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Genetic variation in gonadal impairment in female survivors of childhood cancer: a PanCareLIFE study protocol

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Genetic variation in gonadal impairment in female survivors of childhood cancer: a PanCareLIFE study protocol

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Background Improved risk stratification, more effective therapy and better supportive care have resulted in survival rates after childhood cancer of around 80% in developed countries. Treatment however can be harsh, and three in every four childhood cancer survivors (CCS) develop at least one late effect, such as gonadal impairment. Gonadal impairment can cause involuntary childlessness, with serious consequences for the well-being of CCS. In addition, early menopause increases the risk of comorbidities such as cardiovascular disease and osteoporosis. Inter-individual variability in susceptibility to therapy related gonadal impairment suggests a role for genetic variation. Currently, only one candidate gene study investigated genetic determinants in relation to gonadal impairment in female CCS; it yielded one single nucleotide polymorphism (SNP) that was previously linked with the predicted age at menopause in the general population of women, now associated with gonadal impairment in CCS. Additionally, one genome wide association study (GWAS) evaluated an association with premature menopause, but no GWAS has been performed using endocrine measurements for gonadal impairment as the primary outcome in CCS. Methods As part of the PanCareLIFE study, the genetic variability of chemotherapy induced gonadal impairment among CCS will be addressed. Gonadal impairment will be determined by anti-Müllerian hormone (AMH) levels or alternatively by fertility and reproductive medical history retrieved by questionnaire. Clinical and genetic data from 837 non-brain or non-bilateral gonadal irradiated long-term CCS will result in the largest clinical European cohort assembled for this late-effect study to date. A candidate gene study will examine SNPs that have already been associated with age at natural menopause and DNA maintenance in the general population. In addition, a GWAS will be performed to identify novel allelic variants. The results will be validated in an independent CCS cohort. Discussion This international collaboration aims to enhance knowledge of genetic variation which may be included in risk prediction models for gonadal impairment in CCS.
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S T U D Y P R O T O C O L Open Access
Genetic variation in gonadal impairment in
female survivors of childhood cancer: a
PanCareLIFE study protocol
Anne-Lotte L. F. van der Kooi
1,2,3*
, Eva Clemens
2,3
, Linda Broer
4
, Oliver Zolk
5
, Julianne Byrne
6
, Helen Campbell
6
,
Marleen van den Berg
7
, Claire Berger
8,9
, Gabriele Calaminus
10
, Uta Dirksen
11,12
, Jeanette Falck Winther
13,14
,
Sophie D Fosså
15
, Desiree Grabow
16
, Riccardo Haupt
17
, Melanie Kaiser
16
, Tomas Kepak
18
, Leontien Kremer
3,19
,
Jarmila Kruseova
20
, Dalit Modan-Moses
21,22
, Andreas Ranft
11,12
, Claudia Spix
16
, Peter Kaatsch
16
, Joop S. E. Laven
1
,
Eline van Dulmen-den Broeder
7
, André G. Uitterlinden
4
, Marry M. van den Heuvel-Eibrink
3
and on behalf of the
PanCareLIFE Consortium
Abstract
Background: Improved risk stratification, more effective therapy and better supportive care have resulted in survival
rates after childhood cancer of around 80% in developed countries. Treatment however can be harsh, and three in every
four childhood cancer survivors (CCS) develop at least one late effect, such as gonadal impairment. Gonadal impairment
can cause involuntary childlessness, with serious consequences for the well-being of CCS. In addition, early menopause
increases the risk of comorbidities such as cardiovascular disease and osteoporosis. Inter-individual variability in
susceptibility to therapy related gonadal impairment suggests a role for genetic variation.
Currently, only one candidate gene study investigated genetic determinants in relation to gonadal impairment
in female CCS; it yielded one single nucleotide polymorphism (SNP) that was previously linked with the predicted age
at menopause in the general population of women, now associated with gonadal impairment in CCS. Additionally, one
genome wide association study (GWAS) evaluated an association with premature menopause, but no GWAS has been
performed using endocrine measurements for gonadal impairment as the primary outcome in CCS.
Methods: As part of the PanCareLIFE study, the genetic variability of chemotherapy induced gonadal impairment
among CCS will be addressed. Gonadal impairment will be determined by anti-Müllerian hormone (AMH) levels or
alternatively by fertility and reproductive medical history retrieved by questionnaire. Clinical and genetic data from 837
non-brain or non-bilateral gonadal irradiated long-term CCS will result in the largest clinical European cohort
assembled for this late-effect study to date. A candidate gene study will examine SNPs that have already been
associated with age at natural menopause and DNA maintenance in the general population. In addition, a GWAS will
be performed to identify novel allelic variants. The results will be validated in an independent CCS cohort.
Discussion: This international collaboration aims to enhance knowledge of genetic variation which may be included in
risk prediction models for gonadal impairment in CCS.
Keywords: Childhood cancer survivor, Genetic variations, SNPs, Late effects, GWAS
* Correspondence: a.vanderkooi@erasmusmc.nl
1
Division of Reproductive Endocrinology and Infertility, Department of
Obstetrics and Gynecology, Erasmus MC Sophia Childrens Hospital,
Rotterdam, The Netherlands
2
Department of Pediatric Hematology and Oncology, Erasmus MC Sophia
Childrens Hospital, Rotterdam, The Netherlands
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
van der Kooi et al. BMC Cancer (2018) 18:930
https://doi.org/10.1186/s12885-018-4834-3
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
As a result of continuous improvements in treatment
and supportive care, survival rates after childhood can-
cer have increased over the past decades, now reaching
80% in developed countries. However, the harsh treat-
ment components that have led to increased survival
rates can induce serious long-term complications. One
in every four childhood cancer survivors (CCS) reveals
severe or life-threatening adverse late effects [1], and
three in every four survivors report at least one late
effect [2,3]. In female CCS, apart from radiotherapy
involving the field of the ovaries or pituitary, alkylating
agents are important risk factors for fertility impairment
[47] and damage is dose-dependent [8]. Such toxic
agents can damage the ovarian follicle pool severely,
leading to impaired fertility illustrated by an absent or
substantially shortened reproductive window. Conse-
quently, considering the current tendency in European
countries to postpone childbearing, female survivors may
find themselves involuntarily childless, leading to an in-
creased use of artificial reproductive techniques. The feasi-
bility to reach parenthood is of great significance to both
parents of children with cancer and to CCS, and is an im-
portant determinant of quality of life [914]. In addition,
gonadal impairment or early menopause carries adverse
health risks for women, such as an increased risk for car-
diovascular disease and osteoporosis, which require inten-
sive and long-term medical attention [15].
Variations in long-term gonadal impairment in CCS
who received the same treatment suggest that genetic
variation may be an important determinant of gonadal
impairment in CCS. Currently, only limited information
is available on the role of genetic factors in the develop-
ment of impaired gonadal reserve after childhood cancer
treatment [4]. One single center study has been per-
formed which evaluated seven genetic single nucleotide
polymorphisms (SNPs) in 176 female CCS. These SNPs
were selected based on the fact that they have been
found to be associated with age at menopause in large
genome wide association studies (GWAS) in the general
female population [16,17]. While one of these allelic
variations in the BRSK1 gene (rs1172822) was found as-
sociated with a low anti-Müllerian hormone (AMH)
level in CCS [4], replication of this finding has not been
reported so far. Meanwhile, many more SNPs have been
reported to be associated with reproductive ageing in
the general population coming from large-scale collab-
orative consortia [18,19] but none have yet been investi-
gated in CCS. In order to identify independent genetic
determinants for therapy related gonadal impairment,
substantially sized cohorts with well-documented clinical
as well as treatment data are required. In addition, inde-
pendent replication cohorts must be available to validate
the results. One GWAS [20] has been performed (with
Affymetrix 6.0 SNP array) in the St. Jude Lifetime Cohort
Study (SJLIFE) among 799 ethnically mixed female CCS,
which included an independent replication cohort (geno-
typed with the Illumina Omni5 SNP array) of 1624 women
from the ethnically mixed Childhood Cancer Survivor
Study (CCSS). This GWAS did not identify a genome wide
significant hit, but found a SNP (rs9999820) that was bor-
derline significantly associated (p=3.3*10
7
)withanin-
creased risk of premature menopause, especially in the
subgroup of CCS who had undergone ovarian irradiation.
This haplotype, consisting of 4 SNPs, is associated with in-
creased hippocampal NPYR2 gene expresssion, which is as-
sociated with a neuroendocrine pathway [20]. Noteworthy
is that this GWAS evaluated the genetic variation in (self--
reported) premature menopause, the latest manifestation of
gonadal impairment or ageing.
The PanCareLIFE initiative, a 5-year (20138) EU Frame-
work 7 Programme in the Health Theme originating from
the PanCare project, focuses on the identification of deter-
minants of long-term health of CCS. Specifically, PanCare-
LIFE will evaluate female gonadal impairment, hearing, and
quality of life. Investigators from sixteen partner institutions
from ten European countries have prospectively and retro-
spectively collected data from over 12,000 survivors from
cancer diagnosed before they were 25 years of age.
The current study is part of this European wide
endeavor and focuses on the identification of genetic fac-
tors which play a role in the risk of treatment-induced
gonadal impairment among female childhood cancer
survivors. Its specific objectives are to validate previously
identified genetic polymorphisms associated with
gonadal impairment in female childhood cancer survi-
vors, using a candidate gene approach; and to identify
novel SNPs that are independently associated with
chemotherapy induced gonadal impairment in female
childhood cancer survivors, using a GWAS.
Methods
Inclusion criteria
For the current study we included female adult survivors
(18 years) of childhood cancer, diagnosed before the age
of 25 years, with a follow-up time of at least 5 years after
diagnosis. Eligible survivors had to have been treated with
chemotherapy. Exclusion criteria included radiotherapy
involving both ovaries, defined as bilateral irradiation of
the abdomen below the pelvic crest, or radiotherapy in-
volving the pituitary, defined as cranial or craniospinal ir-
radiation. Furthermore, survivors were not eligible if they
had undergone myeloablative allogeneic stem cell trans-
plantation, with or without total body irradiation.
Study cohort
PanCareLIFE consists of 8 work packages of which 5
focus on scientific work. Work package 4 encompasses
van der Kooi et al. BMC Cancer (2018) 18:930 Page 2 of 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
two parts: WP4a focuses on genetic variation in gonadal
impairment, and WP4b focuses on genetic variation in
ototoxicity. This study addresses work package 4a. For this
work package, adult female CCS were recruited in ten
institutions from seven countries (Fig. 1). The
participating institutions and included numbers were: the
Dutch Childhood Oncology Group (AMC, EMC, LUMC,
UMCG, UMCN, VUmc) (inclusions n= 306), Erasmus
Medical Center Rotterdam (n= 25) and VU Medical Cen-
ter Amsterdam (n= 19) from the Netherlands, Fakultni
Nemocnice Brno (n= 134) and Fakultni Nemocnice v
Motole (n= 86) from Czech Republic, Oslo University
Hospital Departments of Oncology/ Pediatrics (n=107)
from Norway, I.R.C.C.S. Giannina Gaslini (n= 67) from
Italy, Department of Paediatric Oncology/University
Hospital, St-Etienne (n= 64) from France, University
Hospital Muenster (n= 39) from Germany and Sheba
Medical Center (n= 18) from Israel. In total 865 CCS were
included in this study. DNA samples could not be col-
lected in 28 cases, leaving 837 CCS for analysis (Table 1).
Medical ethics approval for the study was obtained
from all relevant local committees and written informed
consent was obtained from all participants.
Data collection
Basic demographic data of all participants (month and
year of birth and of follow-up), diagnostic data (month
and year of diagnosis, type of diagnosis) and full details
of cancer treatment were retrospectively collected from
medical databases and medical records. Data on cancer
treatment comprised of details on surgery, chemother-
apy and radiotherapy, including start and stop dates and
cumulative dosage. All data will be merged at the central
data center in Mainz likewise a former EU funded sister
project PanCareSurFup [21], and will finally be pseudo-
nymized for the investigators of this study.
Gonadal function
The primary outcome of this study is AMH level. Serum
samples were centrifuged, stored at 20 °C and shipped
on dry ice to the VUmc Amsterdam where all AMH
levels were analysed in the same laboratory using an
ultra-sensitive Elecsys AMH assay (Roche Diagnostics
GmbH, Mannheim, Germany) at one time point. Data
on AMH levels were sent to the central data center in
Mainz and merged into the central database and subse-
quently pseudonymized to the investigators. In addition
to the continuous AMH levels, patients will be divided
in two groups based on AMH levels considered relevant
as a proxy for gonadal impairment, considering data on
AMH levels in healthy females measured with the same
assay in the reference laboratory in the VUmc
Amsterdam. These details will be described in detail in
the forthcoming manuscript. In addition, detailed
Fig. 1 Participating institutions throughout Europe and Israel
van der Kooi et al. BMC Cancer (2018) 18:930 Page 3 of 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
information about menstrual history, and/or FSH level,
and/or information on usage of artificial reproductive
techniques will be used to evaluate gonadal impairment.
Genotyping
Blood or saliva samples were obtained for DNA isola-
tion. Blood samples (n= 781) were stored at ≤−20 °C
and shipped on dry ice while saliva kits (n= 56) were
stored and shipped at room temperature. Genomic DNA
was extracted by the salting-out method. The choice of
genotyping array was made after extensive comparison
between all currently available arrays. The Infinium®
Global Screening Array was chosen based on the rich
up-to-date content and its suitability for GWAS includ-
ing rare variants, while also containing clinically relevant
content, including pharmacogenetics.
Statistical considerations
For the GWAS a genetic sample size calculation was
performed to estimate the number of cases required in
the current study [22]. As it is impossible to estimate
the allelic frequencies in our population, the following
assumptions were made for the power calculation: 1) a
high risk allele frequency of 0.2, 2) a genome-wide sig-
nificant significance level (5*10
8
), 3) a cohort size larger
than n= 800 and 4) a case to control ratio of 1:2. Based
on these assumptions, we determined that the number
of recruited patients provided statistical power (80%) to
identify variants with an odds ratio of at least 1.8.
Quality control and imputations
A quality control (QC) protocol containing multiple filters
will be applied to clean the genetic data and to ensure its
quality prior to either imputations or analysis [23]. Both a
SNP and individual call rate filter of 97.5% will be applied
to remove poorly genotyped SNPs and individuals from
the data. Furthermore, a Hardy-Weinberg Equilibrium test
(significance level < 1*10
7
) will be employed to remove
variants containing potential genotyping errors. To ensure
sample quality, samples with extreme heterozygosity,
gender mismatches, and familial relationships will be
assessed and removed. Genetic ancestry of the samples
will be assessed and corrected for using principal
components (PCs).
Finally, imputations will be performed using the
Michigan Imputation Server using default settings [24].
The reference panel chosen for imputations is the
Haplotype Reference Consortium (HRC r1.1) [25]. The
same approach has previously been used in large-scale
population studies such as the Rotterdam Study [26] and
Generation R [27].
Association analysis
For the candidate gene approach we will extract the
genotypes of a list of predetermined SNPs based on pub-
lished literature. The Mann-Whitney U test and the
Kruskal-Wallis test will be employed to compare the dis-
tribution between groups with continuous data. Logistic
regression will be performed to calculate the odds ratio
and 95% confidence interval of the SNPs to assess their
risk of gonadal impairment. This model will adjust for
several confounders: principal component analysis
(PCA) will be used to correct for population stratifica-
tion by modelling ancestry differences between cases
and controls [28]. PCA is a common tool that has been
widely used for the combined analysis of correlated phe-
notypes in genetic linkage and association studies [29].
Furthermore, the model will adjust for cyclophospha-
mide equivalent dose (CED). This measure enables com-
parison of alkylating agent exposure independent of
drug dose distribution within a particular cohort (as the
formerly used alkylating agent dose), permitting
Table 1 Data providing institutions in genetic work package on gonadal impairment
Country Data provider Treatment data DNA samples
The Netherlands Dutch Childhood Oncology Group 306 298
Erasmus Medical Center, Rotterdam 25 24
VU Medical Center, Amsterdam 19 18
Czech Republic Fakultni Nemocnice Brno 134 132
Fakultni Nemocnice v Motole 86 81
Norway Oslo University Hospital 107 107
Italy I.R.C.C.S. Giannina Gaslini 67 64
France Center Hospitalier Universitaire de Saint-Étienne 64 58
Germany University Hospital Muenster, Germany 39 37
Israel Sheba Medical Center, Tel Hashomer 18 18
TOTAL 865 837
Dutch Childhood Oncology Group: Academic Medical Center in Amsterdam (AMC), Erasmus Medical Center in Rotterdam (EMC), Leiden University Medical Center
(LUMC), University Medical Center Groningen (UMCG), University Medical Center Nijmegen (UMCN), VU Medical Center (VUmc)
van der Kooi et al. BMC Cancer (2018) 18:930 Page 4 of 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
comparison across different cohorts [30]. In addition,
linear regression will be performed to calculate the effect
of the SNPs on continuous AMH levels. This model will
include age, in addition to the principal components and
CED. The modifying effect of genetic predisposition on
the association between CED and gonadal impairment
will be also explored.
To identify relevant SNPs from the GWAS that
may be important but do not reach genome-wide sig-
nificance, we will use a suggestive significance level of
p=5.10
6
. After GWAS analysis, we will use the R
script EasyQC [31] to clean the association results
based, amongst others, on minor allele frequency and
imputation quality. The results will then be visualized
and the functional annotation for all leading SNPs
will be identified using the online platform called
Functional Mapping and Annotation of GWAS
(FUMA-GWAS) [32].
Replication
For both the candidate gene approach and GWAS, to
ensure that associations are not a chance finding or an
artifact due to uncontrolled biases, associations will be
replicated within a replication cohort, based on the St.
Jude Lifetime Cohort Study (SJLIFE) from St. Jude
Childrens Research Hospital, Memphis USA [33,34]
and CCSS cohort.
Discussion
This paper outlines the design of one study within the
PanCareLIFE initiative that has two separate research
aims. Female CCS from ten different institutions from
seven European countries will be included to validate
previously identified genetic polymorphisms associated
with gonadal impairment and to identify novel SNPs that
are independently associated with chemotherapy in-
duced gonadal impairment in female CCS.
Sufficiently-sized cohorts are of key importance in
genetic association studies in order to have adequate
power to identify low-risk variants. This is especially of
importance in the evaluation of common traits such as
gonadal function, where many common variants may
operate with small effect sizes. To this end, we per-
formed a power calculation to estimate the required co-
hort size for the current study, based on the estimated
allelic frequency in our population.
It is standard practice in current genetic association
studies to include an independent replication cohort to
validate findings from the initial discovery cohort. How-
ever, few large cohorts exist that have sufficient numbers
of female CCS, let alone with complete data as well as
stored DNA and AMH for analysis. For this project, a
collaboration with the St. Jude Childrens Research Hos-
pital, Memphis USA and CCSS has been initiated. AMH
levels will be measured in the same laboratory with the
same AMH assay for the discovery and replication co-
hort, thus minimizing lab variation. Given the (non-sig-
nificant) GWAS observations in the St Jude discovery
cohort we believe forces must be joined, and we are
therefore actively looking for additional cohorts to in-
clude in this and future international collaborations. We
encourage readers who are aware of such collections to
contact the corresponding author.
Gonadal impairment in CCS can be defined in many
ways [3537], and especially in international collabora-
tions a clear consensus on the definition, as objective as
possible, is needed. A separate work package within the
PanCareLIFE consortium will combine seven criteria
and several different questionnaires to assess clinical go-
nadal status in 20,000 subjects. For the current study,
the primary endpoint AMH was chosen, which will be
evaluated both linear as categorized. The secondary end-
point is gonadal impairment based on detailed informa-
tion about menstrual history, FSH levels and
information on usage of artificial reproductive tech-
niques. AMH has the advantage to be as objective as
possible, in comparison to questionnaire data that may
be prone to recall bias or incorrect information given by
the survivor. In addition, AMH can serve as a reliable
surrogate marker for ovarian function while the primor-
dial follicle pool is not yet depleted [38,39]. The only re-
ported GWAS investigating therapy induced fertility
impairment in CCS, used premature menopause as pri-
mary outcome (clinically assessed in the discovery co-
hort and self-reported in the replication cohort) [20].
Prior to the clinical manifestation of amenorrhea and in-
creased levels of FSH, impaired gonadal function can be
detected by the measurement of lower serum AMH
levels [40]. AMH in females is produced solely in the
ovary by granulosa cells of small growing follicles and is
considered a surrogate marker for ovarian function and
ovarian reserve [38,39]. Like the primordial follicle pool,
AMH levels decrease from adolescence on, until meno-
pause occurs. Even survivors who do not report prema-
ture menopause (or Primary Ovarian Insufficiency, POI,
defined as menopause before the age of 40 years) can
still have a poor ovarian function, potentially resulting in
reduced fertility or a shorter reproductive window (e.g.,
early menopause or menopause between 40 and
45 years). This impairment of gonadal function can be
identified by the evaluation of AMH levels.
In conclusion, we describe the design of a genetic as-
sociation study that will evaluate the association of gen-
etic variability with gonadal impairment in a European
cohort of childhood cancer survivors, with AMH levels
as the primary outcome measure. This international col-
laboration will enhance knowledge of genetic variation
which may be included in risk prediction models for
van der Kooi et al. BMC Cancer (2018) 18:930 Page 5 of 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
gonadal impairment in CCS. In the future, patients with
childhood cancer, parents and survivors may benefit
from better individualized counselling concerning future
fertility options and necessity for fertility preservation.
Abbreviations
AMH: Anti-Müllerian hormone; CCS: Childhood cancer survivors;
CED: Cyclophosphamide equivalent dose; FSH: Follicle stimulating hormone;
GWAS: Genome wide association study; PCA: Principal component analysis;
SJLIFE: St. Jude Lifetime Cohort Study; SNP: Single nucleotide polymorphism
Acknowledgements
We thank all participating survivors of childhood cancer, without whom this study
would not be possible. In addition to the co-authors of this paper, the
PanCareLIFE consortium includes the following project partners: Pintail
Ltd., Dublin, Ireland (Kylie OBrien), Universität zu Lübeck, Germany (Thorsten
Langer), Charité Universitätsmedizin Berlin, Germany (Anja Borgmann-Staudt),
Westfälische Wilhelms-Universität Münster, Germany (Antoinette am Zehnhoff-
Dinnesen), Universität Bern, Switzerland (Claudia Kuehni) and Universitaetsklini-
kum Bonn, Bonn, Germany (Katja Baust). Data is, in addition to the institutions
of the co-authors, provided by: Netherlands Cancer Institute (Floor van Leeu-
wen), Helios Kliniken Berlin Buch, Germany (Gabriele Strauss), Istituto Giannina
Gaslini, Genoa, Italy (Dr. R Haupt, Dr. M-L Garré), Great Ormond Street Hospital
(Alison Leiper), Medizinische Universität Graz, Austria (H Lackner), Uniwersytet
Medyczny w Białymstoku, Bialystok, Poland (Anna Panasiuk, Maryna Krawczuk-
Rybak), Heinrich Heine Universität Düsseldorf, Germany (Marina Kunstreich), Uni-
versität Ulm, Germany (Holger Cario), Klinikum Stuttgart, Olgahospital, Stuttgart,
Germany (Stefan Bielack), Uniwersytet Gdánski, Poland (Joanna Stefanowicz),
University College London Hospital, UK (Victoria Grandage).
Funding
This work was supported by the PanCareLIFE project that has received
funding from the European Unions Seventh Framework Programme for
research, technological development and demonstration under grant
agreement no 602030.
UD: German Cancer Aid grant 108128 to UD; ERA-Net-TRANSCAN consor-
tium, project number 01KT1310, and Euro Ewing Consortium EEC, project
number EU-FP7 602856, both funded under the European Commission
Seventh Framework Program FP7-HEALTH; Stiftung für krebskranke
Kinder in Essen. The funding bodies did not have a role in the design or
collection of data, neither will they have in analysis and interpretation of
data.
Authorscontributions
AvdK wrote the manuscript. AvdK and EC coordinated the study and were
responsible for study logistics. MvdB, CB, GC, UD, AR, JF, SF, RH, TK, JK, EvD
and DM coordinated the study locally and were responsible for study
logistics, patient recruitment, and data collection at the various institutions.
JB, HC, DG, MK, CS and PK were involved in coordination and management
of the central data center and/or PanCareLIFE. LB and OZ were involved in
aspects of genetic statistical analyses. JL, LK, EvD, AU were involved in
aspects of conceptualization and study design. MvdH is the principal
investigator. All authors revised the manuscript critically for intellectual
content and have given final approval of the final manuscript.
Competing interest
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The PanCareLIFE study has been approved by the local ethics committees:
Kantonale Ethikkommission Bern, 362/2015; Comitate Etico Regionale,
507REG2014; Ethical Committee University Hospital Bno, June 11, 2016; Ethics
Committee Fakultni Nemocnice v Motole, Prague; De Videnskabsetiske
Komiteer Region Hovedstaden, H-1-2014-125; Ethikkommission Medizinische
Universität Graz, 27015 ex 14/15; Ethikkommission der Universität Ulm, 160/
17; Ethikkommission der Universität zu Lübeck, 14/181; Ethik-Kommission der
Ärztekammer Westfalen-Lippe und der Westfälischen Wilhelms-Universität
Münster, 2014619; Medische Ethische Toetsings Commissie Erasmus MC;
Medisch Ethische Toetsingscommissie, 2015_202. Written informed consent
was obtained from all participants.
Consent for publication
Not applicable.
PublishersNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Division of Reproductive Endocrinology and Infertility, Department of
Obstetrics and Gynecology, Erasmus MC Sophia Childrens Hospital,
Rotterdam, The Netherlands.
2
Department of Pediatric Hematology and
Oncology, Erasmus MC Sophia Childrens Hospital, Rotterdam, The
Netherlands.
3
Princess Máxima Center for Pediatric Oncology, Lundlaan 6,
3584, EA, Utrecht, The Netherlands.
4
Department of Internal Medicine,
Erasmus MC, Rotterdam, The Netherlands.
5
Institute of Pharmacology of
Natural Products and Clinical Pharmacology, University Hospital Ulm, Ulm,
Germany.
6
Boyne Research Institute, Drogheda, Ireland.
7
Department of
Pediatric Hematology and Oncology, VU Medical Center, Amsterdam, The
Netherlands.
8
Department of Paediatric Oncology, University Hospital,
St-Etienne, France.
9
Epidemiology of Childhood and Adolescent Cancers,
CRESS, INSERM, UMR 1153, Paris Descartes University, Villejuif, France.
10
Department of Paediatric Haematology and Oncology, University Childrens
Hospital Bonn, University of Bonn Medical School, Bonn, Germany.
11
Pediatrics III, West German Cancer Centre, University Hospital Essen, Essen,
Germany.
12
German Cancer Research Centre, DKTK, sites Bonn and Essen,
Germany.
13
Danish Cancer Society Research Center, Copenhagen, Denmark.
14
Department of Clinical Medicine, Faculty of Health, Aarhus University,
Aarhus, Denmark.
15
Department of Oncology, Oslo University Hospital, Oslo,
Norway.
16
German Childhood Cancer Registry, Institute of Medical
Biostatistics, Epidemiology and Informatics, University Medical Center, Mainz,
Germany.
17
Epidemiology and Biostatistics Unit, Istituto Giannina Gaslini,
Genoa, Italy.
18
Czech Republic & International Clinical Research Center
(FNUSA-ICRC), University Hospital Brno, Brno, Czech Republic.
19
Department
of Pediatrics, Academic Medical Center, Emma Childrens Hospital,
Amsterdam, The Netherlands.
20
Motol University Hospital, Prague, Czech
Republic.
21
Chaim Sheba Medical Center, The Edmond and Lily Safra
Childrens Hospital, Tel Hashomer, Israel.
22
Sackler Faculty of Medicine,
Tel-Aviv University, Tel-Aviv, Israel.
Received: 19 March 2018 Accepted: 18 September 2018
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... Advances in childhood cancer treatment have increased cancer survival rates leading to a growing population of childhood cancer survivors (CCS) (Trama et al., 2016). Abdominal-pelvic radiotherapy and alkylating agents may compromise ovarian function (Green et al., 2009;Overbeek et al., 2017;van der Kooi et al., 2017) and reduce survivors' reproductive window. This may manifest as sub-or infertility (Chow et al., 2016;Anderson et al., 2018) and a higher risk of premature menopause (Levine et al., 2018), which in turn may impair quality of life (Langeveld et al., 2004;van den Berg et al., 2007;Duffy and Allen, 2009;Carter et al., 2010;Zebrack et al., 2013;van der Kooi et al., 2019a). ...
... Serum levels of anti-Müllerian hormone (AMH), produced by the granulosa cells of small growing follicles in the ovaries, are related to age at onset of menopause in healthy women (van Disseldorp et al., 2008) and can detect ovarian dysfunction prior to both detectible changes in FSH/LH or oestrogen and clinical manifestations of menopause (van Beek et al., 2007;Nelson et al., 2011;Anderson et al., 2012;Dewailly et al., 2014). In addition, AMH has been demonstrated as a useful and early surrogate marker of reduced ovarian function in cancer survivors (van Beek et al., 2007;Lie et al., 2009;Charpentier et al., 2014;Lunsford et al., 2014;van den Berg et al., 2018;van der Kooi et al., 2019b). ...
... Another strength of this study is the measurement of AMH levels, as a marker for reduced ovarian function, with the same assay at one laboratory, eliminating betweenassay differences. Previous studies demonstrated that alkylating agents are strongly associated with risk of reduced ovarian function as measured by decreased AMH levels in female CCS (Anderson et al., 2012;Thomas-Teinturier et al., 2015;van der Kooi et al., 2017;van den Berg et al., 2018). By using AMH levels as a marker of ovarian function, this study included a fairly substantial number of cases likely at increased risk of reduced fertility or a shorter reproductive window. ...
Article
Full-text available
STUDY QUESTION Do genetic variations in the DNA damage response pathway modify the adverse effect of alkylating agents on ovarian function in female childhood cancer survivors (CCS)? SUMMARY ANSWER Female CCS carrying a common BR serine/threonine kinase 1 (BRSK1) gene variant appear to be at 2.5-fold increased odds of reduced ovarian function after treatment with high doses of alkylating chemotherapy. WHAT IS KNOWN ALREADY Female CCS show large inter-individual variability in the impact of DNA-damaging alkylating chemotherapy, given as treatment of childhood cancer, on adult ovarian function. Genetic variants in DNA repair genes affecting ovarian function might explain this variability. STUDY DESIGN, SIZE, DURATION CCS for the discovery cohort were identified from the Dutch Childhood Oncology Group (DCOG) LATER VEVO-study, a multi-centre retrospective cohort study evaluating fertility, ovarian reserve and risk of premature menopause among adult female 5-year survivors of childhood cancer. Female 5-year CCS, diagnosed with cancer and treated with chemotherapy before the age of 25 years, and aged 18 years or older at time of study were enrolled in the current study. Results from the discovery Dutch DCOG-LATER VEVO cohort (n = 285) were validated in the pan-European PanCareLIFE (n = 465) and the USA-based St. Jude Lifetime Cohort (n = 391). PARTICIPANTS/MATERIALS, SETTING, METHODS To evaluate ovarian function, anti-Müllerian hormone (AMH) levels were assessed in both the discovery cohort and the replication cohorts. Using additive genetic models in linear and logistic regression, five genetic variants involved in DNA damage response were analysed in relation to cyclophosphamide equivalent dose (CED) score and their impact on ovarian function. Results were then examined using fixed-effect meta-analysis. MAIN RESULTS AND THE ROLE OF CHANCE Meta-analysis across the three independent cohorts showed a significant interaction effect (P = 3.0 × 10−4) between rs11668344 of BRSK1 (allele frequency = 0.34) among CCS treated with high-dose alkylating agents (CED score ≥8000 mg/m2), resulting in a 2.5-fold increased odds of a reduced ovarian function (lowest AMH tertile) for CCS carrying one G allele compared to CCS without this allele (odds ratio genotype AA: 2.01 vs AG: 5.00). LIMITATIONS, REASONS FOR CAUTION While low AMH levels can also identify poor responders in assisted reproductive technology, it needs to be emphasized that AMH remains a surrogate marker of ovarian function. WIDER IMPLICATIONS OF THE FINDINGS Further research, validating our findings and identifying additional risk-contributing genetic variants, may enable individualized counselling regarding treatment-related risks and necessity of fertility preservation procedures in girls with cancer. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the PanCareLIFE project that has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 602030. In addition, the DCOG-LATER VEVO study was funded by the Dutch Cancer Society (Grant no. VU 2006-3622) and by the Children Cancer Free Foundation (Project no. 20) and the St Jude Lifetime cohort study by NCI U01 CA195547. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.
... Thorough evaluation of possible genetic determinants mandates large cohorts and independent replication cohorts. Here, we aimed to evaluate the associations between poly-morphisms in candidate genes of CYP enzymes and treatment-related gonadal impairment that have been previously identified in adult patients receiving cyclophosphamide [18,19], in the largest European cohort of CCSs with available DNA [26], and to independently replicate findings within the St. Jude Lifetime Cohort Study (SJLIFE) cohort [27]. ...
... Exclusion criteria included: history of bilateral ovarian radiotherapy (defined as bilateral irradiation of the abdomen below the pelvic/iliac crest), central nervous system (CNS) irradiation, total body irradiation (TBI), or stem cell transplantation. Further details of the study protocol have been published previously [26]. ...
... So far, large-scale GWAS have identified several SNPs, such as rs11668344 (BRSK1), rs365132 (UIMC1) and rs16991615 (MCM8), relevant for age at natural menopause or premature ovarian insufficiency (POI) in the general population [12,[60][61][62][63][64][65]. Results of a European GWAS study in CCSs exploring genetic susceptibility of cancer treatment-related gonadal damage in girls are currently pending [26,28]. ...
Article
Full-text available
Background: Female childhood cancer survivors (CCSs) carry a risk of therapy-related gonadal dysfunction. Alkylating agents (AA) are well-established risk factors, yet inter-individual variability in ovarian function is observed. Polymorphisms in CYP450 enzymes may explain this variability in AA-induced ovarian damage. We aimed to evaluate associations between previously identified genetic polymorphisms in CYP450 enzymes and AA-related ovarian function among adult CCSs. Methods: Anti-Müllerian hormone (AMH) levels served as a proxy for ovarian function in a discovery cohort of adult female CCSs, from the pan-European PanCareLIFE cohort (n = 743; age (years): median 25.8, interquartile range (IQR) 22.1-30.6). Using two additive genetic models in linear and logistic regression, nine genetic variants in three CYP450 enzymes were analyzed in relation to cyclophosphamide equivalent dose (CED) score and their impact on AMH levels. The main model evaluated the effect of the variant on AMH and the interaction model evaluated the modifying effect of the variant on the impact of CED score on log-transformed AMH levels. Results were validated, and meta-analysis performed, using the USA-based St. Jude Lifetime Cohort (n = 391; age (years): median 31.3, IQR 26.6-37.4). Results: CYP3A4*3 was significantly associated with AMH levels in the discovery and replication cohort. Meta-analysis revealed a significant main deleterious effect (Beta (95% CI): -0.706 (-1.11--0.298), p-value = 7 × 10-4) of CYP3A4*3 (rs4986910) on log-transformed AMH levels. CYP2B6*2 (rs8192709) showed a significant protective interaction effect (Beta (95% CI): 0.527 (0.126-0.928), p-value = 0.01) on log-transformed AMH levels in CCSs receiving more than 8000 mg/m2 CED. Conclusions: Female CCSs CYP3A4*3 carriers had significantly lower AMH levels, and CYP2B6*2 may have a protective effect on AMH levels. Identification of risk-contributing variants may improve individualized counselling regarding the treatment-related risk of infertility and fertility preservation options.
... For a practical example on array choice in a new study, we would like to focus on the PanCareLife study [42,43]. In the PanCareLife study, we were interested in both pharmacogenetics as well as GWAS, in order to study the late-life effects of treatment in childhood cancer survivors. ...
Article
Full-text available
Array technology to genotype single-nucleotide variants (SNVs) is widely used in genome-wide association studies (GWAS), clinical diagnostics, and linkage studies. Arrays have undergone a tremendous growth in both number and content over recent years making a comprehensive comparison all the more important. We have compared 28 genotyping arrays on their overall content, genome-wide coverage, imputation quality, presence of known GWAS loci, mtDNA variants and clinically relevant genes (i.e., American College of Medical Genetics (ACMG) actionable genes, pharmacogenetic genes, human leukocyte antigen (HLA) genes and SNV density). Our comparison shows that genome-wide coverage is highly correlated with the number of SNVs on the array but does not correlate with imputation quality, which is the main determinant of GWAS usability. Average imputation quality for all tested arrays was similar for European and African populations, indicating that this is not a good criterion for choosing a genotyping array. Rather, the additional content on the array, such as pharmacogenetics or HLA variants, should be the deciding factor. As the research question of a study will in large part determine which class of genes are of interest, there is not just one perfect array for all different research questions. This study can thus help as a guideline to determine which array best suits a study’s requirements.
... Thus, PanCareLIFE will help to quantify the toll of cancer treatment in relation to specific deficits. Some results were published at virtually the same time as the project ended (e.g., [24][25][26]). ...
Article
Full-text available
PanCareLIFE brought together European partners and is the largest study to have evaluated the issues of fertility impairment, hearing loss, and health-related quality of life in survivors of childhood and adolescent cancer. Successful delivery of the project aims did not evolve solely from scientific qualities. Organizational structure and careful information management were key components for its successful completion and are retrospectively assessed in this paper. PanCareLIFE used cohort studies, case-control studies, clinical evaluation of hearing, and genetic testing to study 32,000 survivors from 25 data providers. A management team implemented the organizational structures, was the decision making body, developed and maintained a communication plan, and supervised deadlines, and made timely decisions. A biostatistics support group and an ethical advisory board were established. A publication committee ensured quality and accuracy of publications and is jointly responsible for the sustainability of the project. The chosen management structure of PanCareLIFE can serve as a blueprint for the management of complex international projects. Apart from the survivors themselves, various target audiences like oncology researchers, health care providers, and policy makers can derive benefits from the project. The results can also be used in oncological frontline therapy to reduce toxicity.
... This issue is being addressed in the PanCareLIFE study, Europe's largest clinical cohort to study the impact of treatment regimens on the quality of life of CCSs. 10 van Santen et al What should be done when potential for future fertility is questioned? Referral to gynecology/reproductive medicine/endocrinology according to local referral pathways) is recommended for postpubertal female CCSs treated with potentially gonadotoxic chemotherapy and/or ovarian irradiation a without signs and symptoms of POI who desire assessment about potential for future fertility (expert opinion/no literature search). ...
Article
Full-text available
Gonadal dysfunction and infertility after cancer treatment are major concerns for childhood cancer survivors and their parents. Uncertainty about fertility or being diagnosed with infertility has a negative impact on quality of survival. In this article, determinants of gonadal damage are reviewed and consequences for fertility and pregnancies are discussed. Recommendations for screening and treatment of gonadal function are provided. These should enable timely treatment of gonadal insufficiency aiming to improve linear growth, pubertal development, and sexual functioning. Options for fertility preservation are discussed.
... Several studies used pediatric cancer registries to perform genomic research including pharmacogenomics, germline predisposition, and genetic risk scoring. The PanCareLIFE registry, which represents childhood cancer survivors across Europe treated with cisplatin, carboplatin, or cranial radiotherapy, performed genotyping to assess for gene variants associated with ototoxicity [47] and infertility [48] . The TARGET database was also used in one study by Kim et al in conjunction with the International Pleuropulmonary Blastoma/DICER1 Registry to study for the presence of germline DICER1 mutations in various types of pediatric cancer [49] . ...
Article
Infertility is a serious early, as well as late effect of childhood cancer treatment. If addressed timely at diagnosis, fertility preservation measures can be taken, preferably before start of cancer treatment. However, pediatric oncologists may remain reluctant to offer counseling on fertility preservation methods, while infrastructure to freeze ovarian tissue has become available and is currently considered standard care for pre- and post-pubertal girls at high risk of gonadal damage. More importantly, risk factors have been identified for cancer treatment-related impairment of gonadal function, and the first successful pregnancies have been reported after auto-transplanted ovarian tissue, which has been harvested from children. Additionally, great progress has been made in the field of ex vivo maturation of oocytes in frozen ovarian tissue, which provides opportunities for those at risk of ovarian micrometastasis. Hence, it is time to counsel girls at risk and make every effort to cryopreserve their ovarian tissue, now more than ever before.
Article
The survival of childhood Wilms tumor is currently around 90%, with many survivors reaching reproductive age. Chemotherapy and radiotherapy are established risk factors for gonadal damage and are used in both COG and SIOP Wilms tumor treatment protocols. The risk of infertility in Wilms tumor patients is low but increases with intensification of treatment including the use of alkylating agents, whole abdominal radiation or radiotherapy to the pelvis. Both COG and SIOP protocols aim to limit the use of gonadotoxic treatment, but unfortunately this cannot be avoided in all patients. Infertility is considered one of the most important late effects of childhood cancer treatment by patients and their families. Thus, timely discussion of gonadal damage risk and fertility preservation options is important. Additionally, irrespective of the choice for preservation, consultation with a fertility preservation (FP) team is associated with decreased patient and family regret and better quality of life. Current guidelines recommend early discussion of the impact of therapy on potential fertility. Since most patients with Wilms tumors are pre‐pubertal, potential FP methods for this group are still considered experimental. There are no proven methods for FP for pre‐pubertal males (testicular biopsy for cryopreservation is experimental), and there is just a single option for pre‐pubertal females (ovarian tissue cryopreservation), posing both technical and ethical challenges. Identification of genetic markers of susceptibility to gonadotoxic therapy may help to stratify patient risk of gonadal damage and identify patients most likely to benefit from FP methods.
Article
With an improvement in the survival rate of cancer patients, chemotherapy-induced premature ovarian insufficiency (POI) is increasingly affecting the quality of life of female patients. Currently, there are many relevant studies using mice as an animal model. However, a large coefficient of variation for weight in mice is not appropriate for endocrine-related studies, compared with rats; therefore, it is necessary to identify an appropriate experimental model in rats. In this study, cyclophosphamide combined with busulfan was used to establish an animal model. We compared several common modeling methods using chemotherapeutic drugs, cisplatin, cyclophosphamide, and 4-vinylcyclohexene diepoxide (VCD), and we found that the combination of cyclophosphamide and busulfan was more effective in establishing a POI model in rats with few side effects by analyzing general physical conditions, pathological tissue sections of heart, liver, lung, spleen, kidney, uterus, and ovary, serum hormone levels, and follicle counts; thus, providing a more reliable model basis for subsequent studies.
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The timing of puberty is a highly polygenic childhood trait that is epidemiologically associated with various adult diseases. Using 1000 Genomes Project-imputed genotype data in up to ∼370,000 women, we identify 389 independent signals (P < 5 × 10(-8)) for age at menarche, a milestone in female pubertal development. In Icelandic data, these signals explain ∼7.4% of the population variance in age at menarche, corresponding to ∼25% of the estimated heritability. We implicate ∼250 genes via coding variation or associated expression, demonstrating significant enrichment in neural tissues. Rare variants near the imprinted genes MKRN3 and DLK1 were identified, exhibiting large effects when paternally inherited. Mendelian randomization analyses suggest causal inverse associations, independent of body mass index (BMI), between puberty timing and risks for breast and endometrial cancers in women and prostate cancer in men. In aggregate, our findings highlight the complexity of the genetic regulation of puberty timing and support causal links with cancer susceptibility.
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