a-standardized-liquid-biopsy-preanalytical-protocol-for-downstream

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

the Biomedical Research Network in Cancer (CIBERONC)

 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. .

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

Inflated pathogenic variant profiles in the ClinVar database.

Recognizing hypoxia in phaeochromocytomas and paragangliomas.

Longitudinal whole-exome sequencing of cell-free DNA for tracking the co-evolutionary tumor and immune evasion dynamics: longitudinal data from a single patient.

Anti-BRAF/EGFR therapy was recently approved for the treatment of metastatic BRAF colorectal cancer (mCRC). However, a large fraction of patients do not respond, underscoring the need to identify molecular determinants of treatment response. Using whole-exome sequencing in a discovery cohort of patients with mCRC treated with anti-BRAF/EGFR therapy, we found that inactivating mutations in RNF43, a negative regulator of WNT, predict improved response rates and survival outcomes in patients with microsatellite-stable (MSS) tumors. Analysis of an independent validation cohort confirmed the relevance of RNF43 mutations to predicting clinical benefit (72.7% versus 30.8%; P = 0.03), as well as longer progression-free survival (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.12-0.75; P = 0.01) and overall survival (HR, 0.26; 95% CI, 0.10-0.71; P = 0.008), in patients with MSS-RNF43 versus MSS-RNF43 tumors. Microsatellite-instable tumors invariably carried a wild-type-like RNF43 genotype encoding p.G659fs and presented an intermediate response profile. We found no association of RNF43 mutations with patient outcomes in a control cohort of patients with MSS-mCRC tumors not exposed to anti-BRAF targeted therapies. Overall, our findings suggest a cross-talk between the MAPK and WNT pathways that may modulate the antitumor activity of anti-BRAF/EGFR therapy and uncover predictive biomarkers to optimize the clinical management of these patients.

Article	Year
Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 29588308	2018
Inflated pathogenic variant profiles in the ClinVar database. Nature reviews. Endocrinology\| PubMed ID: 29858586	2018
Recognizing hypoxia in phaeochromocytomas and paragangliomas. Nature reviews. Endocrinology\| PubMed ID: 32047276	2020
Longitudinal whole-exome sequencing of cell-free DNA for tracking the co-evolutionary tumor and immune evasion dynamics: longitudinal data from a single patient. Annals of oncology : official journal of the European Society for Medical Oncology\| PubMed ID: 33609721	2021
RNF43 mutations predict response to anti-BRAF/EGFR combinatory therapies in BRAF metastatic colorectal cancer. Nature medicine\| PubMed ID: 36097219	2022

Rodrigo Almeida Toledo

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