KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Abstract

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch TM . DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic TM ), real-time PCR (EntroGen TM ) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.

Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer

“…Concerning this level of agreement found by our group and others 7,19,20 between CTCs and primary tumors, it has been hypothesized that the development of resistance to EGFR therapy is caused by rare cells with KRAS mutations that pre exist at low levels in mCRC with apparent KRAS wt. 22 Diaz et al 22 tested this hypothesis by analyzing 24 patients with mCRC, all of them with KRAS wt in primary tumor and resistant to anti-EGFR therapy.…”

“…Again, it can be explained by the cellular heterogeneity found in epithelial malignancies, which can be reflected by the heterogeneity found in CTCs. 7,19,22,23 Another explanation for our findings is the parallel progression model, which suggests that it may be possible to have various metastatic subclones which are disseminated early during disease development and remain dormant for years. 24 Additionally, we tried to correlate our findings with clinicalpathological characteristics of patients.…”

“…Maybe, a viable alternative is to make laser microdissection of ISET membranes. In the case of KRAS, which is detected in 30-40% of patients with CRC, 7 we believe that the laser microdissection is not necessary and contamination with leukocytes is not sufficient to disrupt the reading. The pyrosequencing is a sensitive technique, being able to identify the mutation in a few cells.…”

“…Genotyping CTCs may be 19 using a dielectrophoresis-based device, had analyzed KRAS mutation (exon 2), and observed 50% of concordance between KRAS mutation in CTCs and matched primary tumor of mCRC. Mostert et al 7 found 5 of the 42 (12%) mCRC patients with KRAS mutation (codon 12/13) in CTCs by nested ASB-PCR. This group also observed that 4 of the 5 patients analyzed had the same KRAS mutation in CTC and their metastasis tissue.…”

“…[4][5][6] In colorectal carcinoma, KRAS mutations are present in 30-40% of patients. 7 Activating mutations in KRAS are responsible to anti-EGFR therapy resistance (Cetuximab or Panitumumab) in metastatic colorectal cancer (mCRC). More recently, around 18% of patients with wild type KRAS -exon 2, were find to carry mutation in exon 3, 4 of KRAS gene and exon 2, 3, 4 of NRAS gene.…”