Failure is not final: ctDNA-guided rechallenge therapy in colorectal cancer
Authors: G. Siravegna, A. Bardelli
Journal: Annals of Oncology (2018)

Medical treatment of metastatic colorectal cancer (mCRC), the second tumor type for incidence, relies primarily on chemotherapies [1.Fakih M.G. Metastatic colorectal cancer: current state and future directions.J Clin Oncol. 2015; 33: 1809-1824Crossref PubMed Scopus (324) Google Scholar]. The addition of targeted agents such as the anti-EGFR antibodies cetuximab and panitutumab has improved patients' survival [2.Peeters M. Price T. Biologic therapies in the metastatic colorectal cancer treatment continuum–applying current evidence to clinical practice.Cancer Treat Rev. 2012; 38: 397-406Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. Unfortunately, almost invariably, patients treated with EGFR blockade drugs develop resistance [3.Bardelli A. Siena S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer.J Clin Oncol. 2010; 28: 1254-1261Crossref PubMed Scopus (555) Google Scholar]. From a molecular perspective, acquired resistance to anti-EGFR (epidermal growth factor receptor) treatment is associated with two main mechanisms: the first involves the emergence of activating mutations in EGFR downstream effectors (primarily KRAS, NRAS and BRAF), while the second relies on mutations in the EGFR extracellular domain (ECD) that impair antibody binding to its target [4.Arena S. Bellosillo B. Siravegna G. et al.Emergence of multiple EGFR extracellular mutations during cetuximab treatment in colorectal cancer.Clin Cancer Res. 2015; 21: 2157-2166Crossref PubMed Scopus (178) Google Scholar]. Treatment of patients who respond and then relapse to EGFR blockade drugs remains an unmet clinical need for at least two reasons: first, the molecular bases of relapse are patient-specific and difficult to define as tissue biopsies are not systematically carried out in this setting and have intrinsic risks [5.Krishnamurthy N. Spencer E. Torkamani A. Nicholson L. Liquid biopsies for cancer: coming to a patient near you.JCM. 2017; 6: 3-6Crossref Scopus (54) Google Scholar]; secondly, KRAS and NRAS secondary mutations, which occur in about 30%–40% of the patients, are presently undruggable as the corresponding proteins are recalcitrant to pharmacological blockade [6.McCormick F. K-Ras protein as a drug target.J Mol Med. 2016; 94: 253-258Crossref PubMed Scopus (72) Google Scholar]. As a result, upon failure to chemo plus anti-EGFR therapy, mCRC patients usually undergo additional lines of standard chemotherapy (irinotecan and/or oxaliplatin-based regimens) together with antiangiogenic drugs. None of these treatments is currently based on a molecular rationale. In this issue of Annals of Oncology, Parseghian et al. [7.Parseghian C.M. Loree J.M. Morris V.K. et al.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 (95) Google Scholar] provide evidence that monitoring levels of KRAS and EGFR ECD mutations in circulating tumor DNA (ctDNA) of patients has relevance in this setting. Specifically, their findings suggest that tracking kinetics of resistance mutations in blood can be used to guide additional rounds of anti-EGFR therapy. This study builds on the extraordinary progress made in the ability to measure tumor-derived somatic variants in the blood of patients with solid cancers [8.Schwaederle M. Chattopadhyay R. Kato S. et al.Genomic alterations in circulating tumor DNA from diverse cancer patients identified by next-generation sequencing.Cancer Res. 2017; 77: 5419-5427Crossref PubMed Scopus (68) Google Scholar, 9.Bettegowda C. Sausen M. Leary R.J. et al.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci Transl Med. 2014; 6: 224ra224Crossref Scopus (2774) Google Scholar]. The analysis of ctDNA, commonly referred to as liquid biopsies, enables noninvasive identification of molecular alterations emerging during treatment evolution, allowing real time genetic profiling of the overall disease [10.Siravegna G. Marsoni S. Siena S. Bardelli A. Integrating liquid biopsies into the management of cancer.Nat Rev Clin Oncol. 2017; 14: 531-548Crossref PubMed Scopus (992) Google Scholar, 11.Siravegna G. Bardelli A. Blood circulating tumor DNA for non-invasive genotyping of colon cancer patients.Mol Oncol. 2016; 10: 475-480Crossref PubMed Scopus (47) Google Scholar]. The results presented by Parseghian et al. rely on previous evidence that mutations associated with resistance to anti-EGFR therapy can be detected in ctDNA [12.Misale S. Yaeger R. Hobor S. et al.Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer.Nature. 2012; 486: 532-536Crossref PubMed Scopus (1334) Google Scholar, 13.Diaz L.A. Williams R.T. Wu J. et al.The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.Nature. 2012; 486: 537-540Crossref PubMed Scopus (1285) Google Scholar]. Furthermore, in 2015 we and others reported that mutant KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of anti-EGFR antibodies, indicating that clonal evolution continues beyond clinical progression [14.Siravegna G. Mussolin B. Buscarino M. et al.Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients.Nat Med. 2015; 21: 795-801Crossref PubMed Scopus (579) Google Scholar, 15.Morelli M.P. Overman M.J. Dasari A. et al.Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment.Ann Oncol. 2015; 26: 731-736Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar]. The decay of KRAS mutations in blood upon anti-EGFR therapy withdrawal is an indication of clonal evolution during therapy. However, the exact molecular bases of this process have not yet been elucidated. The most intuitive hypothesis, which is also supported by Parseghian et al., is that following the Darwinian evolutionary theory, cells with the highest fitness (KRAS and EGFR mutant) are able to survive, ultimately leading to therapeutic failure. The fitness of this resistant population may be, however, innately limited, allowing for the rapid (re)growth of the remaining (sensitive/wild-type) cell population. While this could indeed be the case for KRAS activating variants (which renders CRC cells independent from upstream EGFR inhibition), how EGFR ECD mutants negatively affect the fitness of CRC cells in the absence of an EGFR blockade and, as a result, decay in blood, is less intuitive. These questions should be further addressed as they could reveal important cellular mechanisms responsible for clonal competition and fitness in patients receiving treatment with anti-EGFR antibodies. The most important finding of the current study is the accurate determination of the kinetics of the KRAS and EGFR decay in blood, which had so far remained poorly defined. To address this, Parseghian et al. analyzed postprogression ctDNA samples obtained from 135 RAS/BRAF wild-type mCRC patients who underwent EGFR blockade therapy and acquired RAS and/or EGFR mutations during treatment. Two additional cohorts where included (i) a validation dataset, with 73 patients showing ctDNA profile suggestive of prior anti-EGFR exposure with serial blood sampling and (ii) a separate retrospective dataset with 107 cases to evaluate overall response rate and median time to rechallenge therapy. The results revealed that the relative mutant allele frequency of RAS and EGFR mutant clones, decays exponentially within a cumulative half-life of 4.4 months (Figure 1). Interestingly, when individual mutations were analyzed separately, RAS mutant clones were found to decay faster (half-life of 3.7 months), while EGFR ones dropped within 4.7 months. Most importantly, in the retrospective cohort, the longer the time intervals from EGFR blockade discontinuation and time to rechallenge therapy, the higher was the overall response rate. This is relevant as it suggests that future studies exploiting ctDNA kinetics to guide therapy in this setting should be aimed at maximizing RAS/EGFR mutant levels decay before anti-EGFR therapy is reinitiated. Furthermore, the interval between two exposures to EGFR blockade should be taken carefully into account as it impacts the efficacy of the rechallenge therapy. This is also consistent with previous evidence that upon a second round of therapy, resistant clones that had decayed in blood raise again [14.Siravegna G. Mussolin B. Buscarino M. et al.Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients.Nat Med. 2015; 21: 795-801Crossref PubMed Scopus (579) Google Scholar]. Firstly, they further encourage delivering multiple rounds of anti-EGFR rechallenge therapies for mCRC cases who initially respond to this regimen. Secondly, they offer additional support to the use of liquid biopsies (in this case measuring KRAS and EGFR mutant clones), to monitoring tumor kinetics (clones' half-life) and to guide the timing of rechallenge therapies. Notably, clinical experimentations, which build upon clonal kinetics, are already ongoing in this clinical setting. For example, CHRONOS (the Greek God of time; NCT03227926), is aimed at using liquid biopsies to identify mCRC patients originally responsive to anti-EGFR therapy who can then benefit from rechallenge. In CHRONOS patients receive a second round of EGFR blockade drugs based on RAS/BRAF ctDNA kinetics; specifically patients are being rechallenged when RAS/BRAF levels drop by >50% from their original levels. Based on the results of Parseghian et al. it is likely that future studies will also need to consider fine tuning of the time for rechallenge, e.g. by waiting till mutant RAS levels minimally drop (become nondetectable) in blood before reinitiating treatment. Future studies should also include measurement of EGFR ECD mutations given that, as shown in the current manuscript, they decay when EGFR therapy is suspended. This work was supported by European Community's Seventh Framework Programme under grant agreement no. 602901 MErCuRIC (AB); H2020 grant agreement no. 635342-2 MoTriColor (AB); IMI contract no. 115749 CANCER-ID (AB); AIRC 2010 Special Program Molecular Clinical Oncology 5 per mille, project no. 9970 extension (AB); AIRC IG no. 21923 (AB); AIRC Special Program 5 per mille metastases Project no. 21091 (AB). AIRC 3-year fellowship (GS); Roche per la Ricerca grant 2017 (GS).