Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients

Abstract

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

Fig 1. Study flow.

Fig 2. CfDNA-WES quality assessment.

A. WES mean depth correlates logarithmically with input cfDNA quantity. B. High coverage at low depth for each sample.

Fig 3. Tumor mutation load from cfDNA and tDNA are correlated in ctDNA-positive samples.

Samples were classified as ctDNA-positive when at tDNA variants were also found in cfDNA-TGS with variant fraction in the same range, mildly ctDNA-positive when variant percentages was on average 5 times lower than in tDNA, ctDNA-negative when tDNA variants were not found in cfDNA and unknown when no tDNA variant was covered by the cfDNA-TGS panel. Pearson correlation coefficient of the linear regression for ctDNA-positive samples was 0.97. In a few cases, TML was observed to be higher in cfDNA than in tDNA. For example samples P07 (bladder cancer) harbored a 1.78variants per megabase TML in cfDNA compared to 0.34 variants per megabase TML in tDNA, which could be explained by low cellularity of biopsy (10%). The four cases with the strongest difference in TML between cfDNA and tDNA were P16, P17, P18 and P31. They all had a tumor mutation load lower than 5 in cfDNA and higher than 5 in tDNA.

Fig 4. Correlation of variant percentages between duplicate whole exome sequencing of the same cfDNA extract.

All somatic variants in common between each of the three duplicate samples are displayed. Lower right: Correlation of variant frequencies between duplicates for all curated variants (pooled results from patients P13, P16 and P18).

Fig 5. Allele frequencies of mutations.

A and B. All mutations (curated variants) covered by the TGS panel. C. All mutations. D. Fraction of tDNA-WES variants found in cfDNA-WES (a total of 231 variants were found in tDNA-WES considering all samples, including 70 variants in ctDNA-positive samples).

Fig 6. Number of variants in common between cfDNA and both corresponding biopsy sites.

Liquid biopsies were collected on the day before the solid biopsies 1 and 2, except for patient P30, for whom biopsy 1 was collected 5 months before the liquid biopsy.