Original Article
Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue

https://doi.org/10.1016/j.prp.2011.10.002Get rights and content

Abstract

KRAS, BRAF, and PIK3CA mutation testing before administration of anti-epidermal growth factor receptor therapy of metastatic colorectal cancer (CRC) has become important. However, considerable uncertainty exists regarding which detection method can be applied in a reproducible, sensitive, and simple manner in the routine diagnostic setting. We compared the detection rates of KRAS, BRAF, and PIK3CA mutations in 92 routine formalin-fixed, paraffin-embedded CRC specimens by 2 discrete methods: direct sequencing and peptide nucleic acid (PNA)-mediated PCR. The detection rates for KRAS, BRAF, and PIK3CA mutations by direct sequencing were 20.7%, 3.3%, and 1.1%, respectively. PNA-mediated PCR clamping significantly increased the percentages of KRAS, BRAF, and PIK3CA mutations by up to 7.6%, 1.2%, and 5.4%, respectively, compared to the detection rate of regular PCR followed by direct sequencing (p = 0.039, p = 0.250, and p = 0.031, respectively). The tumor volume of discordant cases was not significantly different from concordant cases (56.2 ± 28.7% vs. 67.6 ± 17.9%, p = 0.41), which implies that there is a minor population of mutant alleles in the heterogeneous tumor population. The PNA-mediated PCR clamping method is highly sensitive and is efficiently applicable to the detection of KRAS, BRAF, and PIK3CA mutations in a clinical setting.

Introduction

Locally confined colorectal cancers (CRCs) without lymph node metastasis may be cured by surgery alone, whereas nodal-positive CRCs require adjuvant radio- and/or chemotherapy [22]. Of the latter, approximately 34% of cases will experience a relapse of the disease despite adjuvant therapy [23]. Cetuximab and panitumumab, the monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), are frequently used as second- or third-line chemotherapy for patients with metastatic CRC [8]. They have been shown to be effective in conferring clinical benefits to approximately 10–20% of patients with metastatic CRC [12]. Both molecules bind to the extracellular domain of EGFR, leading to inhibition of its downstream signaling [19]. EGFR is a transmembrane tyrosine kinase receptor triggering the 2 main RAS-RAF-MAPK and PI3K-PTEN-AKT signaling pathways in ligand binding [3]. The wild-type KRAS presents a condition for response to monoclonal antibodies, whereas tumors with mutated KRAS do not respond to this treatment modality [18]. However, KRAS mutations only account for 30–40% of nonresponsiveness [7]. Mutations in other downstream effectors of the EGFR signaling pathway, such as BRAF and PIK3CA, are also responsible for resistance to EGFR-targeted monoclonal antibody therapy [6], [7], [17], [18]. A significant improvement in overall survival and progression-free survival was observed in patients with wild-type KRAS and BRAF tumors following treatment with cetuximab compared with wild-type KRAS and mutated BRAF tumors [6], [17]. Therefore, it is important that KRAS, BRAF, and PIK3CA mutation status be precisely determined to maximize the benefit to the patient in a clinical setting.

Conventional PCR amplification followed by direct sequencing has been the gold standard for detection of KRAS, BRAF, and PIK3CA mutations. However, these conventional PCR-based assays are limited in their ability to identify low levels of mutation-bearing tumor cells. Recently, several advanced alternative methods have emerged to overcome the limitations of conventional PCR-based assays.

The peptide nucleic acid (PNA)-mediated PCR clamping technique is based on PNA, a synthetic DNA analog in which the phosphodiester backbone is replaced by a peptide-like repeat of the (2-aminoethyl)-glycine chain [14]. A perfectly matched PNA/DNA hybrid has higher thermal stability than the corresponding DNA/DNA hybrid; hence, single base-pair mismatch results in a decrease of the melting temperature (Tm) at 9–16 °C [16]. In PCR, PNA hybridized with the target sequence can inhibit primer annealing or chain elongation without interfering with the reactions of mismatched template DNA; therefore, so-called PNA-mediated PCR clamping induces preferential amplification of mutant DNA fragments even in the presence of an excess amount of wild-type DNA [5], [16].

In this study, we compared the detection rate of KRAS, BRAF, and PIK3CA mutations in 92 routine paraffin slide CRC specimens by 2 methods in a clinical setting: direct sequencing and PNA-mediated PCR clamping. The data showed that PNA-mediated PCR clamping demonstrated higher sensitivity for the detection of KRAS, BRAF, and PIK3CA mutations than did direct sequencing.

Section snippets

Materials and methods

This study was based on 100 cases of primary CRCs with lymph node or distant metastasis obtained from pathology files spanning 2007 to 2010. All patients were chemo- and targeted drug naive at the time of surgery. After reviewing a hematoxylin and eosin (H&E)-stained glass slide for each case, the percentage of tumor was estimated as the percentage of tumor cells to non-neoplastic cells by visual inspection, and the largest area of tumor without necrosis or hemorrhage was selected for molecular

Results

We analyzed the overall proportion and association between KRAS, BRAF, and PIK3CA mutations. As a whole, we detected the mutations in 38.0% (35/92) of CRCs through the two methods of PNA-mediated PCR clamping and direct sequencing; 29.3% (27/92) for KRAS, 4.3% (4/92) for BRAF, and 6.5% (6/92) for PIK3CA. Among 35 CRCs, 24 tumors exclusively had the mutation on the KRAS, 3 on the BRAF, and 5 on the PIK3CA. Concomitant mutations of KRAS and BRAF, or KRAS and PIK3CA occurred in each one CRC,

Discussion

For simplicity and reproducibility, we evaluated the potential of PNA-mediated PCR clamping to enhance the detection of KRAS, BRAF, and PIK3CA mutations in clinical CRC samples. PNA-mediated PCR clamping is a modified PCR method that uses optimized PNA probes that bind tightly to wild-type DNA fragments [20]. The tight binding to the wild-type DNA fragments results in no amplification of wild-type DNA, whereas mutated DNA fragments undergo PCR amplification, enabling multiplication of the

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by IN-SUNG Foundation for Medical Research and by a grant (A092255) from the Korea Healthcare Technology R&D Project, Ministry for Health, Korea.

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    These authors contributed equally to this work.

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