Clinical Outcome
One patient had multiple unresectable liver-limited metastases at their first medical examination and was treated with first-line systemic chemotherapy with anti-EGFR therapy (Fig. 1). The primary tumor had no mutations in KRAS, NRAS or BRAF exon 15 (Fig. 2). Overall survival after first-line treatment initiation was 34 months. The patient was administered bevacizumab with cytotoxic agents as the second-line regimen and was rechallenged with anti-EGFR antibody as the third-line chemotherapy. Despite initial tumor shrinkage in the metastases, the patient ultimately developed PD.
Identification of Acquired RAS Mutations by Conventional Sanger Sequencing
In the patient, no activating RAS mutation was observed in the primary tumor biopsy specimen obtained prior to treatment, in primary tumor tissue obtained after 1 year of panitumumab administration, or in a metastatic tumor in the right liver lobe (RL) that responded continuously to panitumumab with first-line systemic chemotherapy and shrank in response to third-line panitumumab rechallenge (Fig. 2). In addition, KRAS sequences in metastatic specimens obtained during autopsy revealed diverse acquired mutations at different metastatic sites, indicating resistance to the systemic chemotherapy used, including anti-EGFR antibody treatment. Acquired activating KRAS mutations resulting in G61Hc, G12R, and G12V were detected by Sanger sequencing in liver segments II (S2), III (S3), and middle segment (MS), respectively. The KRAS G12C, G13D, and G61Hc mutations were also detected in metastases in the left kidney (Kd), in a hepatic lymph node (HN), and in the left lung (Lu), respectively. No samples from primary and metastatic lesions harbored mutations in NRAS or BRAF.
Identification of Acquired RAS Mutations by a PCR-rSSO Method
By Sanger sequencing, acquired mutations were detected in 6 (87%) of the 7 metastatic lesions. Because of the lower sensitivity for the detection of mutant alleles by Sanger sequencing (~ 20%), we could not exclude the possibility of associated metastatic lesions caused by resistant cells already existing in the primary lesion in extremely low numbers.
We further analyzed all samples using the PCR-rSSO method, RASKET, which has high sensitivity in terms of detecting extended RAS mutant alleles at lower frequencies, 1–5%; a sample was considered mutation-positive when the index was estimated over the cut-off value, the sensitivity of which for the mutant allele was 1–5%, for each mutant allele[16].
Using this procedure, KRAS or NRAS mutations with indices higher than the cut-off values were not found in DNA purified from pretreated primary tumor cells (Fig. 3 and Supplementary File 2). Additionally, PCR-rSSO analysis revealed the same mutation spectrum obtained by Sanger sequencing in the patient except for the metastatic lesion in the Kd and the RL. The index of the KRAS G12C allele captured from the Kd by PCR-rSSO analysis was 114, i.e., lower than the conventional cut-off value (300), which might be influenced by the immediate synonymous mutation of A11Ag clearly demonstrated by Sanger sequencing, as previously reported (Fig. 2) [16]. Interestingly, the metastatic tumor in the RL, which responded consistently to panitumumab and showed no mutation by Sanger sequencing, revealed multiple KRAS mutations, Q61Ht, G12A, and G12R, with indices higher than the cut-off values. Although these mutant alleles were not frequent enough to be detected by Sanger sequencing, heterogeneity in RAS mutant cancer cells may exist in the RL tumor mass.
In summary, by Sanger sequencing and PCR-rSSO, the 7 metastatic lesions were found to harbored diverse acquired mutations in the KRAS gene: Q61Ht, G12A, and G12R in RL; G12V in MS; Q61Hc in S2; G12R in S3; G12C in Kd; G13D in HN; Q61Hc in Lu.
Detection of Acquired Mutations in Circulating Cell-Free DNA
We investigated whether acquired RAS mutations could be detected in the plasma samples using PCR-rSSO. The 7 diverse acquired mutations found in the resistant tumors were not detected before or after PD during the initial course of panitumumab treatment (*20 and *105 in Fig. 4 and Supplementary File 3), but only one of the 7 diverse acquired mutations was detected at a significant level following the confirmation of PD during third-line panitumumab rechallenge (*123). The KRAS G12R frequency, which was confirmed in the resistant tumor in S3, was strikingly elevated in circulating cell-free DNA in a plasma sample collected 123 weeks after treatment initiation. Additionally, KRAS G13D in the HN and KRAS Q61Hc in the Lu and S2 were also detected in the plasma sample *123, but the indices were not reached at the cut-off value. All the acquired mutations detected in the plasma sample *123 were not detected in plasma at 20 weeks (*20, approximately as early as 10 months before PD during first-line therapy) or at 105 weeks (*105, when PD was confirmed radiologically during first-line treatment).