Int. J. Radiation Oncology Biol. Phys., Vol. 65, No. 3, pp. 646 – 655, 2006
GENETIC PREDICTORS OF ADVERSE RADIOTHERAPY EFFECTS: THE
ALICE Y. HO, M.D.,* DAVID P. ATENCIO, PH.D.,* SHEILA PETERS, B.A.,* RICHARD G. STOCK, M.D.,*
SILVIA C. FORMENTI, M.D.,§ JAMIE A. CESARETTI, M.D.,* SHERYL GREEN, M.D.,*
BRUCE HAFFTY, M.D.,¶ KAREN DRUMEA, M.D.,ʈ LARISA LEITZIN, M.D.,ʈ ABRAHAM KUTEN, M.D.,ʈ
DAVID AZRIA, M.D., PH.D.,# MAHMUT OZSAHIN, M.D., PH.D.,**
JENS OVERGAARD, M.D., D.M.SC., F.A.C.R., F.R.C.R.,†† CHRISTIAN N. ANDREASSEN, M.D.,††
CYNTHIA S. TROP, M.D.,‡‡ JANELLE PARK, M.D.,§§ AND BARRY S. ROSENSTEIN, PH.D.*†‡§
Departments of *Radiation Oncology, †Community and Preventive Medicine, and ‡Dermatology, Mount Sinai School of Medicine,
New York, NY; §Department of Radiation Oncology, New York University School of Medicine, New York, NY; ¶Department of
Therapeutic Radiology, Yale University School of Medicine, New Haven, CT; ʈDepartment of Oncology, Rambam Medical Center,
Haifa, Israel; #Department of Radiation Oncology, CRLC Val d’Aurelle, Montpellier, France; **Department of Radiation
Oncology, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland; ††Department of Experimental Clinical
Oncology, Aarhus University Hospital, Aarhus, Denmark; Departments of ‡‡Urology and §§Radiation Oncology, Bronx VA
Purpose: The development of adverse effects resulting from the radiotherapy of cancer limits the use of this treatment
modality. The validation of a test capable of predicting which patients would be most likely to develop adverse
responses to radiation treatment, based on the possession of specific genetic variants, would therefore be of value. The
purpose of the Genetic Predictors of Adverse Radiotherapy Effects (Gene-PARE) project is to help achieve this goal.
Methods and Materials: A continuously expanding biorepository has been created consisting of frozen lympho-
cytes and DNA isolated from patients treated with radiotherapy. In conjunction with this biorepository, a
database is maintained with detailed clinical information pertaining to diagnosis, treatment, and outcome. The
DNA samples are screened using denaturing high performance liquid chromatography (DHPLC) and the
Surveyor nuclease assay for variants in ATM, TGFB1, XRCC1, XRCC3, SOD2, and hHR21. It is anticipated that
additional genes that control the biologic response to radiation will be screened in future work.
Results: Evidence has been obtained that possession of variants in genes, the products of which play a role in
radiation response, is predictive for the development of adverse effects after radiotherapy.
Conclusions: It is anticipated that the Gene-PARE project will yield information that will allow radiation
oncologists to use genetic data to optimize treatment on an individual basis.
2006 Elsevier Inc.
Genetic predictors, Adverse radiotherapy effects, Breast cancer, Prostate cancer.
viewed. In addition, current efforts and techniques used inthe Gene
redictors of A
The term “adverse radiation effects” can generally be de-
(Gene-PARE) project will be discussed as well as future
fined as undesirable clinical and physiologic responses sec-
directions for developing genetic predictors of radiation-
ondary to radiation treatment. In an effort to balance the
eradication of clonogenic tumor cells with minimization ofdamage to surrounding normal tissues, the mechanismsunderlying adverse responses to radiation therapy have been
GENETIC FACTORS AND RADIOSENSITIVITY
studied by both basic scientists and clinicians In thisarticle, both the historical and current literature examining
A variety of patient, tumor, treatment, cellular, and mo-
genetic factors in adverse radiation response will be re-
lecular factors contribute to the variability in severity of
Reprint requests to: Barry S. Rosenstein, Ph.D., Box 1236, Depart-
(BSR); New York State Empire Clinical Research Investigator
ment of Radiation Oncology, Mount Sinai School of Medicine, One
Program Grant; New York State Department of Health Contract
Gustave Levy Place, New York, NY 10029. Tel: (212) 241-9408;
C017931; Swiss Cancer League grants KFS 539-9-1997 and SKL
Fax: (212) 996-8927; E-mail: firstname.lastname@example.org
778-2-1999; The Danish Cancer Society; and The ESTRO
Supported by Department of the Army grants DAMD 17-02-1-
0502, DAMD 17-02-1-0503 and W81XWH-04-0172; American
Received Nov 9, 2005, and in revised form Feb 27, 2006.
Cancer Society Research Scholar Grant RSGT-05-200-01-CCE
Accepted for publication Mar 1, 2006.
The Gene-PARE project ● A. Y. HO et al.
normal tissue reactions exhibited after radiotherapy. Patient
characteristics including age, nutritional status, medica-
Several studies have attempted to define the relationship
tions, body habitus, and coexisting morbidities such as dia-
between in vitro
radiation response and clinically evident
betes or recent surgery all may contribute to radiation tox-
effects by correlating fibroblast radiosensitivity with the
icity Tumor-related factors such as size, histology, and
development of acute and late radiation damage. The un-
tumor grade may also affect the reaction to radiotherapy.
derlying hypothesis of these studies is that genetic differ-
Variation in treatment-related parameters including treated
ences may account for much of the unanticipated severity of
volume, field size, anatomic prescription point, total dose,
acute and chronic radiation reactions exhibited by some
dose per fraction, and use of concomitant chemotherapy
radiotherapy patients. Several studies have reported a cor-
may also contribute to response heterogeneity. Because of
relation between dermal fibroblast radiosensitivity quanti-
the steep dose–response relationship for normal tissues, a
fied by clonogenic survival assays, measuring the SF (i.e.,
small difference in dose could produce divergent outcomes
the surviving fraction after exposure to 2 Gy of X-rays), andthe severity of both early and late effects In
In addition, it has been hypothesized that individual
addition, it has been reported that in vitro
genetic variations may also influence the development of
eration postirradiation may be a useful predictor of wound-
adverse radiation responses Evidence in support of
healing morbidity for patients with soft tissue sarcoma who
this theory was obtained through a study that examined
received preoperative radiotherapy However, in con-
the incidence and time to development of radiation-induced
trast to these positive results, several studies have reported
telangiectasia in a cohort of breast cancer patients. A wide
a lack of correlation between dermal fibroblast SF with
range of values was reported for this patient population
either early or late skin reactions Taken together, these
despite uniform radiation treatment. Consistent with the
studies indicate that skin fibroblast sensitivity correlates
results of previous analyses of radiotherapy patients
only weakly with assessment of radiation-induced skin
it was estimated that approximately 80% to 90% of the
variability was attributed to deterministic effects, possibly aris-ing from potential individual genetic differences, whereas only
10% to 20% of the variation resulted from stochastic events
For assays of normal tissue radiation response, blood is
associated with the random nature of radiation-induced cell
considered to be the tissue of choice because of the ease of
killing in addition to random variations in dosimetry and
collection in a standardized, patient-convenient manner.
However, initial lymphocyte radiosensitivity studies were disappointing with respect to experimental varia-tion, which confuted the predictive power of this assay.
Because the various lymphocyte cell types display different
EFFORTS TO DEVELOP PREDICTIVE ASSAYS
radiation responses, fluctuations in the relative frequency of
FOR NORMAL TISSUE RADIOSENSITIVITY
lymphocyte types cause an apparent shift in radiosensitivity
The development of an in vitro
resulting in large experimental variation However,
capable of predicting the extent of normal tissue damage in
by taking into account cell-type specific radiosensitivities, ithas been reported that CD4 and CD8 T-lymphocyte radio-
radiotherapy patients represents a long-sought goal
sensitivity can discriminate differences in radiation-induced
Despite limited success, the effort to achieve this objective
cytotoxicity between individuals although it is
continues because an assay capable of predicting suscepti-
premature to use such an approach as a predictive assay.
bility for the development of adverse radiation effectswould allow customization of radiotherapy protocols on an
Chromosomal aberrations and micronuclei
individual basis. By doing so, it has been estimated that a
Additional attempts to find suitable assays include anal-
significant improvement in the therapeutic index could be
ysis of fibroblast chromosomal aberrations However,
achieved This work is also reflective of the new era
this technique is time consuming and allows examination of
of “individualized” or “personalized” medicine
only a limited number of cells. Thus, it is considered im-
The goal is therefore to develop a robust, specific assay to
practical for cell types that exhibit slow growth and low
enable individual dose adjustment based on the response of
mitotic indices. Micronucleus induction analysis is another
means of detecting chromosomal damage. Although this
Numerous assays have been proposed to provide the
assay has a well-established role in genetic toxicology
clinician with information that predicts the outcome after
as a means of biomonitoring human populations and as
irradiation and thus guide treatment prescription, but none
a biologic indicator of radiation damage efforts to
have become established in daily practice. Major difficulties
predict radiosensitivity have been inconclusive
limiting the success of these assays are lack of sensitivityand specificity, technical burden of the procedures, poor
characterization of the assayed cells, and the complexity of
Despite multiple and various attempts to develop an assay
capable of predicting which patients are susceptible to de-
I. J. Radiation Oncology ● Biology ● Physics
veloping adverse radiotherapy effects, none of the assays
attempting to “pare away” those individuals from the gen-
examined to date has proved to be consistently sensitive and
eral patient population who are most likely to experience
accurate for the prediction of side effects among patients
pronounced radiation-induced normal tissue damage. Al-
receiving radiation However, new technologies in mo-
though these radiosensitive patients may be better suited to
lecular biology may promote novel strategies for developing
a surgical treatment approach, paradoxically these individ-
a predictive assay with clinical applicability. The use of
uals could alternatively represent a subset of patients who
gene expression arrays that could predict the variation in
are actually optimal candidates for radiotherapy, given that
normal tissue sensitivity to radiation among individuals
their cancers should harbor identical sequence alterations
based on the expression patterns of different genes is cur-
associated with radiosensitivity. This highlights the poten-
rently under investigation. Several studies have demon-
tial for radiotherapy dose modification, as radiosensitive
strated the predictive power of pretreatment expression pro-
tumors theoretically should require lower total treatment
filing for human tumors but similar large-scale
doses than their genetically nonvariant counterparts. Con-
studies on normal tissues to assess the extent of radiation-
versely, for the vast majority of patients who do not possess
induced toxicity have yet to be reported. In addition, a few
genetic variants associated with radiosensitivity, it may be
studies have demonstrated meaningful correlations with
possible to dose escalate and potentially achieve a larger
morbidity by focusing primarily on cytokine responses
Another new molecular approach involves analysis of DNAend-binding complexes that form at DNA double strand
Inclusion of African-American patients
breaks after irradiation. It has been reported that the levels
A unique feature of Gene-PARE that distinguishes it
-containing complexes correlated with cellular radi-
from its European counterpart, the Genetic Pathways for the
osensitivity as measured by the SF Although these
Prediction of the Effects of Irradiation (GENEPI) project
new molecular approaches appear to be promising, it has not
coordinated through the European Society for
yet been determined whether any will have clinical appli-
Therapeutic Radiology and Oncology (ESTRO), as well as
the developing Japanese RadGenomics and the BritishRadiogenomics: Assessment of Polymorphisms for Predict-ing the Effects of Radiotherapy (RAPPER) and Radiation
GOAL OF THE GENE-PARE PROJECT
Complications and Epidemiology (RACE) studies is
To develop an alternative approach to establish an assay
the inclusion of a substantial number of patients of African-
predictive of which patients are most likely to experience
American ethnicity. Based upon currently funded Gene-
radiation-induced complications, a research program has been
PARE studies, it is anticipated that at a minimum, approx-
initiated to identify the genetic factors associated with clin-
imately 500 African-American subjects will be screened for
ical radiosensitivity. To achieve this goal, a broad interna-
genetic variants associated with clinical radiosensitivity.
tional effort has been organized comprising investigators
Screening of these samples may allow identification of
from radiation oncology departments in the United States,
important genetic predictors specific for this population, as
Israel, France, and Switzerland, to create the Gene-PARE
genetic alterations that contribute to enhanced radiosensi-
project Through the studies currently active in Gene-
tivity could differ among ethnicities. Initial results of Gene-
PARE, more than 2000 radiotherapy patients will be
PARE studies suggest that substantial differences exist be-
screened for genetic variants. The primary objective of
tween the genetic factors associated with the development
Gene-PARE is to establish the genetic alterations, the pres-
of adverse radiotherapy effects for African Americans com-
ence of which may confer increased susceptibility for de-
pared with variants correlated with radiosensitivity in the
veloping an adverse response to radiotherapy. Although the
general population . This preliminary finding is consistent
subjects screened to date are primarily breast and prostate
with accumulating pharmacogenomic evidence indicating that
cancer patients, the Gene-PARE tissue biorepository is not
African Americans have a significantly different spectrum of
exclusive to these two types of cancers as it is open to tissue
polymorphisms in genes associated with drug metabolism
samples from patients diagnosed with any form of cancer
compared with those in the general population
treated with radiation. For all patients accrued into Gene-PARE studies, a blood sample is obtained for lymphocyte
DISTINCTION BETWEEN MUTATIONS, SNPs,
isolation and DNA extraction. In addition, frozen lympho-
AND RARE VARIANTS
cytes from patients exhibiting clinical radiosensitivity ornotable genetic characteristics have been used for EBV
Several semantic issues deserve mention. Throughout this
transformation to create permanent cell lines, which are
review, the word “mutation” is generally avoided, as this
being used in assays examining the functional significance
term is often used to signify a particular DNA sequence
variation that exerts a functional impact on the protein
By identifying genetic factors associated with radiosen-
encoded by the gene. Instead, the term “single nucleotide
sitivity, the goal of Gene-PARE is to develop a means to
polymorphism” (SNP) is used to indicate a sequence vari-
predict which patients are at increased risk for complica-
ation in which the less common or minor allele occurs at a
tions secondary to radiation treatment. In this sense, we are
population frequency Ͼ1% The expression “rare vari-
The Gene-PARE project ● A. Y. HO et al.
ant” is used to mean a sequence variation for which the
atively common in this cohort. Based on a logistic regres-
minor allele occurs with a frequency Ͻ1%. Hence, these
sion model, a dose–response using the ED
terms refer only to the prevalence of a minor allele and do
that resulted in a 50% incidence of Grade 3 radiation-
not imply whether a particular genetic variant possesses
induced fibrosis) was generated for these patients. The find-
functional or pathologic significance. The terms “DNA se-
ings of this study suggest a correlation between possession
quence variation” or “genetic variant/alteration” are used to
of the 5557 G¡A variant in ATM
and radiosensitivity as the
signify SNPs and rare variants. The use of “mutation” is
for women who were carriers of this SNP was 52 Gy,
limited to avoid any suggestion as to the functional impact
on the protein encoded by a gene possessing a particular
possess this genetic alteration. These results are consistent
with those of Angele et al.
who found a significantassociation between homozygote carriers of the G¡A tran-sition at ATM
nucleotide 5557 and adverse radiotherapy
ROLE OF ATM IN CLINICAL
responses, as well as a separate study that reported a non-
significant overrepresentation of the ATM
5557 A allele
During the initial years of the Gene-PARE project, sub-
among breast cancer patients with marked alterations in
stantial attention was devoted to study of the ATM
breast appearance after postlumpectomy radiotherapy
and its relationship to radiosensitivity, which has pioneered
In addition, an association was reported between this SNP
the way for examination of other genetic variations as
and late morbidity in prostate cancer patients, although it
predictors of adverse radiation responses. The ATM protein
did not achieve statistical significance because of the small
functions as a protein kinase involved in cellular stress
responses, cell-cycle checkpoint control and DNA repair
Further evidence supporting the relationship between
Loss of these functions may subsequently lead to
sequence variations and radiosensitivity has been ob-
a diminished DNA repair ability and defective cell-cycle
tained for prostate cancer patients treated with iodine-125
checkpoint control. The clinical association between pa-
(125I) brachytherapy The samples for these patients
tients producing nonfunctional ATM protein and the subse-
were obtained from the Mount Sinai Prostate Cancer Patient
quent devastating responses to ionizing radiotherapy have
Tissue Biorepository, which represents a critical resource
been described In addition, cells derived from
for Gene-PARE. This biorepository maintains DNA and
individuals who were heterozygous for a mutation in ATM
frozen blood lymphocytes derived from the approximately
exhibited a radiosensitivity intermediate between persons
2400 prostate cancer patients treated with radiotherapy and
diagnosed with AT and those who were not ATM
followed at this medical center over the past 15 years. A
pilot study involving ATM
screening reported that 10 of the
The initial studies examining the role of ATM
16 subjects (63%) shown to possess sequence variants ex-
in clinical radiosensitivity failed to find a positive correla-
hibited at least one form of adverse response (defined as
tion between ATM
mutation status and the development of
erectile dysfunction, late rectal bleeding, or severe urinary
enhanced normal tissue damage in breast cancer patients
disturbance). In contrast, of the 21 patients who did not
. However, all of these studies used a protein trunca-
harbor an ATM
sequence variation, only 3 (14%) manifested
tion test, which only detects genetic alterations that cause
radiation-induced adverse responses. Nine of the patients
protein truncations. Subsequent to these reports, evidence
with sequence alterations specifically possessed missense
was obtained that missense mutations, which result in
mutations, which encode for amino acid substitutions, and
amino acid substitutions rather than protein truncation, are
are therefore more likely to possess functional importance.
more prevalent in cancer patients and therefore serve as a
In this group, 7 of 9 (78%) exhibited at least one form of
more appropriate type of DNA alteration to measure for
adverse response. In contrast, among the 28 patients who
ascertainment of ATM
did not have a missense alteration, only 6 (21%) displayed
In the first Gene-PARE study examining the role of ATM
any form of adverse response to the radiotherapy.
mutations in susceptibility to radiotherapy-induced morbid-ity, 46 breast cancer patients were screened for ATM
ADDITIONAL RADIOSENSITIVITY CANDIDATE
quence variations It was reported that 3 of 3 (100%)
GENES UNDER STUDY
of the patients who developed a Grade 3/4 subcutaneousreaction, manifested as either fibrosis or soft-tissue necrosis,
Although there is now evidence supporting ATM
missense variants. In contrast, only 3 of the 43
gene associated with clinical radiosensitivity, it is nonethe-
patients (7%) who did not develop this form of severe
less likely that this is not the only gene the alteration of
toxicity harbored this type of ATM
alteration. In a separate
which is responsible for adverse radiotherapy responses.
study, DNA samples isolated from 41 postmastectomy pa-
Additional radiosensitivity candidate genes that have been
tients who were treated with either a hypofractionated or
linked to enhanced radiation responses include TGFB1,
standard radiotherapy fractionation protocol were screened
. TGF␤1, the protein
Because many of these patients received a hypofrac-
encoded by TGFB1
, is a key cytokine involved with the
tionated treatment, radiation-induced skin fibrosis was rel-
regulation of cell growth and immunosuppressive activities.
I. J. Radiation Oncology ● Biology ● Physics
It is also associated with the deposition of extracellular
sion, whereas amino acid substitutions resulting from vari-
matrix proteins and plays a central role in radiation-induced
ants present in exons may alter protein function. Even SNPs
fibrosis The primary function of the XRCC1 protein is
present within noncoding regions could be of significance
to coordinate the activities of the enzymes that perform base
through their affect upon RNA stability or splicing mecha-
excision repair of radiation-induced damage. Cells lacking a
functional XRCC1 protein have demonstrated a hypersen-
The “allelic architecture” of complex traits has received
sitivity to radiation XRCC3 is involved in recom-
significant attention Susceptibility to adverse
binational repair of radiation-induced DNA double strand
radiotherapy responses can be conceptualized through the
encodes the manganese superoxide dis-
two competing theories for the genetic basis of complex
mutatse that represents an important line of cellular antiox-
traits The first theory, the so-called “common dis-
idant defense against the reactive oxygen species induced
ease/ common variant hypothesis,” suggests that the inher-
by irradiation hHR21
is the human homolog of the
ited basis of complex traits is most likely the result of
the encoded protein of which is involved
genetic variants characterized by relatively high allelic fre-
with repair of DNA double stand breaks sister chro-
quencies According to this theory, common SNPs in
a limited number of genes are responsible for the inheri-
To summarize this work, a correlation between radiosen-
tance of complex traits. However, this approach to identify
sitivity and the presence of a Pro/Pro at codon 10 and the
genes associated with complex traits has achieved only
T/T genotype in position Ϫ509 of TGFB1
has been reported
modest success. Therefore, the alternative “rare variant”
A relationship has also been demonstrated between
hypothesis has been proposed, which suggests that a large
codon 16 Val/Ala, XRCC3
codon 241 Thr/Thr
pool of alleles is accountable for the development of com-
codon 399 Arg/Arg genotypes and an increased
plex traits The most realistic model for complex
risk of radiation-induced fibrosis Another study screened
genetic traits likely incorporates aspects of both theories,
three SNPs in XRCC1
and detected an association with
with predisposing alleles of varying population frequencies
radiosensitivity for patients possessing either the codon 194
present in the same and different genes. The Gene-PARE
Arg/Trp alone or in combination with the codon 399 Arg/
project will not be limited by either of these theories, as the
Gln genotype Finally, a T¡C transition at position
approach being used in the studies that constitute this
1440 of the open reading frame of hHR21
has been found in
project routinely involves screening the entire coding por-
6 of 19 radiation-sensitive cancer patients
In aggregate, these studies support the general hypothesis
A question also arises as to the types of mutations that
that genetic factors play a significant role as predictors of
may be associated with clinical radiosensitivity. The studies
adverse radiotherapy responses. It is also important to note
reporting the results of ATM
screening lend support to an
that the postmastectomy radiotherapy breast cancer patients
association between minor sequence alterations, such as
who were screened through Gene-PARE for ATM
SNPs and rare variants, with susceptibility to adverse effects
have also been examined for SNPs in the additional genes
of radiotherapy In contrast, evidence has been
cited above From the results obtained, it appears that
susceptibility to the development of radiation-induced fi-
of pathogenic truncating mutations, which are typically the
brosis depends critically upon the total number of genetic
type of mutation found in individuals with AT appear
variants possessed rather than on any single genetic alter-
not to have been radiosensitive. It is possible that the
ation or gene affected These findings suggest that
presence of a null mutation in one copy of the ATM
clinical normal tissue radiosensitivity should be regarded as
does not confer clinical radiosensitivity, whereas possession
a complex genetic trait that is dependent on the effect of
of a functional but altered ATM protein may result in an
increased risk for the development of an adverse response toradiation treatment.
Cellular radiosensitivity and possession ofgenetic variants
Radiosensitivity and tolerance dose
The Human Genome Project is a well-publicized example
The question may also be raised as to whether a small
of the increasing effort to unravel the genetic variation
difference in cellular survival associated with possession of
underlying complex diseases and traits by illustrating the
genetic variants that confers a relatively small increase in
genetic differences existing between individuals The
cellular radiosensitivity could account for an increased se-
role of SNPs and rare variants, which constitute approxi-
verity in radiation response. In fact, the performance of a
mately 90% of naturally occurring sequence variations, is of
simple calculation demonstrates that this is a likely out-
particular importance SNPs and rare variants are
come. For example, an SF for cells from an individual not
known to potentially affect phenotype, although they have
possessing variants associated with radiosensitivity may be
often been regarded as genetic changes without functional
0.5, whereas for a person possessing genetic variants caus-
significance. However, these sequence alterations may in
ing mild radiosensitivity, the SF could be 0.3. Considering
fact have an important biologic impact as genetic variants
a protocol involving the use of 25 2-Gy fractions, at the
located within regulatory regions could affect gene expres-
completion of treatment, cellular survival would be approx-
The Gene-PARE project ● A. Y. HO et al.
imately 3 ϫ 10Ϫ8 for normal patients whereas it would be8 ϫ 10Ϫ14 for patients possessing radiosensitivity alleles.
This effectively represents the biologic impact of an 88 Gy
total treatment dose for radiosensitive patients compared
with 50 Gy for the patients not harboring such genetic
alterations. This large biologically effective dose could cer-
tainly account for adverse effects from the radiation treat-
ment. In fact, when taking into account the relatively steep
increase in the complication curves for normal tissue re-
sponses and the practice of treating to normal tissue toler-ance, only a small increase in radiosensitivity could result in
a large increase in the probability of normal tissue radiation-
It is also important to note that this small increase in
radiosensitivity may be difficult to detect through routine
cellular radiosensitivity studies, considering the limitations
in accuracy and precision of in vitro
assays. Thus, when
taking into account the steep slope of the normal tissuedose– complication curves, it is likely that a relatively mod-
est, and possibly undetectable effect upon protein function,
resulting in mild cellular radiosensitivity, could still substan-
tially increase the probability for an adverse clinical response.
Thus it may prove difficult or impossible to detect through
functional assays the impact of a genetic variant that causes
Denaturing high-performance liquid chromatography andthe Surveyor nuclease assay
The principal screening techniques for identification of
genetic variants in the Gene-PARE project are denaturing
high-performance liquid chromatography (DHPLC) and the
Surveyor nuclease assay. These are both robust techniques
that can be used to screen any gene in a large population for
single nucleotide substitutions as well as small deletions andinsertions The main advantage of DHPLC lies in
its rapid and accurate identification of polymorphisms and
rare genetic variants in an automated fashion with a high
level of sensitivity and specificity The samples
obtained through Gene-PARE are also being screened using
a complementary methodology that uses Surveyor nuclease
(Trangenomic, Inc., Omaha, NE), which is a mismatch-
specific DNA endonuclease. It is a member of the CEL
nuclease family of plant DNA endonucleases. Surveyor
nuclease cleaves with high specificity at the 3= side of any
mismatch site in both DNA strands, including all base
substitutions and insertion/deletions up to at least 12 nucle-
otides. When mutant and wild-type alleles are mixed,heated, and then cooled to form heteroduplexes, Surveyor
nuclease cleaves the heteroduplex fragments. The cleavage
products are then analyzed using the same HPLC platform
used for DHPLC but performed under nondenaturing con-
ditions. This assay is performed under high sensitivity con-
ditions in which the DNA is stained with a fluorescent probeand detected using a fluorescence detector. Hence the use of
this approach permits the recognition of certain variants that
are difficult to identify using DHPLC, which may require
samples to be run at multiple denaturing temperatures to be
I. J. Radiation Oncology ● Biology ● Physics
detected. A further advantage in the use of the Surveyor
will enable radiation oncologists to take greater advantage
nuclease assay is that it provides information not only as to
of the increasingly powerful and inexpensive methodologies
the presence of a genetic alteration, but also its relative
to sequence DNA in anticipation of the day when patients
position in the DNA fragment being analyzed
diagnosed with cancer arrive at their initial radiation oncol-
Although genotyping assays designed to detect common
ogy consultation armed with their full genome sequenced
SNPs may be less costly to perform, these assays are limited
By identifying genetic predictors of radiosensi-
to detection of already known SNPs and are not designed to
tivity, Gene-PARE may help cancer patients avoid serious
discover new sequence variants. Of greatest importance,
complications that lead to severe morbidity, or even mor-
DHPLC and the Surveyor assay are capable of detecting
tality, arising from organ damage secondary to radiother-
virtually all variants in a gene, rather than just specific
apy. In addition, it could be discovered through this work
that there exists a small radiosensitive portion of the popu-lation and that standard treatment doses are effectivelybeing limited by their radiation tolerance. If these individ-
uals can be identified through genetic screening, it may then
The goal of the Gene-PARE project is to identify the
be revealed that the vast majority of people are more resis-
genetic sequence variants that are predictive for the devel-
tant to radiation than generally assumed. This finding might
opment of adverse effects resulting from radiotherapy. To
permit radiation oncologists to be more aggressive and to
accomplish this objective, a clinical database and bioreposi-
dose escalate, which could translate not only into improved
tory of frozen lymphocytes derived from cancer patients
clinical outcomes for radiotherapy patients but also to more
treated with radiation have been established. DNA isolated
frequently provide safe treatment of relatively radioresis-
from each tissue sample is being screened for variants in
tant cancers. Thus, the results of the research conducted
genes associated with radiation responses. It is expected that
under Gene-PARE will help in the development of a
the results of Gene-PARE will enable the greater use of data
predictive test that will provide individuals faced with a
generated as part of the Human Genome Project and the
diagnosis of cancer, and to their doctors, critical information
emerging field of radiogenomics. In addition, Gene-PARE
that is necessary to reach optimal treatment decisions.
1. Anscher MS, Vujaskovic Z. Mechanisms and potential tar-
13. Jones IM, Thomas CB, Xi T, et al.
The genetic basis for
gets for prevention and treatment of normal tissue injury after
variation in radiation sensitivity in the general population.
radiation therapy. Semin Oncol
2. McBride WH, Chiang CS, Olson JL, et al.
A sense of danger
14. Bourguignon MH, Gisone PA, Perez MR, et al.
from radiation. Radiat Res
epigenetic features in radiation sensitivity. Part II: implica-
3. Stone HB, Coleman CN, Anscher MS, et al.
tions for clinical practice and radiation protection. Eur J Nucl
radiation on normal tissue: Consequences and mechanisms.
Med Mol Imaging
15. Safwat A, Bentzen SM, Turesson I, et al.
4. Denham JW, Hauer-Jensen M. The radiotherapeutic inju-
rather than stochastic factors explain most of the variation in
ry—a complex ‘wound.’ Radiother Oncol
the expression of skin telangiectasia after radiotherapy. Int J
5. Hall EJ. Do no harm—normal tissue effects. Acta Oncol
Radiat Oncol Biol Phys
16. Tucker SL, Geara FB, Peters LJ, et al.
How much could the
6. Bentzen SM, Overgaard J. Patient-to-patient variability in the
radiotherapy dose be altered for individual patients based on
expression of radiation-induced normal tissue injury. Semin
a predictive assay of normal-tissue radiosensitivity? Ra-
1994;4:68 – 80.
7. Jackson A, Kutcher GJ, Yorke ED. Probability of radiation-
17. Turesson I, Joiner MC. Clinical evidence of hypersensitivity
induced complications for normal tissues with parallel archi-
to low doses in radiotherapy. Radiother Oncol
tecture subject to non-uniform irradiation. Med Phys
18. Fletcher GH. Regaud lecture perspectives on the history of
radiotherapy. Radiother Oncol
8. Tucker SL, Turesson I, Thames HD. Evidence for individual
19. Mackay RI, Hendry JH. The modelled benefits of individu-
differences in the radiosensitivity of human skin. Eur J
alizing radiotherapy patients’ dose using cellular radiosensi-
tivity assays with inherent variability. Radiother Oncol
9. Fernet M, Hall J. Genetic biomarkers of therapeutic radiation
sensitivity. DNA Repair (Amst)
20. Evans WE, Relling MV. Moving towards individualized
10. Baumann M, Holscher T, Begg AC. Towards genetic pre-
medicine with pharmacogenomics. Nature
diction of radiation responses: ESTRO’s GENEPI project.
21. Fierz W. Challenge of personalized health care: To what
11. Andreassen CN, Alsner J, Overgaard J. Does variability in
extent is medicine already individualized and what are the
normal tissue reactions after radiotherapy have a genetic
future trends? Med Sci Monit
basis—where and how to look for it? Radiother Oncol
22. Gurwitz D, Livshits G. Personalized Medicine Europe:
Health, Genes and Society: Tel-Aviv University, Tel-Aviv,
12. Andreassen CN. Can risk of radiotherapy-induced normal
Israel, June 19 –21, 2005. Eur J Hum Genet
tissue complications be predicted from genetic profiles? Acta
23. Agren A, Brahme A, Turesson I. Optimization of uncompli-
The Gene-PARE project ● A. Y. HO et al.
cated control for head and neck tumors. Int J Radiat Oncol
biological indicator of radiation damage. Mutat Res
24. MacKay RI, Niemierko A, Goitein M, et al.
42. Geard CR, Chen CY. Micronuclei and clonogenicity follow-
impact of normal-tissue intrinsic radiosensitivity testing. Ra-
ing low- and high-dose-rate gamma irradiation of normal
human fibroblasts. Radiat Res
25. Dubray B, Pavy JJ, Giraud P, et al.
[Predictive tests of
43. Shibamoto Y, Streffer C, Fuhrmann C, et al.
response to radiotherapy. Assessment and perspectives in
sensitivity prediction by the cytokinesis-block micronucleus
1997] (in French). Cancer Radiother
assay. Radiat Res
26. Loeffler JS, Harris JR, Dahlberg WK, et al.
In vitro radio-
44. Champion AR, Hanson JA, Venables SE, et al.
sensitivity of human diploid fibroblasts derived from women
tion of radiosensitivity in established and primary squamous
with unusually sensitive clinical responses to definitive radi-
cell carcinoma cultures using the micronucleus assay. Eur J
ation therapy for breast cancer. Radiat Res
45. Twardella D, Chang-Claude J. Studies on radiosensitivity
27. Oppitz U, Baier K, Wulf J, et al.
The in vitro colony assay:
from an epidemiological point of view– overview of methods
A predictor of clinical outcome. Int J Radiat Biol
and results. Radiother Oncol
46. Alizadeh AA, Eisen MB, Davis RE, et al.
Distinct types of
28. Akudugu JM, Bell RS, Catton C, et al.
diffuse large B-cell lymphoma identified by gene expression
morbidity in STS patients treated with preoperative radio-
therapy in relation to in vitro skin fibroblast radiosensitivity,
47. Pomeroy SL, Tamayo P, Gaasenbeek M, et al.
proliferative capacity and TGF-beta activity. Radiother On-
central nervous system embryonal tumour outcome based on
gene expression. Nature
2002;415:436 – 442.
29. Begg AC, Russell NS, Knaken H, et al.
Lack of correlation
48. van ’t Veer LJ, Dai H, van de Vijver MJ, et al.
of human fibroblast radiosensitivity in vitro with early skin
expression profiling predicts clinical outcome of breast can-
reactions in patients undergoing radiotherapy. Int J Radiat
49. Torres-Roca JF, Eschrich S, Zhao H, et al.
30. Stewart CC, Stevenson AP, Habbersett RC. The effect of
radiation sensitivity using a gene expression classifier. Can-
low-dose irradiation on unstimulated and PHA-stimulated
human lymphocyte subsets. Int J Radiat Biol Relat Stud Phys
50. Sotiriou C, Lothaire P, Dequanter D, et al.
filing of head and neck tumors. Curr Opin Oncol
31. Crompton NE, Ozsahin M. A versatile and rapid assay of
radiosensitivity of peripheral blood leukocytes based on
51. Lonning PE, Sorlie T, Borresen-Dale AL. Genomics in
DNA and surface-marker assessment of cytotoxicity. Radiat
breast cancer-therapeutic implications. Nat Clin Pract Oncol
32. Crompton NE, Miralbell R, Rutz HP, et al.
52. Quarmby S, West C, Magee B, et al.
profiles in irradiated patients with increased toxicity. Int J
of cytokine genes in fibroblasts derived from skin biopsies of
Radiat Oncol Biol Phys
patients who developed minimal or severe normal tissue
33. Crompton NE, Shi YQ, Emery GC, et al.
Sources of varia-
damage after radiotherapy. Radiat Res
tion in patient response to radiation treatment. Int J Radiat
53. Ismail SM, Buchholz TA, Story M, et al.
Oncol Biol Phys
predicted by DNA end-binding complex density, but not by
34. Ozsahin M, Ozsahin H, Shi Y, et al.
Rapid assay of intrinsic
nuclear levels of band components. Radiother Oncol
radiosensitivity based on apoptosis in human CD4 and CD8
T-lymphocytes. Int J Radiat Oncol Biol Phys
54. West CM, McKay MJ, Holscher T, et al.
predicting radiotherapy response: Report and recommenda-
35. Ozsahin M, Crompton NE, Gourgou S, et al.
CD4 and CD8
tions from an International Atomic Energy Agency technical
T-lymphocyte apoptosis can predict radiation-induced late
meeting. Int J Radiat Oncol Biol Phys
toxicity: A prospective study in 399 patients. Clin Cancer
55. Iwakawa M, Imai T, Harada Y, et al.
(in Japanese). Nippon Igaku Hoshasen Gakkai Zasshi
36. Azria D, Gourgou S, Sozzi WJ, et al.
Concomitant use of
tamoxifen with radiotherapy enhances subcutaneous breast
56. Rosenstein BS. ATM Mutations and the Development of
fibrosis in hypersensitive patients. Br J Cancer
Severe Radiation-Induced Morbidity Following Radiother-
apy for Breast Cancer. The Fourth Era of Hope Meeting for
37. Rigaud O, Guedeney G, Duranton I, et al.
the Department of Defense (DOD) Breast Cancer Research
of radiotherapy and chemotherapy on the circulating lym-
phocytes of breast cancer patients. II. Alteration of DNA
57. Holden C. Race and medicine. Science
repair and chromosome radiosensitivity. Mutat Res
58. Brookes AJ. The essence of SNPs. Gene
59. Shiloh Y. ATM and related protein kinases: Safeguarding
38. Miller B, Potter-Locher F, Seelbach A, et al.
genome integrity. Nat Rev Cancer
the in vitro micronucleus test as an alternative to the in vitro
60. Savitsky K, Bar-Shira A, Gilad S, et al.
A single ataxia
chromosomal aberration assay: position of the GUM Work-
telangiectasia gene with a product similar to PI-3 kinase.
ing Group on the in vitro micronucleus test. Gesellschaft fr¨
Umwelt-Mutations-forschung. Mutat Res
61. Lavin MF, Birrell G, Chen P, et al.
ATM signaling and
39. Fenech M. The cytokinesis-block micronucleus technique
genomic stability in response to DNA damage. Mutat Res
and its application to genotoxicity studies in human popula-
tions. Environ Health Perspect
62. McKinnon PJ. ATM and ataxia telangiectasia. EMBO Rep
40. Muller WU, Streffer C, Wuttke K. Micronucleus determina-
tion as a means to assess radiation exposure. Stem Cells
63. Gotoff SP, Amirmokri E, Liebner EJ. Ataxia telangiectasia.
Neoplasia, untoward response to x-irradiation, and tuberous
41. Muller WU, Nusse M, Miller BM, et al.
sclerosis. Am J Dis Child
I. J. Radiation Oncology ● Biology ● Physics
64. Morgan JL, Holcomb TM, Morrissey RW. Radiation reac-
tients with severe late responses to radiation therapy. Cancer
tion in ataxia telangiectasia. Am J Dis Child
84. Cesaretti JA, Stock RG, Lehrer S, et al.
65. Pandita TK, Hittelman WN. Increased initial levels of chro-
variants are predictive of adverse radiotherapy response
mosome damage and heterogeneous chromosome repair in
among patients treated for prostate cancer. Int J Radiat Oncol
ataxia telangiectasia heterozygote cells. Mutat Res
85. Martin M, Lefaix J, Delanian S. TGF-beta1 and radiation
66. Parshad R, Sanford KK, Jones GM, et al.
fibrosis: A master switch and a specific therapeutic target? Int
radiosensitivity of ataxia-telangiectasia heterozygotes. Can-
J Radiat Oncol Biol Phys
cer Genet Cytogenet
86. Marsin S, Vidal AE, Sossou M, et al.
Role of XRCC1 in the
67. Shiloh Y, Parshad R, Sanford KK, et al.
Carrier detection in
coordination and stimulation of oxidative DNA damage re-
1986;1:689 – 690.
pair initiated by the DNA glycosylase hOGG1. J Biol Chem
68. Sanford KK, Parshad R, Price FM, et al.
damage in blood lymphocytes after G2 phase x irradiation, a
87. Thompson LH, West MG. XRCC1 keeps DNA from getting
marker of the ataxia-telangiectasia gene. J Natl Cancer Inst
stranded. Mutat Res
88. Liu Y, Masson JY, Shah R, et al.
RAD51C is required for
69. Paterson MC, MacFarlane SJ, Gentner NE, et al.
Holliday junction processing in mammalian cells. Science
hypersensitivity to chronic gamma-radiation in cultured fi-
broblasts from ataxia-telangiectasia heterozygotes. Kroc
89. Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase mul-
tigene family: A comparison of the CuZn-SOD (SOD1),
70. Weeks DE, Paterson MC, Lange K, et al.
Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures,
chronic gamma radiosensitivity as an in vitro assay for het-
evolution, and expression. Free Radic Biol Med
erozygote identification of ataxia-telangiectasia. Radiat Res
90. McKay MJ, Troelstra C, van der Spek P, et al.
71. Appleby JM, Barber JB, Levine E, et al.
Absence of muta-
conservation of the rad21 Schizosaccharomyces pombe DNA
tions in the ATM gene in breast cancer patients with severe
double-strand break repair gene in human and mouse.
responses to radiotherapy. Br J Cancer
72. Ramsay J, Birrell G, Lavin M. Testing for mutations of the
91. Birkenbihl RP, Subramani S. Cloning and characterization of
ataxia telangiectasia gene in radiosensitive breast cancer
rad21 an essential gene of Schizosaccharomyces pombe
patients. Radiother Oncol
volved in DNA double-strand-break repair. Nucleic Acids
73. Clarke RA, Goozee GR, Birrell G, et al.
Absence of ATM
truncations in patients with severe acute radiation reactions.
92. Pati D, Zhang N, Plon SE. Linking sister chromatid cohesion
Int J Radiat Oncol Biol Phys
and apoptosis: Role of Rad21. Mol Cell Biol
74. Oppitz U, Bernthaler U, Schindler D, et al.
ysis of the ATM gene in 20 patients with RTOG grade 3 or
93. Quarmby S, Fakhoury H, Levine E, et al.
4 acute and/or late tissue radiation side effects. Int J Radiat
transforming growth factor beta-1 single nucleotide polymor-
Oncol Biol Phys
phisms with radiation-induced damage to normal tissues in
75. Weissberg JB, Huang DD, Swift M. Radiosensitivity of
breast cancer patients. Int J Radiat Biol
normal tissues in ataxia-telangiectasia heterozygotes. Int J
94. Andreassen CN, Alsner J, Overgaard M, et al.
Radiat Oncol Biol Phys
normal tissue radiosensitivity from polymorphisms in candi-
76. Concannon P, Gatti RA. Diversity of ATM gene mutations
date genes. Radiother Oncol
detected in patients with ataxia-telangiectasia. Hum Mutat
95. Moullan N, Cox DG, Angele S, et al.
Polymorphisms in the
DNA repair gene XRCC1, breast cancer risk, and response to
77. Gilad S, Khosravi R, Shkedy D, et al.
Predominance of null
radiotherapy. Cancer Epidemiol Biomarkers Prev
mutations in ataxia-telangiectasia. Hum Mol Genet
96. Severin DM, Leong T, Cassidy B, et al.
Novel DNA se-
78. Telatar M, Teraoka S, Wang Z, et al.
quence variants in the hHR21 DNA repair gene in radiosen-
identification and detection of founder-effect mutations in the
sitive cancer patients. Int J Radiat Oncol Biol Phys
ATM gene in ethnic populations. Am J Hum Genet
97. Cavalli-Sforza LL. The Human Genome Diversity Project:
79. Iannuzzi CM, Atencio DP, Green S, et al.
ATM mutations in
Past, present and future. Nat Rev Genet
female breast cancer patients predict for an increase in radi-
98. Lee JE, Choi JH, Lee JH, et al.
Gene SNPs and mutations in
ation-induced late effects. Int J Radiat Oncol Biol Phys
clinical genetic testing: Haplotype-based testing and analy-
sis. Mutat Res
80. Andreassen CN, Overgaard J, Alsner J, et al. ATM sequence
99. Mehrian-Shai R, Reichardt JK. A renaissance of “biochem-
variants and risk of radiation-induced subcutaneous fibrosis
ical genetics”? SNPs, haplotypes, function, and complex
after post-mastectomy radiotherapy. Int J Radiat Oncol Biol
diseases. Mol Genet Metab
100. Erichsen HC, Chanock SJ. SNPs in cancer research and
81. Angele S, Romestaing P, Moullan N, et al.
treatment. Br J Cancer
and cellular response to DNA damage: Association with
101. Lawrence RW, Evans DM, Cardon LR. Prospects and pitfalls
breast cancer risk and clinical radiosensitivity. Cancer Res
in whole genome association studies. Philos Trans R Soc
Lond B Biol Sci
82. Andreassen CN, Alsner J, Overgaard J, et al.
102. Newton-Cheh C, Hirschhorn JN. Genetic association studies
morphisms are associated with risk of late normal tissue
of complex traits: Design and analysis issues. Mutat Res
complications in the breast after radiotherapy for early breast
cancer. Radiother Oncol
103. Hirschhorn JN. Genetic approaches to studying common
83. Hall EJ, Schiff PB, Hanks GE, et al.
A preliminary report:
diseases and complex traits. Pediatr Res
Frequency of A-T heterozygotes among prostate cancer pa-
104. Hirschhorn JN, Daly MJ. Genome-wide association studies
The Gene-PARE project ● A. Y. HO et al.
for common diseases and complex traits. Nat Rev Genet
ysis in hereditary breast and ovarian cancers. Hum Mutat
105. Halushka MK, Fan JB, Bentley K, et al.
Patterns of single-
117. Choy YS, Dabora SL, Hall F, et al.
Superiority of denaturing
nucleotide polymorphisms in candidate genes for blood-
high performance liquid chromatography over single-
pressure homeostasis. Nat Genet
stranded conformation and conformation-sensitive gel elec-
106. Doris PA. Hypertension genetics, single nucleotide polymor-
trophoresis for mutation detection in TSC2. Ann Hum Genet
phisms, and the common disease:common variant hypothe-
118. Jones AC, Austin J, Hansen N, et al.
107. Pritchard JK. Are rare variants responsible for susceptibility
selection for mutation detection by denaturing HPLC and
to complex diseases? Am J Hum Genet
comparison to single-stranded conformation polymorphism
108. Su Y, Swift M. Outcomes of adjuvant radiation therapy for
and heteroduplex analysis. Clin Chem
breast cancer in women with ataxia-telangiectasia mutations.
119. Wagner T, Stoppa-Lyonnet D, Fleischmann E, et al.
J Am Med Assoc
turing high-performance liquid chromatography detects reli-
109. Bremer M, Klopper K, Yamini P, et al.
ably BRCA1 and BRCA2 mutations. Genomics
tivity in breast cancer patients carrying pathogenic ATM
gene mutations: No observation of increased radiation-in-
120. Gross E, Arnold N, Pfeifer K, et al.
Identification of specific
duced acute or late effects. Radiother Oncol
BRCA1 and BRCA2 variants by DHPLC. Hum Mutat
121. Nickerson ML, Weirich G, Zbar B, et al.
110. Gatti RA, Tward A, Concannon P. Cancer risk in ATM
analysis of MET proto-oncogene mutations using DHPLC.
heterozygotes: A model of phenotypic and mechanistic dif-
ferences between missense and truncating mutations. Mol
122. Bernstein JL, Teraoka S, Haile RW, et al.
1999;68:419 – 423.
implementing quality control for multi-center screening of
111. Huber CG, Oefner PJ, Bonn GK. High-resolution liquid
mutations in the ATM gene among women with breast can-
chromatography of oligonucleotides on nonporous alkylated
cer. Hum Mutat
123. Qiu P, Shandilya H, D’Alessio JM, et al.
using Surveyor nuclease. Biotechniques
124. Bannwarth S, Procaccio V, Paquis-Flucklinger V. Surveyor
112. Kuklin A, Munson K, Gjerde D, et al.
Detection of single-
Nuclease: A new strategy for a rapid identification of het-
nucleotide polymorphisms with the WAVE DNA fragment
eroplasmic mitochondrial DNA mutations in patients with
analysis system. Genet Test
respiratory chain defects. Hum Mutat
113. Varghese S, Schmidt-Ullrich RK, Dritschilo A, et al.
125. Shi R, Otomo K, Yamada H. Temperature-mediated hetero-
hanced radiation late effects and cellular radiation sensitivity
duplex analysis for the detection of drug-resistant gene mu-
in an ATM heterozygous breast cancer patient. Radiat Oncol
tations in clinical isolates of Mycobacterium tuberculosis by
denaturing HPLC, SURVEYOR nuclease. Microbes Infect
114. O’Donovan MC, Oefner PJ, Roberts SC, et al.
of denaturing high-performance liquid chromatography as a
126. Janne PA, Borras AM, Kuang Y, et al.
A rapid and sensitive
tool for mutation detection. Genomics
1998;52:44 – 49.
enzymatic method for epidermal growth factor receptor mu-
115. Liu W, Smith DI, Rechtzigel KJ, et al.
tation screening. Clin Cancer Res
performance liquid chromatography (DHPLC) used in the
127. Shendure J, Porreca GJ, Reppas NB, et al.
detection of germline and somatic mutations. Nucleic Acids
plex polony sequencing of an evolved bacterial genome.
116. Arnold N, Gross E, Schwarz-Boeger U, et al.
128. Pennisi E. Biochemistry. Cut-rate genomes on the horizon?
sensitive, fast, and economical technique for mutation anal-
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Cresophene, Unwanted effects If you develop any side effects and they are severe or they get worse, orlast for more than a few days, contact your dentist. Solution for dental use Storing your medicine It is most unlikely that you will be given this medicine to look after. Yourdentist will not use any of the solution after the expiry date printed onthe container. If you have any medici