Ultra deep amplicon sequencing of RAS genes for colorectal carcinoma diagnostics.

Introduction

Tumor DNA testing of KRAS and NRAS (RAS) genes, which are components of the EGFR signaling pathway, is a prerequisite for personalized biological treatment using anti-EGFR drugs such as panitumumab and cetuximab in metastatic colorectal carcinoma (mCRC). The treatment cannot be prescribed in case of mutations in RAS (codons 12, 13, 59, 61, 117 and 146). Detection of increasing number of possible mutations with probebased qPCR is cumbersome while ultra-deep amplicon sequencing (UDAS) has a potential to be suitable method for simultaneous direct detection of all somatic mutation within tested regions and with a defined detection limit. The aim of the study was to optimize and validate RAS sequencing of tumor DNA samples for the detection of all predictive mutations. Tumor DNA testing of KRAS and NRAS (RAS) genes, which are components of the EGFR signaling pathway, is a prerequisite for personalized biological treatment using anti-EGFR drugs such as panitumumab and cetuximab in metastatic colorectal carcinoma (mCRC). The treatment cannot be prescribed in case of mutations in RAS (codons 12, 13, 59, 61, 117 and 146). Detection of increasing number of possible mutations with probe-based qPCR is cumbersome while ultra-deep amplicon sequencing (UDAS) has a potential to be suitable method for simultaneous direct detection of all somatic mutation within tested regions and with a defined detection limit. The aim of the study was to optimize and validate RAS sequencing of tumor DNA samples for the detection of all predictive mutations.

Materials/methods

During more than one year, 423 formalin fixed paraffin embedded (FFPE) tumor or quality control samples were tested. UDAS RAS assays were introduced into laboratory. Assays consist of qPCR with a control of amplicons by melting curves. One RAS assay was based on adapter ligation, other one on simultaneous adapter tagging and fragmentation (tagmentation) of amplicons. After PCR indexing and product purification, the samples from both assays were normalized, pooled and sequenced at MiSeq (IllumiDuring more than one year, 423 formalin fixed paraffin embedded (FFPE) tumor or quality control samples were tested. UDAS RAS assays were introduced into laboratory. Assays consist of qPCR with a control of amplicons by melting curves. One RAS assay was based on adapter ligation, other one on simultaneous adapter tagging and fragmentation (tagmentation) of amplicons. After PCR indexing and product purification, the samples from both assays were normalized, pooled and sequenced at MiSeq (Illumina) at ultra-deep coverage. Results with at least 5% mutation frequency were concluded as „mutation detected“, according to the consensus of the Czech Society of Pathology. na) at ultra-deep coverage. Results with at least 5% mutation frequency were concluded as „mutation detected“, according to the consensus of the Czech Society of Pathology.

Results and conclusions

We observed 95% concordance of therascreenR KRAS RGQ PCR Kit (KRAS codons 12 and 13, CEIVD, Qiagen) and RAS assay results in 180 selected samples. In every RAS sequencing run, KRAS G13D or NRAS Q61L mutation standards were correctly found, confirming reproducibility of the method. Interlaboratory comparison of 12 samples with mutations showed 100 % concordance, yet one of the samples was retested in both laboratories. External quality assessments (EQA) yielded one result differing from consensus out of ten. Upon investigating the cause of this discrepancy with EQA organizers, we found that sample was artificially prepared cell line that did not amplify properly with our primers. Mutation in this sample was correctly identified by modified set of primers.

Overall, out of 391 tested patients samples, 39% were positive for major mutations (KRAS codons 12 and 13). Furthermore, we detected minority mutations in 12% of samples (mainly KRAS codons 61 and 146, and NRAS codon 12 and 61). The relatively high percentage of tumors with minority mutations justifies the SPC-given necessity for their testing. 36 samples which were not possible to be analyzed by tagmentation based assay due to DNA quality, were successfully analyzed by ligation based assay with modified set of primers. Three (3) samples remained untestable.

Taken together we show that UDAS method is a suitable and valid method for a predictive mCRC diagnostics. Shortening of the sequencing area is very beneficial for improving the quality of the results, especially in case of FFPE samples. We envision the automation and/or simplification of protocols would foster broad spread of UDAS based methods to diagnostics laboratories.

The work was supported by grants CZ.1.07/2.3.00/30.0060, C Z . 1 . 0 7 / 2 . 3 . 0 0 / 3 0 . 0 0 4 1 , TE02000058 and LO1304.