STUDY QUESTION: What is the impact of the EuroNet-PHL-C2 treatment for boys with classical Hodgkin lymphoma (cHL) on semen parameters? SUMMARY ANSWER: More than half of the patients (52%, n = 16/31) had oligozoospermia or azoospermia at 2 years from cHL diagnosis; particularly boys treated for advanced-stage cHL had low sperm counts and motility. WHAT IS KNOWN ALREADY: Chemotherapy and radiotherapy to the inguinal region or testes can impair spermatogenesis and result in reduced fertility. The EuroNet-PHL-C2 trial aims to minimize radiotherapy in standard childhood cHL treatment, by intensifying chemotherapy. The present study aims to assess the (gonadotoxic) impact of this treatment protocol on semen parameters and reproductive hormones in boys aged ≤18 years. STUDY DESIGN, SIZE, DURATION: This international, prospective, multi-centre cohort study was an add-on study to the randomized phase-3 EuroNet-PHL-C2 trial, where the efficacy of standard cHL treatment with OEPA-COPDAC-28 (OEPA: vincristine, etoposide, prednisone, and doxorubicin; COPDAC-28: cyclophosphamide, vincristine, prednisone, and dacarbazine) was compared to intensified OEPA-DECOPDAC-21 chemotherapy (DECOPDAC-21: COPDAC with additional doxorubicin and etoposide and 25% more cyclophosphamide). Patients were recruited between January 2017 and September 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligibility criteria included male patients, diagnosed with classical HL before or at the age of 18 years, and treated according to the EuroNet-PHL-C2 protocol in any of the 18 participating sites in the Netherlands, Germany, Belgium, Czech Republic, and Austria. Sperm parameters (sperm concentration, progressive motility, sperm volume, and calculated total motile sperm count) were assessed at diagnosis and 2 years after diagnosis in (post)pubertal boys. Laboratory measurements (serum follicle-stimulating hormone (FSH) and inhibin B) were performed in samples drawn at diagnosis, during treatment (2-3 times), and at 2 years post-diagnosis, and (age-adjusted) analyses were conducted separately for pre-pubertal and (post)pubertal boys. Outcomes were compared between the treatment levels (TL1, TL2, and TL3) and consolidation treatment schemes (COPDAC-28 and DECOPDAC-21). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 101 boys were included in the present analysis: 73 were (post)pubertal (median age 15.4 years, (IQR 14.4; 16.6), 10 TL1, 29 TL2, 34 TL3, 62% of TL2/3 patients received COPDAC-28) and 28 boys were pre-pubertal (median age 9.6 years (IQR 6.6; 11.4), 4 TL1, 7 TL2, 17 TL3, 38% of TL2/3 patients received COPDAC-28). The study included six boys who had received pelvic radiotherapy; none were irradiated in the inguinal or testicular area. At diagnosis, 48 (post)pubertal boys delivered semen for cryopreservation; 19 (40%) semen samples were oligospermic and 4 (8%) were azoospermic. Low sperm concentration (<15 mil/ml) appeared to be related to the HL disease itself, with a higher prevalence in boys who presented with B symptoms (76% vs 26%, aOR 2.3 (95% CI 1.0; 3.8), P = 0.001) compared to those without such symptoms. At 2 -years post-diagnosis, 31 boys provided semen samples for analysis, of whom 12 (39%) boys had oligozoospermia and 4 (13%) had azoospermia, while 22 boys (71%) had low total motile sperm counts (TMSC) (<20 mil). Specifically, the eight boys in the TL3 group treated with DECOPDAC-21 consolidation had low sperm counts and low progressive motility after 2 years (i.e. median sperm count 1.4 mil/ml (IQR <0.1; 5.3), n = 7 (88%), low sperm concentration, low median progressive motility 16.5% (IQR 0.0; 51.2), respectively). Age-adjusted serum FSH levels were significantly raised and inhibin B levels (and inhibin B:FSH ratios) were decreased during chemotherapy in (post)pubertal boys, with subsequent normalization in 80% (for FSH) and 60% (for inhibin B) of boys after 2 years. Only 4 out of the 14 (post)pubertal boys (29%) with low sperm concentrations after 2 years had elevated FSH (>7.6 IU/l), while 7 (50%) had low inhibin B levels (<100 ng/l). In pre-pubertal boys, reproductive hormones were low overall and remained relatively stable during chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The present analyses included sperm and laboratory measurements up to 2 years post-diagnosis. Long-term reproductive outcomes and potential recovery of spermatogenesis remain unknown, while recovery was reported up to 5- or even 10-year post-chemotherapy in previous studies.Boys who were pre-pubertal at diagnosis were still too young and/or physically not able to deliver semen after 2 years and we could not assess a potential difference in gonadotoxicity according to pubertal state at the time of treatment. Overall, the statistical power of the analyses on sperm concentration and quality after 2 years was limited. WIDER IMPLICATIONS OF THE FINDINGS: Results of the semen analyses conducted among the 31 boys who had provided a semen sample at 2 years post-treatment were generally poor. However, additional long-term and adequately powered data are crucial to assess the potential recovery and clinical impact on fertility. The participating boys will be invited to deliver a semen sample after 5 years. Until these data become available, benefits of intensified chemotherapy in cHL treatment to reduce radiotherapy and lower risk for development of secondary tumours should be carefully weighed against potentially increased risk of other late effects, such as diminished fertility due to the increased chemotherapy burden. Boys with newly diagnosed cHL should be encouraged to deliver sperm for cryopreservation whenever possible. However, patients and clinicians should also realize that the overall state of disease and inflammatory milieu of cHL can negatively affect sperm quality and thereby reduce chance of successful fertility preservation. Furthermore, the measurement of FSH and inhibin B appears to be of low value in predicting low sperm quality at two years from cHL treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Dutch charity foundation KiKa (project 257) that funds research on all forms of childhood cancer. C.M.-K., D.K., W.H.W., D.H., MC, A.U., and A.B. were involved in the development of the EuroNet-PHL-C2 regimen. The other authors declare no potential conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

• Klíčová slova
• azoospermia, childhood Hodgkin lymphoma, fertility, follicle-stimulating hormone, gonadotoxicity,
• MeSH
• analýza spermatu * MeSH
• azoospermie farmakoterapie   MeSH
• cyklofosfamid terapeutické užití   MeSH
• dakarbazin terapeutické užití   MeSH
• dítě MeSH
• doxorubicin terapeutické užití   škodlivé účinky   MeSH
• etoposid terapeutické užití   aplikace a dávkování   MeSH
• folikuly stimulující hormon krev   MeSH
• Hodgkinova nemoc * farmakoterapie   metabolismus   MeSH
• inhibiny krev   MeSH
• lidé MeSH
• mladiství MeSH
• motilita spermií účinky léků   MeSH
• oligospermie farmakoterapie   MeSH
• počet spermií MeSH
• pohlavní hormony * metabolismus   MeSH
• prednison terapeutické užití   aplikace a dávkování   MeSH
• předškolní dítě MeSH
• prospektivní studie MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   škodlivé účinky   MeSH
• vinkristin terapeutické užití   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze III MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• cyklofosfamid MeSH
• dakarbazin MeSH
• doxorubicin MeSH
• etoposid MeSH
• folikuly stimulující hormon MeSH
• inhibin B MeSH Prohlížeč
• inhibiny MeSH
• pohlavní hormony * MeSH
• prednison MeSH
• vinkristin MeSH

STUDY QUESTION: What is the impact of the EuroNet-PHL-C2 treatment protocol for children with classical Hodgkin lymphoma (cHL) on gonadal function in girls, based on assessment of serum anti-Müllerian hormone (AMH)? SUMMARY ANSWER: Serum AMH levels decreased after induction chemotherapy and increased during subsequent treatment and 2 years of follow-up, with lowest levels in patients treated for advanced stage cHL. WHAT IS KNOWN ALREADY: Treatment for cHL, particularly alkylating agents and pelvic irradiation, can be gonadotoxic and result in premature reduction of primordial follicles in females. The current EuroNet-PHL-C2 trial aims to reduce the use of radiotherapy in standard childhood cHL treatment, by intensifying chemotherapy. This study aims to assess the gonadotoxic effect of the EuroNet-PHL-C2 protocol. STUDY DESIGN, SIZE, DURATION: This international, prospective, multicenter cohort study is embedded in the EuroNet-PHL-C2 trial, an European phase-3 treatment study evaluating the efficacy of standard cHL treatment with OEPA-COPDAC-28 (OEPA: vincristine, etoposide, prednisone, and doxorubicin; COPDAC-28: cyclophosphamide, vincristine, prednisone, and dacarbazine) versus intensified OEPA-DECOPDAC-21 (DECOPDAC-21: COPDAC with additional doxorubicin and etoposide and 25% more cyclophosphamide) in a randomized setting. Participants were recruited between January 2017 and September 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Female patients aged ≤18 years, treated according to the EuroNet-PHL-C2 protocol for cHL were recruited across 18 sites in the Netherlands, Belgium, Germany, Austria, and Czech Republic. All parents and patients (aged ≥12 years old) provided written informed consent. Serum AMH levels and menstrual cycle characteristics were evaluated over time (at diagnosis, one to three times during treatment and 2 up to 5 years post-diagnosis) and compared between treatment-levels (TL1, TL2, and TL3) and treatment-arms (OEPA-COPDAC-28 and OEPA-DECOPDAC-21). Serum samples obtained from patients after receiving pelvic radiotherapy were excluded from the main analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 104 females, with median age at diagnosis of 15.6 years (IQR 13.7; 17.0), were included in the analysis. Ninety-nine were (post)pubertal. Eighteen girls were diagnosed with an early stage of cHL (TL1) and 86 with intermediate or advanced stage disease (50 TL2 and 36 TL3, 66% received COPDAC-28 and 34% DECOPDAC-21). Five patients received pelvic radiotherapy. Median AMH level at diagnosis was 1.7 µg/l (IQR 0.9; 2.7). After two courses of OEPA chemotherapy, AMH levels decreased substantially in all patients (98% <0.5 µg/l), followed by a significant increase during the consolidation treatment and follow-up. After 2 years, 68% of patients reached their baseline AMH value, with overall median recovery of 129% (IQR 75.0; 208.9) compared to baseline measurement. Five patients (7%) had AMH <0.5 µg/l. In patients treated for advanced stage disease, AMH levels remained significantly lower compared to early- or intermediate stage disease, with median serum AMH of 1.3 µg/l (IQR 0.8; 2.1) after 2 years. Patients who received DECOPDAC-21 consolidation had lower AMH levels during treatment than patients receiving COPDAC-28, but the difference was no longer statistically significant at 2 years post-diagnosis. Of the 35 postmenarchal girls who did not receive hormonal co-treatment, 19 (54%) experienced treatment-induced amenorrhea, two girls had persisting amenorrhea after 2 years. LIMITATIONS, REASONS FOR CAUTION: The studied population comprises young girls with diagnosis of cHL often concurring with pubertal transition, during which AMH levels naturally rise. There was no control population, while the interpretation of AMH as a biomarker during childhood is complex. The state of cHL disease may affect AMH levels at diagnosis, potentially complicating assessment of AMH recovery as a comparison with baseline AMH. The current analysis included data up to 2-5 years post-diagnosis. WIDER IMPLICATIONS OF THE FINDINGS: The current PANCARE guideline advises to use the cyclophosphamide-equivalent dose score (CED-score, as an estimation of cumulative alkylating agent exposure) with a cut-off of 6000 mg/m2 to identify females aged <25 years at high risk of infertility. All treatment-arms of the EuroNet-PHL-C2 protocol remain below this cut-off, and based on this guideline, girls treated for cHL should therefore be considered low-risk of infertility. However, although we observed an increase in AMH after chemotherapy, it should be noted that not all girls recovered to pre-treatment AMH levels, particularly those treated for advanced stages of cHL. It remains unclear how our measurements relate to age-specific expected AMH levels and patterns. Additional (long-term) data are needed to explore clinical reproductive outcomes of survivors treated according to the EuroNet-PHL-C2 protocol. STUDY FUNDING/COMPETING INTEREST(S): The fertility add-on study was funded by the Dutch charity foundation KiKa (project 257) that funds research on all forms of childhood cancer. C.M-K., D.K., W.H.W., D.H., M.C., A.U., and A.B. were involved in the development of the EuroNet-PHL-C2 regimen. The other authors indicated no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

• Klíčová slova
• anti-Müllerian hormone, chemotherapy, childhood Hodgkin lymphoma, gonadotoxicity, late effects, treatment-induced amenorrhea,
• MeSH
• antimülleriánský hormon * krev   MeSH
• cyklofosfamid terapeutické užití   aplikace a dávkování   MeSH
• dítě MeSH
• Hodgkinova nemoc * krev   farmakoterapie   MeSH
• lidé MeSH
• mladiství MeSH
• prospektivní studie MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   škodlivé účinky   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antimülleriánský hormon * MeSH
• cyklofosfamid MeSH

PURPOSE: The purpose of this study is to evaluate the use of fertility-preserving (FP) treatments and fertility counseling that was offered in a cohort of newly diagnosed children with classical Hodgkin lymphoma (cHL). METHODS: In this observational study, boys and girls with cHL aged ≤ 18 years with scheduled treatment according to the EuroNet-PHL-C2 protocol were recruited from 18 sites (5 countries), between January 2017 and September 2021. In 2023, a subset of Dutch participants (aged ≥ 12 years at time of diagnosis) and parents/guardians were surveyed regarding fertility counseling. RESULTS: A total of 101 boys and 104 girls were included. Most post-pubertal boys opted for semen cryopreservation pre-treatment (85% of expected). Invasive FP treatments were occasionally chosen for patients at a relatively low risk of fertility based on scheduled alkylating agent exposure (4/5 testicular biopsy, 4/4 oocyte, and 11/11 ovarian tissue cryopreservation). A total of 17 post-menarchal girls (20%) received GnRH-analogue co-treatment. Furthermore, 33/84 parents and 26/63 patients responded to the questionnaire. Most reported receiving fertility counseling (97%/89%). Statements regarding the timing and content of counseling were generally positive. Parents and patients considered fertility counseling important (94%/87% (strongly agreed) and most expressed concerns about (their child's) fertility (at diagnosis 69%/46%, at present: 59%/42%). CONCLUSION: Systematic fertility counseling is crucial for all pediatric cHL patients and their families. FP treatment should be considered depending on the anticipated risk and patient factors. We encourage the development of a decision aid for FP in pediatric oncology.

• Klíčová slova
• childhood Hodgkin lymphoma, fertility counseling, fertility preservation,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• anti-Müllerian hormone, candidate gene approach, chemotherapy, childhood cancer survivors, cytochrome P450 genes, ovarian function,
• Publikační typ
• časopisecké články MeSH