GONADAL FUNCTION AND FERTILITY IN CHILDREN WITH HODGKIN LYMPHOMA TREATED ACCORDING TO THE EURONET-PHL-C2 PROTOCOL

Survival for children with Hodgkin lymphoma (HL) has increased rapidly over the
years and with current multimodal treatment protocols over 90% survives (1).
However, late effects of treatment, such as second malignant neoplasms,
infertility and premature menopause, are of great concern given their impact on
future health and quality of life. Young cancer survivors, particularly those
with lymphoma diagnosis or radiotherapy to the pelvis (23,34), have an
increased risk of infertility (> 1 year of unsuccessful attempts at conception)
compared with sibling controls. In addition, a premature menopause is a
well-recognised effect of HL treatment (19, 20, 22) which is very relevant in
the light of todays trend to postpone childbearing to the early thirties. Risk
factors for impaired gonadal function include abdominal/pelvic radiotherapy
(23), TBI, and treatment with alkylating agents (Procarbazine in particular)
and the risk increases with cumulative dose (2-5), and older age at time of
treatment in females (6). In adult males with HL, alterations of sperm
characteristics have been described already before treatment (24,25). Potential
causes described are fever (26), stress (27) or inflammatory immune-mediated
mechanisms (28). Twelve months after treatment containing doxorubicin and
Dacarbazine, sperm count recovered to pretreatment values but this was not the
case for patients receiving chemotherapy courses containing cyclophosphamide or
Procarbazine. 24 months post-treatment, azoospermia rates were 22 % and 50 %
respectively (29). After treatment with BEACOPP (containing Cyclophosphamide
and Procarbazine) even 89% of HL patients had azoospermia (30). However,
studies on fertility in boys with HL are lacking. In order to reduce the risk
of adverse late effects, pediatric HL treatment protocols have been adapted
over the years. The use of radiotherapy (RT) was limited in order to prevent
its described secondary malignancies and Procarbazine was replaced by
Dacarbazine to reduce gonadotoxicity (1). Studies in adult HL patients,
however, have shown that gonadal damage was still present after treatment with
Dacarbazine in a subset of patients (3). Since pelvic radiotherapy increases
the risk of infertility (22), it is important to study whether radiotherapy can
be omitted in patients with HL without compromising survival. The current
EuroNet-PHL-C2 treatment protocol will answer that important question in a
subgroup of patients. In this protocol, the use of radiotherapy will be reduced
in the intensified chemotherapy arm (DECOPDAC-21) in contrast with standard
COPDAC-28 courses (for EuroNet-PHL-C2 protocol see figure 1 and 2). However,
the impact of the intensified chemotherapy on gonadal function is unknown. The
intensified DECOPDAC courses are given every 3 weeks instead of every 4 weeks
in the standard COPDAC-arm, allowing less time for gonadal recovery.
Furthermore DECOPDAC-21 consists of a higher dose of (gonadotoxic)
Cyclophosfamide, in addition to Etoposide and Doxorubicin and should therefore
be investigated. The gold standard for assessing gonadal function in
(post)pubertal males is sperm analysis. However, in prepubertal boys and in
boys who are not able to provide a semen sample, surrogate markers are often
used including pubertal staging and serum hormone levels of which the Inhibin
B/FSH ratio (IFR) has shown to be the best marker of male infertility (9,10).
Direct assessment of gonadal function in females is impossible and relies
solely on a combination of surrogate tests. Menstrual history, serum levels of
early follicular phase FSH, estradiol, and Inhibin B, and ultrasound based
antral follicle counts (AFC) and ovarian volume measurements are commonly used
(11-13). However, all these measurements require specific timing within the
menstrual cycle and in girls taking oral contraceptives their value is limited
(37). Recently, anti-Mu*llerian hormone (AMH) has emerged as a promising marker
of ovarian reserve (11-16,35). This hormone is presumed cycle independent, is
stable from 8-25 years (17), has shown to decrease over time in normo-ovulatory
women, long before changes in serum FSH, E2 and inhibin B (12-14), and is the
only current marker reported to be useful in the assessment of ovarian reserve
in prepubertal girls receiving cancer treatment (18). To preserve fertility,
sperm may be banked for those young boys who are physically and emotionally
able. In adolescent females, cryopreservation of oocytes or ovarian tissue are
available options although still experimental and not possible in all centers.
In girls who need to be irradiated at the pelvic region, ovariopexy can protect
the ovaries and preserve gonadal function. Some oncologists administer oral
contraceptives or GnRH analogues as co-treatment to preserve fertility. It is
hypothesized that resting follicles are more resistant to CT and that the
hypo-estrogenic state may decrease utero-ovarian perfusion, resulting in a
decreased exposure of the ovaries to chemotherapy (7, 8). However, reported
effects of GnRH-a on ovarian function are contradicting and should be evaluated
further. In summary, since the use of (gonadotoxic) chemotherapy will be
intensified in the new EuroNet-PHL-C2 protocol, this study will prospectively
study the overall, and treatment arm specific, gonadotoxic potential of the
protocol in boys and girls treated for HL in Europe. In addition, the frequency
and effect of various methods applied as co-treatment to preserve fertility
will be evaluated.

The primary aim of this study is to assess the overall, and treatment arm
specific, gonadotoxic potential of the protocol in both boys and girls treated
for classical Hodgkin Lymphoma in Europe.
Secondary aim is to evaluate the frequency of various methods applied as
co-treatment to preserve fertility in European countries; if number are
sufficient, the effect of these methods on gonadal function will be assessed
subsequently.

More specifically, primarily this study:

1) will prospectively evaluate the gonadotoxic effect of 2, 3, 4 or 6 cycles of
chemotherapy (with or without RT) in boys and girls treated for Hodgkin
Lymphoma according to EuroNet-PHL-C2 protocol.
2) will evaluate the difference in gonadotoxicity between OEPA-COPDAC-28 and
OEPA-DECOPDAC-21 (with or without RT) in both boys and girls treated according
to the EuroNet-PHL-C2 protocol.

Secondarily, the current study:
3) will assess the frequency of oral contraceptives or GnRH-a co-treatment
and/or ovariopexy, given as part of standard patient care in a subset of
patients. Subsequently, in case of sufficient numbers, the impact of these
methods on gonadal function will be studied in young females.
4) will investigate the frequency of sperm banking in young boys, the
differences in frequency between countries, and the reasons for doing or not
doing so.
5) will investigate the frequency of cryopreservation of ovarian cortical
tissue or oocytes in young females, the differences in frequency between
countries, and the reasons for doing or not doing so.
6) will evaluate patient and parental experience on provided fertility care
and counseling in a subset of patients (patients treated in the Netherlands).

General study design and study population This study is set up as a European,
multi-center, prospective, observational cohort study, in children treated for
classical Hodgkin Lymphoma according to the EuroNet-PHL-C2 protocol between
2016 and 2021. Children diagnosed with Hodgkin Lymphoma before the age of 18
years, in participating centers for pediatric oncology in 5 European countries,
will be asked to participate in the study after approval of the
country-specific ethics committee. The study will be initiated in the
Netherlands and Belgium first and then expanded to Austria, Czech Republic and
Germany. In order to perform all follow-up measurements of this study (see
table 1 below), patients will be included during a period of five years
(2017-2021). It is expected that approximately 700 patients will be eligible
during the 5 year inclusion period, based on number of patients diagnosed per
country: Netherlands: 27 patients/year, Belgium 11 patients/year, Germany 140
patients/year, Czech Republic 20 patients/year and Austria 20 patients/year.
With a participation rate of approximately 50%, we expect to include over 300
newly diagnosed patients. The number of semen samples collected, however, will
be lower than 150, i.e. in prepubertal boys it is not possible, but also some
pubertal boys who are willing to provide a sample, will not be able to do so;
the estimated % of boys who will provide semen for analysis is considered to be
approximately 30%. Written informed consent for participation in the current
study will be obtained from all patients and their parents prior to inclusion
in the study. Participation in the study does not require extra visits for the
patients to the hospital. Data collection Relevant clinical data according to
the EuroNET-PHL-C2 protocol will be collected for all children with HL treated
according to this protocol by the participating institutes of approximately 20
different European countries using CRFs. These data will be entered and stored
in a central database at the EuroNet-PHL study office in Leipzig using the
infrastructure which was already in place for the data collection relevant for
the previous EuroNet-PHL-C1 protocol. The EuroNet-PHL-C2 database will contain
all clinical data, for instance treatment center and country, date of birth,
gender, date of diagnosis, stage of disease, tumor site and regions involved
(nodes and extra nodal involvement), treatment level (1,2, or 3), randomization
group, detailed treatment data (chemotherapy per cycle: the dates and doses of
each chemotherapeutic agent administered, RT: dose at each radiation site),
dates of recurrence, dates of diagnosis of second malignancies, medical
history, toxicity/complications. Furthermore, MRI*s of children will be stored
at the central EuroNet-PHL Study office, University Leipzig for central review
of the response assessment of the Hodgkin treatment (EuroNet-PHL-C2 protocol).
For the current study, relevant clinical data of all patients will be extracted
from the central EuroNet-PHL-C2 study database. R elevant data: • Treatment
center and country, date of birth, gender • Date of diagnosis, stage of
disease, tumor site and regions involved (nodes and extra nodal involvement) •
Treatment level (1,2, or 3), randomization group, detailed treatment data
(chemotherapy per cycle: the dates and doses of each chemotherapeutic agent
administered; RT: dose at each radiation site) • Dates of recurrence, dates of
diagnosis of second malignancies • Medical history Furthermore, additional data
for this study will be collected by the centers participating in the study on
additional CRF*s from diagnosis onwards until 5 years post-diagnosis/the age of
18 years (T5, see table 1). Data will be collected during regular visits in the
context of patient care and/or will be retrieved from medical records. In
girls, completed CRF*s will contain information on • menstrual cycle (menarche
yes/no, length and regularity of menstrual cycle) • use of hormonal
contraception(if yes, type, dose and frequency will be recorded) • GnRH-a co
treatment (if yes, type, dose and frequency will be recorded) • menopausal
status of the mother, and age at menopause if applicable. • height, weight ,
pubertal stage according to Tanner criteria, • smoking, drinking alcohol, •
whether ovariopexy (when iliac/pelvic lymph node region is to be irradiated)
was performed, • whether ovarian cortex or oocytes were harvested for
cryopreservation (yes/no, if no: why not), For boys, data will be collected
accordingly on • height, weight , pubertal stage according to Tanner criteria,
• testicular volume measured with the Prader orchidometer, • smoking, drinking
alcohol, • Whether or not sperm is banked for fertility preservation and if not
(while Tanner stage 4 and/or testicular volume >15 mls as measured by Prader
orchidometer), why not . These data will be sent to VU University medical
Center and stored anonymously in the statistical (SPSS) database. In order to
be able to trace data to an individual subject, a subject identification code
list will be used which will be safeguarded by the principal investigator. To
accomplish the highest achievable participation rates and to ensure solid data
collection, the National chair of participating countries will recruit local
champions/dedicated pediatric oncologists who will keep in close contact with
the PhD student as well as the PI*s. The PhD student will provide the relevant
CRF*s to the local champions well timed and will be responsible for the return
of the CRF*s and proper storage of the data. A core working group consisting of
the PI*s and the local champions/dedicated oncologists will be established,
monitoring of the progress and content of the study on a regular basis i.e.
accrual charts for each institution. Blood sampling Blood samples from both
boys and girls will be collected during regular visits at several time points
to assess in blood values over time within and between treatment arms (table
1). In females 6 ml of extra blood will be drawn for measurement of
anti-Mullerian hormone (AMH); timing within the menstrual cycle i.a. will be
documented. In males 6 ml of extra blood will be drawn to assess serum levels
of Inhibin B, and FSH. Blood for AMH (in girls only) and FSH and Inhibin B (in
boys) should be collected in a serum-separating tube. To enhance and accelerate
the clotting process, invert the tube at least 6-12 times immediately upon
filling. Leave the tube for an hour to coagulate. After 1 hour the tube should
be spinned for 10 minutes 1800g. Then the serum should be divided between 3
tubes, each containing at least 500-750 µL serum (and labelled with date of
blood sampling and unique patient number ). Serum should subsequently be frozen
at -20 °C locally in each participating center. Frozen sera will be transported
(frozen; on dry ice) in batch to VUmc for central evaluation on set occasions.
In all children some serum and blood (DNA) will be stored for future research
into reproductive function and ovarian reserve outcomes. Ovarian volume, uterus
dimensions and testicular volume: In girls, ovarian volume as well as uterus
dimensions (max length and max width) will be assessed retrospectively from
MRI*s that are performed in the context of patient care by the PhD appointed to
the project. This in order to assess changes over time within treatment arms
and between treatment arms. In boys, these MRI*s will be used to measure
testicular volume. These MRI*s are stored for central review of response
assessment of the Hodgkin disease at the central EuroNet-PHL Study office,
University Leipzig. Semen sampling: All young boys who are physically and
emotionally able to provide a semen sample will be asked to do so on three
occasions during the study (before treatment and 2&5 years post diagnosis;
table 1). Semen analysis will be performed on site in fresh semen. Semen
samples will be evaluated for sperm volume, sperm count, sperm forward motility
according to World Health Organisation guidelines. Conditions of disturbed
sperm quality will include hypospermia (volume <1.5 mls), oligozoospermia
(sperm concentration <15 x 106/ml), asthenozoospermia (sperm with forward
motility < 32 %), and theratozoospermia (sperm with normal morphology <4 %).
Total motile sperm count will be calculated by multiplying the sample volume by
the sperm concentration and the % motile sperm (31) since this is considered as
the best indicator of male infertility. Fertility questionnaire A subset of
patients (included in the Netherlands, provided separate informed consent) will
receive a fertility questionnaire during follow-up. The questionnaire will be
directed to the parents/guardians of the patients, containing questions on: -
fertility counseling: was fertility counseling offered, and if so: what was the
timing, and who was the interlocutor (pediatric oncologist, specialized nurse,
gynecologist/urologist) - fertility preservation: was fertility preservation
offered and if so: did they eventually choose to perform a treatment -
participation of the child: did their child actively participate in the
fertility counseling and were they incolved in the decision-process whether to
use available fertility preservation methods - satisfaction and experience: do
they feel like they received sufficient information on fertility and available
fertility preservation methods. In addition, patients who were aged 12 years or
older at time of diagnosis will be asked to fill in a short questionnaire
themselves. Patients will be asked whether they can remember conversations
about fertility, how they experienced these conversations (was it important to
them and was it understandable) and whether they currently of formertly feel
worried about their fertility. Parents of boys and girls will receive distinct
versions of the questionnaire. The questionnaire will be sent out at a single
timepoint (2023), at approximately 1.5-3 years post-diagnosis for most
patients. One full-time researcher (PhD.-student: *AIO*) is requested for a
period of four years. Under the supervision of the principal investigators, the
researcher will coordinate and conduct the study and will be responsible for
the day-to-day management of the project.

The current study will be performed in children with Hodgkin disease and who
will be treated according to the upcoming EuroNet-PHL-C2 protocol. This
EuroNet-PHL-C2 protocol aims to reduce the use of radiotherapy in order to
reduce secondary malignancies by intensifying the use of (gonadotoxic)
chemotherapy. It is unknown whether this will lead to (increase of) gonadal
damage. This is particularly relevant to the population of HL patients of whom
the majority will survive. Therefore this European, multi center, prospective,
observational study will be performed. Data will be collected before start of
treatment as well as during and following treatment during regular visits to
the hospital. The risks associated with participation in the current study can
be considered negligible and the patient burden low. For the blood samples only
some extra blood (max 1-2 ml per sample, maximum 7 samples) will be drawn. We
do not expect to induce a lot of stress in boys when a second or third semen
sample, 2/5 years after treatment will be requested.
A subset of patients aged 12 years or older (at time of diagnosis) and parents
will be asked to fill in a paper questionnaire during follow-up at a single
time point (Dutch participants who provided informed consent to participate in
this survey). Questions can be considered personal as they comprise patient
experience and satisfaction. Participants are allowed to skip questions if they
do not wish to answer certain questions. Nevertheless, the questionnaire is
short (time to complete is estimated at 10-15 for parents and 5 minutes for
children) and mostly contains multiple choice questions.

This study will reveal the overall, and treatment arm specific, gonadotoxic
potential of the EuroNET-PHL-C2 treatment protocol in both boys and girls
treated for classical Hodgkin Lymphoma in Europe, and will provide information
on whether and which patients are at risk of gonadal damage, which has been
associated with a reduced quality of life if a survivor finds him or herself
unable to procreate later in life.
In addition, the frequency of various methods applied as co-treatment in
females (GnRH-analogues, oral contraceptives and/or ovariopexy) to preserve
fertility will be evaluated and when numbers are sufficient the effect of these
methods on gonadal function will be studied. Furthermore, this will evaluate
the frequency of banking of sperm, cryopreservation of ovarian cortical tissue
or oocytes, and will provide insight in reasons for not doing so. Data on
patient- and parental experience on provided fertility care and counseling will
be collected in a subset of patients (patients treated in the Netherlands)

The results of the current study will allow us to better inform future patients
at risk of (persistent) gonadal damage and their parents about the risk of the
current Hodgkin lymphoma treatment and methods that can be applied to preserve
fertility. In addition, it can help us to inform current female survivors at
risk for a premature menopause about their possible reduced fertile life span,
which may have implications for the timing of parenthood if they wish to have
children in the future. This is particularly relevant given the trend of
postponing childbearing to the early thirties in several countries throughout
Europe. Also, if fertility appears to be reduced after treatment in women who
still have a natural cycle there may still be options for fertility
preservation in survivorship. Fertility preservation in survivorship might help
alleviate the distress often associated with the urgency to start a family due
to an imminent premature menopause, particularly if a woman is still very young
and/or without a partner. When gonadal damage will be demonstrated in the boys
who underwent treatment according to the EuroNet-PHL-C2 protocol, they might be
reassured because their semen was also cryopreserved prior to treatment.
Furthermore, if we find a protective effect of hormonal contraception,
GnRH-analogues or ovariopexy in females, these co-treatments should be
implemented in girls as standard supportive care during Hodgkin treatment.
Moreover, if we find that methods that can be used to preserve fertility are
applied infrequently in patients at risk of gonadal damage, this information
will be used to inform and educate pediatric oncologists.
All in all, the risks associated with participation in the current study can be
considered negligible and the patient burden low. Given this negligible risk
and low burden, and the fact that this study has the potential to have major
consequences for future HL patients as well as for current HL survivors, this
study is considered to have a favourable risk-benefit assessment .