Oncofertility: State of the art for young people with cancer

Oncofertility: State of the art for young people with cancer Professor W Hamish Wallace Consultant Paediatric Oncologist, Edinburgh Scotland, UK hamish.wallace@nhs.net Swedish Society for Paediatric Oncologists, Stockholm 28 January 2015

Improved Five Year Survival (1966-2000)

Increasing numbers of five year UK survivors by current age Skinner, Wallace & Levitt, Lancet Oncology, 2006

Risk assessment for Fertility preservation Intrinsic factors Heath status of patient Consent (Patient/Parent) Assessment of ovarian reserve Extrinsic factors Nature of predicted treatment High/Medium/Low/Uncertain Risk Time available Expertise available Wallace WH, Critchley HOD & Anderson RA. JCO, 2012

Infertility - Risk Factors Ü RT to HPA or a field that includes testes/ovaries Ü Busulphan Ü BCNU Ü CCNU Ü Cyclophosphamide Ü Ifosfamide Ü Melphalan Ü Mustine Ü Nitrogen mustard Ü Procarbazine Ü Thiotepa Ü Chlorambucil Ü Cytarabine The pre-pubertal gonad is not protected

Radiation-induced ovarian damage Human oocyte (Primordial follicle) Ü LD 50 < 2 Gy Wallace, Thomson, Kelsey. (2003) Hum Reprod.

Risk of infertility Low risk (<20%) Medium risk High risk (>80%) ALL Wilms tumour Brain tumour Sx, RT < 24Gy Soft tissue sarcoma (stage1) Hodgkin s Lymphoma HL(Low stage) AML Osteosarcoma Ewing s sarcoma STS: stage II/III Neuroblastoma NHL Brain tumour RT>24Gy HL (High Stage) Total Body Irradiation Pelvic/testes RT Chemo pre BMT Metastatic Ewing's HL (Pelvic RT) Wallace, Anderson, Irvine. Lancet Oncology 2005

The Wallace-Kelsey Model (Five parameter asymmetric double-gaussian cumulative curve) ESHRE, Lille, 2012 Wallace &Kelsey (2010) PloS ONE

Ovarian reserve: Conception to Menopause Wallace &Kelsey (2010) PloS ONE

Ovarian reserve: Conception to Menopause Bella Anna Wallace &Kelsey (2010) PloS ONE

Effective ovarian sterilizing doses of radiotherapy with increasing age

Prediction of Ovarian Reserve (AMH) Ü Anti Mullerian Hormone (AMH) is an important product of the adult ovary, produced by the granulosa cells of small growing follicles Ü AMH has little variation across and between menstrual cycles Ü AMH is the best currently available marker of the number of small-growing follicles in the ovary Ü But there was no validated reference model for AMH available Anderson, Nelson, Wallace (2011) Maturitas

A validated model of serum anti-mullerian hormone (AMH) from conception to menopause Kelsey et al. PLoS ONE 2011

AMH in childhood cancer AMH (ng/ml) 2.5 2.0 1.5 1.0 0.5 0.0 Pre Cycle 1 Cycle 2 Cycle 3 22 girls age 0.3-15yr 17 prepubertal ** ** *** *** Cycle 4 Cycle 5 Cycle 6 AMH (ng/ml) AMH (ng/ml) Brougham et al 2012 JCE&M 3 2 1 0 3 2 1 0 Medium/low risk Pre End Recovery High risk ** * ** Pre End Recovery

AMH in 3 girls with cancer AMH (ng/ml) 1.0 0.8 0.6 0.4 0.2 0.0 0 25 50 75 3.0 Age 1.2; neuroblastoma Weeks 2.0 1.0 Age 2.4; rhabdomyosarcom 0.0 0 50 100 150 200 2.0 1.5 Age 14.6: Hodgkin s lymphoma 1.0 0.5 0.0 0 50 100 150 Weeks Brougham et al 2012 JCE&M

Summary Ü AMH is detectable before puberty Ü AMH falls rapidly during cancer treatment in both pre-pubertal and pubertal girls Ü AMH levels recover in those patients at low/ medium risk of gonadotoxicity Ü AMH fails to recover in those at high risk. This could be indicative of future reproductive impairment Brougham et al 2012 JCE&M

Pretreatment anti-müllerian hormone predicts for loss of ovarian function after chemotherapy for early breast cancer. sensitivity 98.2% specificity 80.0% for correct classification of amenorrhoea n=75 Anderson and Cameron 2011 JCE&M Anderson et al 2013 Eur J Cancer

Fertility preservation options: established and experimental

FERTILITY RISK ASSESSMENT (Includes Intrinsic and Extrinsic factors) MALE FEMALE Pre-pubertal Pubertal Post-pubertal Patient Assessment Pre-pubertal Post-pubertal Able to produce a suitable semen sample Intervention Ovarian stimulation NO YES Testis biopsy Testis biopsy/ Gamete extraction Ovarian biopsy Partner/ Donor sperm Testis Tissue Cryopreservation Sperm Cryopreservation Storage Ovarian Tissue Cryopreservation Oocyte Cryo Embryo Cryo Experimental Established

Key features of the 3 options for fertility preservation for women Ü Embryo cryopreservation Ü Established but require time and a partner Ü Oocyte cryopreservation Ü Established but require time and hormone stimulation (success rate per oocyte low) Ü Ovarian tissue cryopreservation Ü Minimal delay Ü No lower age limit Ü Surgical procedure Ü Allows for future developments

Ovarian tissue cryopreservation: World- wide experience * At least 39 pregnancies worldwide after othotopic reimplantation of frozen thawed ovarian cortex * Success rate is unclear as the denominator is unknown * No pregnancies reported following the reimplantation of ovarian tissue harvested pre- pubertally * Young children are potentially ideal candidates Donnez, J. & Dolmans, M. M. Nat. Rev. Endocrinol. 9, 735 749 (2013)

Children born from transplanta7on of frozen/thawed ovarian 7ssue 8 8 1 2 5 3 All Normal Babies weight and dura7on Orthotopic >> heterotopic All except for one is a result of a slow- freezing protocol An es7mated excess of 150 transplanta7ons have been performed 3 3 1 3 1 1

Cryopreservation: European experience Ü Three centres ( Denmark, Spain and Belgium) Ü 60 cases of orthotopic reimplantation. Ü Of these women, 11 (21%) became pregnant Ü Six have delivered 12 healthy babies. Ü Restoration of ovarian activity was observed in 93% of the patients between 3.5 months and 6.5 months after grafting Ü The mean duration of ovarian function after transplantation is ~4 5 years but can persist for up to 7 years. Donnez, J. et al. Fertil. Steril. 99, 1503 1513 (2013).

Reduc7on in FSH and LH following transplanta7on of frozen/thawed ovarian 7ssue in Danish pa7ents 100 90 80 70 60 FSH LH IU/L 50 40 30 20 10 0 0 4 8 12 16 20 24 28 32 Weeks a5er transplanta;on

Ovarian Cryopreservation & Ovarian Function Edinburgh experience in children (< 18 yrs) 1996-2012

Cryopreservation of ovarian cortical tissue Edinburgh criteria Selection criteria (1995, modified 2000) Ü Age < 35 years Ü No previous chemotherapy/radiotherapy if age >15 years Ü Mild, non gonadotoxic chemotherapy if < 15 years Ü A realistic chance of surviving five years Ü A high risk of ovarian failure Ü Informed consent (parent and where possible patient) Ü Negative HIV and Hepatitis serology Ü No existing children

The normative validated model of ovarian volume throughout life Kelsey TW, Dodwell SK, Wilkinson AG, Greve T, Andersen CY, et al. (2013) Ovarian Volume throughout Life: A Validated Normative Model. PLoS ONE 8(9): e71465. doi:10.1371/journal.pone.0071465

15 year, population-based analysis of criteria for ovarian cryopreservation Female cancer patients age <18 at diagnosis 01/01/1996-30/6/2012 n = 410 = cryopreservation offered. = reasons for not having tissue cryopreserved. = patients in study eligible for ovarian function evaluation. Offered cryopreservation n = 34 Not offered cryopreservation n = 376 Tissue cryopreserved n = 20 Procedure declined n = 13 Procedure unsuccessful n = 1 Deceased n = 1 Deceased n = 81 <12 years old n = 91 Deceased n = 1 Poor communication n = 1 Uterine factor n = 1 Parental choice n = 2 Too unwell n = 9 On COCP n = 17 <12 years old n = 4 <12 years old n = 1 Deceased n = 3 Still on treatment n = 4 On COCP n = 1 <12 years old n = 2 Lost to follow-up n = 1 n = 14 n = 6 n = 141 Walllace WH et al. 2014 Lancet Oncology Insufficient information on follow-up n = 42 Do the Offered group have a higher prevalence of POI?

Cumulative incidence of POI Not offered Offered 15-year probability 35% [95% CI 10 53] vs 1% [0 2] p<0.0001 Hazard ratio 56.8 [95% CI 6.2 521.6] at 10 years Walllace..and Anderson 2014 Lancet Oncology

Conclusion Ovarian cryopreservation was offered to 9% of our patients, and performed in 5% The procedure was safe and without complications No patients have asked for re- implantation of their tissue to date All patients who have thus far developed premature ovarian insufficiency were identified except one patient The Edinburgh Selection Criteria have proved to be helpful in selecting those patients at highest risk of POI Wallace WH..and Anderson 2014 Lancet Oncology

Reimplantation? Ü It is important to be aware that reimplantation of ovarian cortical tissue is a separate procedure at a time distant from the treatment of the original cancer Ü Consent for harvesting ovarian tissue from children often will have been obtained from their parents Ü Informed consent for reimplantation can be obtained from the patients at a much later date when they are competent to assess the complex issues themselves.

Ewings sarcoma localised T 7 Vertebrae (Age 12) unexpected contamination of ovarian biopsy CD99

Re- implantation or IVG and maturation? Ü Contamination of the cryopreserved tissue with malignant cells, particularly in haematological malignant disease shown in a rodent lymphoma model to cause recrudescence of the original disease Ü Oocyte maturation in vitro, followed by IVF, would eliminate this risk Antral development from in vitro grown human primordial follicles within 10 days Telfer et al., 2008: A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin. Human Reproduction 23: 1151-1158 Telfer et al. (2008) Human Reproduction

The Uterus

Normative model for uterine volume from birth to 40 years. The r 2 is 0.859. 2.4 2.2 2 1.8 Uterine Volume (cm 3 logadjusted) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Age (years) Observed Uterine Volume Predicted Uterine Volume 95% Conf Lim for Model 95% Prediction Limit Kelsey et al. unpublished

Uterine volume and age at irradiation (TBI) 30 25 20 15 10 5 0 2 4 6 8 10 12 14 Age at Irradiation (years) Bath et al. BJOG (1999)

Uterine function after cancer treatment Ü No reports of uterine damage due to chemotherapy Ü Radiotherapy: Ü Uterine damage, manifest by impaired growth and blood flow. Ü Uterine volume correlates with age at irradiation. Ü Exposure of the pelvis to radiation is associated with an increased risk of miscarriage, mid-trimester pregnancy loss, PPH, pre-term birth and low birth weight.

Vitruvian man Leonardo da Vinci 1490

Sertoli Cell

Hormone levels and semen concentration in relation to the number of MOPP cycles in male long-term survivors of childhood Hodgkin s. van Beek R D et al. Hum. Reprod. 2007;22:3215-3222

Radia7on- induced tes7cular damage Germinal epithelium >1.2Gy azoospermia

Radia7on- induced tes7cular damage Leydig cell function Ü Dose received by testis P <0.05 Ü Time Interval after radiotherapy P <0.05 Ü Age at treatment NS Li, Kelsey, Wallace (unpublished data)

Males: Fer7lity preserva7on Ü Young men who can produce semen should have the opportunity of sperm banking before treatment begins Ü Sperm retrieval should be considered if the chances of infer7lity are high and the testes are >10mls Ü Storage of gametes is governed by the HFE act 1990 Ü WriWen informed consent from a competent male is required Ü There is currently no established op7on to preserve fer7lity in the pre- pubertal boy.

Isolated human sperm cells (1500x) Albert Tousson Nikon Small world

Cryopreservation of pre-pubertal testis tissue prior to cancer treatment Ü Boys undergoing cancer treatment with >80% risk of infertility Ü Biopsy to be taken with routine procedure Ü Storage by Tissue Services according to mature or immature protocol Ü Small piece of tissue to be used for research Ethical Approval Granted September 2013

Human Testis Xenografting

Challenges Ü Provide fertility counseling to all young patients with cancer Ü Cryopreserve ovarian and pre- pubertal testicular tissue from the right (high risk) patients Ü Define the success rate of the procedures Ü Develop IVG/M as a safe alternative to re- implantation through basic research

Acknowledgements Richard Anderson David T Baird Tom Kelsey Evelyn Telfer Marie McLaughlan Alice Grove Smith Lucy Li Phoebe Wright Sarah Dodwell Rod Mitchell Louise Bath Chris Kelnar Angela Edgar Mark Brougham Fraser Munro Merrill McHoney

References Ü [1] R. Skinner, W. H. Wallace, G. A. Levitt, and U. K. C. s. C. S. G. L. E. Group, "Long-term follow-up of people who have survived cancer during childhood," Lancet Oncol, vol. 7, pp. 489-98, Jun 2006. Ü [2] W. H. Wallace, R. A. Anderson, and D. S. Irvine, "Fertility preservation for young patients with cancer: who is at risk and what can be offered?," Lancet Oncol, vol. 6, pp. 209-18, Apr 2005. Ü [3] W. H. Wallace, H. O. Critchley, and R. A. Anderson, "Optimizing reproductive outcome in children and young people with cancer," J Clin Oncol, vol. 30, pp. 3-5, Jan 1 2012. Ü [4] W. H. Wallace and T. W. Kelsey, "Human ovarian reserve from conception to the menopause," PLoS One, vol. 5, p. e8772, 2010. Ü [5] W. H. Wallace, A. B. Thomson, and T. W. Kelsey, "The radiosensitivity of the human oocyte," Hum Reprod, vol. 18, pp. 117-21, Jan 2003. Ü [6] R. A. Anderson, S. M. Nelson, and W. H. Wallace, "Measuring anti- Mullerian hormone for the assessment of ovarian reserve: when and for whom is it indicated?," Maturitas, vol. 71, pp. 28-33, Jan 2012.

References Ü [7] R. A. Anderson and D. A. Cameron, "Pretreatment serum anti-mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer," J Clin Endocrinol Metab, vol. 96, pp. 1336-43, May 2011. Ü [8] R. A. Anderson, M. Rosendahl, T. W. Kelsey, and D. A. Cameron, "Pretreatment anti-mullerian hormone predicts for loss of ovarian function after chemotherapy for early breast cancer," Eur J Cancer, vol. 49, pp. 3404-11, Nov 2013. Ü [9] M. F. Brougham, P. M. Crofton, E. J. Johnson, N. Evans, R. A. Anderson, and W. H. Wallace, "Anti-Mullerian hormone is a marker of gonadotoxicity in preand postpubertal girls treated for cancer: a prospective study," J Clin Endocrinol Metab, vol. 97, pp. 2059-67, Jun 2012. Ü [10] J. Donnez and M. M. Dolmans, "Fertility preservation in women," Nat Rev Endocrinol, vol. 9, pp. 735-49, Dec 2013. Ü [11] T. W. Kelsey, P. Wright, S. M. Nelson, R. A. Anderson, and W. H. Wallace, "A validated model of serum anti-mullerian hormone from conception to menopause," PLoS One, vol. 6, p. e22024, 2011. Ü [12] J. Donnez, M. M. Dolmans, A. Pellicer, C. Diaz-Garcia, M. Sanchez Serrano, K. T. Schmidt, et al., "Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation," Fertil Steril, vol. 99, pp. 1503-13, May 2013.

References Ü [13] T. W. Kelsey, S. K. Dodwell, A. G. Wilkinson, T. Greve, C. Y. Andersen, R. A. Anderson, et al., "Ovarian volume throughout life: a validated normative model," PLoS One, vol. 8, p. e71465, 2013. Ü [14] W. H. Wallace, A. G. Smith, T. W. Kelsey, A. E. Edgar, and R. A. Anderson, "Fertility preservation for girls and young women with cancer: population-based validation of criteria for ovarian tissue cryopreservation," Lancet Oncol, vol. 15, pp. 1129-36, Sep 2014. Ü [15] E. E. Telfer, M. McLaughlin, C. Ding, and K. J. Thong, "A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin," Hum Reprod, vol. 23, pp. 1151-8, May 2008. Ü [16] R. D. van Beek, M. Smit, M. M. van den Heuvel-Eibrink, F. H. de Jong, F. G. Hakvoort- Cammel, C. van den Bos, et al., "Inhibin B is superior to FSH as a serum marker for spermatogenesis in men treated for Hodgkin's lymphoma with chemotherapy during childhood," Hum Reprod, vol. 22, pp. 3215-22, Dec 2007. Ü [17] R A Anderson, R T Mitchell, T W Kelsey, N Spears, E E Telfer, W H B Wallace; Cancer treatment and gonadal function: experimental and established strategies for fertility preservation in children and young adults,the Lancet Diabetes and Endocrinology, 2015. Ü [18] W. H. Wallace, T. W. Kelsey, and R. A. Anderson, "Ovarian cryopreservation: experimental or established and a cure for the menopause?," Reprod Biomed Online, vol. 25, pp. 93-5, Aug 2012.

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