In mCRC, patients harboring RAS and BRAF mutations do not benefit from anti-EGFR therapies, such as cetuximab[12-14]. Due to the heterogeneity of tissue samples, the mutational status may be different intralesionally and interlesionally. In recent years, analyzing mutations in plasma-derived ctDNA has been used to provide a more comprehensive overview of the mutational landscape compared to analyses of primary and/or metastatic tumors. Consequently, ctDNA analysis, as an alternative approach with a minimally invasive nature, has been gradually used to guide management and monitor treatment response in some cancers, including CRC[3,15]. In this study, mutations in plasma were measured in 126 Chinese patients with mCRC by a panel of 4 genes (197 hotpots),including KRAS, NRAS, BRAF, and PIK3CA, using a NGS approach.
Our results showed that the concordance rate of RAS/BRAF mutations between plasma and tissue was 78.6%, which was in line with that found in previous studies [15-17]. In a subgroup of 92 patients from the CAPRI-GOIM clinical trial, NGS analysis of both tumor tissue and plasma identified a concordance rate of 78.3%[18], similar to our results. In our study, 11 patients had mutations in tissue that were not detected in plasma. Nine of these patients had a low tumor burden and their cfDNA concentrations ranged from 4 to 10 ng/ml, which was less than the median concentration. A low amount of tumor DNA could hypothetically lead to false-negative results in plasma. In the other two patients with high tumor load, their metastatic sites were predominantly the lymph nodes, bone and peritoneum. In such patients, these metastatic tumors might release less DNA into the circulation than metastatic tumors at other sites. For the subgroup of 16 patients with mutations in plasma but no mutations in tissue, we propose three possible reasons. First, the discrepancy may have resulted from the different sampling times and heterogeneity of tissue specimens. Eleven of these patients had their primary tumor removed before blood sample collection. Therefore, the testing results in plasma might have indicated the mutational status of metastases originating from a different clone. Indeed, the mutational signature of KRAS has been reported to be different between primary tumors and metastatic lesions, with an inconsistency rate ranging from 5% to 32%[19]. Second, sequencing analysis was performed by different approaches in our routine clinical work-up. Mutations in plasma were measured by a panel covering 197 hotpot mutations using NGS approach, while mutations in tissue were assayed by a panel containing only 17 hotpots using ARMS technology. Some rare mutations detected in plasma, such as KRAS p.V14I, BRAF p.K601E, and PIK3CA p.E545G, were not covered by the sequencing panel of tissue. Third, the sensitivity of variant detection used in plasma was 0.2%, while the ARMS technology used in tissue had an analytical sensitivity of approximately 1%, which could also have led to false-negative results in tissue.
How can anti-EGFR treatment be guided using the testing results from both plasma and tissue? It is generally believed that patients with any RAS/BRAF mutations in either tumor tissue or plasma have less treatment benefit than patients who have no mutations. A prospective multicenter clinical study [20] reported that 59%, 11.8% and 14.4% of KRAS, NRAS and BRAF mutations were found in plasma, while 44%, 8.8% and 7.2% were found in tissue respectively. Even patients who showed RAS wild-type in tissue failed to respond to anti-EGFR therapy when mutations were present in their plasma, with the incidence of disease progression as high as 56% [20]. Therefore, in our study, the majority of these patients with RAS/BRAF mutations in tissue and/or in plasma did not receive anti-EGFR treatment, except one (patient No. 25, in Supplemental Table 4). After surgical removal of this patient’s RAS wild-type primary tumor, only a small number of KRAS-mutated subclones were found in his plasma with a mutational frequency of 0.26%. Considering that there were only a few mutated clones in his plasma, we treated him with cetuximab plus chemotherapy, and the PFS was up to 12 months, indicating that he could still have clinical benefit from anti-EGFR therapy. Therefore, whether low frequency mutation in plasma (0.2-1%) is a contraindication for anti-EGFR treatment needs further study. In addition, polyclonal mutations were more frequently detected in plasma than in tissue (10.3% versus 0.9% in our study), which emerged mostly in the form of double mutations in KRAS/PIK3CA or NRAS/PIK3CA. Such polyclonal mutations may influence mechanisms of resistance, as reported in some other studies [21]. Consequently, the baseline assessment of the mutational status in plasma can identify additional mutated patients and further improve patient selection for anti-EGFR treatment.
This study further analyzed the correlation between cfDNA and tumor burden. We found that cfDNA concentration was positively correlated with the serological levels of CEA, CA199, NSE, LDH, and the sum of tumor diameters (including all tumors assessable). However, the correlation might not be tumor-specific because cfDNA comprises both healthy and tumor DNA. Therefore, we further found that the mutational frequency in plasma was also correlated with the levels of CA199 and LDH and the sum of tumor diameters. It was indicated that cfDNA concentration and mutational frequency could serve as quantitative tools for assessing tumor burden, which has been supported by some other studies [4]. In this study, we also found that patients with a higher cfDNA concentration had shorter PFS and OS than those with a lower cfDNA concentration, with an optimal threshold of 17.91 ng/ml. Another potential application of cfDNA testing is to dynamically evaluate therapeutic effects and predict recurrence [7]. As seen in Figure 2E, we found that an early decrease in cfDNA concentration was related to a good therapeutic effect. Thomsen et al also reported that a low level of cfDNA after chemotherapy, prior to radiological imaging evaluation, was associated with a low risk of progression[22].
In this study, the liver, lung and lymph nodes were the most common metastatic sites in mCRC regardless of specific mutations. Additionally, we also observed some interesting metastatic patterns in patients harboring rare mutations in this study. For example, one patient (patient No. 40 in Supplemental Table 4) with a high-frequency (28%) Q61K mutation in exon 3 of NRAS had mandible metastasis, which is a rare metastatic site in clinical practice. Patient No. 31, carrying both the KRAS p.V14I and KRAS p.G12A mutations, had metastasis mainly in soft tissues, such as the chest wall, anterior sacrum, and psoas major muscle. Patient No. 39, with the K117R mutation in exon 4 of KRAS, had only systemic lymph node metastases, and his cfDNA concentration was more than 50 ng/ml, but his PFS was more than 20 months. We believe that these patients with rare mutations might have unique mechanisms of metastasis that further influence survival, which might be an interesting problem worthy of further research.
There are some limitations of this study. First, correlations between rare mutations and metastatic sites could not be made because of the small sample size. Second, the mutational status in tissue and plasma were sequenced with panels used in our daily routine practice, covering only four genes, and these panels need to be optimized to obtain more information with low cost for every patient.