Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 2;13(1):143.
doi: 10.1186/s12920-020-00804-y.

Highly multiplexed quantifications of 299 somatic mutations in colorectal cancer patients by automated MALDI-TOF mass spectrometry

Chang Xu  1   2 , Danli Peng  2   3 , Jialu Li  2   3 , Meihua Chen  2   3 , Yujie Hu  2   3 , Mingliang Hou  2   3 , Qingjuan Shang  2   3 , Qi Liang  2   3 , Jie Li  2   3 , Wenfeng Li  4 , Xiaoli Wu  5 , Changbao Liu  6 , Wanle Hu  6 , Mao Cai  6 , Huxiang Zhang  7 , Guorong Chen  7 , Lingling Yu  8 , Xiaoqun Zheng  2   8 , Feizhao Jiang  1 , Ju Luan  9   10 , Shengnan Jin  11   12 , Chunming Ding  13   14
Affiliations

Highly multiplexed quantifications of 299 somatic mutations in colorectal cancer patients by automated MALDI-TOF mass spectrometry

Chang Xu et al. BMC Med Genomics. .

Abstract

Background: Detection of somatic mutations in tumor tissues helps to understand tumor biology and guide treatment selection. Methods such as quantitative PCR can analyze a few mutations with high efficiency, while next generation sequencing (NGS) based methods can analyze hundreds to thousands of mutations. However, there is a lack of cost-effective method for quantitatively analyzing tens to a few hundred mutations of potential biological and clinical significance.

Methods: Through a comprehensive database and literature review we selected 299 mutations associated with colorectal cancer. We then designed a highly multiplexed assay panel (8-wells covering 299 mutations in 109 genes) based on an automated MADLI-TOF mass spectrometry (MS) platform. The multiplex panel was tested with a total of 319 freshly frozen tissues and 92 FFPE samples from 229 colorectal cancer patients, with 13 samples also analyzed by a targeted NGS method covering 532 genes.

Results: Multiplex somatic mutation panel based on MALDI-TOF MS detected and quantified at least one somatic mutation in 142 patients, with KRAS, TP53 and APC being the most frequently mutated genes. Extensive validation by both capillary sequencing and targeted NGS demonstrated high accuracy of the multiplex MS assay. Out of 35 mutations tested with plasmid constructs, sensitivities of 5 and 10% mutant allele frequency were achieved for 19 and 16 mutations, respectively.

Conclusions: Automated MALDI-TOF MS offers an efficient and cost-effective platform for highly multiplexed quantitation of 299 somatic mutations, which may be useful in studying the biological and clinical significance of somatic mutations with large numbers of cancer tissues.

Keywords: Colorectal cancer; MALDI-TOF mass spectrometry; Multiplex detection; Somatic mutation.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Representative MS results showing paired tumor and adjacent normal tissues from frozen and FFPE tissues. Shown in (a), (b), and (c) are three different mutations in the TP53, APC and KRAS genes from three frozen tissues, (d), (e) and (f) show the mass spectra for three FFPE tissues. Mutant allele frequency can be estimated by comparing the peak signals for the mutant and wild type alleles. Wt: wild type, Mut: mutation
Fig. 2
Fig. 2
Evaluation the performance of the MS assay. The DNA mixture samples with 5 and 10% mutation were prepared to analyze the sensitivity and accuracy of the assay. Among them, 19 assays achieved a 5% sensitivity for mutations, and 16 assays achieved a 10% sensitivity. Here we show 4 assays that could achieve a 5% sensitivity
Fig. 3
Fig. 3
Mutation profiles quantified by the multiplex CRC panel. Each colored bar represents one mutation (orange) or two mutations in the same gene (blue). The heights of the colored bars represent mutant allele frequencies
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Stratton MR, Campbell PJ, Futreal PA. The cancer genome. Nature. 2009;458(7239):719–724. doi: 10.1038/nature07943. - DOI - PMC - PubMed
    1. Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B, Hersey A, Oprea TI, Overington JP. A comprehensive map of molecular drug targets. Nat Rev Drug Discov. 2017;16(1):19–34. doi: 10.1038/nrd.2016.230. - DOI - PMC - PubMed
    1. Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH, Dechaphunkul A, Imamura F, Nogami N, Kurata T, Okamoto I, Zhou C, Cho BC, Cheng Y, Cho EK, Voon PJ, Planchard D, Su WC, Gray JE, Lee SM, Hodge R, Marotti M, Rukazenkov Y, Ramalingam SS, Investigators F. Osimertinib in untreated EGFR-mutated advanced non-small-cell lung Cancer. N Engl J Med. 2018;378(2):113–125. doi: 10.1056/NEJMoa1713137. - DOI - PubMed
    1. Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, Ychou M, Bouche O, Landi B, Louvet C, Andre T, Bibeau F, Diebold MD, Rougier P, Ducreux M, Tomasic G, Emile JF, Penault-Llorca F, Laurent-Puig P. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 2008;26(3):374–379. doi: 10.1200/JCO.2007.12.5906. - DOI - PubMed
    1. Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, Juan T, Sikorski R, Suggs S, Radinsky R, Patterson SD, Chang DD. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26(10):1626–1634. doi: 10.1200/JCO.2007.14.7116. - DOI - PubMed

Publication types

Substances