Shifts in Intra-Clonal Dynamics Rather Than Novel Mutations Are the Main Engine Driving Tumor Evolution in Relapsed CLL
Authors: Landau Dan-Avi , Stojanov Petar , Lawrence Michael S , Sougnez Carrie , McKeena Aaron , Carter Scott , Gabriel Stacey , Neuberg Donna , Hacohen Nir , Lander Eric S , Brown Jennifer R. , Getz Gad , Wu Cathy 
Published: October 2019

Abstract
Abstract 284
Tumor evolution is a complex process, and is the biologic underpinning of disease progression, resistance to therapy and relapse. Using whole-exome sequencing (WES) of sequential samples from patients with relapsed chronic lymphocytic leukemia (CLL) treated with conventional chemotherapy, we studied genetic tumor evolution of cancer relapse.
We performed WES using paired-end reads on DNA from two peripheral blood-derived CLL tumor samples at least one year apart and on germline DNA for 20 patients. Here we report the analysis of tumor exomes from the first seven patients, of whom 6 had relapsed disease after chemotherapy and one untreated patient without intervening therapy between samples. All samples had a tumor purity that exceeded 90%. Sequencing coverage was >86% of target territory, with 132x depth obtained for all samples. In total, 187 coding region mutations (124 nonsynonymous, 63 synonymous) were identified (median: 21 somatic mutations/patient; range: 10–64), not including Ig gene mutations which were >80% clonal and remained clonally stable in our cohort.
We measured the abundance of specific mutations in each patient tumor to assess clonality. An allelic frequency of 0.3–0.6 likely represents heterozygous mutations in most or all tumor cells (‘clonal') while a frequency of <0.3 represents mutations in a subset of tumor cells (‘subclonal'). Overall, 118 (63%) somatic mutations were clonal, and their allelic frequency remained unchanged in the relapse samples. 65 (35%) mutations were subclonal (average allelic frequency 0.13±0.075). Ten subclonal mutations, found in 3 of 7 initial samples, evolved into clonal mutations in the relapse samples, compared with only a single opposite occurrence where a clonal mutation became subclonal (p< 0.005, FDR q<0.01). The remaining 4 of 7 tumors showed only minor shifts in allelic frequencies over time, and included the individual who did not receive chemotherapy between samples.
In Patient A, a subclone with three mutations appeared to expand to become the dominant clone, with a change in allelic fraction from an average of 0.17 (0.14–0.23) to an average of 0.43 (0.41–0.46) (p<0.000001). Two of three mutations were non-silent and are likely cancer drivers: NRAS (Q61R, found in 38/38 samples in COSMIC- Catalogue of Somatic Mutations in Cancer, Sanger Institute), and a cancer related gene PLK1. The third mutation is likely a passenger mutation as it was a synonymous mutation in ADAM18. In Patient B, a subclone containing a novel, recently identified driver in CLL, SF3B1, became the dominant clone that included additional mutations in cancer-related genes, CSMD1 and KIAA1199 (change in allelic fraction from an average of 0.16 (0.12–0.18) to an average of 0.37 (0.35–0.38) (p<0.001)). In another example, Patient C, a TP53 mutation increased in allelic frequency from 0.18 in the initial sample to 0.69 in the relapse sample (p<0.005). Analysis of copy number variation (CNV) by CapSeg (a novel algorithm that examines CNV from WES) revealed this change in allelic frequency to be coupled with a ploidy change in del(17p) from 0.8 to 0.5, consistent with a loss of both alleles.
Only one sample demonstrated the appearance of novel mutations with relapse (Patient C), with 19 new mutations (13 non-silent, 3 Silent) of a total of 46 appearing at relapse. All however were subclonal, and thus less likely to have driven tumor relapse. A comparison of the 10 mutations that were selected by chemotherapy to all other mutations demonstrated an enrichment in mutations seen in the COSMIC database (p<0.05), which hints at a higher proportion of cancer drivers in this set. Our ongoing analyses are focused on the association of clinical features with copy number variation and changes in gene expression.
In summary, our analysis of serial exomes from seven patients provided important insights into the genetic evolution of CLL under the selective pressure of chemotherapy. We demonstrate a significant change in clonal dynamics in one half of treated patients, which suggests that relapsed disease following treatment is driven by expansion of subclones under the selective pressure of chemotherapy rather than by novel mutagenesis. This observation may have clinical implications, as it suggests that pre-treatment WES may allow not only for the delineation of current genetic abnormalities, but through investigation of subclonal mutations, may also predict genetic evolution in future relapse.

Disclosures:
No relevant conflicts of interest to declare.