3. EPIDEMIOLOGY/BEHAVIORAL SCIENCE

ANNUAL REPORT FOR JOINT PROJECT

1. Title of Project: Molecular Epidemiological Characteristics of Lung and Colon Cancer Development among Atomic Bomb Survivors
2. Duration: April 1, 2004 — March 31, 2006
3. Project Organization
   1. Japanese Principal Investigator (JPI)
      Kei Nakachi (Chief, Radiation Effects Reseach Foundation/ Depatment of Radiobiology/Molecular Epidemiology)
   2. U.S. Principal Investigator (USPI)
      Curtis C. Harris (Chief, National Cancer Institute Division of Basic Sciences, Laboratory of Human Carcinogenesis)
   3. Research Associate (Japanese / U.S.)
      Masataka Taga (Reseach Scientist, Radiation Effects Research Foundation/ Depatmnet of Radiobiology/Molecular Epidemiology)
4. Number of Participants
   1) Japanese side:  10  persons 2) US side:  2  persons
5. Number of Exchanges in JFY 2004
   1. from Japan to US
      Masataka Taga (Radiation Effects Research Foundation)
      Duration: March 1^st,’05~(February 28,’06)
      Host Institution: National Cancer Institute
   2. from US to Japan: None
6. Activities of RA in JFY2004
   Most cancer tissue specimens from atomic-bomb survivors used for this study are long-term preserved formalin-fixed paraffin-embedded ones. Dr. Taga has developed several molecular techniques specifically for the analyses of these archival tissue specimens, such as new primer sets designed for loss-of-heterozygosity (LOH) analysis and also for whole genome amplification of DNA using DOP-PCR with a newly modified protocol. He examined TP53 and EGFR mutations with 18 lung cancer tissue specimens from atomic-bomb survivors in Japan and started collaborative work with Dr. Harris’s group at the Laboratory of Human Carcinogenesis, NCI from March 1 in 2005, using DNA samples extracted from those lung cancer specimens, on the basis of the approvals from the Ethical Committees at both U.S. NCI and Hiroshima University.
7. Status Report of Project Implementation and Scientific Achievements
   (1) To date, a total of 34 lung cancer tissue specimens have been collected by the MHLW (Ministry of Health, Labor, and Welfare, Japan) study group (P.I. Dr. Tahara and Prof. Yasui), and these are now available for this study. Information about exposed radiation dose and clinicopathological data of these lung cancer cases were also collected. A total of 120 colorectal cancer tissue specimens from atomic-bomb survivors and non-exposed persons have also been found in 2004 to be used for this study, and they will be analyzed in 2005.
   (2) Most of these lung and colorectal cancer tissue specimens from atomic-bomb survivors were archival formalin-fixed paraffin-embedded (FFPE) ones: DNA extracted from archival FFPE specimens was fragmented and modified. Therefore, we have developed various methods for the DNA/RNA extraction, amplification, and molecular analyses of those archival specimens, including heat pre-treatment of DNA samples in borate buffer for PCR, whole genome amplification of fragmented DNA, and new primer sets for microsatellite markers. In addition, for efficient screening of TP53 and EGFR mutations in lung cancer, we developed the use of denaturing high performance liquid chromatography (DHPLC).
   (3) Lung cancer Although we plan to examine 1) somatic mutations in TP53, EGFR and other genes, 2) methylation status of CDKN2A, RARB, RASSF1A and other genes, and 3) LOH at 3p, 9p and 17p, and 4) microsatellite instability, we first started analyzing in TP53 and EGFR mutations. In 2004, we examined 13 lung adenocarcinoma specimens and found TP53 and EGFR mutations to occur in 5 (38%) and 4 (30%), respectively, along with the analysis of 5 lung cancer specimens other than adenocarcinoma.
   Colon cancer We plan to examine mutations and methylation of MHL1 and APC in addition to the above-mentioned items except for LOH. We have completed the development of necessary experimental techniques in 2004, along with preliminary analysis using cell lines.
8. Any Comments: None