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White paper: Oncofertility in pediatric patients with Wilms tumor

Abstract 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 prepubertal, potential FP methods for this group are still considered experimental. There are no proven methods for FP for prepubertal males (testicular biopsy for cryopreservation is experimental), and there is just a single option for prepubertal 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.


| INTRODUCTION
The survival rate of childhood cancer has increased tremendously over the past decades. Since the overall survival of patients with Wilms tumor (WT) is currently around 90%, nearly all patients treated for WT reach reproductive age and thus the impact of therapy on future fertility must be considered. [1][2][3][4][5] Chemotherapy and radiotherapy are established risk factors for gonadal damage 6,7 and both may be part of WT treatment. 8,9 Globally, most patients have been treated according to protocols from either the National Wilms Tumor Study Group (NWTSG)/Children's Oncology Group (COG) or Societe Internationale D'oncologie Pediatrique (SIOP). [10][11][12][13] Although most patients diagnosed with WT are prepubertal, fertility preservation (FP) options have recently become available for young patients. However, since some of these FP methods are still experimental, they have been largely reserved for patients at high risk of gonadal damage. [14][15][16][17][18][19] This manuscript aims to provide an overview of the available evidence on the risk of gonadal damage after WT treatment, including the patient perspective, the options for fertility preservation, ethical and genetic considerations and recommendations concerning FP in patients with WT.

| OVERVIEW OF THE ISSUE OF FERTILITY IMPORTANCE TO CANCER PATIENTS
When considering FP for pediatric cancer patients, it is vital to understand the patient and family vantage point (Table S1). While future fertility is generally important to most patients and caregivers, FP is not universally discussed nor undertaken prior to initiation of oncologic therapy, as the immediate focus is on achieving cure. Unfortunately, if FP is not discussed prior to treatment, this may negatively impact the utilization and success rate of FP techniques.
Several surveys have identified attitudes of parents of children with cancer as well as the adolescent and young adult (AYA) patient population toward FP in the setting of a cancer diagnosis. [20][21][22] These surveys uncovered that nearly all AYA patients and parents are aware of a significant risk of infertility related to cancer therapy, and that FP is important to most of this population. However, only about 20% were willing to take actions toward preserving fertility. 21 This finding is known as the intention-behavior gap. Nearly half of AYA patients reported limited access, such as being unaware of the options and/or cost concerns, as the reason for not making FP arrangements despite financial support by philanthropic organizations, or public or private health plans being regionally variably available. Insurance coverage of FP costs is usually limited to the procedure and not the storage of gametes, and some insurance plans may not cover all of the procedure costs. Health-related concerns are prevalent and impair access to FP, noted by about one third of male AYA patients, and over half of female AYAs. These concerns include personally not wanting to delay treatment, physician advising against treatment delay, and concerns about the effect of cancer therapy on future offspring. Personal reasons such as not wanting children or feeling too young to consider such a decision were also noted in about a third of patients. 22 Research studying shared decision making in adolescents and parents of young patients with WT is lacking.
In addition, it is well-established that many patients regret deciding not to pursue FP. 20,23,24 The level of regret tends to be higher among those who believe that the opportunity for FP was not discussed or was discussed at a time when it was too late to effectively act on it. 20 Over half of surveyed patients reported feeling a moderate to high amount of concern that infertility has negatively affected their emotions, relationships, and feeling of self-worth. Additionally, patients who identified themselves as having higher concerns about fertility were more likely to suffer from depression and lower-quality of life. 25 It has been reported that most male cancer patients/survivors do not feel sufficiently informed and postpubertal boys strongly desire information on FP options. 26,27 Similarly, female cancer patients feel it is important to discuss fertility, preferably shortly after diagnosis. [28][29][30][31][32] A recently published guideline by the International Guideline Harmonization Group (IGHG) states that current standard care should include informing all pediatric cancer patients and their families on their relative risk of gonadal damage, including the low-risk group. 10,11,33 As such, counseling is paramount and clear discussion of the experimental nature of any intervention must be emphasized.
Taken together, these findings underscore that FP is important to pediatric cancer patients and survivors, and should be discussed earlyon, when options for FP are greatest. The intention-behavior gap highlights the importance of providing adequate counseling, to support the desire for FP, and help develop that into a behavior that accomplishes that goal when feasible. While not all children and their parents will elect to proceed with FP, there is strong evidence that future regret is greatly reduced when families feel that they have made an informed decision. 25,34 3 | OVERVIEW OF WT THERAPY AND ONCOLOGIC OUTCOMES Although most patients with WT are cured with surgery and two-drug chemotherapy with very low gonadotoxic potential, 9 almost all patients with relapse will be exposed to intensive therapy, typically including alkylating agents. Notably these patients require counseling again at the time of relapse, providing an opportune time to discuss FP options. 17 Four-year event free survival (EFS) for Stages II to IV anaplastic WT with current COG/SIOP treatment regimens is 68%, 35 and long-term survival for higher risk (HR) relapsed favorable histology Wilms tumor (FHWT) who were previously treated with the combination of vincristine, dactinomycin and doxorubicin is around 50%. 36 The evolution of risk stratification has outlined subgroups of WT patients for whom reduction of therapy decreases long-term treatment-related morbidity exemplified by patients with very low risk favorable histology Wilms tumor (FHWT, age <2 years and tumor weight <550 g) who achieved excellent outcomes with surgery alone. 37 On the other hand, both COG and SIOP treatment regimens for patients identified as having HR disease can increase the possibility of infertility, with exposure to alkylators, carboplatin and radiation therapy. COG protocols showed that augmentation of therapy leads to improved outcomes among patients with Stage III and IV FHWT whose tumors harbor combined loss of heterozygosity (LOH) for 1p and 16q, with the use of regimen M (five-drug chemotherapy with vincristine, dactinomycin, doxorubicin, etoposide and cyclophosphamide). 38 SIOP protocols use high-dose doxorubicin, cyclophosphamide, carboplatin and etoposide for HR patients with identified risk factors of postchemotherapy HR histology, incomplete lung metastasis response, and blastemal-predominant histology with high blastemal volume. These therapy regimens have reduced the risk of relapse for these patient groups, avoiding the use of marked intensification of therapy at relapse, but increasing the exposure to gonadotoxic agents during initial therapy. 10,39 Tables 1 and 2 summarize the most recent published outcomes and current treatment protocols from cooperative trials (COG and SIOP) for WT patients.

| GENERAL IMPACT OF WT CHEMOTHERAPY ON FERTILITY
The COG treatment approach to WT comprises upfront tumor resection whenever feasible, usually followed by risk-adapted chemotherapy and, in certain circumstances, radiation treatment. In comparison, apart from specific clinical-radiological features, the SIOP-Renal Tumor Study Group (RTSG) advocates preoperative chemotherapy followed by risk-adapted treatment after surgery. The differences in the COG and SIOP treatment approaches may present different logistic (timing) opportunities for FP. SIOP usually starts preoperative chemotherapy immediately after radiological or histological confirmation. This regimen does not contain gonadotoxic agents so FP is not likely to be needed at that time. Postoperative RT and chemotherapy can usually be well anticipated, allowing a window in time to achieve FP, possibly combined with the tumor nephrectomy. Notably, COG protocols currently mandate initiating chemotherapy within 14 days after surgery/biopsy, and the stage and histology results may only be available after 10 to 12 days. This may leave only a short window for decision-making and FP prior to the start of chemotherapy, even in willing patients and parents. However, prior chemotherapy is not an absolute contra-indication for ovarian tissue cryopreservation and testicular biopsy. The impact of chemotherapy on future fertility is determined by the cumulative doses of chemotherapy agents utilized.  (Tables 3 and 4).
Of note, patients with relapsed WT usually receive doses of cyclophosphamide, ifosfamide, doxorubicin, and radiation therapy, sometimes including a high dose chemotherapy regimen or a stem cell transplant, that places them at high risk of infertility regardless of the specific chemotherapy regimen utilized. 46 Tables 4 and 5 combine the fertility risk associated with both chemotherapy and radiation modalities.

| FERTILITY RISKS FOR FEMALE WT SURVIVORS
In relation to the age of exposure to gonadotoxic agents, ovarian damage from chemotherapeutic agents can result in delayed/absent/ arrested puberty in pre-or peripubertal patients and diminished ovarian reserve (DOR) or premature ovarian insufficiency (POI), and infertility in postpubertal individuals. 47 The evidence describing effects on reproductive outcomes is mostly based on retrospective data, which makes it difficult to determine the exact effects of individual chemotherapy agents. 48 However, alkylating agents, such as cyclophosphamide and ifosfamide, have a clear impact on female reproductive health in a dose-related manner when given either alone or in combination. 49 The most important predictors of risk are the cumulative dose of radiotherapy and alkylating agents, and the patient's age at the time of therapeutic exposure. 50  Lifetime Cohort study of 921 female cancer survivors, POI is associated with administration of high-dose cyclophosphamide. 53 The cyclophosphamide equivalent dose (CED) and alkylating agent dose score (AAD) are two methods used to calculate the cumulative dose of alkylating agents (Text S1). 54,55 Multivariable analysis showed independent associations between POI and CED ≥8000  Note: In Table 2 it is specified to which risk groups the regimens AV, AVD, AV-1, AV-2 and HR are given.  55 The impact was dose related, with fertility decreasing with increased dose. When CED was categorized by quartile, female survivors diagnosed between 1970 and 1999 who were exposed to the upper quartile (≥11 295 mg/m 2 ) had a lower likelihood of pregnancy than those not exposed (HR = 0.85; 95% CI, 0.74-0.98; P = .023). 56 Notably, the evidence regarding the threshold for ovarian damage is scarce, and ranges from 6000 to 12 000 mg/m 2 . 11 Currently a CED score of >6000 mg/m 2 is classified as high risk. 10 reported a significantly decreased likelihood of fathering a child compared with those not exposed. 56 Notably, although irreversible gonadal impairment may occur, in some patients with azoospermia before or after treatment, recovery is seen over time in sperm production. 60 Standard of care RT in the COG and SIOP protocols is described in Retrograde ejaculation, anejaculation Erectile dysfunction Azoospermia, oligospermia occur due to radiation and anthracycline induced cardiotoxicity. 18 In SIOP-RTSG, approximately 25% of patients receive abdominal RT. 64 In most patients the pelvic area is not included in the radiation field but 20% of irradiated patients (5% of the total number of patients with WT) are also treated on a HR protocol which includes cyclophosphamide. A radiation boost is delivered to any micro-or macroscopic residual abdominal disease. 65 In SIOP, no radiation is needed in local Stage I with anaplasia. 64,66 Whole lung RT is given for intermediate risk (IR) tumors with no complete remission (CR) after preoperative chemotherapy and/or metastasectomy and to all HR tumors. (Table 6).  68 In the first single center study of VMAT, excellent locoregional control could be achieved by this technique. 69 Unfortunately, gonadal toxicity was not formally assessed in these two papers, but this risk is predicted to be reduced.
There are limited published reports detailing the impact of RT on fertility in WT survivors, and most of these include small patient numbers. In a study of 23 prepubertal children aged 6 months to >4 years following therapy for WT, 1500 to 3000 cGy hemiabdominal RT or WART resulted in serum hormone levels which indicate gonadal damage in both males and females compared with normal controls and those receiving chemotherapy only. 70 An analysis of testicular function of 10 young adult WT survivors who received 268 to 983 cGy to the testes from WART without chemotherapy revealed all of these men to have decreased testicular volume compared to "normal males of the same age" and eight of the nine with evaluable sperm counts had oligo-or azoospermia. 71 The impact of RT prior to puberty on ovarian size, based on ultrasound analysis, was conducted on 18 female WT survivors, 14 of whom were evaluated postpuberty. 72 Of 10 postpubescent females treated with 400 to 4096 cGy flank RT, five had a small or not visible ipsilateral ovary; the ovaries of all three treated with 2100 to 3000 cGy WART were small or not seen. Of note, the uterine length was also decreased in the postpubertal females treated with WART. In general, cases with major tumor rupture that require WART are most at risk. 10,11,17 In a more recent questionnaire-based analysis of male fertility in a large retrospective cohort of 6224 childhood cancer survivors participating in the CCSS, 429 of whom had WT, testicular RT dose >750 cGy was significantly associated with decreased likelihood of being able to establish paternity compared with those not receiving RT. 61 This study identified the subgroup with younger age at diagnosis (0-4 years), in which most WT subjects fall, to be associated with higher likelihood of being able to sire a child. This analysis, however, did not separate survivors by cancer type and is confounded by variable chemotherapy exposure.
In addition to potential impact on gonadal function, late effects of RT to the abdominopelvic region in young children may impair normal growth and development of the irradiated pelvic bones, vasculature and organs including the uterus, which are critical to successful gestation (  77 The relative risk (RR) of perinatal mortality (RR 7.9; P < .0001) and low birthweight (RR 4.0; P < .0001) was significantly higher in the mothers irradiated for WT than expected for pregnancy outcomes for white women in the United States at that time. 77 In another study of 47 WT survivors, 43 of whom received abdominal RT, 78 female WT survivors had a more than 4-fold increased risk of adverse birth outcomes, including preterm birth and birth defects, compared with sibling controls and wives of male WT survivors. The addition of chemotherapy did not modify this risk. Adverse pregnancy outcomes following RT for WT in the above studies have been attributed to uterine fibrosis, impaired placentation, vascular insufficiency, altered bone growth, scarring/adhesions, and/or genitourinary malfunction. 50,[76][77][78] The most important factor for a successful pregnancy after pelvic radiotherapy is the cumulative dose to the ovaries and uterus and the age of the patient at the time of radiation. 74 Prepubertal age at time of antineoplastic therapy exposure has been associated with a lower risk of ovarian failure, with mathematical models suggesting this finding may be due to increased follicular reserve in these very young patients. 55,79 While younger age is considered protective for gonadal damage, the growth and function of the uterus may be impaired due to the radiation. Radiation to a prepubertal uterus may lead to incomplete pubertal growth and development. This may pose difficulties regarding embryo implantation or fetal growth (to term).
Continued efforts to limit RT dose to the adjacent organs-at-risk, including but not limited to the gonads, is essential to improve reproductive success in this patient population. Advances in molecular biology and imaging as well as increased international collaboration between COG and SIOP will be very beneficial in this respect. 80 An example of this partnership is the monthly HARMONIzation and CollAboration (HARMONICA) meetings in which SIOP and COG collaborate on numerous projects including plans for a unified approach to FP in children with renal tumors. Refining flank field and dose exposures in a manner that optimizes cancer control while concurrently limiting gonadal toxicity is also a topic amenable to international discussion. In addition, education and counseling of the parents of these young patients about the risk-benefit ratio of tumor control and late fertility risks, as well as early involvement of endocrinology/ fertility experts, is critical to optimize outcomes and expectations. 81 8 Females with preoperative tumor rupture and most relapsed WT patients are at especially high risk of gonadal damage due to radiation and chemotherapy intensification. In these cases, fertility counseling is mandatory and FP procedures may be considered. Fertility risk is generally triaged early after initial diagnosis, however, sometimes the definitive treatment plan and subsequent gonadal damage risk is determined after the assessment of the initial treatment period. The response determines the treatment intensity and potential impact on Currently, future success of pregnancy after auto-transplantation of prepubertally harvested ovarian tissue is under extensive investigation. [99][100][101] As the effect of OTC on the future ovarian function is still unknown, OTC is limited to patients with a high risk of infertility. 102 Additionally, dormant malignant cells may be present in the harvested ovarian tissue sample, which complicates auto-transplantation. 11 However, promising preclinical research is being conducted to ensure Ovarian tissue cryopreservation F I G U R E 2 Female fertility preservation options. † Only in selected cases receiving abdominal radiotherapy (RT) with an ovary in the RT field. *In rare cases, older patients with a partner may want to opt for embryo cryopreservation. However, for most Wilms tumor patients this will not be an option.^Currently, no strong evidence exists that hormonal suppression has a protective effect on gonadal damage in children. However, it can be used in addition to other fertility preservation methods decision made by parents may be influenced by their own interests. 118 The possibility may arise that the view of a maturing child may differ from the view of his or her parents. Especially in the case of OTC, the fact that 50% of the ovarian reserve is removed and stored, automatically reducing that child's ovarian reserve by 50%, and that future efficacy of use of the material is uncertain need to be considered and weighed. 119

| THE IMPACT OF GENETIC SUSCEPTIBILITY ON FP
The most important known risk factors for treatment-related gonadal damage in WT are use of alkylating agents (boys and girls) and full abdominal radiotherapy (girls). However, previously published work shows that female patients with similar oncologic treatment at the same age can have variable gonadal damage. 81,120 This interindividual variability suggests that genetic factors modify gonadotoxicity of the treatment. 81,120,121 So far, large-scale genome wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs), such as rs11668344 (BRSK1), rs365132 (UIMC1) and rs16991615 (MCM8), relevant for age at natural menopause or POI in the general population. [121][122][123][124][125][126][127] In contrast, only two GWAS studies have been performed to explore genetic susceptibility of cancer treatment-related gonadal damage in girls. [128][129][130] Brooke et al identified and replicated a common haplotype associated with increased prevalence of premature menopause among childhood cancer survivors exposed to ovarian RT. 128 Results of a European GWAS study are currently pending.

BRSK1
(rs11668344) appears to be a relevant SNP in childhood cancer survivors treated with 8000 mg/m 2 cyclophosphamide or more. 120,121 It is hypothesized that the presence of the BRSK1 SNP leads to a less efficient DNA damage response system and this may result in more damage caused by the alkylating agents in healthy cells, including the gonadal cells. 121 In addition, SNPs in cytochrome (CYP) 450 genes have been shown to be associated with cyclophosphamide metabolism and ovarian function, and recently also with anti-Müllerian hormone (AMH) levels in CCSS. [131][132][133] The field of genetic susceptibility as it relates to oncofertility is new and should be further explored.
The role of genetic variation in male infertility in the general population is still unclear and has not been studied extensively in male childhood cancer patients. 134 Although evidence is limited, it should be mentioned during counseling that genetic susceptibility may contribute to the risk of future infertility in childhood cancer when discussing fertility in newly diagnosed and especially relapsed WT patients.

| CONCLUSIONS
Fertility concerns in WT survivors may be related to treatment (surgical intervention, chemotherapy, and/or radiotherapy) or underlying patient-specific risk factors (including syndromes associated with WT development). Fortunately, the reproductive organs are rarely directly affected by surgical intervention for primary WT. Unfortunately, the young age of most children diagnosed with WT makes fertility preservation prior to treatment difficult, although both testicular and ovarian tissue harvest have been described. The absence of effective and widely available methods to preserve fertility in very young patients undergoing cancer treatment may place an emotional burden on families. It should not be assumed that parents will initiate a discussion about fertility preservation and thus, initiation of this discussion by the treating team together with an offer of a consult from an oncofertility team, where available, should be a standard of care for all patients with WT.
In summary, with improved survival for children treated for WT there is an associated risk of late effects including fertility impairment. Refinements in oncologic treatments and an understanding of late effects help to limit morbidity. However, patient, family, and clinician education on fertility preservation in this population is necessary to provide the best and most holistic care possible. Our goal is that this review serves as a statement that we must turn our focus to this area as stated by G.J. D'Angio, "cure is not enough." 135