7.9), 97 (33%) patients received treatment and 81 (28%) deaths were registered among newly diag- nosed patients. Compared to the Danish nation-wide cohort, fewer patients were older than 65 years (58.6% vs. 71.2%) and a lower prevalence of Binet stage B/C disease (15.3% vs. 20.6%) as well as a lower prevalence of del(17p) (2.4% vs. 6.1%) were seen in this cohort. Except for age, there were more high-risk features in the 61 patients at time of treatment compared to the newly diag- nosed patients (Table 1).
Improved robustness of low burden TP53 mutation detection
Robust detection of low burden TP53muts was ensured by combining a DMA and an SEM. For DMA, the ratio of variants called in both undiluted and diluted samples from the same patient (dilution ratio, DR) and the reference allele frequency of a known cell line mutation used for dilution (dilution grade, DG) were plotted (Online Supplementary Figure S3). The proximity to a line with a slope of one between the DG and adjusted DR defined true mutations for DMA. For SEM, we modeled frequent variants (observed ≥20) according to each unique genomic position and nucleotide change, while infrequent variants (observed <20) were modeled according to each unique nucleotide change only. VAFs were fitted to gamma distri- butions allowing for identification of true mutations using Bonferroni correction (Online Supplementary Figures S4 and S5). For the full study cohort of 308 patients, 98 and 116 TP53muts were called by DMA and SEM, respectively (Online Supplementary Tables S4 and S5, and Online Supplementary Figure S6). Using an LOD of 1% VAF, we obtained 100% consistency between DMA and SEM for determination of true mutations (Online Supplementary Table S3C). Between 0.3-1% VAF, 32 true positive TP53muts were further identified while excluding four variants only detected by either DMA or SEM (Online Supplementary Table S3B). Reporting TP53muts as low as 0.2% VAF, 10 additional true positive TP53muts could be identified, while 26 mutations only identified by either DMA or SEM were excluded (Online Supplementary Tables S3A and S4). Validating the first 30 low burden TP53muts identified, all were confirmed by ddPCR with high corre-
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