Pancreatic cancer diagnosis by sequencing, issues such as cost and time
On January 21, the Sapporo Higashitokusu Hospital Conference announced the construction of a six-color multiplex digital PCR assay method to include and detect major driver mutations (KRAS and GNAS hotspot mutations) associated with pancreatic cancer carcinogenesis with a small number of reactions. This research was carried out by a research group including Yusuke Ono, Chief Scientist (Director of the Genome Diagnostics Research Department) at the Medical Research Institute of the same hospital, and Professor Yusuke Mizukami (Director of the Intractable Disease Research Project at the Intractable Diseases and Immunology Genome Research Center, National Institute of Biomedical Innovation, Health and Nutrition, and concurrently Director of the Cancer Biology Research Department, Medical Research Institute, Sapporo Higashi-Tokushukai Hospital), Asahikawa Medical University Department of Gastroenterology, Department of Internal Medicine. Published in “Chemistry”.
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Due to the progressive nature and difficulty in diagnosing pancreatic cancer, early detection and effective treatment are urgently needed.
On the other hand, digital PCR (dPCR) is a quantitative PCR technology characterized by absolute quantification and high sensitivity, and when compared to sequencing, it is a low-cost reaction and exhibits high detection power even when detecting mutations in minute amounts or fragmented nucleic acid samples. PCR system (Bio-Rad) significantly increases the number of targets that can be measured at one time compared to conventional two-color detection, making effective multiplex analysis possible.
The research group there has developed a new diagnostic method that maximizes the time required for dPCR to detect major driver mutations in pancreatic cancer (KRAS, GNAS genes).
Two types of 6-color multiplex dPCR assay methods have been constructed, compatible with screening and analysis of 14 major types.
In this study, we constructed two six-color multiplex dPCR assays to detect KRAS and GNAS hotspot mutations associated with pancreatic cancer. Using a cleaning method based on the f principle, we aim to detect a wide range of mutations in KRAS codons 12/13, 61, and GNAS codon 201, not just those variants. Both methods applied a dPCR reaction system that uses a two-color dye indicator to detect mutant or wild type, examined combinations of judgments and reaction conditions to obtain a large amount of information using six dye colors, and evaluated detection sensitivity using various specimens: sample DNA, formalin-fixed paraffin-embedded (FFPE) tissue-derived DNA, and liquid biopsy DNA.
PlexScreen achieves high sensitivity detection, PlexID can measure 14 types of specific mutations simultaneously
In PlexScreen-dPCR, we designed Hotspots and References covering KRAS codons 12/13, 61, and GNAS codon 201 to optimize multiplex dPCR conditions, resulting in 7 variants of KRAS G12/13, 5 variants of KRAS Q61, and GNAS. Two mutations in R201 were clearly detected as a single fluorescent mutation cluster on a two-dimensional plot, demonstrating that this is a highly sensitive method with a maximum detection limit of mutation frequency of 0.03-0.06% (cell line-derived genomic DNA).
On the other hand, in the PlexID-dPCR method, KRAS G12D/G12V/G12R/G12A/G12C/G12S/G13D/Q61H(A>T)/Q61H(A>C)/Q61K/Q61L/Q61R, GNAS The 14 types of mutations of R201C/R201H and the wild type of each gene were combined using six color dyes, and the conditions were optimized to clearly separate clusters on a two-dimensional plot so that various mutations could be measured simultaneously in a single reaction.
Both assays were verified with 35 samples, enabling detection and quantification with the same accuracy as sequencing.
These PlexScreen- and PlexID-dPCR assays were measured using genomic DNA extracted from 23 specimens of pancreatic cancer-derived FFPE tissue (tumor resection and fine needle biopsy) or 12 body fluid specimens, and the results showed a high concordance rate with previously obtained mutation information, and the quantitative mutation frequency with both methods showed a strong correlation with target sequencing (PlexScreen-dPCR: R2=0.985, PlexID-dPCR: R2=0.970). These findings demonstrated that both assays have reliable diagnostic performance to detect and quantify genetic and GNAS mutations (including rare mutations) with high concordance and correlation.
Can be used for applications such as screening and precise diagnosis, and is also expected to be applied to other cancer types
The results of this research are that the two distinctive assays, PlexScreen-dPCR and PlexID-dPCR, have the potential to serve as versatile analysis methods for the diagnosis of pancreatic cancer. It was then shown that this is a perceptive point in the selection of subjects that leads to early detection, including medical examinations, and frequent recurrence monitoring through liquid biopsies.
“In the future, by applying the design of this multicolor multiplex dPCR measurement system and developing digital PCR gene panels for short-term cancer types such as lung cancer, colorectal cancer, and breast cancer, we expect to expand the scope of personalized treatment by improving time and cost efficiency compared to large-scale sequencing.