Toppan Printing Co., Ltd. (hereafter Toppan Printing; head office: Chiyoda Ward, Tokyo; President & CEO: Naoki Adachi) has developed a new analysis technique called the "Fluorescent PHFA (Preferentially Homoduplex Formation Assay)" method for the gene analysis of cancerous tissue. As the result of the study with the University of Chicago, it has been shown that this new technique makes it possible to analyze the genotype of cancerous tissue taken from actual patients at low cost and with a high degree of accuracy.

With the existing "direct sequence technique" for gene analysis, it is considered difficult to accurately detect genetic mutation of less than 30% in the cancerous tissue extracted. With the Fluorescent PHFA method, which uses precise thermal treatment, it is possible to detect genetic mutation of just 1% with a high level of reproducibility and cost has been reduced due to the fact that it does not use expensive enzyme reagents.
This research was announced at the 8th AACR/JCA joint conference in Hawaii held by the American Association for Cancer Research and the Japan Cancer Association.

Background
Personalized medicine provides effective treatment and administration of drugs to each individual on a scientific basis with consideration of the genetic background of the patient. With this type of medicine, the quality of life of each patient is improved and it is expected that in Japan it will lead to the reduction of medical costs arising from the side-effects of drugs.
In particular, it is now understood that there are differences in how effective medicines are for each individual due not only to individual differences in congenital gene polymorphism but also to acquired gene mutations. Therefore, gene analysis is receiving attention as a method for measuring the effectiveness of medicines for each individual. Research is underway to clarify the relationship between genetic mutation and the effectiveness of medicines for cancers of various organs.
If a low cost test for these gene mutations with a high level of reproducibility becomes common in actual medical practice, it will become possible to implement effective treatment matched to the individual’s medical condition.

In recent years, testing carried out before treatment through molecularly targeted anticancer drugs has received attention as a method for realizing personalized medicine. Molecularly targeted anticancer drugs are a very effective method of treatment with which side affects can be minimized but it has become clear that even with cancer of the same organ, there are cases where it is not effective due to differences in the genetic mutation of the cancerous tissue. Therefore, it will become important to administer highly effective molecularly targeted anticancer drugs after taking a sample of cancerous tissue from the patient and examining the genetic mutation of the cancer through gene analysis.
However, with gene analysis of cancerous tissue, it is necessary to detect only the genes of the cancer cells from a sample that also contains normal cells and the level of sensitivity has not been sufficient, with cases of detection not being possible using existing analysis techniques. In addition, the high cost of testing has become an obstacle for widespread use as the gene analysis technique is complicated and the cost of reagents is high.

In order to resolve this issue, Toppan Printing has established a new gene analysis technique - the "Fluorescent PHFA" method. Low cost testing will be possible because the new technique uses an extremely simple principle to analyze mutations simply through thermal treatment.
In the study with the University of Chicago, it has been confirmed that the accuracy of the Fluorescent PHFA method is higher than that of traditional analysis techniques.

Future developments
First, Toppan Printing will proceed with development of gene analysis testing kits using the Fluorescent PHFA method for research institutions. This new gene analysis technique is expected to also be applicable to fields other than cancer treatment, and in the future the company will target the commercialization and sale of specialist devices through joint initiatives with organizations such as RIKEN GENESIS CO.,LTD. (*1) a group company that undertakes contract gene analysis business.

The device development in this research is supported by NEDO’s "Grant for Practical Application of Industrial Technology" from August 2009 to February 2011.

Overview of the study with the University of Chicago
Using the University of Chicago’s Department of Medicine’s numerous clinical specimens, verification is being carried out comparing the Fluorescent PHFA method with several existing gene analysis techniques. The mutations of the subject cancer-critical genes investigated from cancerous tissue are EGFR genes (15 types of mutation), KRAS genes (7 types of mutation), NRAS genes (2 types of mutation) and BRAF genes (1 type of mutation), a total of 25 types, and the research has already been successful in identifying all the genetic mutations from 150 samples using Fluorescent PHFA. It is also anticipated that through analyzing the correlation between the medical information that corresponds to the clinical specimens and the mutations, it will be possible to make new and helpful medical discoveries.

Overview of "Fluorescent PHFA"
Two-strand unlabeled DNA amplified from the sample through fluorescently modified two-strand labeled DNA and PCR is denatured thermally and the respective DNA is made into single-strand form. Then by gently reducing the temperature, an operation is performed to form the two-strand DNA once again, and by measuring the changes in the intensity of the fluorescence that arise due to the strand displacement that occurs between the labeled DNA and the unlabeled DNA, it is possible to detect differences as small as a cancer-cell-originated single base of a gene existing only in a small amount. Excluding the time for the gene amplification reaction, the time needed for detecting mutation is around 15 minutes, and Toppan Printing believes that in the future it will be possible to reduce this to around 1 minute. For more detailed information on the PHFA response principle, refer to Nucleic Acids Reserch, 1994,Vol.22,No.9 1541-1547.


(*1) RIKEN GENESIS CO.,LTD.
With a foundation of the SNP typing technology developed by the RIKEN Center for Genomic Medicine, RIKEN GENESIS was established on October 15, 2007 through joint investment by RIKEN Venture Capital Co. Ltd. (President: Akito Arima) and Toppan Printing. In technical collaboration with RIKEN, the company provides SNP typing analysis services, engages in business for analysis systems and related services, and targets the improvement of each individual’s quality of life through the realization of personalized medicine.

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