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Diagnostic Strategies toward Clinical Implementation of Liquid Biopsy RAS/BRAF Circulating Tumor DNA Analyses in Patients with Metastatic Colorectal Cancer

2020; Elsevier BV; Volume: 22; Issue: 12 Linguagem: Inglês

10.1016/j.jmoldx.2020.09.002

1943-7811

Iris van ’t Erve, Marjolein J.E. Greuter, Karen Bolhuis, Daan C.L. Vessies, Alessandro Leal, Geraldine R. Vink, Daan van den Broek, Victor E. Velculescu, Cornelis J.A. Punt, Gerrit A. Meijer, Veerle M.H. Coupé, Remond J.A. Fijneman,

Genetic factors in colorectal cancer

Detection of KRAS, NRAS, and BRAF mutations in tumor tissue is currently used to predict resistance to treatment with anti–epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (mCRC). Liquid biopsies are minimally invasive, and cell-free circulating tumor DNA (ctDNA) mutation analyses may better represent tumor heterogeneity. This study examined the incorporation of liquid biopsy RAS/BRAF ctDNA analyses into diagnostic strategies to determine mCRC patient eligibility for anti-EGFR therapy. Tumor tissue and liquid biopsies were collected from 100 mCRC patients with liver-only metastases in a multicenter prospective clinical trial. Three diagnostic strategies incorporating droplet digital PCR ctDNA analyses were compared with routine tumor tissue RAS/BRAF mutation profiling using decision tree analyses. Tissue DNA mutations in KRAS, NRAS, and BRAF were present in 54%, 0%, and 3% of mCRC patients, respectively. A 93% concordance was observed between tissue DNA and liquid biopsy ctDNA mutations. The proportion of patients with RAS/BRAF alterations increased from 57% to 60% for diagnostic strategies that combined tissue and liquid biopsy mutation analyses. Consecutive RAS/BRAF ctDNA analysis followed by tissue DNA analysis in case of a liquid biopsy–negative result appeared to be the most optimal diagnostic strategy to comprehensively determine eligibility for anti-EGFR therapy in a cost-saving manner. These results highlight the potential clinical utility of liquid biopsies for detecting primary resistance to anti-EGFR–targeted therapies. Detection of KRAS, NRAS, and BRAF mutations in tumor tissue is currently used to predict resistance to treatment with anti–epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (mCRC). Liquid biopsies are minimally invasive, and cell-free circulating tumor DNA (ctDNA) mutation analyses may better represent tumor heterogeneity. This study examined the incorporation of liquid biopsy RAS/BRAF ctDNA analyses into diagnostic strategies to determine mCRC patient eligibility for anti-EGFR therapy. Tumor tissue and liquid biopsies were collected from 100 mCRC patients with liver-only metastases in a multicenter prospective clinical trial. Three diagnostic strategies incorporating droplet digital PCR ctDNA analyses were compared with routine tumor tissue RAS/BRAF mutation profiling using decision tree analyses. Tissue DNA mutations in KRAS, NRAS, and BRAF were present in 54%, 0%, and 3% of mCRC patients, respectively. A 93% concordance was observed between tissue DNA and liquid biopsy ctDNA mutations. The proportion of patients with RAS/BRAF alterations increased from 57% to 60% for diagnostic strategies that combined tissue and liquid biopsy mutation analyses. Consecutive RAS/BRAF ctDNA analysis followed by tissue DNA analysis in case of a liquid biopsy–negative result appeared to be the most optimal diagnostic strategy to comprehensively determine eligibility for anti-EGFR therapy in a cost-saving manner. These results highlight the potential clinical utility of liquid biopsies for detecting primary resistance to anti-EGFR–targeted therapies. Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world.1Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2018; 68: 394-424Crossref PubMed Scopus (44890) Google Scholar Systemic treatment with therapeutic monoclonal antibodies directed against epidermal growth factor receptor (EGFR) can be offered to patients with metastatic CRC (mCRC). Exclusion criteria include the presence of a mutation in either KRAS, NRAS, or BRAF, or a right-sided location of the primary tumor.2Douillard J.-Y. Oliner K.S. Siena S. Tabernero J. Burkes R. Barugel M. Humblet Y. Bodoky G. Cunningham D. Jassem J. Rivera F. Kocákova I. Ruff P. Błasińska-Morawiec M. Šmakal M. Canon J.L. Rother M. Williams R. Rong A. Wiezorek J. Sidhu R. Patterson S.D. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer.N Engl J Med. 2013; 369: 1023-1034Crossref PubMed Scopus (1625) Google Scholar,3Tejpar S. Stintzing S. Ciardiello F. Tabernero J. Van Cutsem E. Beier F. Esser R. Lenz H.-J. Heinemann V. Prognostic and predictive relevance of primary tumor location in patients with RAS wild-type metastatic colorectal cancer: retrospective analyses of the CRYSTAL and FIRE-3 trials.JAMA Oncol. 2017; 3: 194-201Crossref PubMed Scopus (415) Google Scholar Approximately 40% to 45% of the mCRC patients harbor a somatic RAS mutation,4Hsu H.-C. Thiam T.K. Lu Y.-J. Yeh C.Y. Tsai W.-S. You J.F. Hung H.Y. Tsai C.-N. Hsu A. Chen H.-C. Chen S.-J. Yang T.-S. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients.Oncotarget. 2016; 7: 22257-22270Crossref PubMed Scopus (91) Google Scholar,5Therkildsen C. Bergmann T.K. Henrichsen-Schnack T. Ladelund S. Nilbert M. The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer: a systematic review and meta-analysis.Acta Oncol. 2014; 53: 852-864Crossref PubMed Scopus (279) Google Scholar and 5% to 12%, a BRAF mutation.6Yaeger R. Chatila W.K. Lipsyc M.D. Hechtman J.F. Cercek A. Sanchez-Vega F. Jayakumaran G. Middha S. Zehir A. Donoghue M.T.A. You D. Viale A. Kemeny N. Segal N.H. Stadler Z.K. Varghese A.M. Kundra R. Gao J. Syed A. Hyman D.M. Vakiani E. Rosen N. Taylor B.S. Ladanyi M. Berger M.F. Solit D.B. Shia J. Saltz L. Schultz N. Clinical sequencing defines the genomic landscape of metastatic colorectal cancer.Cancer Cell. 2018; 33: 125-136.e3Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 7Souglakos J. Philips J. Wang R. Marwah S. Silver M. Tzardi M. Silver J. Ogino S. Hooshmand S. Kwak E. Freed E. Meyerhardt J.A. Saridaki Z. Georgoulias V. Finkelstein D. Fuchs C.S. Kulke M.H. Shivdasani R.A. Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer.Br J Cancer. 2009; 101: 465-472Crossref PubMed Scopus (245) Google Scholar, 8Fakih M.G. Metastatic colorectal cancer: current state and future directions.J Clin Oncol. 2015; 33: 1809-1824Crossref PubMed Scopus (323) Google Scholar, 9Tran B. Kopetz S. Tie J. Gibbs P. Jiang Z.-Q. Lieu C.H. Agarwal A. Maru D.M. Sieber O. Desai J. Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer.Cancer. 2011; 117: 4623-4632Crossref PubMed Scopus (497) Google Scholar Analysis of RAS/BRAF mutation status before the subscription of anti-EGFR therapy has therefore been implemented in routine clinical practice to predict who will not benefit from this antibody treatment. DNA from tumor tissue is used to assess the RAS/BRAF mutation status within each hospital using its own routine diagnostic technique. Although tumor tissue DNA mutation analysis is the gold standard, obtaining tissue is invasive, and it does not always accurately represent the genetic heterogeneity of the tumor.10Sveen A. Løes I.M. Alagaratnam S. Nilsen G. Høland M. Lingjærde O.C. Sorbye H. Berg K.C.G. Horn A. Angelsen J.-H. Knappskog S. Lønning P.E. Lothe R.A. Intra-patient inter-metastatic genetic heterogeneity in colorectal cancer as a key determinant of survival after curative liver resection.PLoS Genet. 2016; 12: e1006225Crossref PubMed Scopus (40) Google Scholar Moreover, it may require complex logistics to obtain (previously) resected tumor tissue or a burdensome procedure to obtain new tissue biopsies. mCRC patients have among the highest levels of circulating tumor DNA (ctDNA), making it possible to detect mutations in liquid biopsies at high sensitivity.11Bettegowda C. Sausen M. Leary R.J. Kinde I. Wang Y. Agrawal N. et al.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci Transl Med. 2014; 6: 224ra224Crossref Scopus (2751) Google Scholar, 12Strickler J.H. Loree J.M. Ahronian L.G. Parikh A.R. Niedzwiecki D. Pereira A.A.L. McKinney M. Korn W.M. Atreya C.E. Banks K.C. Nagy R.J. Meric-Bernstam F. Lanman R.B. Talasaz A. Tsigelny I.F. Corcoran R.B. Kopetz S. Genomic landscape of cell-free DNA in patients with colorectal cancer.Cancer Discov. 2018; 8: 164-173Crossref PubMed Scopus (148) Google Scholar, 13Thierry A.R. Mouliere F. El Messaoudi S. Mollevi C. Lopez-Crapez E. Rolet F. Gillet B. Gongora C. Dechelotte P. Robert B. Del Rio M. Lamy P.-J. Bibeau F. Nouaille M. Loriot V. Jarrousse A.S. Molina F. Mathonnet M. Pezet D. Ychou M. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA.Nat Med. 2014; 20: 430-435Crossref PubMed Scopus (483) Google Scholar, 14Phallen J. Sausen M. Adleff V. Leal A. Hruban C. White J. et al.Direct detection of early-stage cancers using circulating tumor DNA.Sci Transl Med. 2017; 9: eaan2415Crossref PubMed Scopus (529) Google Scholar Previous studies have shown a high concordance between tumor tissue and liquid biopsy RAS mutation status in CRC patients.15Normanno N. Esposito Abate R. Lambiase M. Forgione L. Cardone C. Iannaccone A. Sacco A. Rachiglio A.M. Martinelli E. Rizzi D. Pisconti S. Biglietto M. Bordonaro R. Troiani T. Latiano T.P. Giuliani F. Leo S. Rinaldi A. Maiello E. Ciardiello F. CAPRI-GOIM InvestigatorsRAS testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with first-line FOLFIRI plus cetuximab in the CAPRI-GOIM trial.Ann Oncol. 2018; 29: 112-118Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16Grasselli J. Elez E. Caratù G. Matito J. Santos C. Macarulla T. Vidal J. Garcia M. Viéitez J.M. Paéz D. Falcó E. Lopez Lopez C. Aranda E. Jones F. Sikri V. Nuciforo P. Fasani R. Tabernero J. Montagut C. Azuara D. Dienstmann R. Salazar R. Vivancos A. Concordance of blood- and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer.Ann Oncol. 2017; 28: 1294-1301Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 17García-Foncillas J. Tabernero J. Élez E. Aranda E. Benavides M. Camps C. Jantus-Lewintre E. López R. Muinelo-Romay L. Montagut C. Antón A. López G. Díaz-Rubio E. Rojo F. Vivancos A. Prospective multicenter real-world RAS mutation comparison between OncoBEAM-based liquid biopsy and tissue analysis in metastatic colorectal cancer.Br J Cancer. 2018; 119: 1464-1470Crossref PubMed Scopus (43) Google Scholar In these studies, tumor tissue DNA mutation analyses were performed centrally using a uniform approach. However, concordance may differ in clinical practice because previously resected tumor tissue material might not be representative for the current tumor, and analyses may be performed using diverse approaches in different hospitals. Previous studies in lung cancer demonstrated the clinical applicability of liquid biopsy ctDNA testing above tumor tissue DNA testing, both regarding mutation detection and turnaround time.18Sabari J.K. Offin M. Stephens D. Ni A. Lee A. Pavlakis N. et al.A prospective study of circulating tumor DNA to guide matched targeted therapy in lung cancers.J Natl Cancer Inst. 2019; 111: 575-583Crossref PubMed Scopus (64) Google Scholar,19Leighl N.B. Page R.D. Raymond V.M. Daniel D.B. Divers S.G. Reckamp K.L. Villalona-Calero M.A. Dix D. Odegaard J.I. Lanman R.B. Papadimitrakopoulou V.A. Clinical utility of comprehensive cell-free DNA analysis to identify genomic biomarkers in patients with newly diagnosed metastatic non-small cell lung cancer.Clin Cancer Res. 2019; 25: 4691-4700Crossref PubMed Scopus (231) Google Scholar Moreover, it was recently shown that continuous monitoring of RAS and EGFR mutations may select patients for rechallenge of anti-EGFR treatment, which may be less demanding using liquid biopsies than tissue biopsies.20Parseghian C.M. Loree J.M. Morris V.K. Liu X. Clifton K.K. Napolitano S. Henry J.T. Pereira A.A. Vilar E. Johnson B. Kee B. Raghav K. Dasari A. Wu J. Garg N. Raymond V.M. Banks K.C. Talasaz A.A. Lanman R.B. Strickler J.H. Hong D.S. Corcoran R.B. Overman M.J. Kopetz S. Anti-EGFR-resistant clones decay exponentially after progression: implications for anti-EGFR re-challenge.Ann Oncol. 2019; 30: 243-249Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar In the present study, the concordance between RAS/BRAF mutation status was assessed in tumor tissue and liquid biopsies of mCRC patients in a clinical setting. Thereby, the diagnostic assay costs and proportion of mCRC patients eligible for anti-EGFR treatment were compared by evaluating four diagnostic strategies using decision tree analyses. Prior to treatment, tumor tissue and liquid biopsies were collected from 100 unresectable liver-only mCRC patients participating in the CAIRO5 (Treatment Strategies in Colorectal Cancer Patients With Initially Unresectable Liver-Only Metastases) multicenter clinical trial (ClinicalTrials.gov; https://clinicaltrials.gov/ct2/show/NCT02162563).21Huiskens J. van Gulik T.M. van Lienden K.P. Engelbrecht M.R.W. Meijer G.A. van Grieken N.C.T. Schriek J. Keijser A. Mol L. Molenaar I.Q. Verhoef C. de Jong K.P. Dejong K.H.C. Kazemier G. Ruers T.M. de Wilt J.H.W. van Tinteren H. Punt C.J.A. Treatment strategies in colorectal cancer patients with initially unresectable liver-only metastases, a study protocol of the randomised phase 3 CAIRO5 study of the Dutch Colorectal Cancer Group (DCCG).BMC Cancer. 2015; 15: 365Crossref PubMed Scopus (45) Google Scholar The trial was approved by a medical ethical committee, and all patients signed written informed consent for study participation as well as blood and tissue collection for translational research. Prior to randomization, tissue DNA mutation analyses for RAS/BRAF were performed in the participating hospitals following routine clinical practice. Different methods were used ranging from a variety of next-generation sequencing panels (77% of the patients) to Sanger sequencing (22%) and high-resolution melting analysis (1%). Tissue mutation analyses were performed on DNA isolated from the primary tumor for most patients because tissue from metastases was rarely available (91% versus 9%, respectively). Supplemental Table S1 shows a detailed overview of the patient and sample characteristics. Prior to treatment, 10 mL of blood was collected using a cell-stabilizing BCT tube (Streck, La Vista, NE) in the participating hospitals and collected centrally at the Clinical Chemistry laboratory at the Netherlands Cancer Institute. Upon arrival, cell-free plasma was collected in a two-step centrifugation process: 10 minutes at 1700 × g followed by 10 minutes at 20,000 × g, and stored at −80°C within an average of 2 days after venapunction. Cell-free DNA was isolated using the QIAsymphony system (Qiagen, Hilden, Germany) with an elution volume set to 60 μL. The concentration of cell-free DNA was between 0.174 and 36.6 ng/μL measured using the Qubit dsDNA High-Sensitivity Assay (Thermo Fisher Scientific, Waltham, MA). RAS/BRAF mutation analyses using extracted cell-free DNA from plasma were performed with the droplet digital PCR (ddPCR; Bio-Rad Laboratories, Hercules, CA). For these analyses, the ddPCR KRAS G12/G13 (#1863506), ddPCR KRAS Q61 (#12001626), ddPCR KRAS A146 (#10049550), and the ddPCR BRAF V600 (#12001037) screening kits were used according to the manufacturer's instruction, making use of 1 μL of the multiplex assay, 11 μL of the ddPCR Supermix for Probes (No dUTP) (Bio-Rad Laboratories), 9 μL of sample, and 1 μL of nuclease-free water. All measurements were performed in duplicate and included a blank (nuclease-free water) and an in-house positive control. Data were analyzed using the QuantaSoft software version 1.6.6 (Bio-Rad Laboratories). Individual wells with <10,000 total events (droplets) were excluded from the analysis. In the nine cases when T, and Q61H 183A>C mutations), assay 3 = ddPCR KRAS A146T Assay (#10049550, detecting A146T mutations), assay 4 = ddPCR BRAF V600 Screening Kit (#12001037, detecting V600E, V600K, and V600R mutations). 1 only§Includes personnel costs per sample.€2.42/$2.86€36.81/$43.44€3.37/$3.98€7.76/$9.16€50/$59 Assays‡Assay 1 = ddPCR KRAS G12/G13 Screening Kit (#1863506, detecting G12D, G12V, G13D, G12A, G12C, G12R, and G12S mutations), assay 2 = ddPCR KRAS Q61 Screening Kit (#12001626, detecting Q61K, Q61L, Q61R, Q61H 183A>T, and Q61H 183A>C mutations), assay 3 = ddPCR KRAS A146T Assay (#10049550, detecting A146T mutations), assay 4 = ddPCR BRAF V600 Screening Kit (#12001037, detecting V600E, V600K, and V600R mutations). 1+2+3+4§Includes personnel costs per sample.€2.13/$2.51€36.81/$43.44€2.96/$3.49€6.82/$8.05€195/$230Costs are depicted in 2018 euros and in 2018 US dollars using the purchasing power parity for that year (1 euro = 1.18 US dollars).cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; ddPCR, droplet digital PCR; NA, not applicable; NGS, next-generation sequencing.∗ Material costs/run and equipment costs/year are based on the current throughput of 16 samples per run (including two controls) and one run per week.† Personnel costs/run, material costs/run, and equipment costs/year are based on an addition of 100 samples (one assay strategy) or 400 samples (four assay strategy) per year to the current throughput of 20 samples per run, two runs a week.‡ Assay 1 = ddPCR KRAS G12/G13 Screening Kit (#1863506, detecting G12D, G12V, G13D, G12A, G12C, G12R, and G12S mutations), assay 2 = ddPCR KRAS Q61 Screening Kit (#12001626, detecting Q61K, Q61L, Q61R, Q61H 183A>T, and Q61H 183A>C mutations), assay 3 = ddPCR KRAS A146T Assay (#10049550, detecting A146T mutations), assay 4 = ddPCR BRAF V600 Screening Kit (#12001037, detecting V600E, V600K, and V600R mutations).§ Includes personnel costs per sample. Open table in a new tab Costs are depicted in 2018 euros and in 2018 US dollars using the purchasing power parity for that year (1 euro = 1.18 US dollars). cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; ddPCR, droplet digital PCR; NA, not applicable; NGS, next-generation sequencing. Using the decision trees (Figure 1), the expected proportion of patients that are eligible for anti-EGFR therapy and overall expected diagnostic costs per patient were calculated for each strategy. Note that patients in whom a RAS/BRAF mutation was detected were considered not eligible for anti-EGFR treatment. In addition, the minimum and maximum turnaround time from the first analysis to the result was assessed for each strategy based on current routine diagnostic practice within the Netherlands Cancer Institute. A probabilistic sensitivity analysis was conducted to evaluate the impact of uncertainty on the expected proportion of patients considered eligible for anti-EGFR therapy as well as costs for each strategy. A beta distribution was assigned to the proportion of analyses in which a mutation was detected. For the total costs of each strategy, a uniform distribution was used with the assumption that the costs varied by 10% at most. Each strategy was then evaluated using 1000 randomly selected parameter sets. Based on tumor tissue DNA analyses, 54% of patients harbored a KRAS mutation and 3% harbored a BRAF mutation (Supplemental Table S1). No NRAS mutations were observed. Based on the outcome of the tissue mutation analysis, a ddPCR assay was performed to confirm the RAS/BRAF mutation status in liquid biopsies. Tumor tissue DNA and liquid biopsy ctDNA analysis for KRAS/BRAF mutation status showed a 93% concordance (Figure 2). In 4% of patients, there was a discordant result with no mutation detected in the liquid biopsy, comprising three KRAS G12D and one KRAS G12C mutations. In 3% of the patients, a mutation was detected using the KRAS G12/13 liquid biopsy assay, which had not been detected by the tumor tissue analysis. Thereby, the tumor tissue mutation testing was not enriched for one particular assay, for example, Sanger sequencing. The potential of liquid biopsy ctDNA analyses for the determination of anti-EGFR treatment eligibility in mCRC patients was assessed by evaluating four diagnostic strategies using decision tree analyses (Figure 1). For each strategy, the expected proportion of anti-EGFR–eligible patients and overall expected assay costs per patient were calculated. In the tumor tissue–only analysis, reflecting current clinical practice (strategy A), 57% of the patients were not eligible for anti-EGFR therapy. This proportion increased to 60% for strategies B and C, which both combined liquid biopsy with tissue mutation analysis. In strategy D, the liquid biopsy–only strategy, 56% of patients were not eligible for anti-EGFR therapy. Based on the decision-analytic model, current practice was the most expensive diagnostic strategy at €579/$683 per patient. The costs of combination strategies B and C were €457/$539 and €401/$473 per patient, respectively. Strategy D, in which patients were only evaluated with liquid biopsy testing, was the least costly strategy at €195/$230 per patient. The turnaround time for the (ct)DNA analysis was 3 to 5 days for the tumor tissue–only analysis (strategy A), 1 to 10 days (average of 5 days) for the combination strategies (B and C), and 1 to 5 days for a liquid biopsy–only analysis (strategy D). In the probabilistic sensitivity analysis (Figure 3), the liquid biopsy–only strategy always led to the highest proportion of patients considered eligible for anti-EGFR therapy, whereas this was lowest for the combination strategies in almost all parameter sets (>99%). With respect to costs, the liquid biopsy–only strategy was consistently the cheapest strategy, whereas the tissue-only mutation analysis strategy was the most expensive strategy in all analyses. Systemic treatment with monoclonal antibodies directed against EGFR is one of the therapeutic options considered for mCRC patients with wild-type RAS and BRAF status. Here, the authors demonstrated that analysis of RAS/BRAF mutations in liquid biopsy ctDNA from liver-only mCRC patients offers an attractive and cost-saving complementary approach to tissue mutation analyses, suited for clinical implementation. This is the first study investigating the diagnostic opportunities for liquid biopsy–based strategies to determine RAS/BRAF mutation status from a cost perspective. The observed RAS and BRAF mutation rates in this cohort (54% and 3%, respectively) conformed with what is reported in the literature.24Peeters M. Kafatos G. Taylor A. Gastanaga V.M. Oliner K.S. Hechmati G. Terwey J.-H. van Krieken J.H. Prevalence of RAS mutations and individual variation patterns among patients with metastatic colorectal cancer: a pooled analysis of randomised controlled trials.Eur J Cancer. 2015; 51: 1704-1713Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 25Bläker H. Alwers E. Arnold A. Herpel E. Tagscherer K.E. Roth W. Jansen L. Walter V. Kloor M. Chang-Claude J. Brenner H. Hoffmeister M. The association between mutations in BRAF and colorectal cancer-specific survival depends on microsatellite status and tumor stage.Clin Gastroenterol Hepatol. 2019; 17: 455-462.e6Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 26Venderbosch S. Nagtegaal I.D. Maughan T.S. Smith C.G. Cheadle J.P. Fisher D. Kaplan R. Quirke P. Seymour M.T. Richman S.D. Meijer G.A. Ylstra B. Heideman D.A.M. de Haan A.F.J. Punt C.J.A. Koopman M. Mismatch repair status and BRAF mutation status in metastatic colorectal cancer patients: a pooled analysis of the CAIRO, CAIRO2, COIN, and FOCUS studies.Clin Cancer Res. 2014; 20: 5322-5330Crossref PubMed Scopus (387) Google Scholar The high concordance between liquid biopsy and tumor tissue RAS/BRAF mutation status shown in this study is comparable to other studies. Overall, the concordance between liquid biopsy and tumor tissue for RAS mutation status in mCRC patients fluctuates between 78% and 93%.27Molinari C. Marisi G. Passardi A. Matteucci L. De Maio G. Ulivi P. Heterogeneity in colorectal cancer: a challenge for personalized medicine?.Int J Mol Sci. 2018; 19: 3733Crossref Scopus (87) Google Scholar Less research has been performed to investigate the concordance for BRAF mutation status.13Thierry A.R. Mouliere F. El Messaoudi S. Mollevi C. Lopez-Crapez E. Rolet F. Gillet B. Gongora C. Dechelotte P. Robert B. Del Rio M. Lamy P.-J. Bibeau F. Nouaille M. Loriot V. Jarrousse A.S. Molina F. Mathonnet M. Pezet D. Ychou M. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA.Nat Med. 2014; 20: 430-435Crossref PubMed Scopus (483) Google Scholar In this study, there were three BRAF mutant cases, all of which had concordant results. Several discordant results in the comparison of tumor tissue and liquid biopsy RAS mutations were observed in this study. Tumor heterogeneity might be responsible for the fact that KRAS mutations were found in liquid biopsies but were missed in tumor tissue analyses (3% of the samples), but these are rare events,27Molinari C. Marisi G. Passardi A. Matteucci L. De Maio G. Ulivi P. Heterogeneity in colorectal cancer: a challenge for personalized medicine?.Int J Mol Sci. 2018; 19: 3733Crossref Scopus (87) Google Scholar,28Diaz Jr., L.A. Sausen M. Fisher G.A. Velculescu V.E. Insights into therapeutic resistance from whole-genome analyses of circulating tumor DNA.Oncotarget. 2013; 4: 1856-1857Crossref PubMed Scopus (32) Google Scholar consistent with the notion that these gene alterations typically occur early in tumorigenesis.29Rajagopalan H. Bardelli A. Lengauer C. Kinzler K.W. Vogelstein B. Velculescu V.E. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status.Nature. 2002; 418: 934Crossref PubMed Scopus (1047) Google Scholar Another possibility is that a fraction of these discordant results is associated with false-positive alterations due to clonal hematopoiesis. Previous studies have shown that cancer genes such as TP53 and KRAS can be mutated in white blood cells at the time the liquid biopsy is drawn.30Hu Y. Ulrich B.C. Supplee J. Kuang Y. Lizotte P.H. Feeney N.B. Guibert N.M. Awad M.M. Wong K.-K. Jänne P.A. Paweletz C.P. Oxnard G.R. False-positive plasma genotyping due to clonal hematopoiesis.Clin Cancer Res. 2018; 24: 4437-4443Crossref PubMed Scopus (212) Google Scholar Mutations that were detected in tumor tissue but missed in the liquid biopsies in the present study (4% of samples) might be explained by limited tumor ctDNA shedding into the circulation.31Diehl F. Schmidt K. Choti M.A. Romans K. Goodman S. Li M. Thornton K. Agrawal N. Sokoll L. Szabo S.A. Kinzler K.W. Vogelstein B. Diaz Jr., L.A. Circulating mutant DNA to assess tumor dynamics.Nat Med. 2008; 14: 985-990Crossref PubMed Scopus (1740) Google Scholar These discordant observations support the added value of diagnostic strategies combining liquid biopsy RAS/BRAF mutation testing with tumor tissue RAS/BRAF mutation testing in liquid biopsy–negative cases as described in the present study, as well as for several studies in lung cancer.18Sabari J.K. Offin M. Stephens D. Ni A. Lee A. Pavlakis N. et al.A prospective study of circulating tumor DNA to guide matched targeted therapy in lung cancers.J Natl Cancer Inst. 2019; 111: 575-583Crossref PubMed Scopus (64) Google Scholar,19Leighl N.B. Page R.D. Raymond V.M. Daniel D.B. Divers S.G. Reckamp K.L. Villalona-Calero M.A. Dix D. Odegaard J.I. Lanman R.B. Papadimitrakopoulou V.A. Clinical utility of comprehensive cell-free DNA analysis to identify genomic biomarkers in patients with newly diagnosed metastatic non-small cell lung cancer.Clin Cancer Res. 2019; 25: 4691-4700Crossref PubMed Scopus (231) Google Scholar The decision tree analysis showed that the combination strategies consisting of mutation analyses of liquid biopsy ctDNA followed by tumor tissue DNA analyses in liquid biopsy–negative cases were cheaper compared with the current clinical practice, that is, a tissue-only mutation analysis. In addition, the accuracy of determining patient eligibility for anti-EGFR therapy increased in these combination strategies, although this was accompanied by an increase in turnaround time for the liquid biopsy–negative patients. This increase in turnaround time will not interfere with patient care, because treatment is generally started 2 weeks after the first patient visit. Compared with the current diagnostic standard, the liquid biopsy–only strategy had a shorter turnaround time at lower costs, although patients in whom the RAS/BRAF mutation was missed using the liquid biopsy–only assay compared with the current diagnostic standard would receive futile treatment leading to needless burden due to side effects and high costs of anti-EGFR treatment.32Fakih M. Vincent M. Adverse events associated with anti-EGFR therapies for the treatment of metastatic colorectal cancer.Curr Oncol. 2010; 17 Suppl 1: S18-S30PubMed Google Scholar,33Sepulveda A.R. Hamilton S.R. Allegra C.J. Grody W. Cushman-Vokoun A.M. Funkhouser W.K. Kopetz S.E. Lieu C. Lindor N.M. Minsky B.D. Monzon F.A. Sargent D.J. Singh V.M. Willis J. Clark J. Colasacco C. Rumble R.B. Temple-Smolkin R. Ventura C.B. Nowak J.A. Molecular biomarkers for the evaluation of colorectal cancer: guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology.J Mol Diagn. 2017; 19: 187-225Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Altogether, a stepwise combination of liquid biopsy and tumor tissue RAS/BRAF mutation status would likely lower the overall costs of diagnostic testing as well as health care costs by making a better selection of putative anti-EGFR responders. Anti-EGFR therapy has shown to be effective in a limited percentage (10% to 20%) of patients who are wild type for RAS and BRAF.34Bertotti A. Sassi F. 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Insights into therapeutic resistance from whole-genome analyses of circulating tumor DNA.Oncotarget. 2013; 4: 1856-1857Crossref PubMed Scopus (32) Google Scholar In this light, a stepwise analysis consisting of a combination of the highly prevalent KRAS G12/13 mutation in liquid biopsy followed by a broader tumor tissue mutation analysis panel for the KRAS liquid biopsy–negative patients is an approach that anticipates more extensive future mutation analyses. In conclusion, this study showed that liquid biopsy analyses have high concordance with tissue biopsy RAS/BRAF mutation analyses in liver-only mCRC patients. By modeling various diagnostic strategies, the authors have demonstrated that liquid biopsy testing for RAS/BRAF mutations in ctDNA offers an attractive and cost-saving complementary approach to current routine diagnostic tissue DNA mutation analyses, ready for clinical implementation. We thank Mirthe Lanfermeijer, Dorothé Linders, Kalpana Ramkisoensing, Pien Delis-van Diemen, Margriet Lemmens, Anne Bolijn, and Marianne Tijssen for laboratory assistance. We acknowledge the Core Facility Molecular Pathology & Biobanking of the Netherlands Cancer Institute for supplying biobank material. I.v.t.E., C.J.A.P., G.A.M., V.M.H.C., and R.J.A.F. conceptualized the study; I.v.t.E., M.J.E.G., V.M.H.C., and R.J.A.F. designed the study; I.v.t.E., M.J.E.G., K.B., and D.C.L.V. acquired the data; I.v.t.E. and M.J.E.G. analyzed and interpreted the data; I.v.t.E. drafted the manuscript; I.v.t.E., M.J.E.G., K.B., D.C.L.V., A.L., G.R.V., D.v.d.B., V.E.V., C.J.A.P., G.A.M., V.M.H.C., and R.J.A.F revised the manuscript for intellectual content; and I.v.t.E. and R.J.A.F. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. 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