Ultrasensitive Detection of Multiplexed Somatic Mutations Using MALDI-TOF Mass Spectrometry

Abstract

Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy.

Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice

“…11 Sanger capillary sequencing has long been the gold standard for DNA sequence analysis; however, it does not offer the high sensitivity required to detect somatic mutations at allelic frequencies less than ~20%. 12 Numerous comparisons of technology platforms for EGFR 13 14 and KRAS (mainly in CRC [15][16][17][18][19][20][21][22][23] ) mutation testing conclude that quantitative PCR (qPCR)-based methods offer the sensitivity, tissue economy and turnaround time required by physicians to guide treatment decisions. The advent of next-generation sequencing (NGS) platforms offers pathology laboratories an unparalleled insight into the cancer genome including de novo detection of variants as well as known actionable targets.…”

Somatic mutations and immune checkpoint biomarkers

“…Using the MALDI-TOF mass spectrometry and Mas-sARRAY technology, multiplex panels such as Onco-Carta, LungCarta, OncoFOCUS and UltraSEEK have been developed. 52 Other groups have developed customized panels using MALDI-TOF technology to detect for somatic mutations in NSCLC. 53 This technology is based on PCR amplification and allele-specific single-based primer extension.…”

Circulating Cell Free Tumor DNA Detection as a Routine Tool forLung Cancer Patient Management

“…UltraSEEK (high-throughput, multiplexed, ultrasensitive mutation detection) is a Mass-spectrometry-based technology that has been designed to address the limited multiplexing ability of conventional PCR approaches [52]. Briefly, a multiplex-PCR is first performed to amplify several genes, biotinylated probes, specific of the targeted mutations, are then used to generate biotinylated-mutated-strand.…”

“…Briefly, a multiplex-PCR is first performed to amplify several genes, biotinylated probes, specific of the targeted mutations, are then used to generate biotinylated-mutated-strand. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is finally used to detect presence of mutations [52]. …”