**5.3 CtDNA in mCRC: genotyping to monitor for targeted treatment response and resistance**

The role of genomic alterations and their evolution in both the development and progression of CRC have culminated in the realisation that serial genotyping of the primary tumour, and its secondaries, is ideally required if we want to succeed in personalising patient care with precision [80]. Unfortunately, patients with mCRC who do not harbour a somatic *RAS* mutation pre-treatment, will typically develop acquired resistance to anti-EGFR therapy in a matter of months after initially showing response. There is a battery of pre-clinical and clinical evidence which points to the acquisition of molecular mechanisms of resistance associated with aberrations in the *RAS*-*MEK*-*mitogen-activated protein kinases* (*MAPK*), pathway [2, 42, 81, 82].

Longitudinal ctDNA analysis can be used in this setting with high diagnostic precision to detect both primary resistance and early molecular changes that may confer acquired resistance. Several translational studies have successfully employed ctDNA to illustrate and verify the emergence of *RAS* mutations as a mechanism of acquired resistance to anti-EGFR therapy.

In a phase II trial of anti-EGFR antibody, panitumumab in mCRC, serial prospective plasma analysis detected more emergent *RAS* mutations than serial tissue biopsies, suggesting that the former may be more comprehensive in evaluating global tumoural heterogeneity [83]. In a small retrospective study of 10 mCRC patients who developed resistance to anti-EGFR therapy (cetuximab or panitumumab) in combination with chemotherapy, Misale et al. demonstrated that the onset of the emerging *KRAS* mutations was detected in serum ctDNA analysis as early as 10 months prior to radiological reporting of disease progression [2]. In this study, ctDNA analysis was also explored in a separate cohort of patients who were receiving chemotherapy alone (control group). No acquired *KRAS* mutations were identifiable at disease progression [2]. In the same year, Diaz et al. also demonstrated the feasibility of using serum ctDNA to identify emerging resistance to panitumumab in a prospective cohort of 28 patients [81]. Thirty-eight percent of patients whose tumours were initially *KRAS* wild type developed detectable *KRAS* mutations in their sera, three of whom developed multiple different *KRAS* mutations. These were detected approximately 5 months before radiological progression [81]. Another study subsequently made the intriguing observation that *KRAS* clones can fluctuate under the selective pressure of anti-EGFR therapy implying that there may be a role for 'pulsing' or re-challenging with anti-EGFR therapy [84].

Furthermore, in a prospective study of 108 patients, treated in the third line setting with cetuximab and irinotecan, Spindler et al. investigated the quantitative correlation between plasma cfDNA with tumour-specific plasma mutant *KRAS* levels [85]. This study revealed that (i) the majority of *KRAS* mutations that were detected in tumours were also found in the plasma, (ii) there was a strong correlation between cfDNA and plasma mutant *KRAS* levels, and (iii) high levels of plasma mutant *KRAS* were associated with 0% disease control rate [85]. More recently, a large retrospective exploratory analysis used BEAMing technology to identify *KRAS*, *PIK3CA* and *BRAF* mutations in the plasma ctDNA of 503 patients who enrolled in the CORRECT trial of regorafenib, a multi-kinase inhibitor in refractory mCRC [66]. Tumour-associated *KRAS* mutations were readily detected with BEAMing of plasma DNA and were identified in 48% of patients who had previously received anti-EGFR therapy and whose archival tumour tissues were KRAS wild type [66].

Beyond *KRAS* mutations and amplifications, acquired genetic aberrations in other genes have been found to potentially lead to anti-EGFR therapy resistance, albeit in smaller subsets of patients. For example, emerging *EGFR* extracellular domain (ECD) mutations which lead to impaired antibody binding were found to be a resistance mechanism to anti-EGFR therapy in approximately 20% of patients. Interestingly, these mutations tend to arise later than *RAS* mutations during therapy, and patients with *EGFR* ECD mutations had greater and more durable response to anti-EGFR therapy than patients with *RAS* mutations [86]. Interestingly, a phase I trial of a third generation EGFR-targeting agent that binds multiple regions of the EGFR ECD demonstrated efficacy in patients with *EGFR* ECD mutations and acquired resistance to prior EGFR blockade [87]. Other genomic alterations linked to acquired resistance to EGFR blockade include *MET and ERBB2* amplifications [88, 89] and mutations in *NRAS*, *BRAF* and *PIK3CA* [6]. *ERBB2* amplification was found in the plasma in four out of eight *RAS* wild type patients who derived no clinical benefit from anti-EGFR treatment, suggesting that it may also be a source of primary resistance [84].

Another innovative study provided proof-of-principle that parallel analysis of patient-derived xenografts and ctDNA allowed the identification of resistance mechanisms to a pan-tropomyosin-related kinase (TRK) inhibitor in mCRC, with validation in preclinical models [90]. In interpreting these translational findings, it is important to note that typically, multiple complex molecular abnormalities emerge rather than a singular clone and an overlap exists between abnormalities associated with primary and secondary resistance [6].

CtDNA genotyping has now paved the way for prospective clinical trials which aim to evaluate a range of targeted agents in mCRC and their resistance mechanisms. However, significant knowledge gaps exist in the field, including lack of standardisation of ctDNA techniques, clinical relevance of minority clones detected (for example, no threshold for *KRAS* allele frequency has been established to predict anti-EGFR therapy resistance) and it remains to be proven that changing treatment strategy according to ctDNA findings improves patient outcomes [6]. Challenges notwithstanding, it is foreseeable that in the near future, ctDNA genotyping may be used longitudinally to (i) identify *RAS* wild type patients with mCRC who may be suitable for anti-EGFR antibodies, (ii) dynamically assess treatment response, (iii) identify patients who are developing acquired resistance, (iv) delineate resistance mechanisms to therapy, and (v) discover new druggable targets.
