| Abstract |
Approximately 40-50% of patients with colorectal cancer (CRC) harbor KRAS mutations and another 5-10% harbor BRAF mutations. The presence of these mutations is predictive for absence of benefit to anti-EGFR agents. Nevertheless, the emergence of resistance is inevitable due to multiple factors, such as the acquisition of KRAS mutations. As a result, proper patient selection and early therapy adaptation could lead to the optimization of patient management. Circulating tumor cell (CTC) enumeration is prognostic in metastatic CRC (mCRC), while their genotypic analysis carries predictive information. ISET (Rarecells, France) selects CTCs based on their size and it has been shown to capture diverse cellular populations in mCRC. The aim of this study was the genotypic analysis of CTCs captured by ISET.
2. Patients and methods
This prospective observational study, aiming to enumerate and characterize at the genomic level CTCs captured by ISET from patients with mCRC, was conducted at the Medical Oncology department, University Hospital of Heraklion. Following consent, CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated based on previously published morphological criteria,
using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay.
3. Results
In total, 28 patients were enrolled, 15 wild type (WT), 8 with a KRAS exon 2 mutation (6 harbored the G12D and 2 the G12V mutation), 1 with an exon 4 mutation (A146T), 1 NRAS and 3 with a BRAF mutation. 57 samples were collected; in all the samples, at least 1 CTC / 1 ml was identified, with a median of 6 CTCs / 1 ml (range, 1 – 32) and a mean of 8,22 CTCs / 1 ml. Both the number of CTCs and the number of clusters of 2 or more CTCs increased from baseline to response evaluation and then decreased; only differences in CTC clusters were statistically significant. Among the WT patients, 9/15 (60%) had at least one positive for a mutation sample (in total, 11/28 – 39.2% of samples). 3/11 (27%) patients had a positive baseline sample, while 3/8 (37.5%) samples at progression harbored a mutation. Out of these 3 patients, no previous mutations had been detected and 2 of them had been treated with an anti-EGFR agent. The most frequently detected mutations were G13D and G12C (n=3). Out of the 21 samples from patients with RAS mutated tumors, the G13D mutation was detected 6 times and G12V and G12C once. Finally, out of the 6 samples from 3 patients with a BRAF mutation, no such mutation could be detected in CTCs using Sanger sequencing. However, in one patient the STK11 F354L was detected in both CTCs and the primary tumor. The detection of mutations at baseline was not found to be prognostic for patient outcomes.
4. Discussion
In this study, the kinetics of CTCs isolated with ISET did not follow the disease trajectory as assessed by imaging. These results are in accordance with a published study from another group. Possible reasons are the low number of enrolled patients, the subjective nature of CTC characterization, the uncertain relative importance of the CTC populations that are captured by ISET and the phenomenon of CTC mobilization under the effect of systemic chemotherapy. This study serves as a proof-of-principle regarding the possibility for the genomic characterization of CTCs captured by ISET. The detection of mutations concerned patients with both WT and mutated primary tumors and was achieved in all the phases of first line therapy. The same point mutation identified in the primary tumor could not be identified in the patient’s CTC sample. Possible reasons could be methodology-related, due to the different methods used to detect mutations in the primary tumors and the CTCs or, lastly, this could be an accurate representation of the true molecular heterogeneity of the disease. Despite this study’s weaknesses, the genomic characterization of CTCs captured by ISET is feasible and could provide information regarding the heterogeneity and the emergent resistance while under treatment with an anti-EGFR agent before it is clinically detected. The standardization of the methodology is necessary before it is evaluated in larger scale clinical trials
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