A Quantitative Analysis of Subclonal and Clonal Gene Mutations before and after Therapy in Chronic Lymphocytic Leukemia

Abstract

Purpose: Chronic lymphocytic leukemia (CLL)-associated gene mutations that influence CLL cell fitness and chemotherapy resistance should increase in clonal representation when measured before therapy and at relapse.

Experimental design: To uncover mutations associated with CLL relapse, we have performed whole-exome sequencing in a discovery cohort of 61 relapsed CLL patients identifying 86 recurrently mutated genes. The variant allele fractions (VAF) of 19 genes with mutations in ≥3 of 61 cases were measured in 53 paired pre- and posttreatment CLL samples sorted to purity using panel-based deep resequencing or by droplet digital PCR.

Results: We identify mutations in TP53 as the dominant subclonal gene driver of relapsed CLL often demonstrating substantial increases in VAFs. Subclonal mutations in SAMHD1 also recurrently demonstrated increased VAFs at relapse. Mutations in ATP10A, FAT3, FAM50A, and MGA, although infrequent, demonstrated enrichment in ≥2 cases each. In contrast, mutations in NOTCH1, SF3B1, POT1, FBXW7, MYD88, NXF1, XPO1, ZMYM3, or CHD2 were predominantly already clonal prior to therapy indicative of a pretreatment pathogenetic driver role in CLL. Quantitative analyses of clonal dynamics uncover rising, stable, and falling clones and subclones without clear evidence that gene mutations other than in TP53 and possibly SAMHD1 are frequently selected for at CLL relapse.

Conclusions: Data in aggregate support a provisional categorization of CLL-associated recurrently mutated genes into three classes (i) often subclonal before therapy and strongly enriched after therapy, or, (ii) mostly clonal before therapy or without further enrichments at relapse, or, (iii) subclonal before and after therapy and enriching only in sporadic cases. Clin Cancer Res; 22(17); 4525-35. ©2016 AACR.

Figure 1. Frequent enrichment of mutations in TP53 and SAMHD1 after therapy in CLL

Displayed are variant allele fractions (VAFs) for A: TP53 (N=7 CLL carrying a combined total of 11 mutations, CLL90 carried 2 mutations and CLL117 carried 3 mutations) and, B: SAMHD1 (N=4 CLL carrying a combined total of 8 mutations; CLL 17 carried 4 mutations and CLL 218 carried 2 mutations; the two declining SAMHD1 VAFs are both from CLL 17) in paired CLL samples procured before and after therapy. VAFs for TP53 were not corrected for presence of del17p. C: Representative droplet digital PCR results for NEG: unmutated control DNA, PRE: CLL DNA procured pre-therapy and POST: CLL DNA procured post-therapy. Red numbers indicate the mean VAFs based on duplicate measurements. Results for two representative CLL cases are shown each for TP53 and SAMHD1.

Figure 2. Evidence for an early pathogenetic role of various recurrent gene mutations in CLL

Displayed are variant allele fractions (VAFs) for A: NOTCH1; B: SF3B1; C: XPO1; D: NXF1; E: MYD88; F: ZMYM3; G: CHD2; H: POT1 and, I: FBXW7 in paired samples procured before and after therapy. Black: CLL samples untreated at first measurements; Red: CLL samples that had relapsed at first measurement and that received additional therapy followed by relapse.

Figure 3. Candidate gene drivers of CLL relapse

Displayed are variant allele fractions (VAFs) for A: ATP10A; B: ATRX; C: CACNA1E; D: FAM50A; E: FAT3 and, F: MGA in paired samples procured before and after therapy. Black: CLL samples untreated at first measurements; Red: CLL samples that had relapsed at first measurement and that received additional therapy followed by relapse.

Figure 4. CLL clone dynamics in individual patients identifies rising, stable and falling clones and subclones post therapy

A-C: All CLL cases were untreated at first measurements. A: CLL cases with rising subclones; B: CLL cases with stable clones; C: CLL cases with complex clone dynamics including rising and falling clones. D: CLL samples that had relapsed at first measurement and that received additional therapy.