3. EPIDEMIOLOGY/BEHAVIORAL SCIENCE

RESEARCH 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)
      Dr. Kei Nakachi (Chief, Department of Radiobiology / Molecular
      Epidemiology, Radiation Effects Research Foundation)
    2. U.S. Principal Investigator (USPI)
      Dr. Curtis C. Harris (Chief, Laboratory of Human Carcinogenesis, Division of Basic Sciences, National Cancer Institute)
    3. Research Associate (Japanese / US)
      Masataka Taga (Research Scientist, Department of Radiobiology /Molecular Epidemiology, Radiation Effects Research Foundation)
    4. List of Other Project Participants (except for PIs and RA)

      Japanese-side:
      Eiichi Tahara (Senior Consulting Scientist, Radiation Effects Research
      Foundation (RERF)
      Kazunori Kodama (Chief, Department of Epidemiology, RERF)
      Kiyohiro Hamatani (Senior Researcher, Dept. of Radiobiology/Molecular Epidemiology, RERF)
      Hidetaka Eguchi (Research Scientist, Dept. of Radiobiology/Molecular Epidemiology, PERF)
      Kazue Imai (Research Scientist, Dept. of Radiobiology/Molecular Epidemiology, RERF)
      Reiko Ito (Research Scientist, Dept. of Radiobiology/Molecular Epidemiology, PERF)
      Shizue Izumi (Associate Professor, Dept. Of Computer Science and Intelligent Systems, Faculty of Engineering, Oita University)
      Wataru Yasui (Professor, Graduate School of Biomedical Sciences, Hiroshima University)

      US-side:
      Perwez Hussain, Ph.D. (Staff Scientist, Laboratory of Human Carcinogenesis, NCI, NIH)
  4. Activities of Research Associate (RA)
    1. Duration in US: From March 1, 2005 to February 28, 2006
    2. Institution Visited in US: Laboratory of Human Carcinogenesis, US National Cancer Institute
    3. Scientific Results/Achievements
      Dr. Taga was assigned as an RA to Dr. Harris’s laboratory for one year and achieved the following items:
      1. Dr. Taga analyzed TP53 and EGFR mutations among 28 lung cancer cases occurred in atomic bomb survivors along with KRAS mutation among 68 lung cancer cases.
      2. Dr. Taga sensitively detected gene mutations by combining direct sequencing with SSCP polyacrylamide gel electrophoresis method established at Dr. Harris’s laboratory, specifically for the DNA samples containing a small portion of mutated gene.
      3. Dr. Taga performed quality control tests by using same DNA samples and comparing mutations found in Japan with those in U.S.
      4. In order to compare lung cancer from atomic-bomb survivors, Dr. Taga also contributed for the analysis on radon-associated lung cancer at Dr. Harris’s laboratory.
  5. Purpose and Content of Project
    Objectives: The purpose of this study is to elucidate the molecular characteristics of radiation-associated lung and colon cancers among atomic-bomb survivors, in terms of a stratified case-study design and comprehensive molecular analysis of cancer and noncancer tissue specimens among atomic-bomb survivors and those in the general population.
    Study samples: A network of local pathologists in Hiroshima was recently organized by E. Tahara and W. Yasui, and it commenced the systemic collection of cancer, precancerous, and noncancer tissue specimens from newly diagnosed cancer patients as well as archival specimens. This study analyzed these tissue specimens together with clinico-epidemiological data upon approval by the ethical committees of Hiroshima University and RERF.
    Methods: To elucidate the molecular characteristics of radiation-associated lung cancer among atomic-bomb survivors, we conducted molecular analysis of genetic alterations in lung cancer among atomic-bomb survivors and control (non-exposed) subjects by intensive collaboration between Japanese and U.S. researchers. The data obtained from this molecular analysis were further analyzed in relation to radiation dose. Dr. Taga was assigned to the U.S.NCI for one year as a research associate and participated in the allotted study of U.S. researchers.
    We also commenced the analysis of colon cancer among atomic-bomb survivors and prepared for the future collaborative study with U.S. researchers, specifically focusing on microsatellite instability and radiation exposure. However, we gave priority to the analysis of lung cancer.
  6. Status Report of Project Implementation
    Project items
    1. Collection of tissue specimens
    2. Preparation of DNA and RNA for molecular analysis
    3. Supply of a part of DNAs upon approval of the ethical committee
    4. Development of mutation screening system by DHPLC
    Implementation
    We have developed a method for efficiently amplifying DNA and RNA using those extracted from the archival tissue samples, in terms of lesser-mediated microdissection technique and improved RT-PCR amplification. Using these methods, we prepared good-quality DNA from cancerous and non-cancerous tissues for this collaborative study. Upon approval by the ethical committees of Hiroshima University and RERF, we sent the DNA samples of 28 lung cancer cases to the laboratory of Dr. C.C. Harris with Dr. M. Taga, RA. TP53 and EGFR mutations in 28 lung cancer cases were analyzed at the laboratory of Dr. C.C. Harris along with KRAS mutation analysis on 68 lung cancer cases (including those obtained after the assignment of Dr. Taga); the analysis of TP53 and EGFR mutations in 32 lung cancer cases (collected after the assignment of Dr. Taga) has been completed at RERF.

    5. Dispatch of a research associate
    Implementation
    Dr. M. Taga had been assigned to the laboratory of Dr. C.C. Harris for one year as a research associate to investigate mutations in the TP53, EGFR, and KRAS genes in lung cancers among atomic-bomb survivors.

    6. Molecular analysis
    Implementation
    DNA samples of 60 lung cancer cases were analyzed in the U.S. and Japan,
    comprising 37 non-exposed cases (radiation dose=0), 19 exposed cases (dose>0), and 4 cases with unknown dose. Examined were somatic mutations in the TP53 (exons 5-8), KRAS (codons 12, 13, 61), and EGFR (exons 18, 19, 21) genes: 20, 5, and 14 cases were found to carry mutated TP53, KRAS, or EGFR gene, respectively. The mutation status of these genes was further studied in relation to radiation exposure. We also commenced analysis of microsatellite instability (MSI) in colon cancer of atomic-bomb survivors and prepared for further collaboration with U.S. researchers.


    7. U.S.-Japan seminar
    Implementation
    We held the U.S.-Japan Cooperative Cancer Research Seminar on "molecular epidemiological characteristics of lung and colon cancer development among atomic-bomb survivors," at NIH, in Bethesda, on February 23 and 24, 2006.
  7. Seminar
    1. Title: Molecular Epidemiological Characteristics of Lung and Colon Cancer Development Among Atomic Bomb Survivors.
    2. Period: February 23-24, 2006
    3. Site: Room 1207, Building 40, National Cancer Institute
    4. Participants: Total 16 (Japan side 8 , US side 8 , Other country 0 )
    5. Other Country Participants: None
    6. . Agenda, Topics and Scientific Achievements
      Monday, February 22, 2006
        Discussion about the study plan
       
      Thursday, February 23, 2006
      1:00 PM Introduction
      Eiichi Tahara, Radiation Effects Research Foundation (RERF)
      Curtis Harris, NCI
      1:15 PM Overview of RERF Molecular Oncology and Immunology Studies
      Kei Nakachi, Radiation Effects Research Foundation (RERF)
      Epidemiological Overview
      Kazue Imai, Radiation Effects Research Foundation (RERF)
      1:45 PM Radon, Secondhand Smoke, Glutathione-S-transferase M1 and Lung Cancer Risk among Women
      Michael Alavanja, NCI
      2:15 PM Radiation-Associated Thyroid Carcinogenesis in A-bomb Survivors
      Kiyohiro Hamatani, Radiation Effects Research Foundation (RERF)
      2:45 PM - 3:15PM   BREAK
      3:15 PM SNP Analysis of Lung Cancer in Case-Control Molecular Epidemiology Studies
      Leah Mechanic, NCI
      3:45 PM Molecular Immuno-Epidemiology on the Health Effects of Radiation
      Tomonori Hayashi, Radiation Effects Research Foundation (RERF)
      4:15 PM p53 suppresses homologous recombination triggered during DNA replication by interaction with RPA to maintain genome stability
      Simon Powell, Washington University in St. Louis
       
      Reception
       
      Friday, February 24, 2006
      8:30 AM EGFR Mutatiuon in Japanese Lung Cancer in Relation to the CA-repeat Polymorphism in Intron 1
      Naoko Sueoka, Saga University
      9:00 AM Nicotine Addiction and Tobacco-Related Harm
      Peter Shields, Georgetown University
      9:30 AM - 10:00AM   BREAK
      10:00 AM p53, EGFR and K-ras Mutation in Lung Cancer Among A-bomb Survivors
      Masataka Taga, Radiation Effects Research Foundation (RERF)
      10:30 AM Molecular Analysis on Colorectal Cancer in A-bomb Survivors
      Hidetaka Eguchi, Radiation Effects Research Foundation (RERF)
      11:00 AM Cox-2, bystander Response and Low dose Radiation Risk Assessment
      Tom Hei, Columbia University Medical Center
      11:30 AM The Role of DNA Repair and Cell Cycle Checkpoints in Cancer Susceptibility:Phenotypic Analysis of Cancer Risk in Molecular Epidemiology Studies
      Yun-Ling Zheng, Georgetown University
      12:00 PM - 1:00PM   LUNCH
      1:00 PM - 2:00 PM  General Discussion
  8. Research Results of Project
    The scientific achievements of this project comprise four items: 1) Development of molecular oncology analytical techniques, 2) dispatch of a research associate (RA), 3) scientific results of this project?molecular patterns of genetic alterations found in lung and colon cancers of atomic-bomb survivors, and 4) a U.S.-Japan cancer research seminar.
    1. Development of molecular analytical techniques. Since the molecular analysis of this project uses long-term-preserved formalin-fixed paraffin-embedded tissue samples of atomic-bomb survivors, we have developed a method for efficiently amplifying DNA and RNA using those extracted from the archival tissue samples, in terms of lesser-mediated microdissection technique and improved RT-PCR amplification. Using these methods, we prepared good-quality DNA from cancerous and non-cancerous tissues for this collaborative study. Upon approval by the ethical committees of Hiroshima University and RERF, we sent the DNA samples of 28 lung cancer cases (11 exposed and 17 non-exposed to atomic radiation, collected by that time) to the laboratory of Dr. C.C. Harris, U.S. NCI, with Dr. M. Taga, RA.
    2. Dispatch of RA. Dr. M. Taga, a junior scientist of the Cell Biology Laboratory of our department, had been assigned to the laboratory of Dr. C.C. Harris for one year (from March 2005 to February 2006) to investigate mutations in the TP53, EGFR, and KRAS genes in lung cancers among atomic-bomb survivors, in close collaboration with researchers in that laboratory. During his stay, he also contributed to the molecular analysis of radon-associated lung cancers for comparison with lung cancers of atomic-bomb survivors.
    3. Scientific results.
      3-1. Lung cancer. TP53 and EGFR mutations in 28 lung cancer cases were analyzed at the laboratory of Dr. C.C. Harris along with KRAS mutation analysis on 68 lung cancer cases (including those obtained after the assignment of Dr. Taga); the analysis of TP53 and EGFR mutations in 32 lung cancer cases (collected after the assignment of Dr. Taga) has been completed at RERF. Combining the cancer cases analyzed in both U.S. and Japan, the following results were obtained.
      A total of 68 lung cancer samples have been collected as of March 1, 2006; 18 were freshly frozen samples, while 50 were archival formalin-fixed paraffin-embedded samples. Among them, 60 DNA samples were analyzed in the U.S. and Japan, comprising 37 non-exposed cases (radiation dose=0), 19 exposed cases (dose>0), and 4 cases with unknown dose. The median dose for the 19 exposed cases was 26.5 mGy. Examined were somatic mutations in the TP53 (exons 5-8), KRAS (codons 12, 13, 61), and EGFR (exons 18, 19, 21) genes: 20, 5, and 14 cases were found to carry mutated TP53, KRAS, or EGFR gene, respectively. In adenocarcinoma and squamous cell carcinomas, TP53 mutation frequency among cases with dose>0 was higher than that among those with dose=0: 53% (8/15) vs. 26% (8/31), P=0.07. Of 8 cases with dose>0, 2 cases (25%) carried double mutations in the TP53 gene, in contrast to no double mutations found in cases with dose=0. Since double mutations of this gene have been reported rare (about 2%) in lung cancers of the Japanese general population, it is notable that double mutations in the TP53 gene seem more frequent in radiation-exposed lung cancer than was expected.
      When TP53 gene mutations were categorized into two groups: G:C>T:A transversion or deletion type and all other types, 7 cases with the former type of TP53 gene mutations showed higher median radiation dose than that of 13 cases with the other mutation types (27 mGy vs. 0 mGy, P= 0.003), suggesting that certain TP53 gene mutation types may be associated with radiation exposure. On the other hand, we observed no association between KRAS or EGFR gene mutations and radiation exposure. Further analysis with an increased number of cases, specifically those with high radiation dose, is needed, along with consideration of other risk factors (e.g., cigarette smoking).
      3-2. Colon cancer. We also commenced analysis of microsatellite instability (MSI) in colon cancer of atomic-bomb survivors and prepared for further collaboration with U.S. researchers. To summarize the preliminary results in a total of 30 colon cancer patients, MSI-positive colon cancer was frequently observed at proximal sites in female atomic-bomb survivor patients, as was the case in sporadic colon cancer in non-exposed populations. However, the median radiation dose in patients with MSI-positive colon cancer was found to be higher than that in MSI-negative patients (P=0.07), suggesting a possible association of radiation exposure and MSI.
    4. U.S.-Japan seminar. We held the U.S.-Japan Cooperative Cancer Research Seminar on "]molecular epidemiological characteristics of lung and colon cancer development among atomic-bomb survivors," at NIH, in Bethesda, on February 23 and 24, 2006 (proceedings of this seminar are attached). The seminar was organized by Drs. C.C. Harris and K. Nakachi with seven U.S. participants and eight Japanese participants (including Dr. Taga). The results of this collaborative study were presented at the seminar, together with those of studies on thyroid cancer in atomic-bomb survivors, ERFR mutations in sporadic lung cancer in Japan, genome approach to radiation-associated carcinogenesis, radon-associated lung cancer in women, SNP analysis of lung cancer, p53 and regulation of homologous recombination, and COX-2 and bystander response to low-dose radiation. This seminar was very successful for both U.S. and Japanese researchers, ensuring further development of the collaborative study.

    Scenes at the U.S.-Japan seminar

    Scenes at the U.S.-Japan seminar
  9. Papers and Publications (Project-related papers that have or will be published)
    (1) Paper
    Hamatani, K, Eguchi, H, Takahashi, K, Koyama, K, Mukai, M, Ito, R, Taga, M, Yasui, W, Nakachi, K. “Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens” J. Histochem. Cytochem., in press.

    (2) Presentations
    1. Takahashi K, Hamatani K, Eguchi H, Taga M, Ito R, Imai K, Soda M, Arihiro K, Hayashi Y, Nakata Y, Nakachi K. "Molecular epidemiological characteristics in radiation-associated papillary thyroid cancer" The 6th Annual Meeting of Japanese Society of Cancer Molecular Epidemiology, 21 May 2005
    2. Eguchi H, Taga M, Ito R, Hamatani K, Katayama H, Kodama K, Tahara E, Matsumura S, Yasui W, Nakachi K. "Molecular epidemiological analysis of lung and colorectal cancers among atomic bomb survivors (I)" The 64th Annual Meeting of the Japanese Cancer Association, 14 -16 September 2005
    3. Hamatani K, Takahashi K, Eguchi H, Taga M, Ito R, Imai K, Arihiro K, Nakachi K. “Molecular epidemiological characteristics of papillary thyroid cancer as radiation-associated cancer” The 64th Annual Meeting of the Japanese Cancer Association, 14-16 September 2005
    4. Takahashi K, Hamatani K, Eguchi H, Taga M, Ito R, Imai K, Cologne J, Soda M, Hayashi T, Abe K, Fujihara M, Arihiro K, Hayashi Y, Nakata Y, Nakachi K. "Point mutation of BRAF gene in papillary thyroid cancer developed from atomic bomb survivors (II)" The 7th Annual Meeting of Japanese Society of Cancer Molecular Epidemiology, 19-20 May 2006
    5. Hamatani K, Eguchi H, Takahashi K, Taga M, Ito R, Cologne J, Soda M, Hayashi T, Abe K, Fujihara M, Arihiro K, Hayashi Y, Nakachi K. “ RET/PTC rearrangement in papillary thyroid cancer developed from atomic bomb survivors” The 7th Annual Meeting of Japanese Society of Cancer Molecular Epidemiology, 19-20 May 2006
    6. Taga M, Eguchi H, Hamatani K, Ito R, Imai K, Cologne JB, Katayama H, Kodama K, Matsumura S, Yasui W, Nakachi K. "Mutations of p53, K-ras, and EGFR genes in lung cancer from atomic bomb survivors" The 7th Annual Meeting of Japanese Society of Cancer Molecular Epidemiology, 19-20 May 2006
    7. Eguchi H, Hamatani K, Taga M, ito R, Imai K, Cologne JB, Katayama H, Kodama K, Matsumura S, Yasui W, Nakachi K. "Micorsatellite in colorectal cancer developed from atomic bomb survivors" The 7th Annual Meeting of Japanese Society of Cancer Molecular Epidemiology, 19-20 May 2006
    8. Hamatani K. "Papillary thyroid carcinogenesis among atomic-bomb survivors in relation to radiation exposure" 36th Annual Meeting of European Environmental Mutagen Society, July 2-6, 2006
    9. Eguchi H, Taga M, Nakachi K, Hamatani K. "Improvement of PCR and RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens" The 12th International Federation of Placenta Associations Meeting. September 6-9, 2006
  10. Any Comments: None