Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies

Abstract

Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown. We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts. WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the /VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our and functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice. Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. .

Whole exome sequencing of cell-free DNA – A systematic review and Bayesian individual patient data meta-analysis

“…In total, 20 studies were included in this review of which the individual patient data of 12 studies were included for the meta-analyses of SNV sensitivity and agreement ( Fig. 1) [5,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33].…”

Longitudinal whole-exome sequencing of cell-free DNA unravels the metastatic evolutionary dynamics of BRCA2-mutated breast cancer

“…In addition to recent technical advances 17 , our longitudinal WES-cfDNA approach generated biological insights of the patient's disease course from relapse until near to the time of her death. We found a core of 31 mutations and specific SCNA profiles that remained throughout her disease development from the PT, LR and through treatment for metastatic disease, comprising a period of 14 years.…”

“…DNA isolated from cells in the buffy coat served as a germline control. Libraries were prepared using 15ng of DNA following the WES workflow using the Illumina HiSeq4000 platform as described in Toledo, Garralda et al 2018 17 .…”

“…Aiming to overcome these limitations, new liquid biopsy approaches such as total cfDNA whole-exome sequencing (WES-cfDNA) have been recently developed and shown high concordance with tumor whole-exome sequencing [15][16][17] . This WES-cfDNA is able to non-invasively depict the genomic landscape of advanced cancer, monitor known circulating tumor mutations and pinpoint acquired somatic mutations at the time of cancer progression possibly associated with cancer therapy resistance.…”

New genetic variations discovered in KRAS wild‐type cetuximab resistant chinese colorectal cancer patients

“…In addition, there are recent studies in RAS/BRAF/PIK3CA WT mCRC patient's germline and plasma cell free DNA samples which were sequenced in whole exomes (WES‐cfDNA) to explore mechanisms of antivascular drug resistance. Interestingly, it was found that VEGFR2 undergoes somatic mutation in various tumor types, and VEGFR2 mutants may be an important factor in promoting cancer and are closely related to sensitivity against antiangiogenic drugs 15 …”