Ovarian function after autologous bone marrow ... - Nature

Bone Marrow Transplantation, (1998) 22, 989–994  1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt

Ovarian function after autologous bone marrow transplantation in childhood: high-dose busulfan is a major cause of ovarian failure C Teinturier1, O Hartmann2, D Valteau-Couanet2, E Benhamou3 and PF Bougneres1 1

Department of Pediatric Endocrinology, Hopital Saint-Vincent de Paul, Paris; 2Department of Pediatric Oncology; 3Department of Biostatistics, Institut Gustave Roussy, Villejuif, France

Summary: We studied pubertal status and ovarian function in 21 girls aged 11–21 years who had earlier received 1.2– 13 years (median 7 years) high-dose chemotherapy and autologous BMT without TBI for malignant tumors. Ten of them were given busulfan (600 mg/m2) and melphalan (140 mg/m2) with or without cyclophosphamide (3.6 g/m2). Eleven others did not receive busulfan. Twelve girls (57%) had clinical and hormonal evidence of ovarian failure. Among nine others who had completed normal puberty, six had normal gonadotropin levels, one had elevated gonadotropin levels and two had gonadotropin levels at the upper limit of normal. The 10 girls who received busulfan all developed severe and persistent ovarian failure. High-dose busulfan is therefore a major cause of ovarian failure even when given in the prepubertal period. These findings emphasize the need for long-term endocrine follow-up of these patients in order to initiate estrogen replacement therapy. Keywords: ovarian function; high-dose chemotherapy; bone marrow transplantation; busulfan; childhood

Increasing numbers of children who receive hematopoietic stem cell transplants (HSCT) for malignancies now survive. It is therefore important to evaluate ovarian toxicity secondary to the therapeutic regimens. New chemotherapy protocols will have to aim towards effecting long-term survival with minimal late morbidity. Chemotherapy is used alone or in combination with total body irradiation. Total body irradiation, used in the preparative regimens for hematologic malignancies, is deleterious to gonadal function.1,2 Myeloablative therapy consisting of cyclophosphamide, busulfan or melphalan, has been used as an alternative to avoid the side-effects of irradiation on endocrine functions and growth.1,3,4 Cytotoxics are known to induce ovarian toxicity in adults, dependent upon type of agent, dosage, administration schedule and patient age.5–7 Much less is known about the effects of high-dose chemotherapy on ovarian function in children.2,8,9 We report an analysis of

Correspondence: Dr C Teinturier, Service d’Endocrinologie Pediatrique, Hopital Saint-Vincent de Paul, 82, Avenue Denfert-Rochereau, 75014 Paris, France Received 18 February 1998; accepted 8 July 1998

ovarian function in long-term survivors following myeloablative chemotherapy and bone marrow transplantation. Patients and methods We screened all long-term female survivors older than 11 years who had received high-dose chemotherapy and autologous bone marrow transplantation without abdominal or pelvic radiation for malignant tumors at Institut Gustave Roussy. Twenty-one girls fulfilled these criteria. At time of transplantation, they were aged 2–17 years (median 9 years). Pubertal development was evaluated clinically according to Tanner.10 Fourteen girls were prepubertal (stage 1), two were undergoing puberty (stage 3) and five had regular menses (stage 5). No hormonal evaluation was carried out before chemotherapy, but pubertal stages of all the patients were appropriate for their age. The study took place 1.2–13 years (median 7 years) after bone marrow transplantation and discontinuation of chemotherapy. At time of study, girls were aged 11.5–21 years (median 14.5 years). Serum concentrations of 17␤estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured. Samples from menstruating girls were obtained during the follicular phase of cycle. In six girls, hormones were measured before and 20, 40, 60 and 90 min after an intravenous injection (100 ␮g/m2) of gonadotropin releasing hormone (Stimu-LH; Roussel, Paris, France). Serum FSH and LH concentrations were measured using commercial immunoradiometric (IRMA) kits. Chemotherapy regimens Patients had all received different conventional chemotherapies before entering the consolidation phase. Twelve girls were treated for metastatic neuroblastoma, four for Ewing’s sarcoma, three for lymphoma (Hodgkin’s disease in one), one for a metastatic rhabdomyosarcoma and another for a malignant ovarian germinal tumor. The cumulative doses of alkylating agents used before transplantation are detailed in Table 1, as well as clinical characteristics of the 21 girls. Nineteen girls received cyclophosphamide (median cumulative doses 7.3 g/m2, range 2–16.7), seven received ifosfamide (median cumulative doses 18 g/m2, range 11.5–45) and two received other alkylating agents (procarbazine, lomustine). Ten girls were prepared for the transplant with busulfan

Ovarian function after BMT in childhood C Teinturier et al

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Table 1

Clinical characteristics of the 21 girls and cumulative doses of alkylating agents administered during conventional chemotherapy

Case No.

Age at transplantation (years)

Pubertal stage (Tanner)10

Diagnosis

Alkylating agents (g/m2)

No. of cycles

1 2 3 4 5 6 7 8

4 2 8 2 5.8 4.7 3.8 14.8

1 1 1 1 1 1 1 3

Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma Rhabdomyosarcoma Neuroblastoma Hodgkin’s disease

8 6 5 6 4 10 7 9

9

12.7

1

Ovarian germinal tumor

10 11 12 13 14 15 16

7.5 14.2 4.7 7.8 5.7 9.2 13.2

1 5 1 1 1 1 5

Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma Ewing’s sarcoma Ewing’s sarcoma

17

12.2

1

Lymphoma

18

13.5

5

Ewing’s sarcoma

19 20

14.5 17.3

5 5

Neuroblastoma Ewing’s sarcoma

21

14.5

3

Lymphoma

CY 9 CY 8.5 CY 8.5 CY 9 CY 3 IFO 45 CY 9.8 CY 3.5 IFO 12.5 PCZ 9.4 CY 4 IFO 11.5 CY 8 CY 9 CY 4.5 CY 3 IFO 18 CY 10 CY 5.3 IFO 18.4 CY 7 Lomustine 0.7 CY 5 IFO 18 CY 16.7 CY 2 IFO 19 CY 7.3

5 8 9 6 6 6 10 8 10 8 8 8 7

CY = cyclophosphamide; IFO = ifosfamide; PCZ = procarbazine; lomustine = 1-(2-chloroethyl)-3cyclohexyl-1-nitrosourea.

(600 mg/m2) given orally for 4 consecutive days, combined with melphalan (140 mg/m2) with or without cyclophosphamide (3.6 g/m2) given intravenously on 2 consecutive days or other drugs depending on the tumor type (Table 2). The other 11 girls received no busulfan, but melphalan (140–180 mg/m2) combined with other drugs depending on tumor type (Table 2). Results The pubertal stage and biological features of the patients at time of study are presented in Table 3. Nine girls had normal pubertal development including six with menses. However, 12 other girls had clinical evidence of ovarian failure, with no pubertal development in six, arrested development of puberty in two others and secondary amenorrhea in the last four girls (Table 3). Serum gonadotropin and estradiol levels were in the menopausal range in all these cases: basal FSH values were above 20 IU/l (normal range ⬍12), and estradiol values remained below 100 pm/l. Hormonal status of the other nine girls was normal, except for one (case No. 11) who had elevated plasma concentrations of LH and FSH despite persistence of menses (Table 3). These results were confirmed by three hormonal evaluations over the last 4 years. In two other girls (case Nos 3 and 4), FSH levels were at the upper limit of normal values. The gonadotropin releasing hormone test confirmed

ovarian failure in the six girls tested, as already shown by their high basal FSH values. Ovarian function following busulfan The 10 girls who had received busulfan (case Nos 12–21), all had evidence of ovarian failure based on lack of pubertal development and raised basal serum gonadotropin concentrations. Pubertal development had not progressed since bone marrow transplantation (no sexual development has occurred in five, no progression in puberty in one) and the four girls who had menses before transplantation remained amenorrheic with a follow-up of 3–5 years after transplantation. Basal LH values ranged from 9.7 to 36 IU/l (mean 23.1, normal range ⬍8) and FSH from 26 to more than 70 IU/l (normal range ⬍12). Basal oestradiol values were low (⬍100 pm/l) in all these patients. Ovarian function after administration of conditioning regimens without busulfan Eleven patients had not received busulfan, but other combinations including melphalan in 10 girls. Only three had evidence of ovarian failure (case Nos 1, 8, 11) (Table 2). One (case No. 8) was treated for Hodgkin’s disease during puberty and received high cumulative doses of alkylating agents (procarbazine 9.4 g/m2, cyclophosphamide 3.5 g/m2, ifosfamide 12.5 g/m2 and carmustine 300 mg/m2). Another


Table 2 girls Case No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Details of preparative regimens and ovarian function in 21

Preparative regimens

No. of grafts

TM CTM CaM CTM CTM VCM CTM BEAM Cy-E-Cis M CTM BuMCy BuMCy BuMCy BuM BuM BuM BuMCy BuM BuM BAM

1 1 1 2 2 2 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1

Ovarian failure ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ? ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

TM = teniposide 1 g/m2, melphalan 180 mg/m2; CTM = carmustine 300 mg/m2, teniposide 1 g/m2, melphalan 180 mg/m2; CaM = carboplatin 1.8 g/m2, melphalan 180 mg/m2; VCM = vincristine 6.5 mg/m2, carmustine 200 mg/m2, melphalan 180 mg/m2; BEAM = carmustine 300 mg/m2, etoposide 800 mg/m2, aracytine 800 mg/m2, melphalan 140 mg/m2; CyECis = cyclophosphamide 6.4 g/m2, etoposide 1750 mg/m2, cisplatin 200 mg/m2; M = melphalan 180 mg/m2; BuMCy = busulfan 600 mg/m2, melphalan 140 mg/m2, cyclophosphamide 3.6 g/m2; BuM = busulfan 600 mg/m2, melphalan 140 mg/m2; BAM = busulfan 600 mg/m2, aracytine 1.8 g/m2, melphalan 140 mg/m2.

(case No. 11) had evidence of partial ovarian failure with persistence of menses but elevated serum FSH. She was treated after puberty for a neuroblastoma with cumulative doses of cyclophosphamide of 9 g/m2 and two courses of high-dose chemotherapy (carmustine 600 mg/m2). Her treatment was similar to that administered to patients 4 and 7. However, unlike the previous child, these two girls were treated before 4 years of age. Puberty developed normally and hormonal evaluations showed normal serum FSH at ages 12 and 13.8 years. The third patient (case No. 1) has no sexual development at the age of 12.5 years, elevated serum FSH (Table 3) and cardiac failure. She was treated at 4 years of age for a neuroblastoma. Comparison between the two groups with and without busulfan The group treated with busulfan had a significantly higher incidence of ovarian failure (100 vs 27%, P ⬍ 0.002) (Yates test). Other statistical calculations were not viable because the number of patients in each group was too small. Girls treated with busulfan were aged 4.6–17 years (median 12.7) at time of transplantation vs 2–14.8 years (median 5.8) in the others. Among the 11 girls who did not receive busulfan, eight were prepubertal compared to five out of 10 in the busulfan-treated group. Conventional chemotherapy regimens were different in the two groups (Tables 1 and 2).

Table 3 Pubertal development according to Tanner.10 Plasma gonadotropin concentrations and serum basal estradiol in 21 girls after bone marrow transplantation Case No.

Age (years)

Pubertal stage

Estradiol (pm/l)

LH (IU/l)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

12.5 11.5 11.5 13.8 12.8 12.5 12 16.8 19.7 20 21 12 12.3 14 15.2 14.5 14.6 15.5 16.7 18.8 15

1 5 M⫹ 1 5 M⫹ 2 4 5 M⫹ 3 5 M⫹ 5 M⫹ 5 M⫹ 1 1 1 1 5 M⫺ 1 5 M⫺ 5 M⫺ 5 M⫺ 3

100 ⬍36 ⬍36 210

2.5

215 315 ⬍36 48 362 95 40 ⬍36 ⬍36 58 ⬍36 44 70 ⬍36 48 106

1.7 4.8 ⬍0.5 1.6 2.3 11 4.5 3.5 9.3 32 11 28 28 22 31 17.5 36 16.1 9.7

FSH (IU/l) 21.8 2.5 13 12.2 7 4.6 2 41.7 8.5 2.7 27 ⬎70 39 ⬎70 42 ⬎70 ⬎70 56 ⬎70 56 26

LH = luteinising hormone: Normal values ⬍8 IU/l in follicular phase. FSH = follicle stimulating hormone. Normal values ⬍12 IU/l in follicular phase. M⫹ = with menses; M⫺ = amenorrhea.

Discussion Ovarian failure during childhood results in no pubertal development and ultimately sterility. The relative contribution of high-dose chemotherapy to ovarian failure is difficult to establish, since the majority of patients treated for malignant disease received conventional chemotherapy before bone marrow transplantation, and often alkylating agents known to induce ovarian toxicity in adult women. The manifestations of ovarian failure in these women are amenorrhea, high serum gonadotropin levels and histologic features similar to those observed after radiation therapy, ie reduction in numbers of ova and primordial follicles together with stromal fibrohyalinization.7,11 The incidence of amenorrhea depends on chemotherapeutic agent and dose, and patient’s age at time of treatment, younger women being less likely to develop premature ovarian failure.5–7 Severe impairment of gonadal function is relatively rare after conventional chemotherapy for acute leukemia. The spinal component of craniospinal irradiation seems to be the major risk factor, but cyclophosphamide may also be contributory.12–15 Ten percent of girls have sustained asymptomatic ovarian damage after conventional chemotherapy for acute lymphoblastic leukemia (ALL) and are likely to develop premature menopause.12 In long-term survivors of childhood ALL, irradiation of the ovarian zone was found to be the main gonadotoxic treatment.13 However, a high prevalence of primary gonadal damage followed high doses of cyclophosphamide, cytosine-arabinoside and lomustine.14

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Table 4

Studies of ovarian function after chemotherapy with busulfan in childhood

Author/Ref.

n

Diagnosis

Conventional chemotherapy

Preparative regimens

Ovarian failure (n)

De Sanctis28

15

thalassaemia

no

Bu 14 mg/kg Cy 200 mg/m2

12

Sanders29

3

hematologic malignancies

yes


3

Liesner3

1

acute myeloid leukemia

yes


1

Lopez-Ibor32

1

chronic myelogenous leukemia

Bu (1.5 mg/d)

Michel33

2


yes

2

Thibaud34

5

miscellaneous tumors

yes

Bu 16 mg/kg Cy 120–200 mg/kg Bua Cya Melphalan 140 mg/m2

Giorgiani30

2


yes

Bua Melphalana Cya

2

1

5

a Doses not specified. Bu = busulfan; Cy = cyclophosphamide.

A high incidence of ovarian failure with MOPP and MVVP regimens for Hodgkin’s disease,5–7 but not with the ABVD regimen has been reported.16,17 Amenorrhea is often transient in young patients and persistent in older patients after chemotherapy for Hodgkin’s disease.5 Young women treated for Hodgkin’s disease may be at risk of premature menopause.7,18 Byrne et al18 report a significantly increased relative risk of early menopause in cancer survivors treated with either radiotherapy or alkylating agents alone. According to these authors, 42% of women treated with alkylating agents and subdiaphragmatic radiotherapy were menopausal by the age of 31. The prevalence of ovarian dysfunction is high after treatment of brain tumors, as a direct effect of radiation to the spinal field19 or chemotherapy with nitrosourea.20,21 The majority of women surviving bone marrow transplantation are amenorrhoeic and infertile following a combination of chemotherapy and TBI.2,15,22–26 Even in children, it is unclear whether ovarian failure is caused mainly by high-dose chemotherapy, TBI, conventional chemotherapy before myeloablative treatment, or the combination of all three.26 Graft-versus-host disease can also play a toxic role after allogeneic transplantation.27 Little is known about ovarian damage due to high-dose chemotherapy alone in children. For this reason, our study assessed ovarian function in young girls who received highdose chemotherapy and bone marrow transplantation without TBI or abdominal or pelvic radiotherapy. Grafts were autologous so graft-versus-host disease was not implicated in ovarian damage. Because long-term survival resulted in these children, we were able to document late ovarian toxicity following chemotherapy administered in early infancy. It was difficult to estimate the precise contribution of each agent in causing ovarian damage since drug combinations and cumulative doses of alkylating agents differed in each case. Nevertheless, all the girls who received busulfan developed severe and persistent ovarian damage whatever age they were and whether or not they were undergoing

puberty at the time of chemotherapy. Every girl who received busulfan also received melphalan, but 7/10 girls who received only melphalan had normal ovarian function. Busulfan has been reported to influence gonadal function in children and adults.3,28–31 Among 73 women aged 14– 57 years at transplant who received busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg), only one recovered normal ovarian function.31 In the literature, we identified 29 girls who received busulfan in the prepubertal or pubertal period; 26 had signs of ovarian failure3,28–30,32–34 (Table 4). In a study of 15 girls aged 9–17 years treated for thalassemia with allogenic bone marrow transplantation, De Sanctis et al28 demonstrated ovarian failure in 12 girls after a busulfan–cyclophosphamide conditioning regimen, without conventional chemotherapy. Thalassemic patients often sustain impaired gonadal function, but in this series ovarian failure is likely the result of the toxic effects of busulfan itself. The role of cyclophosphamide in ovarian failure at the doses used in BMT (120–200 mg/kg) is not clear since some children who receive cyclophosphamide alone as transplant preparation for aplastic anemia or a malignant disease can have normal puberty and normal serum gonadotropin levels, as well as ovarian failure and impaired pubertal development.2,23,34 It is unlikely that ovarian damage, 1.2–13 years (median 7) after high-dose chemotherapy in our series is reversible. Prolonged follow-up after transplantation, however is necessary, since young adults below the age of 30 years rarely recover menstruation and fertility 3–7 years after transplantation.2,29,31,34–36 As dividing cells are more sensitive to the cytotoxic effects of alkylating agents than are quiescent cells, inhibiting the pituitary–gonadal axis could render the germinal epithelium less vulnerable to the cytotoxic effects of chemotherapy. Gn-RH agonists inhibit chemotherapyinduced ovarian follicular depletion in the rat and in Rhesus monkeys.37,38 In women treated for Hodgkin’s and non-


Hodgkin’s lymphoma, over 93% of surviving patients in the Gn-RH-a group resumed ovulation and menses spontaneously, whereas less than 40% resumed normal ovarian cyclic activity in the group without Gn-RH-a.39 This suggests that cotreatment with Gn-RH-a protects against ovarian failure during chemotherapy and may explain why ovarian failure is far more frequent when chemotherapy is administered after the onset of puberty. In conclusion, high-dose busulfan is a major cause of ovarian failure in girls conditioned with high-dose chemotherapy, even during the prepubertal period. Longer followup is necessary to determine whether gonadal damage is definitive. Girls who have received high-dose busulfan should be carefully followed-up so that ovarian failure can be detected and estrogen replacement therapy administered accordingly. Acknowledgements We thank Lorna Saint-Ange for reviewing the manuscript.

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