A rapid, sensitive, reproducible and cost-effective method for mutation profiling of colon cancer and metastatic lymph nodes

Abstract

Background: An increasing number of studies show that genetic markers can aid in refining prognostic information and predicting the benefit from systemic therapy. Our goal was to develop a high throughput, cost-effective and simple methodology for the detection of clinically relevant hot spot mutations in colon cancer.

Methods: The Maldi-Tof mass spectrometry platform and OncoCarta panel from Sequenom were used to profile 239 colon cancers and 39 metastatic lymph nodes from NSABP clinical trial C-07 utilizing routinely processed FFPET (formalin-fixed paraffin-embedded tissue).

Results: Among the 238 common hot-spot cancer mutations in 19 genes interrogated by the OncoCarta panel, mutations were detected in 7 different genes at 26 different nucleotide positions in our colon cancer samples. Twenty-four assays that detected mutations in more than 1% of the samples were reconfigured into a new multiplexed panel, termed here as ColoCarta. Mutation profiling was repeated on 32 mutant samples using ColoCarta and the results were identical to results with OncoCarta, demonstrating that this methodology was reproducible. Further evidence demonstrating the validity of the data was the fact that the mutation frequencies of the most common colon cancer mutations were similar to the COSMIC (Catalog of Somatic Mutations in Cancer) database. The frequencies were 43.5% for KRAS, 20.1% for PIK3CA, and 12.1% for BRAF. In addition, infrequent mutations in NRAS, AKT1, ABL1, and MET were detected. Mutation profiling of metastatic lymph nodes and their corresponding primary tumors showed that they were 89.7% concordant. All mutations found in the lymph nodes were also found in the corresponding primary tumors, but in 4 cases a mutation was present in the primary tumor only.

Conclusions: This study describes a high throughput technology that can be used to interrogate DNAs isolated from routinely processed FFPET and identifies the specific mutations that are common to colon cancer. The development of this technology and the ColoCarta panel may provide a mechanism for rapid screening of mutations in clinically relevant genes like KRAS, PIK3CA, and BRAF.

Trial registration: ClinicalTrials.gov: NSABP C-07: NCT00004931.

Figures

Figure 1

Methodology for mutation detection. Genomic DNA from the samples is amplified by PCR, resulting in copies of both mutant and wildtype alleles. Shrimp Alkaline Phosphatase removed excess nucleotides from the sample wells. Primer extension was performed using terminator nucleotides A, C, T, G, each with distinct masses. This linear amplification results in sequences proportional to the alleles that can be distinguished by mass spec (Maldi-Tof Separation).

Figure 2

Spectra for cell lines UACC-893 and A2058. The expected positions for the unexteneded primer (UEP), and the extension products (Mutant and WT) from assays PIK3CA_9 and BRAF_15 in cell lines UACC-893 and A2058, respectively, are indicated with red dashed lines. The proportion of peak areas and the specific base is also shown. Assays PIK3CA_9 and BRAF_15 detected mutations in PIK3CA at amino acid position 1047 and in BRAF at amino acid position 600, respectively. Other peaks included in these spectra as result of multiplexing but not part of the designated assays are indicated as grey dashed lines.

Figure 3

Fractional unextended primer versus input DNA. The range and the average for the percent of unextended primer for different amounts of input DNA into the PCR reactions are shown. The number of samples used in each category was 4 for 1-3 ng, 9 for 3-9 ng, 13 for 9-13 and 210 for 14 ng.

Figure 4

Sensitive detection of mutations in clinical FFPE samples with the Sequenom platform. Small mutant but definitive peak illustrating a PIK3CA-1047R mutation in approximately 5% of the sample DNA is shown.

Figure 5

Quantification of the sensitivity with a cell line mixing experiment. Spectra of MCF-7 cells (mutant) alone or mixed with SKBR3 cells (WT) are shown. Percents are based on the ng amounts of DNA. This assay detects an E545K mutation in PIK3CA.

Figure 6

MET mutation is amplified. The proportion and position (blue dashed lines) of mutant and wt alleles are shown. The MET_1 assay detects R970C mutations in MET.