1
Radiation Effects Research Foundation News and Views
Hiroshima and Nagasaki, Japan

Volume 18, 2007

Table of Contents
From the Editors ......................................................................................................................................... 1
Letter to the Editor ...................................................................................................................................... 1
RERF NewsThe 33rd Scientific Council Meeting .................................................................................................... 2
The 41st Board of Directors Meeting ................................................................................................... 3
Staff News ............................................................................................................................................. 5
Honors and Awards Received by RERF Scientists ............................................................................... 6
Report on Conferences and Workshops
Results of the Health Effects Study of the Children of A-bomb Survivors Announced,
by Akihiko Suyama ........................................................................................................................... 8
Science Articles
Solid Cancer Incidence among Atomic Bomb Survivors, 1958–1998, by Dale L. Preston et al. ........ 9
Use of Stored Sera for Detection of Hepatitis B and C Viruses, by Waka Ohishi et al. ..................... 14
Molecular Features of Colorectal Cancer Developing among Atomic Bomb Survivors with
Emphasis on Microsatellite Instability—An Interim Report, by Hidetaka Eguchi et al. ............... 19
Human Interest Notes
The “Old Boys’ Club”—Report on a Series of Interviews with Former ABCC Employees .............. 33
Facts and Figures
Data Processing and Management System for Adult Health Study, by Hiroaki Katayama ............... 41
Research Protocols and Publications
Research Protocols .............................................................................................................................. 43
Recent Publications ............................................................................................................................. 44

This newsletter is published by the Radiation Effects Research Foundation (formerly the Atomic
Bomb Casualty Commission), established in April 1975 as a private, nonprofit Japanese foundation.
It is supported by the government of Japan through its Ministry of Health, Labour and Welfare andthat of the United States through the National Academy of Sciences under contract with the
Department of Energy.
RERF conducts research and studies—for peaceful purposes—on the medical effects of radiation on humans with a view toward contributing to the maintenance of the health and welfare of
atomic-bomb survivors and to the enhancement of the health of all mankind.
Editor-in-Chief: Thomas Seed, Associate Chief of ResearchTechnical Editor: Yuko Ikawa, Public Relations & Publications Office
Editorial Policy
Contributions to Update receive editorial review only and do not receive scientific peer review.
The opinions expressed herein are those of the authors only and do not reflect RERF policies orpositions.
Contact: Mailing address: RERF Public Relations & Publications Office, 5-2 Hijiyama Park,
Minami-ku, Hiroshima 732-0815 Japan
Telephone: 81-82-261-3131; Facsimile: 81-82-263-7279
Internet: www.rerf.jp

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From the Editors, Letter to the Editor

Haikei!
Well, here we are once again…trying to catch up
on our reporting of RERF’s various research
activities over the past several years.
Faithful readers…gambatte kudasai (keep yourchin up)…we’re close to our objective! More
importantly, we’re trying to…and believe that weare succeeding…in making significant improvements in the quality of our reports, as well as
making the reports more enjoyable to the reader.
Toward this end, we have included in thisvolume three very interesting and timely science
articles: the first by Dale Preston and colleagues,
concisely and elegantly summarizes the findings oftheir recent landmark study in which radiationassociated cancer (solid tumors) risks were updated,documented, and characterized for various organsystems of body; the second article by Waka Ohishi,
reports on a technical breakthrough in using archived
frozen sera diagnostic virology; and the third article
by Hidetaka Eguchi, summarizes his recent findingson the molecular biology (genomic instability) ofradiation-associated colorectal cancer in A-bomb
survivors.
In addition, we have included a “Facts and Figures”

piece on the Information Technology Department’s“Data Processing and Management System for Adult
Health Study” (by Hiroaki Katayama). Also, we
would like to point out a special “Human Interest”
article that we believe that you will enjoy, namely
an interview with five very distinguished RERF retirees who recalled their early days working at ABCC
in Hiroshima and Nagasaki.
So as always, please enjoy this volume, and ofcourse feel free to drop us a line and let us know
your thoughts on our reporting of RERF’s many
activities.

I regularly read “RERF Update” and am very
impressed with its continuingly improved quality. It
is clear that all those involved in the creation of thispublication are continuously striving to achieve an
impeccable product. The recent addition of ‘Science
Articles’ and ‘Human Interest Notes’ are especially
noteworthy. I have read the ‘Science Articles’ of thelast two volumes (16 and 17) and am thrilled by thestandard of published papers.
In volume 17 the first science article titled “Thefrequency of XPA heterozygotes bearing a foundermutation among individuals who resided in
Hiroshima and Nagasaki” by Hirai et al. has elegantly
investigated the frequency of heterozygotes bearing
a specific mutation in the Xeroderma pigmentosum
group A (XPA) gene in a cohort from Hiroshima andNagasaki. XP is a rare autosomal recessive disorderwith high incidence of UV-related skin cancer associated with the impaired ability to repair UV-induced
DNA damage due to a defective translesional DNA
polymerase. The authors have developed a simple
polymerase chain reaction restriction fragment length
polymorphism (PCR-RFLP) in their laboratory to
identify XPA heterozygotes bearing the foundermutation in a cohort of 1,020 individuals who

resided in Hiroshima or Nagasaki. Primer set extending into intron 3 and exon 4 of the XPA gene (theDNA region that includes the founder mutation) wasused for PCR amplification and the amplification
product was a 61 bp fragment. If the founder mutation was present, the amplified 61 bp DNA wascleaved into two fragments (25 and 36 bp) with
AlwN1 digestion. This is the first molecular genetic,
population based study focusing XP heterozygotes.
It demonstrated that the frequency of XPA heterozygotes bearing the founder mutation was 1 in 113 (9out of 1,020; 0.88%) among Hiroshima and Nagasaki
population. This estimate suggests that there may beapproximately 1 million carriers of the XPA foundermutation in the 120 million population of Japan. The
frequency of XPA heterozygotes observed in thisstudy suggests that the risk of skin cancer in XPA
heterozygotes can be elucidated.
The second science article in volume 17 is by
Cullings et al. and titled “The introduction of
dosimetry system DS02 and its impact on survivor
doses and related risk estimates.” This article statesthat the new dosimetry system, DS02, makes a moreaccurate estimate of the received dose because it
takes more factors into account. It improves upon

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Sincerely,

Thomas M. Seed
Editor in Chief

Yuko Ikawa
Technical Editor

RERF Update Volume 18, 2007

RERF News2the old DS86 by taking into account leakage in thecasing of the bombs that would lower the amount ofradiation given off, the shielding effect of low structures, which were ignored by DS86, and the shielding of various pieces of furniture inside buildings,
enhancing the “globe” method of shielding calculation, as well as taking into account the shielding fromthe terrain (features such as hills). The authors notedthat only the terrain shielding produced a substantial effect on the doses received, whereas all the othermodifications made only slight changes. Furthermore, DS02 improves on determining the bodies selfshielding effect for neutron radiation, as seen by
Tbody,n being about 6% to 9% less in DS02 than in
DS86. The DS02 system makes use of GIS withmaps, aerial photography, and survivor shielding
history drawings to better predict the correct dose
received by a person exposed to radiation than theprevious system.

The 33rd Scientific Council meeting, co-chaired
by Dr. Clarice Weinberg (U.S. National Institute ofEnvironmental Health Sciences) and Dr. Ohtsura
Niwa (Kyoto University), was convened on March
27–29, 2006 in Hiroshima to review the scientificprogram of RERF. The Council meeting was opened
by Vice Chairman Dr. Roy E. Shore’s announcement
and memoriam of the death of Dr. Robert Miller,
who was an eminent scientist in the fields of pediatrics, genetics and cancer epidemiology and made anumber of outstanding scientific contributions in theearly days of ABCC. Dr. Toshiteru Okubo, the Chairman of RERF, then welcomed the Councilors andreported on administrative issues faced by the Foundation over the past year. Thanks was extended tothree executive members, Dr. Burton G. Bennett, Dr.
Eiichi Tahara, and Dr. Senjun Taira, whose terms ofservice were completed in the last year. Dr. Okubo
discussed the budget cut by the Japanese government, which inevitably will necessitate reducing thenumber of personnel, possibly affecting future
activities of the Foundation. Dr. Okubo alsoannounced that, following the recommendation oflast year’s Scientific Council, a Blue Ribbon Panel
will convene later this year to review and makerecommendations regarding the status and future of
RERF’s scientific programs. This is a particularly
timely action, given the multiple challenges andissues faced by the Foundation. Subsequently, overviews of RERF departmental research and several
more detailed reports of new research findings were
presented, along with a presentation by Director
Takanobu Teramoto on RERF’s newly upgradedpublic relations and communications program. Councilors met informally with individual departmentson the second afternoon.

the positive actions taken by RERF in response to
the recommendations made by the Council theprevious year. The Council made a number ofgeneral recommendations as well as ones applicableto the research programs in specific departments. The
principal general recommendations and several
departmental ones with general implications were:• Interdepartmental communication and data
sharing are improving but more attention to thiswill enhance the utility of information andscientific productivity, especially consideringthat complex diseases and systems biology are
increasingly important. Develop methods tolink RERF data sources, while respecting theconstraints of the new privacy laws.
• They applauded the recent establishment of amonthly colloquium as a mechanism for openly
discussing work-in-progress in a highly informal and multidisciplinary context.
• The process of evaluating proposed ResearchProtocols needs to be streamlined if this can bedone without sacrificing quality.
• They encouraged the development of mechanisms to reward and improve research productivity of each of the scientists.
• Improvements need to be made in electronic
access to scientific journals.
• Careful attention should be paid by the Foundation to past recommendations.
• Scientists should be encouraged to apply foroutside grants to support research, insofar as
they fit well within the mission of RERF. This
will help to enhance the visibility of theorganization, build synergistic collaborative
relationships, and improve the science.
• Recruitment remains a critical issue in all
departments in RERF, particularly given that
senior scientists are coming to retirement ages.
Those individuals have played crucial roles as
leaders in the research and often as the interface to the outside radiation community. Efforts

The Scientific Council expressed endorsement of
the core mission of RERF and support for researchin line with its mission. It was impressed by progress
made by RERF since the last year and acknowledged
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Dr. Vijay K. SinghAFRRI (Armed Forces Radiobiology ResearchInstitute)
Bethesda, MD, USA

RERF Update Volume 18, 2007

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RERF Newsshould be made to find creative ways to forestall impending losses of key scientists due togovernmental policies on mandatory retirement. Recruitment of younger investigators is
especially critical for the future of the Foundation.
• They strongly endorsed the partnerships RERF
has established in epidemiology and biostatistics with the University of Washington and
Kurume University. This kind of relationship(and its future extensions) can benefit theradiation research community by promoting
collaborations and developing scientists who
are knowledgeable in radiation risk assessment.
• Try to increase communication and collaboration with other RERF departments in order toacquire relevant statistical, clinical, epidemiologic and/or basic-research expertise early inthe design and conduct of the research.
• Efforts should be continued on the F1 cohort
studies with wide and sustained collaboration.
These studies are particularly important for
examining the prevalence of various noncancer diseases.
• Efforts were applauded to increase the size of
the Adult Health Study cohort for those who
were under the age of five at the time of thebombing.
• The positive relationship between radiation andnon-cancer diseases and conditions calls forstudies to clarify the underlying mechanisms.
• The update of the papers on cancer incidence
in the Life Span Study and in the in utero
exposed cohort will be of wide scientific interest and should be submitted to high impact
journals.
They suggested that the pending Blue Ribbon
Panel:• Address the long-term mission of RERF, assurvivors expire (around 2040) and the F 1cohort drops below 10% around 2055.
• Assess the assumption that at that point in timeradiation effects will still be relevant to scienceand society.
• If radiation health effects are deemed important, consider themes for a future RERF, such

The 41st meeting of the Board of Directors washeld at the RERF Auditorium during June 21–22,
2006. Valuable opinions were actively exchanged
regarding RERF’s future vision for the next 10–20
years, including the issues of personnel reduction
requested by the Japanese government and budgetReturn to Table of Contents

as: 1) preparedness for nuclear accidents or
terrorism and hazards of medical irradiation;2) human systems biology (gene-environmental interactions in the origins of disease); and3) risk assessments for environmental hazards
to health, including the role of the humangenome in modifying responses to environmental factors.
• Address logistic details for supporting a reaffirmed RERF, such as: binational versus multinational sponsorship, sources of funding, etc.
• Propose a long-term strategy to maintain theprestigious role of RERF and its leadership in
the radiation risk research communities.
RERF Scientific Councilors
Dr. Ohtsura Niwa, Professor, Radiation Biology
Center, Kyoto University, Co-chairperson
Dr. Clarice Ring Weinberg, Chief, Biostatistics
Branch, Environmental Diseases and Medicine
Program, National Institute of Environmental
Health Sciences, Co-chairperson
Dr. Shinkan Tokudome, Professor, Health Promotion and Preventive Medicine, Nagoya City
University Graduate School of Medical Sciences
Dr. Teruhiko Yoshida, Chief, Genetics Division,
National Cancer Center Research InstituteDr. Yoshiharu Yonekura, Director, Biomedical
Imaging Research Center, University of Fukui
Dr. Katsushi Tokunaga, Professor, Department of
Human Genetics, Graduate School of Medicine,
University of Tokyo
Dr. Joel S. Bedford, Professor, Department ofRadiological Health Sciences, Graduate Facultyof Cellular and Molecular Biology, Colorado
State University
Dr. Theodore L. DeWeese, Professor and Chair,
Department of Radiation Oncology andMolecular Radiation Sciences, The Johns
Hopkins University School of Medicine
Dr. Marianne Berwick, Professor and Chief, Division of Epidemiology, Department of Internal
Medicine, University of New Mexico
Dr. John J. Mulvihill, Professor of Pediatrics,
University of Oklahoma Health Sciences Centerary actions. In particular, in relation to the “Senior
Review Panel on Future Planning for RERF,” to beestablished in autumn of the same year, RERF
requested the Board members to provide advice
regarding the Foundation’s future vision. Many ofthe visiting directors present at the Board meeting
RERF Update Volume 18, 2007

RERF News
stated that RERF should continue its program as a
research institution, rather than as a radiation information center.
RERF’s future vision has been repeatedly
discussed as one of the organization’s top-priority
issues. During this year’s Board meeting, there was
lively discussion regarding this issue, as well as about
the daunting challenges facing RERF. The main
topics discussed during the two-day meeting arebriefly described below.
In his status report on RERF at the beginning of
the meeting, Dr. Toshiteru Okubo, RERF Chairman,
stated that negotiations about the U.S.-Japan fiveyear agreement on the management of RERF were
held in Tokyo in November 2005 and that establishment of a new Senior Review Panel during 2006 formaking recommendations regarding the future of
RERF was included in the agreement. He also
reported that at the first meeting of the Forum for
Institutes Engaged in A-bomb Survivors’ Medical
Care held in May 2006 in Hiroshima at the requestof Dr. Shizuteru Usui, President of the Hiroshima
Prefectural Medical Association, proposals weremade to further strengthen the partnership among
local institutes engaged in medical care for A-bomb
survivors and that the results of deliberations by theForum would be conveyed to the Senior Review
Panel as opinions representing the local community.
Thus, arrangements are being made to provide
an opportunity for the U.S. and Japanese governments to discuss RERF’s future vision, while efforts
are under way to collect and summarize relevant
opinions of the local community. Behind these
activities is the harsh reality facing RERF. Whatfollows is a report from the RERF side regarding itsdifficult situation and proposed countermeasures.
During the next four years, employment capacity
will be decreased by six persons per year. The salaries of RERF employees will be decreased according to the rate of decrease applied to government
employees. However, the most important policy forcontinuation of RERF’s research activities is theretaining of research staff. With this in mind, every
effort has been made to minimize the effects of thecurrent situation on research activities. Measurestaken include (1) maintenance of the existing fixed
number of research scientists, (2) preparation of
medium- to long-term personnel plans for recruitment of leading research scientists, and (3) introduction of a new system for employment of researchscientists with fixed terms of appointment.
With regard to the above report about RERF and
its difficult set of circumstances, some of the visiting directors voiced their opinions that, since RERF
is a research institution jointly funded by the U.S.
and Japan, the Board should discuss how U.S.
government ideas regarding RERF can be betterreflected in the management of the Foundation, andReturn to Table of Contents

4
that, since U.S. funds account for 40% of all its
operational funds, RERF should request the Ministry of Health, Labour and Welfare (MHLW) to
exempt RERF from across-the-board application ofthis policy of personnel reduction. It was decided
that the results of deliberations of the Board of
Directors would be conveyed to the Japanese
government.
It was also reported at the meeting that, although
the number of participants in the Adult Health Study(AHS), a program of major interest in future plans
for research, will decline to almost zero around 2040,
AHS has provided useful information regarding
human health, unavailable from studies of mortality
or cancer incidence only, and that long-term continuation of the program would be meaningful.
As for public relations activities to increase awareness among the public inside and outside the country regarding RERF’s research activities, it wasreported that, in accordance with the recommendations of last year’s Scientific Council meeting, thePublic Relations and Publications Office wasestablished in the Hiroshima Laboratory and aperson in charge of public relations was placed in
the Nagasaki Laboratory, to ensure that PR activities are strengthened and PR strategies established.
With regard to the FY2006 research activities
report, there was a question about whether or not theF1 Clinical Study (FOCS) should be continued as alongitudinal study. In response to this, some of thevisiting directors, while admitting that FOCS wasan invaluable study, pointed out the necessity of
conducting close review before starting the longitudinal study.
Finally, a discussion ensued concerning election/
appointment of officers, with Dr. Takashi Yanagawa(professor with special appointment, Biostatistics
Center, Kurume University) and Dr. David G. Hoel
(distinguished university professor, Department of
Biometry and Epidemiology, Medical University ofSouth Carolina) elected as successors for two
Scientific Councillors (Dr. Shinkan Tokudome and
Dr. Clarice Ring Weinberg), whose terms were set
to expire June 30, 2006. The terms of office of the
new Scientific Councillors will be five years, to June30, 2011.
It was decided that the next Board of Directors
meeting would be held at the Nagasaki Laboratory
for the three days from June 20 to 22, 2007.
As a whole, at this year’s Board meeting, there
were many items that could not be discussed without future plans in place. In this sense, establishmentof the Senior Review Panel, which was strongly
recommended by the Scientific Council last year andfor which the Board of Directors expressed support
last year, is attracting much attention, because thepanel will have a large impact on the future of RERF.

RERF Update Volume 18, 2007

RERF News

List of Participants
Directors:
Dr. Toshiteru Okubo, Chairman
Dr. Roy E. Shore, Vice Chairman and Chief ofResearch
Mr. Takanobu Teramoto, Permanent Director
Mr. Masaaki Kuniyasu, Former Ambassador Extraordinary and Plenipotentiary to the Republicof Portugal
Dr. Yasuhito Sasaki, Vice President, International
University of Health and Welfare
Dr. Senjun Taira, Permanent Director, Japanese
Association of Quarantine Inspection Hygiene
Dr. Paul L. Ziemer, Professor Emeritus, Purdue
University
Dr. James D. Cox, Professor and Head, Division ofRadiation Oncology, University of Texas M.D.
Anderson Cancer Center
Dr. John E. Burris, President, Beloit College
Supervisors:
Dr. Tomio Hirohata, Professor Emeritus, Faculty of
Medicine, Kyushu University
Mr. David Williams, Senior Financial Advisor, National Academy of Sciences
Scientific Councilor:
Dr. Ohtsura Niwa, Professor, Radiation Biology
Center, Kyoto University

As in any organization change is inevitable; RERF
is no exception. The scientific staff continues to
evolve, with new faces appearing, old faces disappearing, and some faces simply changing features.
During the period from April 2006 to March 2007,
there have been four new additions to the scientificstaff, along with four departures. In addition, threelong term employees retired, but were subsequently
rehired under new contracts of employment. First,
in terms of the new “faces,” Dr. Phillip Ross arrived
in August of last year, leaving his former consulting
and teaching posts in the U.S., and assumed his newrole here as Chief of Statistics. We feel very fortunate to have Dr. Ross onboard, as he has considerable managerial experience and an academic background in a wide-range of statistical analyses. In
addition, Dr. Kengo Yoshida and Dr. Ritsu Sakata,both bright, highly motivated, and energetic researchers, joined RERF in April of 2006 and are now gainfully employed in the Department of Radiobiology/
Molecular Epidemiology and the Departments ofStatistics and Epidemiology, respectively. We fully
expect to see great things coming from the work of
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5
Representatives of Supporting Agencies:
Mr. Katsunori Hoshi, Chief, Relief Budget Unit,
General Affairs Division, Health Service Bureau,
Ministry of Health, Labour and Welfare
Dr. Joseph F. Weiss, Japan Program Manager,
Office of Health Studies, U.S. Department ofEnergyMr. Yuriy R. Fedkiw, Science and Technology
Officer, Embassy of the United States of America(acting Minister-Counselor for Joyce Rabens)
Dr. Kevin D. Crowley, Director, Board on Nuclear
and Radiation Studies, Division on Earth and Life
Studies, National Research Council, National
Academy of Sciences
Dr. Evan B. Douple, Scholar, Board on Nuclear andRadiation Studies, Division on Earth and Life
Studies, National Research Council, National
Academy of Sciences
RERF:
Mr. Eiji Akimoto, Chief of Secretariat
Dr. Thomas M. Seed, Associate Chief of Research
Dr. Kazunori Kodama, Chief Scientist/Chief,
Department of Epidemiology
Dr. Nori Nakamura, Chief Scientist/Chief, Department of Genetics
Mr. Douglas C. Solvie, Associate Chief of Secretariat

these scientists. The Clinical Studies Department has
also benefited from the arrival in April (of 2006) ofa more seasoned physician/clinical researcher,namely Dr. Ikuno Takahashi. Dr. Takahashi’s medical specialty is in internal medicine with a clinicalresearch focus in the area of cardiovascular disease.
So much for the “new faces”; what about the“changing faces.” There are three: Dr. Nori Nakumra,
Mimako Nakano, and Kazuo Ohtaki. All of thesestaff members spent sizable fractions of their careers
here at RERF working in the area of cytogenetics.
Following their retirements, all were reemployed
under new guidelines established here at RERF. Dr.
Nakano and Dr. Ohtaki have been rehired as AdjunctResearch Specialists within the Cytogenetics Laboratory, whereas Dr. Nakumara has assumed a rangeof administrative duties under the title of Chief Scientist. Regardless of the mechanism by which thesevery talented and experienced researchers were
reemployed, we are delighted that we were able toretain these individuals and their most valued
services.
During this period, however, RERF lost some
RERF Update Volume 18, 2007

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RERF News
outstanding researchers who over the years madesignificant contributions to the RERF’s science. Dr.
Kiyoto Ashizawa, very talented clinical researcher
in the Department of Clincial Studies in Nagasaki,
received and accepted a very nice offer of a position
as Chief of the Endocrinology Department at
Saiseikai Hospital in Nagasaki. In addition to Dr.
Ashizawa, the very prominent cancer scientist, Dr.

Eiichi Tahara, who was serving as “Senior Consulting Scientist” resigned his post in June of 2006.
It should be noted that Dr. Tahara ably served
RERF in various senior administrative capacities,
including Permanent Director and Chief of Research.
His foresight on a number of leading-edge biomedical research areas will be missed.

There is an American saying, “When it rains, it
pours,” and RERF investigators received a virtual
cloudburst of honors. In the fall of 2006 no fewer
than five RERF researchers won important awards
for their research contributions from various scientific societies in Japan and abroad. Below, eachresearcher will briefly describe the award he or she
received.
In addition to the above scientific awards, Dr.
Kazunori Kodama, RERF Chief Scientist, wasappointed Chairman of the Japan Epidemiological
Association (JEA) for the term of three years at the
JEA’s 17th annual meeting held in Hiroshima on
January 26–27, 2007, for which he served as chairman. Dr. Kodama’s appointment is an honor also for
RERF, which has focused on epidemiologicalresearch over many years, and we are pleased toreport this news in the RERF Update.

with the aim of studying genetic instability.
Currently working as an associate senior technician at the Cell Biology Laboratory, Department ofRadiobiology/Molecular Epidemiology, I have beenworking as a returning student at the Division ofIntegrated Medical Science, Programs for Biomedical Research, Graduate School of Biomedical
Sciences, Hiroshima University, since April 2003 (ajoint program between RERF and Hiroshima University). Since starting my study at Hiroshima
University, I have been conducting research basedon the theme “Study of genetic instability inducedby radiation exposure,” with tutelage and support
from Dr. Yoshiaki Kodama (Genetics Department),Dr. Yoichiro Kusunoki (Radiobiology/Molecular
Epidemiology Department), and related technicians.
I would therefore like to take this opportunity to
express my gratitude to these special individuals.

Excellent-presentation Award at the 49th
Meeting of the Japan Radiation
Research SocietyFirst Morii Award from the Japan
Osteoporosis Society

Kanya Hamasaki, Department ofRadiobiology/Molecular Epidemiology

I received an excellent-presentation award at the49th meeting of the Japan Radiation Research Society, held at Hokkaido University for three days from
September 6–8, 2006. The excellent-presentation
award was first established at this meeting and waspresented to the 12 posters of distinguished quality
selected from among a total of 192 posters withpresentation themes representing the various fields
related to radiation research (radiation effects,
radiation response, lesion/repair, radio-therapeutic
biology, radiation epidemiology, environmental
effects, among others). At the meeting, I made a presentation about the “Study on genetic instability inperipheral blood lymphocytes among the A-bomb
survivors,” in which we cloned A-bomb survivor
peripheral blood T cells in vitro and analyzed, with
the M-FISH method, each of the clones obtained for
determination of frequency of additional chromosomal mutations occurring in the cultivation process
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Saeko Fujiwara, Chief
Department of Clinical Studies, Hiroshima

I was honored to receive the first Morii Award
from the Japan Osteoporosis Society (JOS) at its
FY2006 meeting, held in Tokyo over three days, from
October 12 through 14. This award is based on adonation made by the family of Dr. Kosei Morii, who
was the chairman of JOS and passed away last year,
and is presented to a principal author of the mostdistinguished clinical research paper among the JOS
members. The paper selected for the award was “Heelbone ultrasound predicts non-spine fracture in Japanese men and women,” Osteoporosis Int 16:2107–
12, 2005.
Although ultrasound bone assay had been widelyused for examination of osteoporosis in Japan, no
reports had been published regarding whether or not
the technique could be used to predict fracture risk.
Evidence for the Japanese population was acquired
for the first time in our study, and that was the major
reason for this work being selected for the award.
This study was conducted jointly by RERF,
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RERF News
Kawasaki Medical College, and Hamamatsu Medical College, and I served as coordinator of the study.
The study, however, could not have been completed
without the considerable efforts made by the late Dr.
Shoichiro Fujita, who conducted the statistical analysis, and those of Ms. Naomi Masunari, research
assistant of the Department of Clinical Studies.

Hypertension Research-Novartis Award
for Best Research Paper
Masazumi Akahoshi, Chief
Department of Clinical Studies, Nagasaki

The 21st meeting of the International Society ofHypertension was held in Fukuoka on October 15–
19, 2006. At the general meeting of the JapaneseSociety of Hypertension (JSH), held during the same
period, on October 18, I received the sixth Hypertension Research-Novartis award for the best
research paper. This award is presented to the best
research paper of the year selected from among thepapers published in the JSH newsletter Hypertension Research. The paper for which I was presented
with the award was titled “Basic characteristics ofchronic hypotension cases: A longitudinal followup study from 1958 through 1999.” As the title ofthe paper suggests, our study was based on data
accumulated in the Adult Health Study.
This study was conducted to ascertain if analyzing the characteristics of those whose blood pressure did not increase with age would further clarify
the factors involved in advancing blood pressure withage. As a result, we found 92 individuals who had
lived long with chronic hypotension and whose bloodpressure had not advanced with age. Their characteristics were slight build, slow pulse, low bodytemperature, and anemia. It was considered that thesecharacteristics were induced by decreased tensionof the sympathetic nervous system, and from this, it
was further assumed that the blood pressure increase
with age seen among modern individuals was notbiological inevitable, but something originating fromincreased tension of the sympathetic nervous system.

JSH Award from the International
Society of Hypertension
Yoshimi Tatsukawa, Research Scientist
Department of Clinical Studies, Hiroshima

The 21st meeting of the International Society ofHypertension (ISH 2006) was held in Fukuoka during October 15–19, and I received the JSH award,

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7
which is presented to a young Asian researcher, for
our study “Relationship between white blood cellcount and incidence of hypertension among a Japanese population.” This society, which meets every
two years, was held in Japan for the first time in 18
years, and this time it was convened jointly with thefifth meeting of the Asian and Pacific Society ofHypertension (APSH) and the 29th meeting of theJapanese Society of Hypertension (JSH). At this last
meeting of ISH, quite a few awards were established
for young researchers, including the award presented
to me.
It has been suggested in recent years that chronic
inflammation is involved in arteriosclerosis as a
causal factor. In our presentation, we used whiteblood cell (WBC) counts (an inflammatory marker)
from the Adult Health Study (AHS) population in
1965–67, and followed up the association between
WBC counts and development of hypertension for along period of about 40 years. Almost no other study
exists in which a follow-up of a large-scale population like that of AHS has been conducted, and I feelthat the society considered this point when deciding
to present the award to us.

Poster Prize at the International Association of Cancer Registries
Nobuo Nishi, Chief, Tumor andTissue Registry Office
Department of Epidemiology, Hiroshima

I attended the 28th Annual Meeting of the International Association of Cancer Registries, held in
Goiania, Brazil, from November 7 to 10, 2006, andmy presentation was awarded a poster prize. Unlike
most other international association meetings,
this association’s meeting is held every year, usuallyin a developing country. Also attending from RERF
were Dr. Midori Soda (executive member for the
association’s Asian region), Assistant Chief of the
Department of Epidemiology in Nagasaki, and Dr.
Hiroaki Katayama, Chief of the Department ofInformation Technology.
My poster titled “Socioeconomic differences incancer mortality, incidence and survival in Japan”
was the first one introduced during the presentationof poster awards. It was indicated that my poster was
given high marks because it was well organized,
effectively using tables albeit with no graphs.
Dr. Hans Storm from Denmark, one of the poster
judges, said that he had read abstracts prior to themeeting and viewed mainly posters with the mostintriguing contents, in order to choose several posters to be awarded from among nearly 100.

RERF Update Volume 18, 2007

Conference and Workshop Report

8

Akihiko Suyama, Chief, Departmentof Epidemiology, Nagasaki
RERF has conducted for seven years from 2000–
2007 a health effects study of the children of A-bomb
survivors to investigate whether there is any relationship between the prevalence rates of adult-onset
multi-factorial diseases (hypertension, diabetes
mellitus, hypercholesterolemia, myocardial infarction, angina pectoris, and stroke) and parental radiation exposure.
It is considered that multi-factorial diseases
develop due to both genetic factors and lifestylefactors. Since the prevalence rates of multi-factorialdiseases increase in adulthood, it is meaningful to
try to evaluate the association of these diseases with
underlying genetic events of radiation-exposed parents. To our knowledge, this health effects study isthe first to investigate such association in humans.
In planning, and later conducting this study, committees of outside experts were established to investigate the appropriateness of study objectives andplans as well as the ethical issues involving study
methods. These committees included: the ScientificCommittee (chairman: Dr. Tadao Shimao, Consultant to the Japan Anti-Tuberculosis Association), theEthics Committee (chairman: Dr. Hiraku Takebe,
Visiting Professor of Kinki University Interdisciplinary Graduate School of Science and Technology),
and the Analysis Subcommittee (chairman: Dr.
Norihiko Hayakawa, Professor Emeritus ofHiroshima University). Thus far, there have been fivemeetings of the Scientific Committee, four meetingsof the Ethics Committee, six joint meetings of the
Scientific and Ethics Committees, and four meetingsof the Analysis Subcommittee.
At the fourth meeting of the Analysis Subcommittee held on February 27, 2007, it was reported
that the initial goal of 10,000 clinical exam participants had been achieved (more precisely: 11,951, a

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participation rate of 48.4% out of 24,673 for whom
the mail survey was conducted), thereby generating
data with sufficient statistical power. The reportconcluded that analysis of association between
radiation exposure and a combination of six multifactorial diseases (including diabetes and hypertension, conducted by taking into account such lifestylefactors as drinking and smoking) failed to detectincreased risk. It was also reported that a negative
association was observed between paternal dose andprevalence rate of these multi-factorial diseases,
which was significantly low, among male children.
However, it was considered that careful interpretation of this finding would be necessary.
While the Scientific and Ethics Committees
accepted the analysis results of the Analysis Subcommittee, they recommended that the clinical healthstudy of the fixed population be continued in aprospective manner, taking into account that the
average age of the study population was still young,at 48.6 years. This recommendation was madedespite the fact that no association was identified
between parental exposure and health effects among
the offspring. As the RERF’s health effects study of
the A-bomb survivors’ children had attracted much
public attention since its start, the above conclusions
were explained to the press on February 28, using avideoconferencing system connecting Hiroshima andNagasaki and reported nationwide.
Thus, the study conducted in a cross-sectional
format for seven years since 2000, marked the endof one stage of what is anticipated to be a continuing
evaluation. We would like to express our heartfelt
appreciation to the committee members for their
dedication to the study, as well as to the children of
A-bomb survivors for their kind cooperation.

RERF Update Volume 18, 2007

9

Science Articles: Solid Cancer Incidence

Solid Cancer Incidence among Atomic Bomb
Survivors, 1958–1998
Dale L. Preston,1 Elaine Ron,2 Shoji Tokuoka,3 Sachiyo Funamoto,4 Nobuo Nishi,5Midori Soda,6 Kiyohiko Mabuchi,2 Kazunori Kodama5
1

HiroSoft International Corporation; 2Division of Cancer Epidemiology and Genetics,
U.S. National Cancer Institute; 3Consultant, Departments of 4Statistics, 5Epidemiology(Hiroshima), and 6Epidemiology (Nagasaki), Radiation Effects Research Foundation
Although a great deal is already known about the
carcinogenic risks from acute exposure to externalradiation, the long-term follow-up of the Life Span
Study (LSS) of atomic-bomb survivors continues toprovide new insights into and raises additional questions about the nature of radiation effects on cancer
risk. We have just published the second comprehensive report on solid cancer and nervous system tumor
incidence in the LSS.1 (Solid cancer includes all
malignancies other than leukemia and other malignancies of the lympho-hematopoietic system.) The
main objectives of our study were to quantify cancer risks attributable to radiation; to explore the shapeof the dose-response; to assess how risk is modified
by age, time, gender and other factors; to help clarify
site-specific differences in risk patterns; and to identify issues and cancer sites needing further research.
The LSS cancer incidence studies are an important complement to the analyses of cancer mortality
in the cohort because they include much more dataon less-fatal cancers, such as cancers of the breast,
thyroid and skin, which are radiation sensitive, but
are under-represented on death certificates. Tumor
registry diagnoses are based on a review of data frommany sources and are substantially more accurate
and detailed than cause-of-death data gleaned fromdeath certificates. Furthermore, the date of cancer
incidence is a better indicator of disease onset andbiologically more relevant than date of death fromcancer which is influenced by stage at diagnosis andtreatment.
With follow-up from 1958 through 1998, the newreport adds 11 years of follow-up, and many more
cancer cases. In contrast to the first LSS cancer incidence report in 1994,2 the so-called “not in city”(NIC) group was included in the new analyses
because the addition of about 25,000 cohort members considerably improves the precision of thedescriptions of baseline cancer risk patterns. With
this addition and the extended follow-up time, thestudy includes 24% more person-years and 56%
more cancer cases than the 1994 report.
The LSS cohort includes 120,321 people who had
been born at the time of the bombs and were alive
and living in Hiroshima or Nagasaki in late 1950,
but who were not otherwise selected on the basis ofhealth status. About 95,000 cohort members were
within 10 km of the epicenter and about 25,000 were

Figure 1. The Life Span Study Cohort: description of how the analytic cohort was defined.

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RERF Update Volume 18, 2007

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Science Articles: Solid Cancer Incidence
Table 1. Life Span Study cancer incidence analysis cohort* : percentage surviving on
December 31, 1998 by age-at-exposure and gender
Male

FemaleTotal

Age atexposure

People% alive

People% alive

People% alive

<10
10-19
20-29
30-39
40-49
≥50

11,196
10,374
3,299
5,224
6,912
5,897

89%
74%
47%
17%
1%
0%

11,478
12,68910,964
10,595
9,170
7,629

94%
87%
72%
36%
5%
0%

22,67423,063
14,263
15,81916,082
13,526

91%
81%
66%
30%
4%
0%

All ages

42,90247%

62,525

54%

105,427

51%*Limited to people who were alive and reported to be cancer free as of January 1, 1958

NIC at the time of the bombings. The new incidence
analyses were based on 105,427 LSS cohort members who have estimated DS02 organ doses and who
were alive and not known to have had cancer on
January 1, 1958 (Figure 1). As indicated in Table 1,
51% of the study cohort members were alive at theend of follow-up on December 31, 1998. Slightly
more than 40% of the cohort members were younger
than 20 years of age at the time of the bombings,
and about 85% of these cohort members were aliveat the end of follow-up.
The whole body doses received by individual LSS
cohort members ranged from no exposure to morethan 4 Gy, with a mean dose of about 0.25 Gy amongcohort members with doses of 5 mGy or more. The
analyses in this report were based on DS02
organ dose estimates. The DS02 dosimetry system,
which was adopted by RERF in 2003, provides
individual organ dose estimates for 95% of thecohort members.3 DS02 incorporates refinements inthe estimates of the bomb yields and source terms aswell as new information on shielding effects on
exposure.4 These improvements resulted in slightly
increased gamma doses and decreased neutron doses,
but these changes have little impact on risk estimates.5 Risk estimates for all solid cancers as a groupwere based on weighted colon dose estimates while
site-specific analyses were based on doses to therelevant target tissue.
Cases were identified through the LSS Tumor
Registry that includes all cancers and benign tumorsof the nervous system diagnosed and reported to theHiroshima and Nagasaki Tumor Registries since
1958 among LSS cohort members. The Hiroshima
and Nagasaki Tumor Registries actively ascertain
cancers in Hiroshima City and Nagasaki Prefecturefrom all large hospitals in their catchment areas andincorporate information from tissue registries thatkeep records of histological findings and the location of slides and other materials. The LSS Tumor
Registry also includes information about cancer listed
as the cause of death on death certificates for LSS
members throughout Japan. This information is
obtained through RERF’s routine mortality followup. Commonly used indices of cancer registry
Return to Table of Contents

quality indicate that the Hiroshima and Nagasaki
registry data are of high quality with regard to thecompleteness and accuracy of diagnoses, and previous analyses demonstrated that there is no radiationdose bias in case ascertainment.6
Analyses were restricted to first primary tumors
to prevent confounding from exposure to high-doseradiation from treatment for a first cancer andpossible detection bias in cancer survivors who generally remain under close medical surveillance. The
data were summarized in a highly stratified personyear (rate) table. The stratifying factors included city,
gender, dose, age at exposure, attained age, anddistance from the hypocenter. We used Poisson
regression to estimate the baseline rates and theexcess relative risks (ERR’s) and excess absolute
rates (EAR’s) of all solid cancers combined and of
individual cancer sites. The excess relative risk (ERR)
describes the proportional change in the cancer incidence rate relative to the baseline (0 dose) rate as afunction of dose and other factors (such as gender,
attained age, or age at exposure). The excess absolute rate (EAR) describes the difference between therate for an exposed group and the baseline rate forthat group. ERR’s are summarized as the ERR per
Gy while EAR’s have units of excess cases per
10,000 person-years per Gy. Since the ERR and EAR
depend on dose and can vary with age at exposure,gender, attained age, and other factors, summary
results are presented in terms of standardized values,
typically the gender-averaged ERR (EAR) per Gy
at age 70 for a person exposed at age 30.
In order to adjust for migration of LSS membersout of the Tumor Registries’ catchment area, we had tolimit analyses to those cancers diagnosed within thecatchment areas and we used gender, age, and timedependent residence probabilities to reduce person
years. Residence probabilities were estimated using
data on Adult Health Study exam contacting results.
Between January 1958 and December 1998,
17,448 eligible first primary solid cancers wereidentified among the 105,427 LSS cohort members
included in the current analyses (Table 2). The mean
age at diagnosis was 67.4 years and 54% of thecancers occurred among females, although 59% of
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Science Articles: Solid Cancer Incidence

Table 2. Distribution of selected solid cancers identified between January 1958 and December 1998 among
LSS cohort members
Site

Cases

TOTAL
17,448Stomach
4,730
Lung
1,759
Colon
1,516
Liver
1,494
Breast
1,082
Cervix
859
Rectum838
Bladder469
Thyroid471
Non-melanoma skin330
Nervous system
281
Other
3,619

% femaleMean age at
diagnosis

54
46
41
54
40
100
100
50
33
81
63
67
52

67.4
67.7
71.2
69.3
67.0
59.8
60.0
68.0
70.6
60.4
72.4
62.6
68.7

the cohort is female. As expected, stomach cancer,
which has a very high incidence in Japan, was themost common cancer site. Lung cancer was the next
most frequent cancer, but the number of cases was
substantially smaller. Males predominated in bothof these cancers. There also were over 1,000 casesof cancers of the colon, liver and breast.
Table 3 provides information on the dose distribution and cancer risks in the LSS. It can be seen thatthe dose distribution is highly skewed: dose estimates
were less than 200 mGy for about 75% of the almost
45,000 cohort members with dose estimates above
5 mGy, whereas survivors with doses over 1 Gy
account for less than 5% of survivors in this group.
The data in Table 3 also demonstrate a strong dose
response and indicate that there are a considerable
number of radiation-associated cancers (~160) in the
5 to 200 mGy dose range. At doses of 1 Gy or morealmost half of the cancer cases identified among survivors were associated with their radiation exposure.
For all solid cancers combined, we observed alinear dose response using weighted colon dose as

the representative dose. There was a statisticallysignificant dose response trend in the 0–0.15 Gy
range which was similar to that estimated for theentire dose range (Figure 2).
Both the ERR per gray (ERR/Gy) and EAR per
10,000 person years per Gy (EAR/104 PY Gy) wereabout 50% higher for women than men. Whengender-specific cancers were excluded from theanalyses, the ERR/Gy remained significantly larger
for females than males, but there was no genderdifference using an EAR model. Figure 3 uses
gender-averaged risks to illustrate how the excess
risk varies with age-at-exposure and attained age.
The ERR/Gy decreased with increasing age at
exposure and attained age. The EAR/104 PY Gy also
decreased with increasing age at exposure, but it
increased with increasing attained age. Indeed, witha 25% increase in follow-up, we estimated a 50%
increase in the number of radiation-associatedcancer cases indicating that the radiation-effect onsolid cancer incidence persists throughout life.
Statistically significant dose-responses were seen
for most cancer sites, including oral cavity, esophagus, stomach, colon, liver, lung, non-melanoma skin,breast, ovary, bladder, nervous system, and thyroid(Figure 4). ERR’s for cancers of the pancreas, prostate, and renal cell were non-significantly elevated
but were consistent with the risk for all solid cancers
as a group. Our data also suggest that the radiationrelated risks for cancers of the rectum, gallbladder,and uterus may be lower than those for all solidcancers combined. There was evidence, however, thatradiation exposure during childhood or adolescence
may elevate the risk of developing cancer of the bodyof the uterus.
Assessing site-specific cancer risks is importantbecause biologically it is almost certain that variation in site-specific risks exists. But, even in a studyof over 100,000 people, the number of cases for most

Table 3. LSS solid cancers 1958–1998. Subject, person-year, and cases with fitted-excess andattributable-fraction estimates by dose category.
Dose
category †Subjects

Mean
distance (m)Person
years

Cases

FittedexcessAttributablefraction (%)

Not in city
< 0.005
0.005 – 0.1
0.1 – 0.2
0.2 – 0.5
0.5 – 1
1–2
2+

25,42735,54527,789
5,527
5,935
3,173
1,647
564

–
3969
2114
1608
1430
1260
1118
934

680,744918,200
729,603145,925
153,886
81,25141,412
13,711

3,994
5,603
4,406
968
1,144
688
460
185

0
3
81
75
179
206
196
111

0
0.0
1.8
7.6
15.7
29.5
44.2
61.0

Total

105,427

2,764,732

17,448853

10.7‡

Note: Estimates of fitted excess cases are based on an ERR model with a linear dose response
effect modification by gender, age at exposure and attained age. All not-in-city subjects wereused in the modeling, but the baseline risk model allows for city-specific differences in thelevel of the baseline risks for the not-in-city group.
† Weighted adjusted colon dose in Gy.
‡ Attributable fraction among people who were in the cities with doses greater than 0.005 Gy

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RERF Update Volume 18, 2007

12

Science Articles: Solid Cancer Incidence

Figure 2. All solid cancer fitted linear dose response and dose category
specific ERR estimates.

Figure 3. Age-dependence for the gender-averaged solid cancer ERR (left panel) and EAR (right panel) for
exposure ages of 10, 30, and 50 years.

Figure 4. Site specific ERR estimates with 90% confidence intervals. The ERR’s are genderaverage and correspond to the fitted risk at age 70 for a person exposed to 1 Gy
at age 30. The solid vertical line indicates no excess risk and the dashed verticalline represents the standardized ERR for all solid cancers as a group.

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RERF Update Volume 18, 2007

Science Articles: Solid Cancer Incidence
individual cancer sites is relatively small making it
difficult to identify statistically significant differencesin risk estimates or patterns. Although current analyses suggest some differences, much of the observed
variability is consistent with random variation.
Figure 5 illustrates gender differences in the ERR
estimates for selected sites. The large gender differences seen for bladder and lung cancer are particularly striking. To some extent the higher ERR for
women than men for these smoking-related cancers
reflects the relatively high prevalence of smoking
among men in the LSS cohort and low prevalence
among women. The gender difference for colon cancer is also remarkable and in the opposite direction(men had greater risks than women) for bladder andlung cancer. The reasons for this difference are unclear.
We also assessed radiation effects in five broadlyclassified histological groups, including adenocarcinoma, squamous cell carcinoma, other epithelial
cancers, sarcomas, and other non-epithelial cancers.
Over 96% of the solid tumors were epithelial tumors
with adenocarcinoma accounting for about 60% of
the epithelial tumors. ERR’s per Gy of similar
magnitude, from 0.3 to 0.48, were seen for the

Figure 5. Gender-specific excess relative risk estimates for all solid cancers and selected sites.

13
different histologic groups.
In summary, more than half a century after theatomic bombings in Hiroshima and Nagasaki, astatistically significant increased risk of cancer incidence is still apparent among radiation-exposed
members of the LSS. Among the survivors with dosesof 5 mGy or more an estimated 10% of the solidcancers can be attributed to radiation exposure. A
larger number of the radiation-associated excess solidcancers are expected to occur in the cohort over the
next 15 to 20 years. The shape of the dose response
for solid cancers as a group continues to be welldescribed by a linear model with no evidence ofsignificant non-linearity. EAR’s for all solid cancers
combined increased throughout life for all ages, while
ERR’s decreased with increasing age at exposure andattained age. Excess risks for all solid cancers werehigher for women than men, however when sexspecific cancers were excluded the difference in the
EAR was reduced substantially. Lifetime solidcancer risk estimates were about 20 times higher thanthose observed in earlier studies of leukemia. While
patterns of organ (or site)-specific risks generally
were similar to those seen in the first cancer incidence study, they have become clearer for several
cancers. High ERR’s were found for cancers of the
bladder, breast, and lung, whereas high EAR’s wereseen for cancers of the stomach, breast, colon, andlung. Accumulating data from further follow-up willcontinue to provide important new information on
the levels and temporal patterns of the excess risk,
especially for survivors younger than 20 years of age
at the time of the bombings. Additional follow-up
will also enable us to explore radiation-associatedrisks for rare cancer sites that didn’t have adequate
number of cases to study previously, and help clarify
the variability in risk patterns for specific cancer sites.
Finally, additional site-specific incidence studies
incorporating pathology reviews and biomarker
studies will add to our knowledge on the radiationsensitivity of specific cell types.

References
1.

Preston DL, Ron E, Tokuoka S, Funamoto S, Nishi N, Soda M, Mabuchi K, Kodama K. Solid cancer incidence
in atomic bomb survivors: 1958–1998. Radiat Res 168:1-64, 2007.

2.

Thompson DE, Mabuchi K, Ron E, Soda M, Tokunaga M, Ochikubo S, Sugimoto S, Ikeda T, Terasaki M, et al.
Cancer incidence in atomic bomb survivors. Part II: Solid tumors, 1958–1987. Radiat Res 137:S17-67, 1994.

3.

Cullings HM, Fujita S, Funamoto S, Grant EJ, Kerr GD, Preston DL. Dose estimation for atomic bomb survivor
studies: its evolution and present status. Radiat Res 166:219-54, 2006.

4.

Young RW, Kerr GD, eds. Reassessment of the Atomic Bomb Radiation Dosimetry for Hiroshima and Nagasaki—
Dosimetry System 2002. Radiation Effects Research Foundation, Hiroshima, Japan, 2005.

5.

Preston DL, Pierce DA, Shimizu Y, Cullings HM, Fujita S, Funamoto S, Kodama K. Effect of recent changes inatomic bomb survivor dosimetry on cancer mortality risk estimates. Radiat Res 162:377-89, 2004.

6.

Mabuchi K, Soda M, Ron E, Tokunaga M, Ochikubo S, Sugimoto S, Ikeda T, Terasaki M, Preston DL, Thompson
DE. Cancer incidence in atomic bomb survivors. Part I: Use of the tumor registries in Hiroshima and Nagasaki
for incidence studies. Radiat Res 137:S1-16, 1994.

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RERF Update Volume 18, 2007

14

Science Articles: Use of Stored Sera

Use of Stored Sera for Detection of
Hepatitis B and C Viruses
Waka Ohishi,1 Saeko Fujiwara,1 Gen Suzuki,1,2 Kazuaki Chayama3
1

Department of Clinical Studies (Hiroshima), Radiation Effects Research Foundation;
Department of Environmental Health, National Institute of Public Health; 3Departmentof Medicine and Molecular Science, Division of Frontier Medical Science, Programsfor Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University

2

Introduction
Molecular biological research on hepatitis B
virus (HBV) and hepatitis C virus (HCV) has
advanced remarkably over the last decade, resulting
in the establishment of methodologies for diagnosis, prevention, and treatment of liver diseases.1–4 In
addition, the use of dried blood samples (spotsamples), as well as frozen sera, has enabled essential data to be collected for epidemiological andclinical studies, as well as for the screening of
subjects with HBV or HCV infection.5–8
The Atomic Bomb Casualty Commission, thepredecessor of the Radiation Effects Research Foundation (RERF), established the Adult Health Study(AHS) longitudinal cohort in 1958, comprised ofapproximately 20,000 atomic-bomb survivors andnon-exposed controls that have been examinedbiennially in outpatient clinics in Hiroshima andNagasaki. In 1969, Dr. Joseph L. Belsky, Department
Chief of Medicine at the time, ordered additionallythe assay of Australia Antigen, called HBV surface
antigen (HBsAg) currently, for studies of liver function abnormality among AHS participants, however
sera obtained from them had already been discarded.
From 1969, sera obtained from the AHS participants
have been stored by either freezing or freeze-drying
methods with this as a turning point.9 From 1990,
their samples have been prepared and stored by both
rapid freezing and by freeze-drying methods. In total,
multiple serum samples from 16,000 subjects are
currently stored at RERF, and represent a valuable
resource for studies of the development of large
variety of diseases.
Biochemical assays using frozen and freeze-driedsera showed that protein, lipid, and electrolyte
levels are relatively stable in both types of sera, butthat enzyme levels were more variable in the freezedried sera than in the frozen sera.9 Nonetheless, thefreeze-dried sera have some value, because they canbe stored economically at room temperatures on arelatively long-term basis and are therefore easier tohandle than frozen sera, especially for shipping.
In this report we describe our attempts toReturn to Table of Contents

validate the use of the frozen and freeze-dried sera
for serological and molecular biological detectionof HBV and HCV in the AHS longitudinal cohort.
Preparation of frozen and freeze-dried serum
samples
The usefulness of stored sera for serological andmolecular biological assays of HBV and HCV
depends on both preparative methods and storage
condition. Therefore, storage methods for serum
samples should be optimized for specific purposes;i.e., for epidemiological and clinical studies, forsimple screening, etc. It is well documented that HCV
RNA is easily degraded by ribonuclease (RNase),
which exists in saliva and perspiration.10 Further, theability to detect HCV RNA decreases by repeatedfreezing and thawing of sera.10 For detection of HBVand HCV, therefore, each serum sample should be
dispensed into small volumes at the time of specimen procurement and stored for long-term conservation at –80°C in order to avoid cycles of freezing
and thawing of single large samples.
First, we used the following procedure for the
preparation of frozen serum samples.11 Serum wasdispensed into four equal volumes and aliquotted into
1.5-ml polypropylene tubes, and stored at –80°C until
use. These serum samples were thawed by leaving
them at room temperature for 30 minutes and thenmixed well by inversion before use.
Second, we used the following procedure for the
preparation of freeze-dried serum samples.11 A 0.4ml portion of the serum was stored in a glass tube at–80°C, and after one week of storage, these samples
were freeze-dried in batches, sealed automaticallyto maintain a vacuum, and stored at room temperature (20 to 25°C) until needed. The moisture content
of freeze-dried serum samples was normally
maintained at less than 0.5%, because moisture
content of freeze-dried serum samples of less than
1% was necessary to retain and assay various
antibodies of interest.9 These serum samples werereconstituted with the volumetric method using
diethyl pyrocarbonate (DEPC)-treated MilliQ water
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Science Articles: Use of Stored Sera
and mixed well before used.

used as supplementary assay to avoid missing HBVcarriers such as HBsAg-negative individuals with
HBs gene escape mutants or HBsAg-low titerindividuals. We did not compare the results of theanti-HBc Ab measured by EIA in frozen and freezedried serum samples with those measured by PHA
in fresh serum samples, because the sensitivity ofthe test for anti-HBc Ab is greatly affected by themethodology.14 Therefore, we compared the EIA
results for anti-HBc Ab in frozen serum samples withthose in freeze-dried serum samples.15 Concordancein measurement of anti-HBc Ab among frozen andfreeze-dried serum samples was not complete, but
was satisfactory. Frozen serum samples of twocases that tested positive for anti-HBc Ab wereindeterminant in the freeze-dried serum samples. On
the other hand, anti-HBc Ab results of a frozen
serum sample of one case proved to be indeterminant,while a freeze-dried serum sample tested negative.
For these three cases with discrepant results, themeasured values for the anti-HBc Ab specimens thatyielded positive or negative result were close tobeing indeterminant (Table 1).
As a whole, both frozen and freeze-dried sera canbe useful for serological assays of HBV and HCV.
We can conclude that serological assays using storedsera may be reliable for detection of HBV and HCV.
Moreover, use of more sensitive molecular biological assays might well compensate for the loss ofserological reactivity in stored sera.

Serological assays in stored serum samples
To assess the usefulness of stored sera for serological detection of HBV and HCV, we selected at
random 12 HBsAg-positive individuals and 25 antiHCV antibody (Ab)-positive individuals11 among
6,121 AHS participants who underwent hepatitis
screening from 1993 through 1995.12,13 Their serum
samples had been processed and stored by both thefreezing and freeze-drying methods.
Detection of HBsAg, anti-HCV Ab, and anti-HBc
Ab
The assays of HBsAg and anti-HCV Ab using
fresh serum samples were measured by reverse
passive hemagglutination (R-PHA) test kit and thesecond-generation PHA test kit, in hepatitis screening from 1993 through 1995. 12,13 The assays of
HBsAg and anti-HCV Ab using frozen and freezedried serum samples were measured by enzyme
immunoassay (EIA) and the second-generation EIA,
respectively.11
Comparison of qualitative detection of HBsAg
and anti-HCV Ab using fresh, frozen, and freezedried serum samples showed good agreement, though
the detection methods of HBsAg and anti-HCV Ab
in frozen and freeze-dried serum samples were
historically different from those in fresh serum
samples. The results indicated that freeze-dried sera,
as well as frozen sera, are interchangeable with thoseof fresh sera.11 This study also showed that freezedried sera are interchangeable with frozen sera in
the qualitative detection of HBsAg and anti-HCV
Ab (Table 1).
Anti-HBc Ab is present during the acute, convalescent, and chronic phases of HBV infection, andpersists for many years. Anti-HBc Ab has long been

Molecular biological assays in stored serum
samples
To assess the usefulness of stored sera formolecular biological detection of HBV and HCV,
we also used frozen and freeze-dried serum samples
obtained from above-mentioned 12 HBsAg-positiveand 25 anti-HCV Ab-positive individuals.11

Table 1. Detection of HBsAg, anti-HBc Ab, and anti-HCV Ab by EIA in stored sera
Results of freeze-dried
serum samples

Results of frozen serum samples
positive

negative

HBsAg
positive
negative

12
0

0
25

Anti-HBc Ab*
positive
negativeindeterminant

24
0
2

0
8
0

Anti-HCV Ab
positive
negative

24
0

1**
12

indeterminant

Concordance
(%)
100

0
1
2

92

97

*Measured values for anti-HBc Ab of ≥70%, <50%, and 50≤ <70% were defined as positive,
negative, and indeterminant, respectively.
**Anti-HCV Ab titer is very low and the detection of HCV RNA was negative with highly
sensitive qualitative PCR.

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16

Science Articles: Use of Stored Sera
Genotyping of HBV and HCV
HBV and HCV genotypes have been recognized
as important viral factors related to pathologicalstatus, effectiveness of antiviral treatments, and prognosis of liver disease.1–3,16 Therefore, we have tested
the suitability of stored sera for HBV and HCVgenotyping. 15 Serum DNA was extracted fromeither 100 µl of frozen or reconstituted freeze-dried
serum samples using QIAamp DNA Mini Kit(Qiagen, Tokyo, Japan), and serum RNA wasextracted from 100 µl of frozen or reconstituted
freeze-dried serum samples using SepaGene RV-R
(Sanko Junyaku Co., Tokyo, Japan). The prepared
RNA was reverse transcribed with random primers
and reverse transcriptase (ReverTra Ace, TOYOBO
Co., Tokyo, Japan). HBV and HCV genotypes were
determined by polymerase chain reaction (PCR)
using type-specific primers in regions of the pre-S1
through S genes of HBV17 and the NS5 gene of
HCV,18 respectively.
HBV genotypes could be identified in ninefrozen and ten freeze-dried serum samples, all ofwhich were found to be genotype C. HCV genotype
1b was identified in 17 frozen and 16 freeze-dried

serum samples. In addition, HCV genotype 2a wasidentified in each of four frozen and four freeze-dried
serum samples tested. (Table 2).
The observed discordance presumably was notdue to differences in genotype, but rather due todifficulties in PCR amplification. Since all discordant cases were borderline, or undetectable, in thehighly sensitive, qualitative PCR (Table 3), thediscordance seemed to be attributable to variationsin the detection limits of PCR, arising from the minimal amounts of DNA and RNA. Alternatively, it ispossible that the HBV DNA and the HCV RNA had
been degraded during prolonged storage of either thefrozen or freeze-dried sera. However these findings
showed that both frozen and the freeze-dried sera
may be useful for HBV and HCV genotyping.
Quantitative detection of HBV DNA and HCV RNA
We compared the efficiencies of HBV-DNA quantification in HBsAg-positive frozen and freeze-dried
serum samples. Quantification of HBV DNA was
performed using real-time PCR and fluorescenceresonance energy transfer probes and primers thatwere designed for a highly conserved X region.15 In

Table 2. Genotyping of HBV and HCV in stored sera
Freeze-dried
serum samples

No. of frozen serum samples with
HBV and HCV genotype results

HBV genotypeC
Not detectedC90

Not detected1
2*

HCV genotype1b2a
Not detected

1b
16
0
1

2a
0
4
0

Concordance(%)
92

Not detected
0
0
4**

96

*HBV-DNA concentration was low for detecting some genotypic determinants.
**The detection of HCV RNA was negative with highly sensitive qualitative PCR.

Table 3. Establishment of detection limit for HCV RNA using qualitative PCR
assay
HCV RNA (IU/ml)*

Estimated inputof HCV RNA (IU)**

No. positive/No. tested(% Positive ratio)

4,700,000
940,000
188,000
37,600
7,520
1,504
301
100
60
0

47,000
9,400
1,880
376
75
15
3
1
0.6
0

8/8 (100%)
8/8 (100%)
8/8 (100%)
8/8 (100%)
8/8 (100%)
8/8 (100%)
8/8 (100%)
8/8 (100%)
4/8 ( 50%)
0/8 ( 0%)*A serum sample known as HCV-RNA concentration by Amplicore HCV
monitor test ver. 2.0 (4,700 kIU/ml, genotype 1b) was diluted with serumfrom an HCV-negative individual.
**Estimated input of HCV RNA in each cDNA solution was applied to therespective qualitative PCR assay.

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RERF Update Volume 18, 2007

Science Articles: Use of Stored Sera
terms of reproducibility, our analyses showed theintra-assay coefficients of variation (CVs) of 2.4%
and 2.5% at two known HBV-DNA concentrations,whereas the inter-assay CVs were 1.4% and 2.5%.
Real-time PCR detected HBV DNA in 10 of 12
frozen and freeze-dried serum samples from HBsAgpositive subjects in the 1993–1995 hepatitis screening. The correlation between log10-transformed HBVDNA quantities for pairs of frozen and freeze-dried
serum samples was significant (r = 0.981, P < 0.0001).
We also compared the efficiencies of HCV-RNA
quantification using real-time PCR in anti-HCV-positive frozen and freeze-dried serum samples.11 In
terms of reproducibility, our analyses showed theintra-assay CVs of 6.2% and 2.9% at two known
HCV-RNA concentrations, whereas the inter-assay
CVs were 3.6% and 4.3%. Real-time PCR detected
HCV RNA in 18 of 25 frozen and freeze-dried
serum samples from anti-HCV-positive subjects,
originally identified in the 1993–1995 hepatitis
screening. The correlation between log10-transformed
HCV-RNA quantities for pairs of frozen and freezedried serum samples was significant (r = 0.908, P <0.0001). HCV RNA is easily decomposed by RNase,and its detectability decreases by repeated freezing
and thawing of sera. Therefore, it was expected that
the PCR products of HCV RNA would vary depending on the method and condition of storage. Nevertheless, the results of our study showed that there
were no remarkable differences in our capacity todetect HCV RNA, even when different methodologies and conditions of storage were employed overprolonged periods.
As a whole, both frozen and freeze-dried sera canbe useful for molecular biological assays of HBVand HCV. We can conclude that molecular biological assays using stored sera may be reliable fordetection of HBV and HCV.
Application of stored sera: A nested case-control
study of hepatocellular carcinoma (HCC)
We conducted a nested case-control study of HCC
using stored sera in the AHS longitudinal cohort. The

17
study included 224 HCC cases and 644 controls thatmatched to the cases on gender, age, city, time ofserum storage, and method of serum storage (freezing or freeze-drying methods), and counter-matchedon radiation dose.19
First, HBsAg, anti-HBc Ab, anti-HCV Ab, andHCV RNA were measured as viral factors using
available stored sera of 211 cases and 640 controls
obtained before the HCC diagnosis. Qualitative
detection of HCV RNA was performed by PCR
using two sets of primers corresponding to the 5′untranslated region. 20 The detection limit wasestablished by limiting dilution method.15 This highly
sensitive qualitative PCR assay detected as HCVRNA positive in 100% of serum samples containing
100 IU/ml of HCV RNA, and 50% of the samples
containing 60 IU/ml of HCV RNA (Table 3). HBV
infection status was defined as positive by thedetection of HBsAg, or having a high titer of antiHBc Ab. HCV infection status was defined as
positive by the detection of HCV RNA.
Among HCC cases, 13.7% were found to be HBV
only infections, 62.6% were HCV only infections,and 2.4% were positive for both; while the remaining subjects negative for both. Among the controls,
2.8% were found to be HBV only infections, 6.4%
were HCV only infections, and 0.3% were positive
for both. These distributions of HBV and HCV
infection status were similar to those in previous
reports on Japanese populations.21
Conclusions
The identification of HBV and HCV infections
in participants of AHS cohort can be accomplishedusing both frozen and freeze-dried sera, and this identification can be accurate and reliable depending on
the method, condition and duration of storage, andassociated optimizations. Therefore, it is expectedthat the sera stored for long periods will be usefulfor future viral hepatitis studies, e.g., evolution of
the hepatitis virus as well as the natural history ofviral liver diseases.

References
1. Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, Okanoue T, Yotsuyanagi H, Iino S. A
case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B
and C. Japan HBV Genotype Research Group. Hepatology 33:218-23, 2001.
2. Wai CT, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with better response to interferon therapy
in HBeAg(+) chronic hepatitis than genotype C. Hepatology 36:1425-30, 2002.
3. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, Izumi N, Marumo F, Sato
C. Mutations in the nonstructual protein 5A gene and response to interferon in patients with chronic hepatitis C
virus 1b infection. N Engl J Med 334:77-81, 1996.
4. Chayama K, Tsubota A, Kobayashi M, Okamoto K, Hashimoto M, Miyano Y, Koike H, Kobayashi M, Koida I,
Arase Y, Saitoh S, Suzuki Y, Murashima N, Ikeda K, Kumada H. Pretreatment virus load and multiple amino

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RERF Update Volume 18, 2007

Science Articles: Use of Stored Sera

18

acid substitutions in the interferon sensitivity-determining region predict the outcome of interferon treatment inpatients with chronic genotype 1b hepatitis C virus infection. Hepatology 25:745-9, 1997.
5. Mendy M, Kirk GD, van der Sande M, Jeng-Barry A, Lesi OA, Hainaut P, Sam O, McConkey S, Whittle H.
Hepatitis B surface antigenaemia and alpha-foetoprotein detection from dried blood spots: Applications tofield-based studies and to clinical care in hepatitis B virus endemic areas. J Viral Hepat 12:642-7, 2005.
6. Seeff LB, Miller RN, Rabkin CS, Buskell-Bales Z, Straley-Eason KD, Smoak BL, Johnson LD, Lee SR, Kaplan
EL. 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann Intern Med 132:105-11, 2000.
7. Das PC, de Vries AH, McShine RL, Sibinga CT. Dried sera for confirming blood-borne virus infections (HCV,HTLV-I, HIV & HBsAg). Transfus Med 6:319-23, 1996.
8. Jardi R, Rodriguez-Frias F, Buti M, Schaper M, Valdes A, Martinez M, Esteban R, Guardia J. Usefulness ofdried blood samples for quantification and molecular characterization of HBV-DNA. Hepatology 40:133-9,
2004.
9. Radiation Effects Research Foundation. Symposium on serum storage methods. Hiroshima Igaku [J Hiroshima
Med Assoc] 42:1596-609, 1989. (Japanese)
10. Halfon P, Khiri H, Gerolami V, Bourliere M, Feryn JM, Reynier P, Gauthier A, Cartouzou G. Impact of various
handling and storage conditions on quantitative detection of hepatitis C virus RNA. J Hepatol 25:307-11, 1996.
11. Ohishi W, Fujiwara S, Suzuki G, Kishi T, Sora M, Matsuura S, Hakoda M, Tatsukawa Y, Yamada M, Chayama
K. Feasibility of freeze-dried sera for serological and molecular biological detection of hepatitis B and C viruses. J Clin Microbiol 44:4593-5, 2006.
12. Fujiwara S, Kusumi S, Cologne J, Akahoshi M, Kodama K, Yoshizawa H. Prevalence of anti-hepatitis C virus
antibody and chronic liver disease among atomic bomb survivors. Radiat Res 154:12-9, 2000.
13. Fujiwara S, Sharp GB, Cologne JB, Kusumi S, Akahoshi M, Kodama K, Suzuki G, Yoshizawa H. Prevalence ofhepatitis B virus infection among atomic bomb survivors. Radiat Res 159:780-6, 2003.
14. Ohishi W, Mori N, Tsuge M, et al. Examination about the clinical usefulness of anti-HBc Ab measurement by
Architect. Acta Hepatol Jpn 45: 555-6, 2004. (Japanese)
15. Ohishi W, Fujiwara S, Suzuki G, Chayama K. Validation of the use of freeze-dried sera for the diagnosis ofhepatitis B and C virus infections in a longitudinal study cohort. Mohan RM, ed. Research Advances in Microbiology 7. Global Research Network, Kerala, India, 2007. pp. 1-9.
16. Orito E, Ichida T, Sakugawa H, Sata M, Horiike N, Hino K, Okita K, Okanoue T, Iino S, Tanaka E, Suzuki K,
Watanabe H, Hige S, Mizokami M. Geographic distribution of hepatitis B virus (HBV) genotype in patientswith chronic HBV infection in Japan. Hepatology 34:590-4, 2001.
17. Naito H, Hayashi S, Abe K. Rapid and specific genotyping system for hepatitis B virus corresponding to six
major genotypes by PCR using type-specific primers. J Clin Microbiol 39:362-4, 2001.
18. Chayama K. Genotyping hepatitis C virus by type-specific primers for PCR based on NS5 region. Lau JYN, ed.
Methods in Molecular Medicine, vol. 19: Hepatitis C Protocols. pp 165-73, Humana Press Inc., Totowa, NJ,
1998.
19. Cologne JB, Sharp GB, Neriishi K, Verkasalo PK, Land CE, Nakachi K. Improving the efficiency of nested
case-control studies of interaction by selecting controls using counter matching on exposure. Int J Epidemiol
33:485-92, 2004.
20. Arataki K, Kumada H, Toyota K, Ohishi W, Takahashi S, Tazuma S, Chayama K. Evolution of hepatitis C virus
quasispecies during ribavirin and interferon-alpha-2b combination therapy and interferon-alpha-2b monotherapy.
Intervirology 49:352-61, 2006.
21. Kiyosawa K, Umemura T, Ichijo T, Matsumoto A, Yoshizawa K, Gad A, Tanaka E. Hepatocellular carcinoma:
Recent trends in Japan. Gastroenterology 127:S17-26, 2004.

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RERF Update Volume 18, 2007

19

Science Articles: Molecular Features of Colorectal Cancer

Molecular Features of Colorectal Cancer Developing
among Atomic Bomb Survivors with Emphasison Microsatellite Instability– An Interim Report –Hidetaka Eguchi,1 Kiyohiro Hamatani,1 Masataka Taga,1 Reiko Ito,1 Nobuo Nishi,2
Kazunori Kodama,2 Hiroaki Katayama,3 Eiichi Tahara,4 Shizue Izumi,5,6 Wataru Yasui,7
Kei Nakachi1
Departments of 1Radiobiology/Molecular Epidemiology, 2Epidemiology (Hiroshima),
3
Information Technology, and 5Statistics, RERF; 4Hiroshima Cancer Seminar Foundation; 6Faculty of Engineering, Oita University; 7Department of Molecular Pathology,
Hiroshima University Graduate School of Biomedical Sciences

IntroductionMore than sixty years have passed since theatomic bombings, but the excess relative risk (ERR)of all solid cancers remains high. As indicated in a
recent follow up report on the Life Span Study (LSS)of atomic-bomb (A-bomb) survivors, the effect ofradiation exposure on the risk of solid cancer varies,
sometimes appreciably, for different bodily organs.1
Colon cancer has a high ERR per Sv of 0.72 (95%
confidence interval, 0.29–1.3) for radiation exposure.
Rectal cancer, by contrast, has a relatively low ERR.
The biologic basis of such differences in exposure
risks are unclear. Likewise, the molecular mechanisms by which radiation induces solid cancers,
especially colon cancer, are ill defined.
Apart from colorectal cancers among A-bomb
survivors, development of such cancers after radiotherapy was first reported in 1957.2 Accumulated
information since then revealed that radiotherapyinduced colorectal cancer showed a high frequency
of cancers with mucinous/poorly differentiatedhistology.3,4
Two major molecular events have been tied tocolorectal carcinogenesis, namely chromosomal
instability and microsatellite instability (MSI). HighMSI (MSI-H) is correlated with mucinous/poorly
differentiated histology and a specific sub site of thecolon—frequently the proximal colon.5 We hypothesized therefore, that the MSI phenotype may be
associated with radiation exposure in colorectal
cancer patients of the A-bomb survivor cohort.
Most MSI-H sporadic colorectal cancer cases
showed a loss of DNA repair enzyme MLH1 expression. Methylation of the MLH1 gene promoter isthought to be a major cause for inactivation of theMLH1 gene in sporadic cancer.6 Importantly, methylation of CpG dinucleotides within only small
Return to Table of Contents

regions of MLH1 gene promoter at its proximal
region (–248 to –178 bp) has been shown to beclosely associated with lowered expression of MLH1
mRNA7,8 and protein.9 In addition to DNA methylation, loss-of-heterozygosity (LOH) of the MLH1
gene also contributes to MLH1 gene defects in somesporadic colorectal cancer with MSI.6
During the past decade, a “serrated polyppathway” has been identified and shown to be mechanistically important in terms of colon cancer. This
pathway is initiated with development of hyperplastic polyp in right-sided colon, and is followed byserrated adenoma and invasive carcinoma. Notably,
this pathway is associated with MSI. 10 In theserrated polyp pathway, loss of MLH1 protein
followed by acquisition of MSI has been shown tooccur in the late stages.11 The constitutive activationof the RAS-RAF-MEK-ERK-MAP kinase-signaling
pathway, which is caused by gene mutations of
mainly BRAF and to a lesser extent KRAS, is recognized as an early event of colon tumorigenic
process(es) following the serrated polyp pathway.12
In addition to these mutations, the lack of RASSF2
function (a modulator of apoptosis signal that isdependent upon binding to K-Ras) that is due tomethylation of its gene promoter, has been recently
identified as an important activator of the signaling
pathway in colorectal carcinogenesis.13,14,15
In this article, we present an interim report on ourmolecular oncology study and the MSI phenotype
of colorectal cancer in A-bomb survivors.
Materials and Methods
Study subjects
For molecular analysis of colorectal cancer cases
among A-bomb survivors, 35 study materials of formalin-fixed and paraffin-embedded tissue specimens
RERF Update Volume 18, 2007

20

Science Articles: Molecular Features of Colorectal Cancerof subjects that had undergone surgery or endoscopy
at medical institutions in Hiroshima during 1986–
2001 with clinico-pathological data were obtained
and unlinkably anonymized through the Study Groupon Atomic Bomb Diseases, entrusted by the Ministry of Health, Labour and Welfare (MHLW), Japan.
This study was conducted under approval of the
Genetic and Medical Ethics Commission at
Hiroshima University, and the Human Investigation
Committee and the Ethics Committee for Genome
Research at the Radiation Effects Research Foundation (RERF). A-bomb radiation doses used in thisanalysis were estimated by the recently implemented
DS02 system.16
DNA preparation
Using hematoxylin and eosin stained tissue
sections, a pathologist (RI) performed pathologicalreviews. Two cases did not provide sufficientamounts of non-cancerous tissue. Five-µm tissue
sections were deparaffinized with Hemo-De (Falma,
Tokyo, Japan), stained with Methyl Green (SigmaAldrich, St. Louis, MO) and dissected manually or
using laser microdissection system AS LMD (Leica,
Wetzler, Germany). DNA was extracted from themicrodissected non-cancerous or cancerous regions
using QIAamp DNA Micro Kit (Qiagen, Hilden,
Germany).
Detection of microsatellite instability
To determine the extent of MSI, five microsatellite
markers (BAT25, BAT26, D2S123, D5S346, andD17S250) recommended by a National Cancer
Institute (NCI) workshop on MSI17 were examined.
Another mononucleotide repeat marker NR-2118 was
examined as well, following a recent recommendation by another NCI workshop on MSI.19 Polymerase
chain reaction (PCR) was performed in a 20-µl
mixture containing 5 pmoles of unlabeled primer, 5pmoles of fluorescently-labeled primer, 250 µM ofeach dNTP, 0.5 U of AccuSure DNA polymerase(Bioline Ltd., London, UK), 10 ng of genomic DNA
and 1× reaction buffer supplied by the manufacturer.
PCR conditions consisted of initial denaturation(95°C for 10 min), followed by 60 cycles (denaturation at 95°C for 15 sec, annealing for 15 sec, extension at 72°C for 15 sec). Primer sequences andannealing temperatures are summarized in Supplementary Table 1. The fluorescently labeled amplicons
with CEQ DNA Size Standard Kit-400 (Beckman

Table 1. Clinico-pathological and epidemiological characteristics of the study subjects
This
study
GenderMale (n)Female (n)

13
22

Organ

Colon (n)Rectum (n)2411Location

Proximal (n)Distal (n)1124Histology

Well (n)Moderately (n)Poorly/Mucinous (n)

10
18
7

Age at operation

Mean (yrs)SD
Range (yrs)

73.3
9.4
53, 91

Age at A-bombing

Mean (yrs)SD
Range (yrs)

25.2
10.8
3, 48

Time since A-bombing

Mean (yrs)
SD
Range

48.1
4.6
41, 56

Radiation dose

Median (mGy)
RangeInterquartile range

26
1, 2140
128

LSS*

LSS**

457
351

P†

0.2

80
105
30

0.5

*Data obtained from a LSS report of cancer incidence (1958–1987) by Thompson DE et al.1**Data obtained from a pathological report of colorectal cancer cases among A-bomb survivors (1955-1980) Nakatsuka H et al.32†
Chi-square test.

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RERF Update Volume 18, 2007

Science Articles: Molecular Features of Colorectal Cancer
Coulter Inc., Fullerton, CA) were subjected tocapillary-electrophoresis using DNA sequencer
CEQ8000 (Beckman Coulter). Microsatellites were
judged to be unstable if one or more novel bands
were present in the amplicons of the tumor samples
compared with non-cancerous samples of the same
subjects. A tumor was considered to be MSI-H if
40% or more of the amplified markers were unstable,
MSI-L if fewer than 40% of the markers were
unstable, and MSS if none of the markers was
unstable.
Detection of gene mutations of BRAF and KRAS
genes
BRAF gene mutation-causing amino acid substitution of glutamic acid for valine at codon 600(BRAFV600E) was determined by restriction fragment
length polymorphism (RFLP) using TspR I (NewEngland Biolabs, Ipswich, MA) and direct sequencing, as described previously.20 Exon 2 region of the
KRAS gene was amplified by PCR. PCR was
performed in a 20-µl mixture containing 5 pmoleseach of primer, 250 µM of each dNTP, 0.5 U of
AccuSure DNA polymerase (Bioline Ltd.), 10 ng ofgenomic DNA, and 1× reaction buffer supplied by
the manufacturer. PCR conditions consisted ofinitial denaturation (95°C for 10 min), followed byrounds of cycles (denaturation at 95°C for 15 sec,
annealing for 15 sec, extension at 72°C for 15 sec).
First round PCR was conducted for 60 cycles using
primers KR12-4Fa: 5′-CCT GCT GAA AAT GAC
TGA ATA TAA-3′ and KR12-2R: 5′-AAT GAT TCTGAA TTA GCT GTA TCG-3′. The first PCR product was diluted to 1/1,000 using MilliQ water andsubjected to nested PCR of 25 cycles using primers
M13M3KRASF001: 5′-GTA AAA CGA CGG CCA
GTT GAA AAT GAC TGA ATA TAA ACT TGT G3′, and M13RVNKRASR001: 5′-TGT GGA ATTGTG AGC GGG CTG TAT CGT CAA GGC ACT
C-3′, with the underlined sequences derived froman M13 vector. The PCR products were purified
using MinElute PCR Purification Kit (Qiagen).
Sequencing of the purified fragments was performed
using CEQ Dye Terminator Cycle Sequencing(DTCS) Quick Start Kit (Beckman Coulter Inc.,
Fullerton, CA), M13priM3: 5′-GTA AAA CGA CGG
CCA GT-3′ or M13RVN: 5′-TGT GGA ATT GTG
AGC GG-3′ as a primer, and DNA sequencer
CEQ8000 (Beckman Coulter).
Analysis of DNA methylation of MLH1 andRASSF2 genes
Methylation of the MLH1 and RASSF2 genes was
carried out using combined bisulfite restriction analysis (COBRA).21 Two hundred ng of DNA was treatedwith bisulfite and purified using EZ DNA Methylation Kit (ZYMO RESEARCH, Orange, CA). Ten ngof the bisulfite-converted DNA was subjected toReturn to Table of Contents

21

semi-nested PCR. PCR was performed in a 20-µl
mixture containing 5 pmoles of each primer, 250 µM
of each dNTP, 0.5 U of AccuSure DNA polymerase(Bioline Ltd.), 10 ng of bisulfite-converted DNA,and 1× reaction buffer supplied by the manufacturer.
PCR conditions consisted of initial denaturation(95°C for 10 min), followed by cycles (denaturation
at 95°C for 15 sec, annealing for 15 sec, extension at
72°C for 15 sec), with 55 and 30 cycles for the firstand second rounds of PCR, respectively. For MLH1,
the first round of PCR was conducted using primers
MehMLH1F7: 5′-AAA AAY GAA TTA ATA GGA
AGA GYG GAT-3′ and MehMLH1R12: 5′-CCT
AAA ACR ACT ACT ACC CRC TAC CTA A-3′,
followed by a second round of PCR using primers
MehMLH1F7 and MehMLH1R11: 5′-CTA AAA
CRA CTA CTA CCC RCT ACC TAA AAA-3′. For
RASSF2, the first round of PCR was conducted using primers, MeRASSF2F1: 5′-GGY GGG AGT
TTG GAY GAG T-3′ and MeRASSF2R1: 5′-CCT
CCC TCC CAA ACA ATA CTA-3′, followed by asecond round of PCR using primers MeRASSF2F1
and MeRASSF2R2: 5′-TCC CTC CCA AAC AATACT ATA ACT A-3′. All the primers used forthis methylation analysis were designed with
MethPrimer.22 The resultant PCR products weredigested with restriction enzyme Mlu I (Fermentas,
Vilnius, Lithuania) for MLH1 and HpyCH4 IV (NewEngland Biolabs) for RASSF2. The undigested anddigested PCR products were electrophoresed on 12%
polyacrylamide gel. After staining with SYBR Goldnucleic acid gel stain (Invitrogen, Carlsbad, CA), theimage was visualized under ultraviolet (UV) illumination using ChemImager 5500 (Alpha Innotech, San
Leandro, CA).
Analysis of LOH at DNA mismatch repair enzyme
genes MLH1 and MSH2
LOH in the MLH1 and MSH2 genes was evaluated with three methods based on either microsatellite
markers or single nucleotide polymorphisms (SNP).
Three microsatellite markers, D3S1611, D3S1007,
and D3S1561, were used for MLH1, while D2S378,
D2S391, and D2S136 were examined for MSH2.
Primer sets and annealing temperatures are summarized in Supplementary Table 2. PCR reaction andauto-sequencer detection were the same as thosedescribed for MSI detection.
DNA fragments encompassing SNP locirs9857252, rs1800734, and rs4535177 located 5′ tothe MLH1 gene and rs1981928, rs2347794,
rs3771274, rs6726691, and rs2042649 within the
MSH2 genes were amplified. Detection of SNP was
conducted with RFLP except for rs4535177. Primer
sets, annealing temperatures, and restriction enzymes
are shown in Supplementary Table 3. After digestion of restriction enzymes that are sensitive for the
SNP, the resultant fragments were electrophoresed
RERF Update Volume 18, 2007

22

Science Articles: Molecular Features of Colorectal Cancer

Table 2. Association of MSI status with clinico-pathological and epidemiological actors
MSS(n=15)

MSI-L(n=13)MSS/MSI-L(n=28)

MSI-H
(n=5)

P

GenderMale (n)Female (n)

8
7

5
8

13
15

0
5

0.1*

Organ

Colon (n)Rectum (n)

9
6

8
5

17
11

5
0

0.1*

Location

Proximal (n)Distal (n)

4
11

2
11

6
22

5
0

0.002*

Histology

Well (n)Moderately (n)Poorly/Mucinous (n)

5
9
1

5
6
2

10
15
3

0
1
4

0.004**

Age at A-bombing

Mean (yrs, SD)

72.7 ( 9.0)

73.2 (10.9)

73.0 ( 9.7)

71.0 (5.3)

0.7†

Age at operation

Mean (yrs, SD)

23.7 (10.2)

25.8 (12.1)

24.6 (11.0)

21.0 (4.7)

0.5†Time since A-bombing

Mean (yrs, SD)

49.1 ( 4.5)

47.5 ( 4.2)

48.3 (4.4)

48.2 (6.3)

1.0†*Fisher’s exact test for MSS/MSI-L vs. MSI-H.
**Mann-Whitney’s U test for MSS/MSI-L vs. MSI-H. †Student’s t test.

Table 3. Association of MLH1 gene methylation with clinico-pathological and epidemiologicalfactors and MSI status

MLH1
Unmethylated(n=30)

Methylated
(n=5)

P

GenderMale (n)Female (n)

12
18

1
4

0.6*

Organ

Colon (n)Rectum (n)

20
10

4
1

1.0*

Location

Proximal (n)Distal (n)

7
23

4
1

0.026*

Histology

Well (n)Moderately (n)Poor/Mucinous (n)

10
16
4

0
2
3

0.031**

Age at A-bombing

Mean (yrs, SD)

74.0 ( 9.8)

69.6 (5.8)

0.3†

Age at Operation

Mean (yrs, SD)

25.7 (11.3)

22.2 (7.1)

0.5†Time since A-bombing

Mean (yrs, SD)

48.2 ( 4.6)

47.4 (5.3)

0.7†

MSI

MSS/MSI-L (n)MSI-H (n)

26
2

2
3

0.017**Fisher’s exact test.
**Mann-Whitney’s U test.
†
Student’s t test.

on 12% polyacrylamide gel. Restriction enzymes
Hha I, Pvu II, HpyCH4 V, and Hind III werepurchased from New England Biolabs and Tas I wasfrom Fermentas. SNP information was obtained fromthe National Center for Biotechnology Information(NCBI).
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For rs4535177, the tetra-primer amplification
refractory mutation system (ARMS) method wasapplied for polymorphism detection.23 A first roundof PCR was conducted in a 20-µl mixture containing 10 ng of DNA, 5 pmoles each of primer
Rs4535177OF1: 5′-GAT TAT TTC ACT GGT GGTRERF Update Volume 18, 2007

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Science Articles: Molecular Features of Colorectal Cancer
ATT GGA TT-3′ and Rs4535177OR1: 5′-ACC TTTTCC CTT TGT TTT CCC-3′, 250 µM of each dNTP,
0.5 U of AccuSure DNA polymerase, and 1×reaction buffer. PCR conditions consisted of initialdenaturation (95°C for 10 min), followed by 65
cycles (denaturation at 95°C for 30 sec, 60° for 30
sec, extension at 72°C for 30 sec). After dilution of
the first PCR product 1,000 fold with MilliQ water,a second PCR was conducted using 1.5 pmoles eachof primers Rs4535177OF1 and Rs4535177OR1, 15
pmoles of Rs4535177IF1: 5′-GGA TTT CTG GCC
ATT TCA CA-3′, and 100 pmoles of Rs4535177IR1:5′-ACA AAA CCA CCT GGC TTG AC-3′. PCR conditions consisted of initial denaturation (95°C for 10min), followed by 5 cycles (denaturation at 95°C for
30 sec, 65°C for 30 sec, extension at 72°C for 30 sec
with decreasing annealing temperature 1°C per
cycle), and 30 cycles (denaturation at 95°C for 30
sec, 60°C for 30 sec, extension at 72°C for 30 sec).
The resultant PCR products were electrophoresed on
12% polyacrylamide gel.
Statistical analyses
We compared continuous variables using
Student’s t-test. The difference of proportions incontingency tables was evaluated by Fisher’s exact
test or chi-square test. The Mann-Whitney U test wasused for non-parametrical comparison of continuous and categorical variables between two groups.
The Jonckheere-Terpstra test was used for assessing
non-parametric trends of continuous variables among
three groups. Statistical analyses were conductedwith the use of SPSS software (ver. 12.0J) and the R
language (a language and environment for statistical computing, ver. 2.3.1).24
Results
Clinico-pathological and epidemiologicalcharacteristics of colorectal cancer patients among
A-bomb survivors
Clinico-pathological and epidemiological characteristics of study subjects are shown in Table 1.
The frequency of mucinous/poorly differentiatedcancer (20.0%) was slightly higher than that of a previous report of colorectal cancer among LSS participants who were diagnosed between 1950–1980(13.6%), though the difference was not statisticallysignificant. The mean time that had elapsed since
the atomic-bombings (A-bombings) was 48.1 years.
Association of MSI and radiation exposure
At first, we determined MSI status of 33 colorectal
cancers from A-bomb survivors with both cancerous and non-cancerous tissue available. Represen-

Figure 1. Electrograms of microsatellite markers showing MSI. Representative patterns of electrograms are shown for fluorescently labeled amplicons of DNA samples from tumor andnormal tissue. N, normal; T, tumor.

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RERF Update Volume 18, 2007

Science Articles: Molecular Features of Colorectal Cancer

Figure 2. Association between radiationdose and MSI status. Distributionof radiation dose for each patient
was plotted by MSI status. Numbers in parentheses indicate median values. Mann-Whitney U test
was conducted for MSS/MSI-L vs.
MSI-H.

24

Figure 3. Methylation analysis of MLH1 and RASSF2
genes by COBRA. COBRA was carried out
using bisulfite-treated DNA. M, methylated;
UM, unmethylated. Representative results
are shown. Sample #1 did not show methylation, while sample #2 demonstrated
methylation. (A) Methylation analysis of
MLH1 gene. N, not digested; D, digested
with restriction enzyme Mlu I. (B) Methylation analysis of RASSF2 gene. N, not digested; D, digested with restriction enzyme
HpyCH4 IV.

tative results of analysis of microsatellite markers
showing MSI are shown in Figure 1. As a result, five
MSI-H cases were observed and all of them werefemale (Table 2). MSI status was significantly associated with proximal-location and mucinous/poorly
differentiated histology (P = 0.002 and P = 0.004,
respectively, Table 2), in good agreement with aprevious report on sporadic colorectal cancer. 25
Notably, the median radiation dose of MSI-H
colorectal cancer cases was significantly higher thanthat of MSS/MSI-L cases (232 vs. 21 mGy, P = 0.042,
Mann-Whitney U test; Figure 2), suggesting a possible association between radiation exposure and
MSI.
Gene alterations responsible for MSI
To explore the molecular events responsible for
MSI, we first examined methylation of CpG dinucleotides of the MLH1 gene at the proximal region(–231 to –228 bp). Representative results of COBRA
for MLH1 gene methylation are shown in Figure 3A.
Methylation status of the MLH1 gene was determinedin five cases, and was shown to be significantly
associated with location and histology (P = 0.026
and P = 0.031, respectively, Table 3). Furthermore
and importantly, the median radiation dose of caseswith MLH1 methylation tended to be higher than thatof MLH1-unmethylated cases (134 vs. 19 mGy, P =0.059; Figure 4). Gene methylation was also associReturn to Table of Contents

Figure 4. Association between radiation dose andMLH1 gene methylation. Distribution of radiation dose for each patient was plottedby methylation status. M, methylated; UM,unmethylated. Numbers in parentheses indicate median values. Mann-Whitney U test
was conducted for UM vs. M.

ated with MSI status in this study (P = 0.017, Table3), with three cases showing both methylation of theMLH1 gene and MSI-H phenotype.
In addition to DNA methylation, we also examRERF Update Volume 18, 2007

Science Articles: Molecular Features of Colorectal Cancer

A

25

B

C
Figure 5. Detection of LOH. (A) Detection of LOH was performed
using microsatellite markers. Representative patternsof electrograms are shown for fluorescently labeled
amplicons of DNA samples from cancer and normal
tissue from a patient. Vertical blue arrows indicate theposition of two alleles, while a red arrow indicates theloss of an allele. (B) Detection of LOH was performed
using RFLP. The upper part of the figure shows thename of the SNPID along with the sequence and therestriction enzyme used for detection. W indicates A/Theterozygote. The lower figure demonstrates representative eloctrophoresis of samples. N, not treated;
T, treated with Hind III; D, digested; UD, undigested.
(C) Detection of LOH was performed using tetra-primer
ARMS method. Horizontal bars indicate the positionsof fragments generated by outer primers, G allele
specific fragment and A allele specific fragment, respectively.

ined LOH at the gene loci of MLH1 and MSH2.
Representative results of LOH detection are shownin Figure 5. We found that all five cases showing
MSI-H demonstrated LOH at MLH1, while only one
case showed LOH at the MSH2 gene loci (Table 4).
These data indicate that development of MSI among
A-bomb survivors may mainly be due to both epigenetic and genetic alterations of the MLH1 gene.
Table 4. LOH and methylation of DNA repair genes in 5
MSI-colorectal cancer cases
Case

MLH1 methylation

MLH1 LOH

MSH2 LOH

A
B
C
D
E

Methylated
Methylated
MethylatedUnmethylatedUnmethylated

Loss
Loss
Loss
Loss
Loss

Loss
Retained
Retained
Retained
Retained

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Gene alterations relative to Ras-signaling
To examine whether radiation exposure is associated with MSI-related early molecular events, wenext analyzed mutations of BRAF at codon 600 and
the KRAS gene at codons 12/13, as well as methylation of the RASSF2 gene. Representative results ofdetecting RASSF2 gene methylation are shown in
Figure 3B, while those results of KRAS gene mutations at codons 12/13 are shown in Figure 6. Frequency and types of KRAS mutations are summarized in Table 5, along with data of colorectal cancerfrom a general Japanese population.26 In brief, nosignificant difference of mutation type distribution
was observed.
Results of methylation of the RASSF2 gene andmutations of the KRAS and BRAFV600E genes in 35
colorectal cancer cases are summarized in Table 6.
Although mutual exclusiveness between KRAS andRERF Update Volume 18, 2007

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Science Articles: Molecular Features of Colorectal Cancer

Figure 6. Detection of KRAS gene mutations. Direct sequencing of PCR fragments encompassingcodons 12 and 13 was conducted. Representative results are shown. G12D, substitutionof Gly for Asp at codon 12; G13D, Gly for Asp at codon 13; G12C, Gly for Cys at codon12.
Vertical arrows indicate position of mutated nucleotides.

BRAFV600E mutation-based cancers has repeatedly
been reported in colorectal cancer by several
groups,27,28,29,30 we found one case that possessed both
KRAS and BRAF V600E mutations simultaneously
among a total of 35 examined cases (Table 6).
We subsequently explored the association
between KRAS and BRAFV600E gene alterations andradiation dose. First, the median radiation dose of
the colorectal cancer cases that bore any of these genealterations was significantly higher than the medianradiation dose of cases lacking these alterations (86vs. 9 mGy, P = 0.041; Figure 7A). In addition, we
found a significant increasing trend of radiation dosewith increased number of gene alterations (P = 0.016,
Figure 7B).
This Ras-signal alteration status was also foundto be closely related to not only MSI but also MLH1
gene methylation (P = 0.044, and P = 0.045, respectively; Table 7). In fact, all MSI-H and MLH1methylated cases were found to carry at least one ofthese alterations. These data imply that radiation
exposure may affect early molecular events in thecolorectal carcinogenetic pathway relative to MSI
among A-bomb survivors.
Discussion
In this study, we found for the first time association between MSI-H phenotype and higher radiationdose in colorectal cancer patients among A-bomb
survivors (1986–2001) (Figure 2). Association
between MSI-H phenotype and mucinous/poorly
differentiated histology, which is commonly observed sporadic colorectal cancer,5 was also foundin our study subjects (Table 2). Interestingly, thefrequency of mucinous/poorly differentiated cancer(7/35, 20%) in this study was slightly higher (13.6%)
than that value previously reported in a LSS pathology study (1950–1980), and significantly higher,
albeit marginally so, than that reported for thegeneral Japanese population (83/764, 10.9%).31 The

Table 5. Comparison of frequencies of KRAS gene mutation types between colorectal
cancer patients from A-bomb survivors and general Japanese population
Substitutions

This study

GeneralJapanese*

Amino acid

Nucleotide

(n)

(%)

(n)

(%)

P **

G12D
G12V
G12S
G12A
G12C
G13D
OthersNo mutation
Total

G35A
G35T
G34A
G35C
G34T
G38A
Others

4
1
0
1
1
1
0
27
35

11.4
2.9
0
2.9
2.9
2.9
0
77
100

30
15
5
4
2
12
4
162
234

12.7
6.4
2.1
1.7
1.2
5.1
1.7
69.1
100

0.8

*Data obtained from a report by Nagasaka T et al.26**Fisher’s exact test for 2 × 8 table.

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RERF Update Volume 18, 2007

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Science Articles: Molecular Features of Colorectal Cancer

Table 6. Summary of gene alterations relative to Ras-signaling in 35 colorectal cancers among
A-bomb survivors

RASSF2Unmethylated

Methylated

BRAFV600E

KRAS

Wild (n)Mutated (n)Total (n)BRAFV600E

Wild

Mutated

Total

Wild

Mutated

Total

Total

17
6
23

1
1
2

18
7
25

7
1
8

2
0
2

9
1
10

27
8
35

Figure 7. Association between gene alterations relative to Ras-signaling and radiation dose. (A)Distribution of radiation dose for each patient was plotted by the presence or absence ofgene alterations. Numbers in parentheses indicate median values. Mann-Whitney’s U
test was conducted for absent vs. present. (B) Distribution of radiation dose for eachpatient was plotted by the numbers of gene alterations. Jonckheere-Terpstra test was
conducted for assessing increasing trend of radiation dose among the three groups (onesided).
Table 7. Association of ras-signaling alterations and MLH1 gene methylation or
MSI status
Ras signaling alterations†
Absence

Presence

MLH1
methylation

UM (n)
M (n)

17
0

13
5

MSI

MSS (n)MSI-L (n)MSI-H (n)

9
7
0

6
6
5

PtrendP
0.045**

0.040*

0.044**†

Ras signaling alterations were assessed by mutations of BRAFV600E and
KRAS gene codons 12 and 13, and methylation of RASSF2 gene.
*Cochran-Armitage test,**Fisher’s exact test for MSS/MSI-L vs. MSI-H.

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RERF Update Volume 18, 2007

Science Articles: Molecular Features of Colorectal Cancer
latter finding is in contrast to a previously reported
pathology study on colorectal cancer among A-bomb
survivors who were diagnosed between 1955 and1980, and the absence of a discernable association
between histology and radiation dose.32
A higher frequency of mucinous/poorly differentiated cancer among radiation-associated cancers has
also been reported for colorectal cancer after pelvic
irradiation.4 The time since radiotherapy for thesesubjects appeared to be distributed uniformly fromfive to over 40 years, with a median time of 20 years;
the median time since radiotherapy for developmentof colorectal cancer with mucinous/poorly differentiated histology was significantly longer than thatfor well/moderately differentiated (27 vs. 20 yrs).4
Since the percentage of cancer cases with mucinous/
poorly differentiated histology did not change by age
at diagnosis group among the Japanese general
population,31 the observed increase of time sinceradiotherapy for mucinous/poorly differentiatedcancer may not be due to the late-onset of cancer
with these histological types, but rather due to thecharacteristic features of radiation-induced cancer.
Besides the development of second primarycolorectal cancer following radiotherapy, there wasa report of radiation-associated cancers after radiotherapy of healthy individuals: three healthy Jewish
teenagers in the Auschwitz concentration campduring World War II were exposed to a single massive dose of irradiation as part of sterilization
experiments, and they contracted colorectal cancer
40 years after the irradiation.33
We observed that radiation exposure was associated with gene alterations relative to Ras-signaling
in colorectal cancer among A-bomb survivors(Figure 7). This suggests that acute radiation exposure may affect colorectal carcinogenesis at very
early stages, e.g., initial phases of the serrated polyppathway which is closely related to the MSI phenotype.
To our knowledge only a single report on themolecular oncological analysis of radiation-associated colorectal cancer has been published to date.34
This study involved the genetic analysis of five casesof radiotherapy-associated colorectal cancer andfinding that the tumor specimens lacked MSI. It
should be noted, however, that mucinous/poorly differentiated cancer specimens were not examined, andthat the post-radiotherapy times were relatively short,i.e., between 12 and 26 years with median durationof 14 years. These times compared with the meantime since A-bombing (48.1 yrs) in our current study
and median time since radiotherapy (27 yrs) for Japanese colorectal cancer cases with mucinous/poorly
differentiated histology that had developed after
pelvic irradiation.4
In considering these observations, we think thatan extension of the follow-up period is essential for
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28

a better understanding of the pathological andmolecular features of radiation-associated colorectal
cancer including such cancer among A-bomb survivors.
Association between methylation of the MLH1
gene and radiation dose was observed in this study(Figure 4). All five cases with the MSI-H phenotype
showed LOH at the MLH1 gene (Table 4). In addition, gene alterations relative to Ras-signaling also
showed association with radiation dose (Figure 7).
These results indicate a possible association between
radiation exposure and gene alterations. Inductionof delayed genetic alterations including somatic
mutations, deletions, and chromosome aberrations
by ionizing radiation has been demonstrated byseveral in vitro studies35: radiation-induced, latearising mutations have been detected in descendant
cells of established cell lines with long passage
histories, mammary epithelium, and hematopoietic
cells.36,37,38 To date, several possible factors/processes,
e.g., secretory factors, gap junctions, reactive oxygen species (ROS), etc., have been suggested by invitro studies to mediate cell signals responsible forheritable changes after radiation exposure.39,40,41 In
addition, it has been reported that frequency of AML1
gene mutations in myelodysplastic syndrome patientsamong A-bomb survivors diagnosed in 1995–2001
was significantly higher than that in the general population not exposed to radiotherapy.42 These results
and ours suggest that radiation may induce genetic
alterations that play some role in the developmentof cancers, including colorectal cancers, among
A-bomb survivors.
Effects of radiation on induction of global
hypomethylation have been shown by both in vitro
and in vivo experiments.43,44 On the other hand,recent studies demonstrated that irradiation of cultured cells and mice can also induce hypermetylationof certain genes.45,46 These observations of opposite
epigenetic alterations by irradiation are similar to
those of simultaneous global hypermethylation andgene specific hypermethylation in cancerous tissue.47
Interestingly, the prevalence of p16 gene methylation
significantly increased as a function of plutonium
dose in lung adenocarcinomas of plutonium-exposed
workers in Mayak.48 In the future, comparable testsof dose-dependency should be conducted using themolecular endpoints reported here for cases of Abomb associated colorectal cancers among A-bomb
survivors, but only when sufficient numbers ofsamples become available.
What factor(s) explain the preferential development of colon cancer among A-bomb survivors?
Recent prospective nested case-control studies
revealed an association between chronic low-grade
inflammation (measured by the inflammatory marker
C-reactive protein [CRP]) and development ofcolorectal cancer, i.e., colon cancer more than rectal
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Science Articles: Molecular Features of Colorectal Cancer
cancer.49,50 Chronic low-grade inflammation may be
one of the factors responsible for this noted preferential development of colon cancer over rectal cancer.
A strong correlation between CRP levels and radiation dose has been reported for Adult Health Study(AHS) A-bomb survivor participants, with CRP
levels increasing by about 31% per Gy of estimatedA-bomb radiation.51 Chronic inflammation provides
sustained production of ROS.52 Recently, a mechanistic link between inflammation and alterations of
DNA methylation status has been proposed:
5-chlorocytosine, which is synthesized by chlorination of a cytosine residue by hypochlorous acid
(HClO) released from inflammatory cells, mimics
5-methylcytosine to transfer a methyl-group to acytosine residue within the opposite complementarystrand CpG sequence by DNMT1.53 Considering
these results along with our own, we speculatethat chronic low-grade inflammation caused by
A-bomb radiation may be one factor affecting thepreferential development of colon cancer overrectal cancer among A-bomb survivors.
Figure 8 summarizes our hypothesis of development of radiation-associated colorectal cancer among
A-bomb survivors. Radiation exposure may directly
induce ROS in the irradiated cells or indirectly via

chronic inflammation, resulting in genomic instability and accumulation of mutations and deletions ofgenes associated with colorectal carcinogenesis,
especially those related to the serrated polyp pathway. Radiation-induced inflammation may also
generate HClO, increasing methylation of the genes
associated with the pathway. To generate invasive
carcinoma, a long duration of several decades would
be required. As a result, the proportion of mucinous/
poorly differentiated carcinoma to well/moderately
differentiated cancer may differ by time since Abombing. To test the hypothesis, analysis of molecular alterations in precancerous lesions such as
hyperplastic polyp and serrated adenoma among Abomb survivors will be necessary. Such tissues may
already be available in the autopsy samples collecteduntil 1985 at RERF.
In summary, the results obtained thus far imply
that radiation exposure may influence MSI status andits related epigenetic and genetic alterations incolorectal carcinogenesis among A-bomb survivors,
although further analysis with an increased numberof cases is essential.
(Notes: Supplementary Tables are available uponrequest.)Figure 8. A hypothetical schema of the development of radiation-associated colorectal cancer
among A-bomb survivors.

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Science Articles: Molecular Features of Colorectal Cancer

30

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Science Articles: Molecular Features of Colorectal Cancer

bystander mutagenesis in a three-dimensional culture model. Cancer Res 65:9876-82, 2005.
42. Harada H, Harada Y, Tanaka H, Kimura A, Inaba T. Implications of somatic mutations in the AML1 gene inradiation-associated and therapy-related myelodysplastic syndrome/acute myeloid leukemia. Blood 101:67380, 2003.
43. Kalinich JF, Catravas GN, Snyder SL. The effect of gamma radiation on DNA methylation. Radiat Res 117:18597, 1989.
44. Pogribny I, Raiche J, Slovack M, Kovalchuk O. Dose-dependence, sex- and tissue-specificity, and persistenceof radiation-induced genomic DNA methylation changes. Biochem Biophys Res Commun 320:1253-61, 2004.
45. Kovalchuk O, Burke P, Besplug J, Slovack M, Filkowski J, Pogribny I. Methylation changes in muscle andliver tissues of male and female mice exposed to acute and chronic low-dose X-ray-irradiation. Mutat Res
548:75-84, 2004.
46. Kaup S, Grandjean V, Mukherjee R, Kapoor A, Keyes E, Seymour CB, Mothersill CE, Schofield PN. Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes.
Mutat Res 597:87-97, 2006.
47. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 21:5400-13, 2002.
48. Belinsky SA, Klinge DM, Liechty KC, March TH, Kang T, Gilliland FD, Sotnic N, Adamova G, Rusinova G,
Telnov V. Plutonium targets the p16 gene for inactivation by promoter hypermethylation in human lung adenocarcinoma. Carcinogenesis 25:1063-7, 2004.
49. Erlinger TP, Platz EA, Rifai N, Helzlsouer KJ. C-reactive protein and the risk of incident colorectal cancer.
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50. Otani T, Iwasaki M, Sasazuki S, Inoue M, Tsugane S; Japan Public Health Center-Based Prospective Study
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51. Hayashi T, Kusunoki Y, Hakoda M, Morishita Y, Kubo Y, Maki M, Kasagi F, Kodama K, Macphee DG, Kyoizumi
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53. Valinluck V, Sowers LC. Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1. Cancer Res 67:946-50, 2007.

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RERF Update Volume 18, 2007

33

Human Interest Notes

The “Old Boys’ Club”—Report on a Series ofInterviews with Former ABCC Employees
Well, they jokingly refer to themselves as belonging to the “Old Boys’ Club,” but their good natured
demeanor, quick minds, and lively steps all belie thislabel—belie this membership. However, we’regetting ahead of ourselves in relaying to you how
this article came about. Sometime ago, Ikawa-sanand I wanted to provide the readers of the Updatewith a special treat—a “human interest” story on
what it was like during the early days of ABCC anddecided that an interview (or series of interviews)
with several prominent former ABCC employees
might just do the trick. So with this in mind we set
upon the task (the pleasure) of talking to fivedelightful gentlemen.
On May 17th, we met with three former employees from the Hiroshima Laboratory—Mr. Michael
E. Rappaport, a former senior administrator, Mr. Isao
Moriyama, a former chief of the General Affairs
Section, and Mr. Shizuo Inoue, a former assistantchief of the Secretariat. Approximately a month later,
on June 22nd, following the annual RERF Board of
Directors meeting in Nagasaki, I along with Dr.
Evan Douple of the U.S. National Academy of
Sciences (NAS), had a chance to interview twoformer ABCC employees from the Nagasaki Laboratory, namely Mr. Masayuki Hayashida, former
operations administrator, and Mr. Yoshio Okamoto,a General Affairs Section chief and later the operations administrator.
For both interviews we asked the same basic setof six questions relating to: a) how they came to workat ABCC, b) the lab’s organization, operations, andcontact with notable personnel, c) major operational
problems, including the issue of bi-national natureof the lab and relationships between Hiroshima andNagasaki labs, d) difficulties in contacting andenlisting cooperation of A-bomb survivors, e) treatment and care of study participants, f) thoughts on
RERF’s present state and future directions.
[Editors note: The responses listed and attributed to
the former employees are not all direct quotes, but
often represent paraphrased and interpreted
responses taken from notes collected during theinterviews. We trust that we’ve reported accurately
the intent of the employees’ comments, if not their
exact words.]

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Questions to the “Old Boys” and Their
Comments
How you happened to come to work for ABCC?
Mr. Rappaport recalled that in 1947 he was astaff officer in the office of chief engineer of the
British Commonwealth Occupation Force on
Etajima. The office was responsible for occupation
force construction in Chugoku and Shikoku region
as well as for the Supreme Commander for the
Allied Powers Military Government and other organizations such as ABCC. Later his office moved toKure and there he got to know Dr. Carl Tessmer,
ABCC’s first director who periodically visited his
office to check on the progress of work on thetemporary laboratory at Ujina and maintenance ofrented staff housing. About that time Australian
Forces were drawing down and it was time to thinkof returning to Australia. He had no enthusiasm for
returning to his old job in a copper mine and thenDr. Tessmer offered him a job for one year as an
associate engineer on the construction of the original Hijiyama Laboratory. He accepted it without
hesitation.
His contract was extended for two more years
during which he was in charge of construction of
additional buildings (Buildings G and H) in Hijiyama
using materials sent to Japan for construction of Kureclinic. Early in 1949, NAS created the position of“Business Administrator” responsible for budget andpersonnel reporting to the ABCC director and the
NAS business manager. He was appointed to that
position and continued it until transition from ABCC
to RERF in 1975. Upon his retirement (1986), Mr.
Richard Sperry succeeded him as business administrator.
Moriyama-san told us that he started his work at
ABCC in January 1948. Following his Navy duty,
he searched for gainful employment and a neighborfriend (in Kure) suggested ABCC as a possibility.
The neighbor happened to work at Health Department of Kure City Office, and assisted ABCC in its
survey of control subjects. Moriyama-san said that
ABCC attracted him because “…it was work and it
paid well... .” He initially applied for a position as a
medical technician; but once employed, he worked
as an ABCC “contactor” of survivors for possible
enrollment as study subjects. He worked six days
per week: Monday, Wednesday, and Friday heworked in Hiroshima trying to enroll A-bombexposed survivors; while on Tuesday, Thursday and
RERF Update Volume 18, 2007

Human Interest Notes
Saturday he worked to enroll non-exposed individuals in Kure. Wednesday and Saturday were apparently half days. During this period, ABCC was
located in the two rooms on the 2nd floor of the Red
Cross Hospital in Hiroshima, and at the Kyosai Hospital in Kure. The unit had about 30 staff in total,
including nurses, clerks, and contactors. Dr. Koji
Takeshima, who had a concurrent assignment
between the Red Cross Hospital and ABCC, wasresponsible for the work of the contactors and theirvisits to survivors with epilation. Moriyama-san said
that he visited about 10 or 15 persons a day both inHiroshima and Kure, and of the survivors contacted,
only about half were willing to participate.
Inoue-san indicated that he started work at ABCC
in 1949. Just prior to coming to ABCC, he was
employed by a telephone company on Miyajima. The
opportunity to work at a research facility and to learn
new things was the major factor that attracted him
to ABCC; although, he added that simply having agood job was undeniably important as well. He wasinterviewed by Mr. Hamako, a Japanese-American.
He worked initially as a dispatcher in the “motorpool” which had approximately 100 people, including mechanics and drivers (ABCC Ujina lab). To put
this number into proper perspective, in the 1950 timeframe ABCC had approximately 1,000 employees,with 700 staff in Hiroshima and 300 staff in Nagasaki.
Hayashida-san joined ABCC Nagasaki as a clerk
in 1950 at the tender age of 25 years. He indicatedthat his primary interest in working for ABCC was
the opportunity to learn English, and at the time thelab with its staff of about 150 had a fair number ofAmericans. Just after the war, upon returning home,he had worked part time at a laundry reception
counter of the Allied Occupation Forces office in thecity. Later he was employed at a local fish company(currently affiliated with the large trading company
Mitsui & Co.) doing clerical work, but the company
closed and he was forced to find other employment.

(From left) Mr. Shizuo Inoue, Mr. Isao Moriyama, Ms.
Yuko Ikawa (editor), and Mr. Michael E. Rappaport at
the Hiroshima Laboratory

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34
Due to his mother’s illness, he sought work in the
Nagasaki area [this comment prompted us to ask,
whether his mother’s illness was due to the A-bomb].
He said that his mother was not exposed, due to thefact that toward the end of the war, women and children were evacuated to Shimabara. Hayashida-sanalso went on to say that he was not an A-bomb survivor, but was away from Nagasaki at the time of
the bombing serving in the military.
Okamoto-san joined ABCC Nagasaki in 1956
as a “field investigator” and worked in this capacity
for about a year. He explained that in the 1950
National Census an interview survey was conducted
for A-bomb survivors, and it was from this surveythat A-bomb survivors were identified for possible
inclusion in the ABCC study population. He and theother field investigators (a team of ten) had the
responsibility of locating, visiting, and interviewing
people who were identified as A-bomb survivors.
Using forms of the master sample questionnaire, theteam collected information regarding family
composition and exposure status, as well as medical
information regarding symptoms after exposure to
the bomb. About a year later he was transferred toanother section that was engaged in shielding
surveys. Specially designed questionnaires were used
to document exposure status of the survivors. The
team focused on those survivors (about 12,000 in
total) who were exposed fairly close (within
2,000 m) to the hypocenter. Only about 80% of these
individuals could be interviewed; about 20% were
unavailable due to a variety of reasons (i.e., some
simply declined, others had passed away, or moved
outside Nagasaki City.) These interviews revealed
that about one-third of these individuals were insidetheir own homes at the time of the bombing, while
the remaining people were exposed outside, or
inside concrete buildings or other structures. We
asked “Was there a problem—and issue—concerning the survivor’s uncertainty-of-location and their‘recall’ ?” “Yes…” he replied, but only occasionally. The problem was solved largely by multiple
visits and conducting additional interviews.
We were curious about whether some individuals
were sheltered, or in actual bomb shelters. Again heanswered “…yes…, some individuals were indeed
sheltered, and were spared, despite being fairly close
to the hypocenter.”
Because of his work and interest in shielding anddosimetry, he was sent to the Oak Ridge National
Laboratory (Tennessee, U.S.) for additional education and training. It was during this time frame (circa
1957) that the so-called dosimetry-based Ichiban
Project was in progress, and as a result Okamotosan had a chance to interact with a small numberof U.S. radiation scientists who took turns visiting
Hiroshima and Nagasaki for well over a year andhalf and provided guidance to ABCC on shielding
RERF Update Volume 18, 2007

Human Interest Notes(From left) Dr. Thomas Seed (editor), Mr. Masayuki
Hayashida, Dr. Evan Douple of NAS, and Mr. Yoshio
Okamoto at the Nagasaki Laboratory(e.g., by concrete construction materials) and dosimetry of A-bomb radiation.
Finally we asked whether he had seen the newDS02 report. He indicated that he had seen it.
Can you tell us little about ABCC’s organization,
operations, and your contact with notablepersonnel?
Mr. Rapapport (on the issue of satellite labs)commented that four labs, of identical design, wereoriginally planned: Hiroshima and Kure, with theKure lab serving as the “control lab” for the unexposed cohort; and Nagasaki and Sasebo, with thelatter serving to monitor the control, unexposedcohort. At first there were plans to include subjects
living in the four prefectures of each of the Chugoku
and Shikoku districts, but also in Tokyo and Yokohama
as well. There was a plan to build a lab in Uragami,
near the hypocenter in Nagasaki. An initial surveyof the area was made, but the project never got past
the design stage just as Kure and Sasebo.
Moriyama-san indicated that the plans for theKure lab were cancelled due to budget problems, as
was the Sasebo lab in the Nagasaki area.
Inoue-san followed up by saying that in fact,
operational elements of ABCC did exist in Kure, e.g.,a motor pool with dispatchers. Inoue-san also said
(on the issue of the Hijiyama site) that despite acommon misimpression, the ABCC site was indeed
used militarily by the Japanese, and had full intention of installing anti-aircraft guns on the site.
However, the site was prepared but the guns neverinstalled.
Mr. Rapapport indicated that York and Sawyer(a New York City firm) was original design firm for
the Hijiyama lab. When asked about the interesting“quonset hut” design, and whether it was the only
design considered, he responded that “…at the time,
the thought was that this design was the most appropriate for rapid installation.” Further, he said that the
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35
plans to obtain the construction materials by the U.S.
army in Okinawa fell through, and in the end, theconstruction materials were all secured and transported from the U.S. Although he was not sure of
the exact cost of the facilities, he estimated it at somewhere between $500,000 and $1,000,000. Takenaka
Komuten Co. was awarded the building contract, andwhen the work was completed, the company ended
up winning the “Contractor of the Year” (a Japaneseaward). Mr. Rapapport went on to say that Hijiyama
Hall (the residence hall) by contrast, was designed
by a Japanese architect, Kunio Maekawa, and constructed by Shimizu Kensetsu Co.
Inoue-san commented on the salaries and budget:the annual budget, circa 1950, was approximately$100,000. By 1975 the budget had risen to $8 million, and by 2006 it topped $38 million. Moriyamasan added the comment that the average salary inJapan during the 1950 time frame was 700–800 yen
per month, and that ABCC was offering approximately 1,500 yen per month, in addition to the offer
of a possible 50 hours of paid overtime (that equatedto an additional 1,000 yen extra per month). As consequence, staff recruitment at ABCC during theseearly years was not a problem. The willingness ofpeople to stand on long lines for extended periods
when jobs were offered, attested to the latter. However, all three gentlemen indicated that at the time,
ABCC employment was considered fairly tenuous(with the labor union being organized somewhat laterin 1953): all three chuckled at this thought, sincethese gentlemen (and many others) found life-timesof very stable employment.
ABCC covered the salary difference between
ABCC and the National Institute of Health (NIH)
employees (ABCC: 1,500 yen, NIH: 1,000 yen in1947), until the reorganization in 1975. NIH’s
involvement was deemed essential for ABCC
research operations (in particular for the Life Span
Study and access to “koseki”), as NIH served as avital liaison function for the organization in dealing
with the Ministry of Health and Welfare (MHW) andlocal governments.
Hayashida-san (on the issue of ABCC facilities
in Nagasaki) commented that by the time he was
hired by ABCC (in November of 1950), ABCC
Nagasaki had already (Spring of 1949) relocated to
the Nagasaki Prefectural Kyoiku Kaikan. Prior tothis, ABCC was located inside the Shinkozen Primary School, which had been made into a hospitalduring the war and from which ABCC initiated its
activities. “What about the capacity of the Prefectural Kyoiku Kaikan? Do you remember how many
study subjects visited ABCC per day?” we asked.
Hayashida-san stated that when ABCC was initiallyestablished, approximately 25 to 30 persons visited
it per day, although the number of study participantsvaried considerably. There was a capacity to handle
RERF Update Volume 18, 2007

Human Interest Notes
even more participants, but the number of participants seen each day was intentionally limited so that
more time could be spent for each individual andthat each individual could be treated more respectfully. “I believe that in those days, we accepted, on
average, 20 subjects or so per day.” “Despite ourefforts, some people complained that ABCC wasforceful in bringing study subjects to the institution.
Therefore, with such a complaint in mind, I thinkwe were determined not to specify the time foranyone to visit ABCC for examinations. We asked
our study subjects when they would be available andtold them that an automobile would be sent to themfor transportation to and from ABCC when it wasconvenient for them.”
Mr. Rapapport (on the issue of notable persons)indicated (as did Moriyama-san and Inoue-san) that
he had no first-hand knowledge of the activities of
initial research group that surveyed the bombed
cities and worked out of the so-called “disaster train”
in 1946 under the auspices of the U.S. National
Research Council. [Editor’s note: This group
included two civilian radiobiologists, Drs. Paul
Henshaw and Austin Brues, three U.S. medical
officers, Drs. James Neel, Melvin Block, andFrederick Ullrich. There were several Japanese
researchers/physicians as well, most notably Dr.
Masao Tsuzuki, a prominent professor of surgery atTokyo University.] Nevertheless, Mr. Rapapport provided an interesting comment on several notables
during the subsequent, early period of ABCC. He
related a story he had been told early on about theinvaluable work of Dr. Tsuzuki in persuading local
doctors in both Hiroshima and Nagasaki to assist
ABCC in their attempts to conduct medical followup studies of the A-bomb survivors. Quite understandably, many of the local physicians were either
hesitant, or outright refused, to assist in the effort,
and it was only through Dr. Tsuzuki’s considerable
influence was this problem resolved. Apparently, thefamous doctor, Dr. Takashi Nagai of Nagasaki University, was just one of many doctors that Dr. Tsuzuki
talked into helping ABCC out. Mr. Rappaport pointed
out the contrasting personalities and interests of
ABCC’s first director, Dr. Tessmer and Dr. Neel, theprominent researcher who was largely responsible
for establishing the clinical research agenda at the
new lab. “The differences were pronounced: Dr.
Tessmer was truly dedicated in terms of trying to set
up and to manage the new lab (ABCC), whereas Dr.
Neel seemed more interested in the research, and lessin management.” He also commented that Dr.
Henshaw was the person who named the laboratory,
i.e., the Atomic Bomb Casualty Commission(ABCC), and served as a liaison between NAS and
ABCC. However, except for relatively short visits,
his stay in Hiroshima was limited.
Hayashida-san commented on Dr. Raisuke
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36
Shirabe, former professor of Nagasaki University,
an ABCC personality of note. “…I knew Dr. Shirabe
very well. We were both heavy smokers, and during
teatime starting at 3 o’clock, he would often ask me
to share a smoke with him. During these breaks, I
learned a lot about him and his various activities…”“…having a meticulous nature, Dr. Shirabe madeentries in his diary every day.” “…I believe that Dr.
Shirabe understood ABCC studies in Nagasaki
better than anyone, and was remarkably cooperativein the conduct of the ABCC-related studies.” “Dr.
Shirabe played a key role in ABCC’s early years,
especially in terms of recruiting key physicians andother Nagasaki University graduates for ABCC. Thiswas particularly remarkable in light of the fact that
his two sons fell victim to the atomic bombing while
they were at the Nagasaki University Faculty of
Medicine, and he himself was an A-bomb survivor.”
Hayashida-san commented that Dr. William J. Schullconsidered himself a disciple of Dr. Shirabe. Apparently, Dr. Schull in his first meeting with Dr. Shirabe(at the time ABCC Nagasaki was working out of
the Shinkozen Primary School), he was deeply
impressed by the man and by “…finding such an
excellent physician in Japan.” Hayashida-san further
commented that Dr. Schull’s modesty really
impressed him, as did the remarkable nature of Dr.
Shirabe. In passing, Hayashida-san mentioned thathe thought Dr. George Darling (ABCC’s fifth director) to be “…quite remarkable as well.” However,Hayashida-san seemed to suggest that it was Dr.
Shirabe whom he most admired: “I read Dr. Shirabe’s
diary in an attempt to understand how it was at thetime of the bombing. During the complete confusion following the bombing, while treating the
injured, Dr. Shirabe calmly took notes about thedamage and the weather, including wind direction
and temperature. I had the impression that Dr. Shirabe
was a true scientist.”
Okamoto-san told a different story about Dr.
Shirabe; one he considered less scientific, and somewhat amusing (at least at this point in time). The story
goes that Dr. Susumu Tsunoo, the then president ofNagasaki University, was severely injured due to thebombing and headed north to find shelter. Being agood friend of Dr. Tsunoo, Dr. Shirabe accompanied
him for medical treatment, but despite treatment,
several days later, Dr. Tsunoo died. Dr. Shirabe feeling sick himself (physically sick and mentally anguished about loss of his good friend) consoled (andself-medicated) himself by drinking a lot of shochu(distilled alcohol) in his bidding a good and final
farewell to this world. But surprisingly, he foundhimself feeling better in subsequent days and eventually fully recovered. “In relaying this story to me,
he told me (quite jokingly) that copious amounts ofalcohol might be an effective medicinal for radiation sickness.”
RERF Update Volume 18, 2007

Human Interest Notes
Would you care to comment on the bi-national
nature of ABCC? Were there personnel issues?
…communication problems? …operational
problems? …How was the relationship between
the two ABCC facilities, Hiroshima and Nagasaki?
Inoue-san (on the bi-national issue) “In my
personal opinion, there was no problem at all.” Inouesan indicated that much like today, the Japanese staff
worked very well together with the small contingentof U.S. staff.
Moriyama-san commented on the fact that theJapanese staff lived in locations different from thatof the U.S. staff, perhaps contributing to a sense of a“cultural divide” between the U.S. and Japanese staff.
The U.S. contingent in Kure, for example, lived in
an area meant for personnel associated with theoccupation, i.e., the “Niji-mura.” Interestingly,
ABCC provided free bus service for staff living inthese outer areas.
Mr. Rapapport added to Moriyama-san’s comment by telling us that he was one of those staff
members who lived in a rented house in Kure andused the bus service that Moriyama-san mentioned.
He also said that there was some minimal resentment over differences in the level of positions heldby U.S. and Japanese staff. Many of JapaneseAmericans were bilingual and were placed insupervisory positions.
Hayashida-san and Okamoto-san both said thatin Nagasaki at least, the Japanese and the U.S.
employees got along very well, and there were amazingly few problems. They reminisced about theChristmas parties in which a small group of resident
Americans would invite the Nagasaki staff to join in
their celebrations. Although there was an “oddball”
or two along the way, for the most part, the U.S.
staff fit in quite nicely, as they were generally kind,
had families, and played sports (baseball and swimming) with the Japanese staff.
Hayashida-san remembers that Dr. Darling wasa good leader, and tried his best to get people working together during these early days of ABCC. However, when MHW became actively engaged, theorganization became much more bureaucratic.
ABCC Hiroshima was “…very prescriptive” in its
approach to management, and would dictate very
detailed, specific instructions for various programmatic activities.
Moriyama-san (on the issue of HiroshimaNagasaki relationship) indicated that working relationships and communications between the two labs
were quite good, despite the distance, often with lessthan ideal communication systems (compared to
today’s systems), and with differences in daily
operations. For example, pay day was different inHiroshima (on the 30th of each month) than it wasin Nagasaki (on the 15th), and since personnel
management and salary calculations for both labs
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37
were made in Hiroshima, Moriyama-san had to travel
to Nagasaki twice every month in order to make thesepayrolls, and to conduct a variety of other business
transactions. Each trip required well over a day (atotal 24 hours for each roundtrip).
What were the difficulties in contacting andenlisting the cooperation of the A-bomb survivors
themselves?
Moriyama-san talked about his very early experience while at the Red Cross Hospital and the Ujina
lab in the contacting effort. He visited 10–15 persons a day, and scheduled appointments for clinicalexams a week in advance for these individuals. However, only about a half of them agreed to come to
ABCC. Probably due to the difference in disaster ofA-bombing, persons exposed more distally (livingin the suburbs) tended to be more cooperative.
Inoue-san said that he was not directly involvedin the “contacting work”; however, as a result of his“motor pool” work, he was told by the drivers about
the survivor’s pleasure of being picked up and sent
back home by “fancy limousine,” and by being
treated very nicely by ABCC staff. He indicated thatthe vast majority of the survivors were very cooperative and willing to participate, and this helped
enormously in gathering “subjects” for both ongoing and future studies. Despite this, some survivors
unquestionably felt some pressure to participate dueto the authority and direction of the “occupying” U.S.
military. However, because of the well-recognizedutility of the ABCC studies, feeling good about
participating was less of a problem for most of thesurvivors. Another drawing point was that many of
the survivors were anxious about their health, andthat ABCC would provide free medical exams andconsultations. However, this was countered by theirknowing that only exams, and no treatments, were
to be given. As a result of the latter, some people
harbored bad feelings about ABCC’s operations.
Okamoto-san commented on the problems incontacting and handling of “survivors” at Nagasaki
clinical facilities. We asked “Was it difficult to persuade ‘survivors’ to participate?” “Yes, at times…,”
and “…although the number was small, some people
simply declined to be interviewed, apparently because they did not want to say anything at all about
their experiences.” Okamoto-san indicated that the“contactors” were successful, more often than not,in convincing survivors to participate in ABCC’s
medical program, but many times this would require
visiting individuals several times and trying to communicate the worthiness of ABCC’s medical program. “At times, I would use my wife as an example,
saying that she is also an A-bomb survivor and isenrolled in the program, and believes that the program is highly beneficial not only to the survivors
themselves, but to their children. By quiet, gentle,
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Human Interest Notes
but full explanation of the program’s virtues, we weregenerally successful in getting survivors to understand ABCC’s intent and to have them participate in
the program.”
It sounds to us as if you were acting as a “goodwill ambassador” for the program. “Yes, I believeso…” Subsequently, Okamoto-san told this story
about the same newspaper reporter who would visit
the foundation almost every year, and would incessantly ask “Isn’t RERF taking advantage of A-bomb
survivors by conducting only research?” Okamotosan would explain to the reporter that RERF is notlike that at all—“Some of the RERF employees are
A-bomb survivors, though the number is small, andmore than half of the employees have family members who are A-bomb survivors. We all believe inthe positive nature of the program, otherwise wewould not be so actively engaged and encouraging
our family members to participate.” Okamoto-sanfollowed this comment up by saying “…prejudiceof this kind has been reduced significantly, largely
by enormous effort made by contactors since the
ABCC days to maintain close relationships withstudy subjects.”
The treatment issue referred to previously still
lingers—Can you shed any light on this issue?
Were there, or were not, treatments given to theparticipants?
Moriyama-san, Mr. Rappaport, and Inoue-sanall indicated that at certain periods, under very limited situations, treatments were provided. They suggested that limited treatments were provided whenneeded, and referred to the beds in clinic area andthe small number of patients (about 15) that received
treatments around 1963–1965 when Dr. Darling was
director, but this program did not continue long (e.g.,
Mr. Rapapport cited drugs for leukemia being given
at times because ABCC had access to the newer
medicines coming from the U.S. that were not readily
available in the Hiroshima/Nagasaki regions.
We asked about the thoroughness of the ABCC
doctors in following up on the health status of participants that were patently ill. Inoue-san cited thestories he had heard from the survivors themselves,
about ABCC doctors recommending further consultations with medical specialists at local hospitals
when warranted. He also mentioned being told by
ABCC drivers just how appreciative a number ofsurvivors of ABCC in the providing checkups thatlead to diagnoses and successful treatments of given
life-threatening diseases.
Moriyama-san confirmed Inoue-san’s commentsthat some of the survivors complained that ABCC
did not provide any treatment. However, he addedthat although the services ABCC provided werelimited, they were exceedingly important and usefulto the survivors (in terms of clinical exams andReturn to Table of Contents

38
diagnoses). Importantly, ABCC gave the survivors
much needed instruction and guidance on seeking
and securing proper help on their medical problems.
As a result, the majority of the survivors appreciated this service and the kindness of ABCC staff.
Mr. Rappaport commented that he thought that
relationships between the doctors and the study subjects were good, and that the nurses were very kindto the subjects. He followed this up by saying that at
one point, ABCC actually employed social workers
to help the “survivors” handle personal problems thatthey were facing in daily lives. Considering therarity of “social workers” in Japan during this period, it points to ABCC being a caring and well-meaning institution.
Hayashida-san (on the treatment question) said
that in Nagaskai, A-bomb survivors were contacted
and were brought by automobile to ABCC. There
were about three station wagons and 25 jeeps at
ABCC. “Our contactors visited the homes of A-bomb
survivors and experienced a range of problems, but
always tried to handle the interview and recruitingof the survivors sensitively. The same was true at
ABCC itself, in that the staff treated the survivors
kindly and politely, in a manner totally different fromthe way patients are treated in hospitals in Japan.”Hayashida-san went on to say “I do not know whether
the word ‘patient’ is even correct or not: I once proposed use of the word ‘participants’ in order to avoid
any misunderstanding (of what ABCC was about). I
was instructed by the Personnel Section in Hiroshimaby phone to employ the most friendly and competent persons as receptionists. ABCC regarded the
Reception (of the participants) as an importantsection. I never heard complaints from A-bomb
survivors who visited ABCC about their treatment,
although they seemed to have some feelings about
the damage they suffered from the atomic bombing.”
In following up on Hayashida-san’s comments,
we asked whether or not ABCC Nagasaki had facilities for accommodating persons who were found tohave health problems during the treatment andrecovery phases of their illnesses? Hayashida-san
answered that “…there were no such treatment
facilities at ABCC Nagasaki. However, at ABCC
Hiroshima, there were such facilities, but only for a
relatively short period of time. ABCC was not an
institution that ill people visited. If any disease wasfound, we referred the patients to a physician andprovided them with their own health examination
data.”
Dr. Douple specifically commented that “Aroundthe time when ABCC was established, U.S. andJapanese government officials thought that it would
be unwise for U.S. involvement to disturb the Japanese medical system, within which Japanese physicians had established the style of and rights for
conducting treatment and achieved credibility among
RERF Update Volume 18, 2007

Human Interest Notes
their patients. It was therefore agreed between thetwo parties that ABCC would not conduct treatment
and that, if any health problems were found, patientswould be referred to specific hospitals. A long time
has passed since such decisions were made. Do you
believe that those decisions were appropriate?”
Hayashida-san mentioned “I’ve heard of suchdecisions. I frequently heard complaints from not
only A-bomb survivors, but also other citizens to theeffect that ABCC conducted examinations but
provided no treatment. …However, I think that therelationship between the local medical community
in Nagasaki and ABCC was very good.”
We asked about the often-leveled criticism bysome A-bomb survivors that ABCC was an institution that treated them as guinea pigs. “When study
participants visited ABCC for their health examinations, and they were diagnosed as having a given
disease, were they instructed to visit hospitals? Did
ABCC confirm that those study participants actually visited hospitals?”
Hayashida-san “ABCC physicians wrote referrals for such individuals: they could not specify
hospitals. Therefore, the physicians generally
suggested to those individuals that they go to the
University Hospital, or to hospitals near their residence for additional examinations, or alternatively
offered to write referrals to the survivor’s hospitalof choice.” Participants were given their ABCCgenerated clinical records (e.g., X-ray films etc.) sothat they could provide these records, for review purposes, to the physician(s) of the referred hospital or
clinic. Hayashida-san also commented that temporary medicines were provided in order to help themthrough the transition period from the ABCC
diagnosis to seeing their primary physician at the
local hospital or clinic. Okamoto-san added “…byproviding guidance to A-bomb survivors, physiciansat ABCC improved the institution’s credibility among
the survivors.”
We asked “Why do you think some of the A-bomb
survivors continue to think that ABCC-RERF hastreated them as guinea pigs?”
Okamoto-san “I do not think that there is stillsuch a sentiment among this group. I believe that
there is a high level of understanding and confidence
placed in RERF. If some people think that way evennow, they would not be A-bomb survivors, but people
who do not understand RERF at all and are simply
saying this.”Hayashida-san “In my opinion, those who insisted that ABCC treated A-bomb survivors as guinea
pigs were people who led anti-ABCC campaigns.
When I entered ABCC, no one said such a thing. At
the beginning of the 1970s, pathological autopsies
were performed at ABCC at both Nagasaki andHiroshima facilities. This brought about a story to
the effect that, when an A-bomb survivor died, ABCC
Return to Table of Contents

39
cut the body into pieces and sent the data to the U.S.
I don’t believe such a story. However, the time when
the pathological autopsy program was initiated waswhen people first started to say that ABCC treated
A-bomb survivors as guinea pigs. I think hardly any
one would say such a thing now.”
Hayashida-san’s comment prompted us to add ourown comment on the subject, to the statements madeby Moriyama-san, Inoue-san, and Mr. Rappaportduring the earlier interview in Hiroshima, namelythat the pathological autopsy program may have theroot cause of such negative feelings about ABCC.
Although clearly there was no intention of treating
A-bomb survivors in any way, but with the utmost
respect and dignity, the autopsy program seems tohave produced this very regrettable effect.
What are your thoughts on RERF’s current status
and future activities? The organization is reallyat a “cross roads” and needs to develop long-termplans for its future. Over the past decade or so,
there’s been pressure to relocate RERF to adowntown area. What are your thoughts on this?
Inoue-san (on the Hijiyama site question) statedthat “…relocation has been a long debated issue. Personally, it would be better to move RERF to the new
downtown site…assuming of course that there isadequate financing of the move.” “RERF studies
should be continued for many more years, requiring
new and improved facilities.”Moriyama-san indicated that the RERF
Hiroshima lab should remain on Hijiyama. “ABCC
made history on the Hijiyama site and these quonsets
are historic, very unique, and famous buildings: everyone knows them in Hiroshima.”
Mr. Rappaport commented that “…should theopportunity arise, RERF should move to a new, updated facility, because the present facilities are so
old.”
RERF is entering a critical period in terms ofdeciding what the organization will be 20, 30, 40
years from now. As the study cohorts are rapidly
aging and diminishing in size, funding will become
problematic. What’s in RERF’s future? A single
purpose lab? A multiple purpose organization?
Do you have any thoughts on this issue?
Inoue-san stated that he thought that the cohort
studies should continue just as long as possible.
Moriyama-san added that these studies should
not only continue, but be extended to include
transgenerational studies on second and third generations.
Mr. Rappaport was a little more pragmatic in
his response, by indicating the need to carefully
evaluate the cost/benefit relationships of either
extending the current work or pursuing new studies.
However, he did say that the current studies needed
RERF Update Volume 18, 2007

40

Human Interest Notes
to be continued, but only within the current framework of the existing effort, and only until the longterm health effects of A-bomb irradiation in thesurvivors and their children are fully accounted for.
As an aside, he commented that it must be difficult
for the Department of Energy (DOE)/NAS to continue to support RERF when the competition forresearch funds is so great in the U.S. He also questioned the “…appropriateness and capacity of RERF
to pursue alternative scientific fields of interest; studies of aging or lifestyle diseases do not seem to be a
mission of RERF.” He spoke about a woman who
he knows that was diagnosed at ABCC with cancerof the cervix, and was counseled and guided on
proper medical follow-up of her condition. Following hospitalization, and cancer surgery, she recovered. “This is what RERF should continue to be (asan organization)—helping survivors and their chil-dren through their radiation-associated medical problems.”
Hayashida-san and Okamoto-san (on the issueof RERF’s future direction) “As to the future, after
the survivors are gone, I hope RERF continues asa research institute to study the possible genetic
effects of parental A-bomb irradiation in the F1cohort.”

ABCC Quonset-hut buildings in Hiroshima in 1950

Nagasaki Prefectural Kyoiku Kaikan where ABCC
was located in 1950

Present RERF Hiroshima facilities

Present building where Nagasaki RERF is locatedReturn to Table of Contents

A final charge was given to us “Do not forget toshow the survivors who visited ABCC-RERF forexams that they are appreciated,” suggesting that“…some of today’s younger staff might not fully
appreciate the contributions of the survivors.”
Amen. We agree, we agree, we agree.
(Editors: Thomas Seed and Yuko Ikawa)

RERF Update Volume 18, 2007

41

Facts and Figures

Data Processing and Management System
for Adult Health Study
Hiroaki Katayama, Chief, Department
of Information Technology (ITD)The Adult Health Study (AHS) is one of the
major projects at RERF. AHS was created to collectdisease incidence and health information throughbiennial medical examinations of the Life Span Study(LSS) survivors. The AHS examinations for a
portion of this cohort make it possible not only todetermine the full spectrum of human illnesses andphysiologic disorders, and the incidence patterns forboth cancers and non-cancer diseases in relation toradiation dose, but also to obtain important clinicaland epidemiological information that are not accessible through mortality or cancer incidence followup of the full LSS cohort. In this article, we describe
how the data from AHS cohort are actually collected
and stored in the database.
The AHS was started in 1958, at a time when mostof the office work was done by hand. For instance,
staff of the Clinical Studies Department had to typethe subject’s name and address on each examination
form. Also, all clinical data were coded manually by
the Epidemiology Department, electronically transcribed onto floppy disks, and then eventually savedin the mainframe computer as a sequential text data.
The construction of the RERF network started inearly 1987 using BRANCH 4670 (1 Mbps) and MSNETWORKS. The network system was basicallydesigned for the environment using a NEC mainframe computer. At the beginning of 1988, ITD
started to build the AHS clinical database system.
The ideas that drove the construction of this systemwere the following: first, to reduce the amount ofmanual clerical work required for the collection andrecording of vital records; second, to have an electronic system that not only would minimize manualerrors in the recording and retrieving of vital data,
but also one in which essential data could be accessedmore readily. The major problem in constructing sucha system was the lack of a proper database systemthat was simultaneously shared by various sectionsof the Clinical Studies Department. There were
database systems such as dBase IV and RBASE in
the PC environment, however these were insufficient
to meet the department’s needs. Therefore, ITD had
to create from scratch, an original database system
and various application programs that could be usedin various departmental sections. After the AHS clinical database was constructed, the department staff
could now use the same data, for a multitude of
Return to Table of Contents

purposes, that were managed in a single, unified
database—a database that effectively serves to minimize clerical work associated with record keeping(e.g., eliminating the need to type the AHS participant’sname and other information again and again for thedifferent examination procedures and findings). Most
clinical data, except some of the diagnosis andelectrocardiograph data, were transferred automatically and directly to the database without the needof typing or coding by hand.
In 1997, we completely revamped the AHS
database system. This was needed because of aging
hardware (e.g., PCs and printers) coupled withincreasingly heavy network traffic. We had to modify
an old network system based on MS-DOS to Windows environment, as well as replacing the old AHS
clinical database system with a new one using Sybase
as a database engine.

Figure. Adult Health Study data collection schema

The schematic of the AHS data collection isshown in the Figure. The first step of the AHS datacollection is the scheduling of AHS subjects. The
20,000 AHS subjects are divided into A–Z groups,
with specific groups being scheduled for clinicalexaminations every two years. The AHS clinicalcontact control list is produced from the AHS database according to this grouping. Based on this list,
the Clinical Contacting Section makes a phone call
RERF Update Volume 18, 2007

Facts and Figures
or a visit to a subject to fix the examination date,and puts the schedule into the AHS database. The
contactor not only fixes the visit date and time, butalso gets information such as a need for wheelchairs
and other necessary items. This kind of information,
which is not directly related to the research, is also
put into the AHS clinical database. The Medical
Records Unit in the Clinical Studies Department
accesses the AHS database and prints all necessary
forms for the clinical examination two or three days
prior to the subject’s scheduled date. The forms necessary for the examination are substantial, andamount to almost 50 pages for each AHS participant. The examination items are frequently added ordeleted because of the start and finish of many substudies. Therefore, it is mandatory to produce theexamination forms from blank paper in order to fully
support the various components of ongoing AHS
clinical and research activities. Furthermore, theMedical Records Unit prints two types of daily scheduling lists: one contains just subject’s name, examination items and time, and the other contains additional, detailed items. The simple daily schedulinglist is distributed to the Nursing Section and otherrelated sections. The detailed one is used at thereception desk for the management of AHS subjects.
After the examinations are completed, theinformed consent forms from the AHS subjects arepreserved as medical record in medical charts. Other
selected clinical data (e.g., oral temperature, bloodpressure) are also entered into the database by theClinical Administration Unit and the Nursing
Section, and subsequently transmitted to the AHS
database. Blood samples collected by the Nursing
Section and processed by the Clinical Laboratories
Division are separated into three tubes and stored at
Departments of Clinical Studies, Genetics, andRadiobiology/Molecular Epidemiology, and thosestored at the latter two departments are managed by
the additional databases developed by ITD. Urineand stool specimen data collected using Hitachi

42
HILAS system are also transferred to the AHS database, along with the results of any special examination such as ophthalmologic tests.
X-ray images are transferred to the AHS databasethrough FUJI FCR2000 by the Radiology Division.
Osteoporosis measurement data are sent to the AHS
database directly. All data regarding diagnosis, electrocardiographs, diagnoses of X rays and ultrasonography, pathological findings, and the osteoporosis
questionnaire data are sent to the Coding Unit ofEpidemiology Department. The data that have to beentered by hand are processed through a double
data entry system with validity checks to avoid
typographical errors. Numerous programs have beendeveloped in order to accumulate and retain AHS
data in a smooth and efficient manner. Following
coding and data entry, these data are sent to the AHS
database.
Upon completion of clinical examinations, anattending physician of the Clinical Studies Department documents the diagnoses and provides writtencomments. If the AHS participant needs moredetailed examinations such as CT scanning or
special therapy, the attending physician will write aletter of introduction for the participant for followup care at an appropriate medical institute or hospital. Finally, various reports produced from the AHS
database will be sent by mail to the participant.
All tables in the AHS database are concatenatedby the master file number. Although most clinicaldata will be transferred to the research database, thesystem id-number, which is given by the system
automatically, is used to manage the resource database instead of the master file number. Therefore,
researchers who analyze AHS clinical data cannot
touch the master file number that directly connects
to the personal identification. We are doing every
effort to maintain confidentiality of such personal
identification numbers. All application programs thatare required to use the AHS database are supplied
via network, only to the registered PCs equipped withthe special ODBC driver. To access the AHS database, the specific username and password are
required. They are different from those used toaccess the RERF network. The special encryption
software is also installed into all PCs used to accessthe AHS database. The data contained in these PCs
are encrypted, and staff cannot copy any file into theexternal media.
Since the AHS clinical examination is essential
for RERF research, ITD always works closely andcooperatively with all the departments so that they
can accomplish their research goals in a proper andtimely fashion.

Two-dimensional labels on blood sample tubes serve
to identify each sample, while securing and safeguarding donor anonymity.

Return to Table of Contents

RERF Update Volume 18, 2007

Research Protocols and Publications

Research Protocols Approved in 2006
RP 1-06 Study on Cancer of the Uterus among
A-bomb Survivors in the Life Span Study
Cohort, 1950–2003 (Addendum to RP 8-85)
Tokuoka S, Fujihara M, Matsuo T, Nishisaka T,
Nakajima H, Hirai Y, Nishi N, Soda M, Suyama A,
Ikeda T, Ron E, Preston DL, Mabuchi K, Kodama K
Among the Life Span Study (LSS) cohort of the
Radiation Effects Research Foundation (RERF),
cases of cancer of the uterus (corpus and cervix) thatdeveloped during the period between 1957 and 2003
will be ascertained through the Hiroshima andNagasaki tumor registries, supplemented by uterine
cancer cases based on autopsy and surgical pathology records in major medical institutions in bothcities and RERF file records and death certificate
information from 1950 to 2003. Reported cases will
be classified according to the International Classification of Diseases for Oncology (2003) codes anduterine cancers or misdiagnoses of uterine cancers
will be reviewed by a panel of pathologists forhistological diagnoses, and final diagnoses will beclassified according to the World HealthOrganization’s (WHO’s) Histological Classificationof Tumors (2003). Cases of the cervix classified histologically are investigated with polymerase chain
reaction for human papillomavirus (HPV) infection
and the viral type will be determined. The association between different types of HPV infection andcervical cancer will be investigated. To ascertain
precancerous and early cancerous changes of the
corpus (e.g., endometrial hyperplasia) as well as thecervix (cervical dysplasia), we will cooperate withmedical institutions in Hiroshima and Nagasaki toidentify and collect atypical hyperplastic endometrial lesions detected by clinical uterine scraping, orcervical dysplastic lesions identified by vaginal
cytology, cervical biopsy or cervical conization
among the LSS participants. We will investigate theassociation between estimated radiation doses(DS02) and cancers of the uterine corpus and cervix
by confirmed histological types and sub-types.

43
mary cancers by radiation dose, analyses will also
be stratified by cancer type, treatments for the firstcancer, gender, age at exposure, age at first cancer
diagnosis, time since radiation exposure, and duration between first and second primary cancers. This
study will provide greater insight into the effects ofradiation exposure on cancer risk.

RP 3-06 Research Plan for RERF Studies of the
Potential Genetic Effects of Atomic Radiation:
Hiroshima and Nagasaki. Part 1. Mortality
Study of Children of Atomic Bomb Survivors(Addendum to RP 4-75)
Suyama A, Kasagi F, Grant EJ, Cullings HM, Sakata
R, Shimizu Y, Cologne JB, Kodama K
The “Ethical Guidelines for EpidemiologicalResearch,” revised as of 28 December 2004, stipulate the required items that should be described in a
research protocol (RP). RP 4-75, “Research Plan for
RERF Studies of the Potential Genetic Effects ofAtomic Radiation: Hiroshima and Nagasaki,” consists of five parts. The purpose of this addendum is
to add required descriptions on items such as
personal information protection and methods ofobtaining informed consent to “Part 1. Mortality
Study of Children of Atomic Bomb Survivors,” onesection of the RP. In addition, since much time has
passed since RP 4-75 was approved, changes have
happened to the conduct of the study. Thus, suchchanges and additions concerning “Part 1. Mortality
Study of Children of Atomic Bomb Survivors” will
be described.

RP 2-06 Relationship Between Radiation Exposure and Risk of Second Primary Cancers
among A-bomb Survivors
Li CI, Nishi N, Sugiyama H, Soda M, Sakata R,
Hayashi M, Kasagi F, Suyama, A, Mabuchi K, Davis
S, Kopecky KJ, Kodama K
The primary goal of this study is to evaluate therelationship between radiation exposure and risk of
second primary cancers among A-bomb survivors.
The population base for this study is members of theLife Span Study (LSS), and the outcome of interestis the diagnosis of two primary cancers during follow-up. In addition to evaluating risk of second priReturn to Table of Contents

RERF Update Volume 18, 2007

Research Protocols and Publications

Recent Publications

44
dent: 20 years on. Journal of Radiological Protection 2006; 26:127-40.

(Japanese): The original article is in Japanese.

Akashi S, Hara T, Takamura N, Imaizumi M,
Ashizawa K, Akahoshi M, Aoyagi K, Yamashita
S. Evaluation of thyroid function and iodine
intake in Japanese young adults. Preparation forprophylaxis of stable iodide during radiation emergency. Hiroshima Igaku [Journal of the Hiroshima
Medical Association] 2006 (April); 59(4):395-6.
(Proceedings of the 46th Late A-bomb Effects
Research Meeting, 2005) (Japanese)
Arataki K, Kumada H, Toyota K, Ohishi W,
Takahashi S, Tazuma S, Chayama K. Evolutionof hepatitis C virus quasispecies during ribavirin
and interferon-alpha-2b combination therapy andinterferon-alpha-2b monotherapy. Intervirology
2006; 49(6):352-61.
Arisawa K, Soda M, Akahoshi M, Fujiwara S,
Uemura H, Hiyoshi M, Takeda H, Kashino W,
Suyama A. Human T-cell lymphotropic virus type1 infection and risk of cancer: 15.4 year longitudinal study among atomic bomb survivors in
Nagasaki, Japan. Cancer Science 2006 (June);
97(6):535-9.
Ashizawa K. Epidemiology of Basedow disease andother thyroid diseases. Nippon Rinsho [Japanese
Journal of Clinical Medicine] 2006 (December);
64(12 Special Issue):2194-200. (Japanese)
Asia Pacific Cohort Studies Collaboration (RERF:
Nakachi K). An evaluation of metabolic risks forcoronary death in the Asia Pacific region. Diabetes Research and Clinical Practice 2006 (December); 74(3):274-81.
Asia Pacific Cohort Studies Collaboration (RERF:
Nakachi K). Central obesity and risk of cardiovascular disease in the Asia Pacific region. Asia
Pacific Journal of Clinical Nutrition 2006;
15(3):287-92.
Asia Pacific Cohort Studies Collaboration (RERF:
Nakachi K). The fraction of ischaemic heartdisease and stroke attributable to smoking in the
WHO Western Pacific and South-East Asian
regions. Tobacco Controle 2006 (June); 15(3):1818.
Cardis E, Howe G, Ron E, Bebeshko V, Bogdanova
T, Bouville A, Carr Z, Chumak V, Davis S, ...
Kopecky KJ, Kryuchkov V, Loos A, Pinchera A,
Reiners C, Repacholi M. Shibata Y, Shore RE, et
al. Cancer consequences of the Chernobyl acciReturn to Table of Contents

Cologne JB. Re: When is baseline adjustment
useful in analyses of change? An example witheducation and cognitive change (Letter). American Journal of Epidemiology 2006 (December);164(11):1138-9.
Cullings HM, Fujita S, Funamoto S, Grant EJ, Kerr
GD, Preston DL. Dose estimation for atomic bomb
survivor studies: Its evolution and present status.
Radiation Research 2006 (July); 166(1):219-54.
(RERF Report 7-05)
Cullings HM, Levenson ZB, Funamoto S, Teranishi
S, Noda A. Practical aspects of the DS02 dosimetry system for atomic bomb survivors. Hoshasen
Seibutsu Kenkyu [Radiation Biology ResearchCommunications] 2006 (June); 41(2):154-63.
(Japanese)
Darby S, Hill D, Deo H, Auvinen A, Barros-Dios
JM, Baysson H, Bochicchio F, Falk R, Farchi,
Figueiras A, Hakama M, Heid I, Hunter N,
Kreienbrock L, Kreuzer M, Lagarde F, et al. Residential radon and lung cancer—Detailed resultsof a collaborative analysis of individual data on
7148 persons with lung cancer and 14208 persons
without lung cancer from 13 epidemiologic
studies in Europe. Scandinavian Journal of Work,
Environment & Health 2006; 32(Suppl 1):1-84.
Eguchi H, Nakachi K. Smoking as a risk factor forpancreatic cancer. Nippon Rinsho [Japanese Journal of Clinical Medicine] 2006 (January 28);
64:10-3. (Japanese)
Fujiwara S. Radiation effects on health amongchildren of the A-bomb survivors. Hiroshima Igaku[Journal of the Hiroshima Medical Association]
2006 (April); 59(4):311-4. (Proceedings of the 46th
Late A-bomb Effects Research Meeting, 2005)(Japanese)
Hagiwara N, Mechanic LE, Trivers GE, Cawley HL,Taga M, et al. Quantitative detection of p53
mutations in plasma DNA from tobacco smokers.
Cancer Research 2006 (August 15); 66(16):830917.
Hakoda M, Kasagi F, Kusunoki Y, Matsuura S,
Hayashi T, Kyoizumi S, Akahoshi M, Suzuki G,
Kodama K, Fujiwara S. Levels of antibodies tomicroorganisms implicated in atherosclerosis andof C-reactive protein among atomic bomb survivors. Radiation Research 2006 (August); 166:3606. (RERF Report 2-05)
RERF Update Volume 18, 2007

Research Protocols and Publications
Hamatani K, Eguchi H, Koyama K, Mukai M,
Hayashi Y, Nakachi K. Application of improved
SMART RACE method to RNA extracted fromformalin-fixed and paraffin-embedded thyroid
cancer tissue specimen: Detection of RET/PTC8
in thyroid cancer among atomic-bomb survivors.
Nagasaki Igakkai Zasshi [Nagasaki Medical Journal] 2006 (September 25); 81(Special Issue):3469. (Proceedings of the 47th Late A-bomb Effects
Research Meeting, 2006) (Japanese)
Hamatani K, Eguchi H, Takahashi K, Koyama K,
Mukai M, Ito R, Taga M, Yasui W, Nakachi K.
Improved RT-PCR amplification for molecularanalyses with long-term preserved formalin-fixed,
paraffin-embedded tissue specimens. The Journalof Histochemistry and Cytochemistry 2006 (July);
54(7):773-80. (RERF Report 13-05)
Hayashi T, Imai K, Morishita Y, Hayashi I, Kusunoki
Y, Nakachi K. Identification of the NKG2D
haplotypes associated with natural cytotoxic activity of peripheral blood lymphocytes and cancer
immunosurveillance. Cancer Research 2006 (January 1); 66(1):563-70.
Hirai Y, Kodama Y, Moriwaki S, Noda A, Cullings
HM, MacPhee DG, Kodama K, Mabuchi K,
Kraemer KH, Land CE, Nakamura N. Heterozygous individuals bearing a founder mutation in the
XPA DNA repair gene comprise nearly 1% of theJapanese population. Mutation Research 2006
(October); 601(1-2):171-8. (RERF Report 4-06)
Hirai Y, Kodama Y, Noda A, Nakamura N. Frequency
of carriers bearing founder mutation at the XPAgene in a Japanese population. Hoshasen Seibutsu
Kenkyu [Radiation Biology Research Communications] 2006 (June); 41(2):231-7. (Japanese)
Imaizumi M. Radiation exposure and thyroid disease in Hiroshima and Nagasaki atomic bomb
survivors (Letter). Journal of the American Medical Association 2006 (August 2); 296(5):512-3.
Imanishi R, Seto S, Ichimaru S, Nakashima E, Yano
K, Akahoshi M. Prognostic significance of incident complete left bundle branch block observed
over a 40-year period. American Journal of
Cardiology 2006 (September 1); 98(5):644-8.
(RERF Report 11-05)
Inai K, Shimizu Y, Kawai K, Tokunaga M, Soda M,
Mabuchi K, Land CE, Tokuoka S. A pathology
study of malignant and benign ovarian tumors
among atomic-bomb survivors—Case series
report. Journal of Radiation Research 2006;
47(1):49-59. (RERF Report 4-05)
Return to Table of Contents

45
Iwamoto KS, Yano S, Barber CL, MacPhee DG,
Tokuoka S. A dose-dependent decrease in the fraction of cases harboring M6P/IGF2R mutations inhepatocellular carcinomas from the atomic bomb
survivors. Radiation Research 2006 (December);166(6):870-6.
Kambe M, Matsumura M, Suyama A, Nishi N,
Tsujiyama S, Hyoudou M, Yunokawa M,
Murakami H, Urabe A, Yamane T, Arai H, Ono H,
Watanabe T, Oda T, Soramoto E, Mukai M,
Shimizu H, Nakamura A, Shimamura E, Yoshida
Y, Usui S. Report on the results of the fifteenth
medical examination of atomic bomb survivors
resident in North America. Hiroshima Igaku [Journal of the Hiroshima Medical Association] 2006
(January); 59(1):23-48.
Kanis JA, Johnell O, Oden A, Johansson H, Eisman
JA, Fujiwara S, Kroger H, Honkanen R, Melton
LJ, O’Neill T, Reeve J, Silman A, Tenenhouse A.
The use of multiple sites for the diagnosis ofosteoporosis. Osteoporosis International 2006
(April); 17(4):527-34.
Kanzaki H, Hanafusa H, Yamamoto H, Yasuda Y,
Imai K, Yano M, Aoe M, Shimizu N, Nakachi K,
Ouchida M, Shimizu K. Single nucleotide polymorphism at codon 133 of the RASSF1 gene ispreferentially associated with human lung adenocarcinoma risk. Cancer Letters 2006 (July);238(1):128-34.
Kanzaki H, Ouchida M, Hanafusa H, Yano M, Suzuki
H, Aoe M, Imai K, Shimizu N, Nakachi K, Shimizu
K. Single nucleotide polymorphism of the AXIN2
gene is preferentially associated with human lungcancer risk in a Japanese population. International
Journal of Molecular Medicine 2006 (August);
18(2): 279-84.
Katayama H, Apsalikov KN, Gusev BI, Galich B,
Madieva M, Koshpessova G, Abdikarimova A,
Hoshi M. An attempt to develop a database for
epidemiological research in Semipalatinsk. Journal of Radiation Research 2006; 47 (Suppl
A):A189-97.
Kodama K. Long-term late effects of radiation on
health. Houshasen Jiko Iryo Kenkyukai Kaiho[Bulletin of Japanese Association for Medical
Management of Radiation Accident] 2007 (February 1); No.15:9-11. (Japanese)
Kondrashova TV, Neriishi K, Ban S, Ivanova TI,
Krikunova LI, Shentereva NI, Smirnova IA,
Zharikova IA, Konova MV, Taira S, Tsyb AF. Frequency of hemochromatosis gene (HFE) mutations
RERF Update Volume 18, 2007

Research Protocols and Publications
in Russian healthy women and patients withestrogen-dependent cancers. Biochimica etBiophysica Acta 2006 (January); 1762(1):59-65.
Kubo Y, Kusunoki Y, Hakoda M, Kasagi F, Yamaoka
M, Sora M, Inoue M, Matsuura S, Hayashi T,
Kyoizumi S, Fujiwara S, Akahoshi M, Nakachi
K. Effects of atomic-bomb radiation on humanimmune responses. Report 22: Radiation-dosedependent decreases in the percentages of CD4 Tcell subsets are also apparent for survivors in
Nagasaki. Hiroshima Igaku [Journal of theHiroshima Medical Association] 2006 (April);59(4):409-12. (Proceedings of the 46th Late
A-bomb Effects Research Meeting, 2005) (Japanese)
Kusunoki Y. Principles of flow cytometry (FCM).
Diagnosis of hematopoietic tumors using flow
cytometry. K. Muroi et al. (ed). Osaka: Iyaku Journal Co., Ltd. 2006 (August):pp 9-20. (Japanese)
Kusunoki Y, Hayashi T, Nakachi K. T-cell homeostasis and inflammatory response among A-bomb
survivors. Low-Dose Radiation Exposures andBio-Defense System (Proceedings on the International Symposium on Low-Dose Radiation Exposures and Bio-Defense System, 28-30 Sep. 2005,
Aomori). Tanaka S, Fujikawa F, Oghiso Y (ed).
Aomori: The Institute for Environmental Sciences
2006:pp 13-7.
Kusunoki Y, Yamaoka M, Kubo Y, Hamasaki K,
Hayashi T, Kyoizumi S, Nakachi K. Effects ofatomic-bomb radiation on human immune
responses. Report 24: Measurements of memory
CD4 T-cell subsets using CD43 expression lebels
as markers. Nagasaki Igakkai Zasshi [Nagasaki
Medical Journal] 2006 (September 25); 81(Special Issue):350-3. (Proceedings of the 47th Late
A-bomb Effects Research Meeting, 2006) (Japanese)
Lagarde F. Understanding estimation of time and age
effect-modification of radiation-induced cancer
risks among atomic-bomb survivors. Health Physics 2006 (December); 91(6):608-18. (RERF
Report 15-05)

46
Fujiwara S, Nakachi K. Effects of A-bomb radiation on human immunological response. 23rdreport: Long-term elevation of inflammatory markers. Hiroshima Igaku [Journal of the Hiroshima
Medical Association] 2006 (April); 59(4):413-6.
(Proceedings of the 46th Late A-bomb Effects
Research Meeting, 2005) (Japanese)
Nakachi K, Harris CC, Tahara E. Japan-US cooperative cancer research seminar on molecularepidemiological characteristics of lung and coloncancer development among atomic-bomb survivors, Bethesda, USA, February 23-24, 2006. Cancer Science 2006 (November); 97(11):1279-82.
Nakamura N. Genetic effects of radiation in atomicbomb survivors and their children: Past, present
and future. Journal of Radiation Research 2006
(October 1); 47 Suppl B:B67-73.
Nakamura N. Radiation effects on human health athigh altitudes. Rinsho Kensa [Journal of Medical
Technology] 2006 (November); 50(11):1229-31.
(Japanese)
Nakamura N, Nakano M, Kodama Y, Ohtaki K, Niwa
O, Toyoshima M. Chromosome aberrations do not
persist in the lymphocytes or bone marrow cellsof mice irradiated in utero or soon after birth.
Hoshasen Kagaku [Radiological Sciences] 2006
(July); 49(7):225-9. (Japanese)
Nakanishi S, Yamada M, Tatsukawa Y, Fujiwara S,
Yamane K, Kohno N. Relationship between BMI
or HbA1c and mortality among atomic bomb
survivors. Tonyobyo Gappeisho [Journal of Japan
Society of Diabetic Complication] 2006;
20(2):126-9. (Japanese)
Nakashima E, Fujii Y, Neriishi K, Minamoto A.
Analysis of postoperative cataract prevalence fromatomic-bomb survivors with decision uncertainty.
Nagasaki Igakkai Zasshi [Nagasaki Medical Journal] 2006 (September 25); 81(Special Issue):22932. (Proceedings of the 47th Late A-bomb Effects
Research Meeting, 2006) (Japanese)

Mizuno T, Tokuoka S, Kishikawa M, Nakashima E,
Mabuchi K, Iwamoto KS. Molecular basis of basal
cell carcinogenesis in the atomic-bomb survivor
population: p53 and PTCH gene alterations.
Carcinogenesis 2006 (November); 27(11):228694.

Nakashima M, Miura S, Kondo H, Hayashi T, Wen
CY, Kawai K, Soda M, Matsuo T, Sekine I.
Discrimination of multiple primary cancers fromcancers in multiple sites among atomic bomb survivors. Hiroshima Igaku [Journal of the Hiroshima
Medical Association] 2006 (April); 59(4):341-3.
(Proceedings of the 46th Late A-bomb Effects
Research Meeting, 2005) (Japanese)

Morishita Y, Hayashi T, Nagamura H, Kubo Y,
Kasagi F, Kusunoki Y, Hakoda M, Kyoizumi S,

Nakayama T, Kuroi N, Sano M, Tabara Y, Katsuya
T, Ogihara T, Makita Y, Hata A, Yamada M,

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RERF Update Volume 18, 2007

Research Protocols and Publications

47

Takahashi N, Hirawa N, Umemura S, Miki T,
Soma M. Mutation of the follicle-stimulating
hormone receptor gene 5′-untranslated region associated with female hypertension. Hypertension
2006 (September); 48(3):512-8.

Chekin SY, Bogdanova TI, Tronko MD, Tsyb AF,
Thomas GA, Yamashita S. TP53 codon 72 polymorphism in radiation-associated human papillary
thyroid cancer. Oncology Report 2006 (April);15(4):949-56.

Neriishi K. Rehabilitation program for residents atradiation contaminated area caused by Chernobyl
accident. Houshasen Jiko Iryo Kenkyukai Kaiho[Bulletin of Japanese Association for Medical
Management of Radiation Accident] 2007 (February 1); No.15:15-6. (Japanese)

Seto S, Nagao S, Ozeki S, Tetsuo H, Akahoshi M,
Yano K. Contribution of central nitric oxide to theregulation of blood pressure and sodium balancein DOCA-salt hypertension. Journal of Cardiovascular Pharmacology 2006 (May); 47(5):535-9.

Neriishi K, Nakachi K, Sharp GB, Eguchi H, Grant
EJ, Cologne JB, Land CE, Stevens RG, Izumi S.
Radiation effects on sex hormones and growth
factors in postmenopausal A-bomb survivors: Fundamental analysis in controls of a case control
study of breast cancer in atomic bomb survivors.
Nagasaki Igakkai Zasshi [Nagasaki Medical Journal] 2006 (September 25); 81(Special Issue):2339. (Proceedings of the 47th Late A-bomb Effects
Research Meeting, 2006) (Japanese)
Neriishi K, Nakashima E, Fujiwara S, Akahoshi M.
Postoperative cataract in atomic-bomb survivors;
Dose response relationship, indirect effect factors
and threshold. Hiroshima Igaku [Journal of theHiroshima Medical Association] 2006 (April);59(4):330-2. (Proceedings of the 46th Late Abomb Effects Research Meeting, 2005) (Japanese)
Nishi N, Sugiyama H, Kodama K, Okuno H,
Kuwabara M, Hiramatsu K, Arita K, Yasui W, Usui
S. Mesothelioma as seen in tumor registry in
Hiroshima prefecture and Hiroshima City.
Hiroshima Igaku [Journal of the Hiroshima Medical Association] 2006 (May); 59(5):435-8. (Japanese)
Ohishi W, Chayama K. Diagnosis of and countermeasures for variant strain development. Modern
Medical Treatment of Liver Disease. Toda G, etal. (edit). Tokyo: Advanced Medical Technology
Research Institute 2006 (April):pp 164-9. (Japanese)
Ohishi W, Fujiwara S, Suzuki G, Kishi T, Sora M,
Matsuura S, Hakoda M, Yamada M, Chayama K.
Feasibility of freeze-dried sera for serological andmolecular biological detection of hepatitis B andC viruses. Journal of Clinical Microbiology 2006
(December); 44(12): 4593-5. (RERF Report 8-05)
Rogounovitch TI, Saenko VA, Ashizawa K,
Sedliarou IA, Namba H, Abrosimov AY,Lushnikov EF, Roumiantsev PO,Konova MV,
Petoukhova NS, Tchebotareva IV, Ivanov VK,
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Seto S, Soda M, Nakashima E, Yano K, Akahoshi
M. Longitudinal analysis of blood pressure trends
and prognosis in isolated systolic hypertension inelderly individuals. American Journal of Hypertension 2007 (February); 20(2):134-9. (RERF
Report 12-05)
Sharp GB, Mizuno T, Fukuhara T, Tokuoka S. Lackof association between acute exposure to ionizing
radiation and liver cirrhosis. International Journalof Radiation Medicine 2006 (April); 82(4):23140. (RERF Report 11-03)
Shimizu Y. Solid cancer risk. Hiroshima Igaku [Journal of the Hiroshima Medical Association] 2006
(April); 59(4):298-301. (Proceedings of the 46th
Late A-bomb Effects Research Meeting, 2005)(Japanese)
Suyama A, Kasagi F, Grant EJ, Nishi N, Kodama K,
Cologne JB, Furukawa K, Akahoshi M, Fujiwara
S, Shore RE. Health effects study of the childrenof A-bomb survivors (Abstract). American Journal of Epidemiology 2006 (June 1); 163(11):S253.
(2006 Congress of Epidemiology Abstracts)
Suyama A, Kodama K, Kasagi F, Shimizu Y, Soda
M, Nishi N, Sugiyama H, Grant EJ. Future issues
for the Radiation Effects Research Foundation in
the study of risks from A-bomb radiation. Nagasaki
Igakkai Zasshi [Nagasaki Medical Journal] 2006
(September 25); 81(Special Issue):157-64. (Proceedings of the 47th Late A-bomb Effects Research
Meeting, 2006) (Japanese)
Takahashi K, Eguchi H, Arihiro K, Ito R, Koyama
K, Soda M, Cologne JB, Hayashi Y, Nakata Y,
Nakachi K, Hamatani K. The presence of BRAF
point mutation in adult papillary thyroid carcinomas from atomic bomb survivors correlates withradiation dose. Molecular Carcinogenesis 2007
(March); 46(3):242-8. (RERF Report 3-06)
Takahashi N, Sasaki K, Kodaira M, Satoh Y, Kodama
Y, Sugita K, Katayama H, Tsuyama N. Study for
genetic effects of atomic-bomb radiation by using
RERF Update Volume 18, 2007

48

Research Protocols and Publicationsof a DNA microarray-based comparative genomic
hybridization (array-CGH) method—Summary of
preliminary study. Hiroshima Igaku [Journal of theHiroshima Medical Association] 2006 (April);59(4):417-21. (Proceedings of the 46th Late Abomb Effects Research Meeting, 2005) (Japanese)
Ueda H, Ito M, Eguchi H, Tanaka S, Yoshihara M,
Haruma K, Hatakeyama M, Chayama K. Development of a novel method to detect helicobacter
pylori cagA genotype from paraffin-embedded
materials: comparison between patients withduodenal ulcer and gastric cancer in young Japanese. Digestion 2006; 73(1): 47-53.
Yamada M. Effects of the atomic bomb exposure on
health condition among survivors. RERF Adults
Health Study. Hoshasen Seibutsu Kenkyu [Radiation Biology Research Communications] 2006;
41(2):164-74. (Japanese)

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Publications Using RERF Data
The following publications represent research done by nonRERF scientists based on the data publicly available from
RERF.

Sasaki MS, Endo S, Ejima Y, Saito I, Okamura K,
Oka Y, Hoshi M. Effective dose of A-bomb radiation in Hiroshima and Nagasaki as assessed bychromosomal effectiveness of spectrum energy
photons and neutrons. Radiation and Environmental Biophysics 2006 (July); 45(2):79-91.
Tomita M, Otake M, Moon S. Statistical assessment
on cancer risks of ionizing radiation and smoking
based on Poisson models. Journal of the Korean
Data & Information Science Society 2006 (May);
17(2):581-98.

RERF Update Volume 18, 2007