Whole-exome sequencing of matched germline and plasma cell-free DNA portrays the somatic mutation landscape of refractory metastatic colorectal cancer and identifies mutated KDR/VEGFR2 as new cause of therapy resistance.

Background

Anti-angiogenic therapies have been broadly used in oncology as in treatment of metastatic colorectal cancer (mCRC) patients, however the causes of resistance occurring in the majority of the patients treated remain largely unknown.

Methods

Whole-exome sequencing libraries of matched leukocyte and basal cfDNA samples (WES-gcfDNA) were generated and sequenced on HiSeq4000. KDR/VEGFR2 mutations were cloned and stably expressing CRC cell lines used for xenograft studies. Biochemical kinase assays were carried out to assess the impact of the VEGFR2 mutants on the inhibition of VEGFR2 kinase activity by cabozantinib, lenvatinib, axitinib and dovitinib. Patient-derived Avatar model was treated with multiple anti-angiogenic drugs. Somatic mutation landscapes depicted by WES-gcfDNA and the standard tumor WES (obtained later on) were compared.

Results

We investigated a RAS/BRAF/PIK3CA wild-type mCRC patient highly refractory to (sequentially): FOLFIRI-cetuximab; FOLFOX-bevacizumab; afatinib-cetuximab (phase-1 trial); oncolytic adenovirus monotherapy (phase-1 trial); capecitabine-bevacizumab; and finally regorafenib. No radiological or clinical benefit was observed after any of these treatments and the patient died due to his progressive disease within 14 months. WES-gcfDNA enabled us to identify the KDR/VEGFR2 L840F mutation exclusively in the cancer sample. Using the methods described above we obtained comprehensive experimental data showing that L840F decreases efficiency of TKIs, promote tumor growth and confer strong in vivo resistance to numerous anti-angiogenic therapies, including bevacizumab and VEGFR2 inhibitors. Other KDR/VEGFR2 somatic mutations we retrieved from cancer sequencing projects showed similar oncogenic and resistant phenotype.

Conclusions

Our study introduces WES-gcfDNA as a robust noninvasive gene discovery platform capable of portraying the somatic mutation landscape of metastatic cancer patients from blood sample solely. Moreover, we characterize a previously unexplored oncogenic and cancer therapy modulating role of VEGFR2 mutants.

Clinical trial identification

Legal entity responsible for the study

Rodrigo Toledo, Manuel Hidalgo

Funding

None

Disclosure

All authors have declared no conflicts of interest.