Clinical Endocrinology (1995) 42, 153-159

Reduced growth hormone secretion with maintained periodicity following cranial irradiation in children with acute lymphoblastic leukaemia

B. Lannerlng., S. Rosberg', 1. Marky., C. Moellt and K. Albertsson-Wikiand* 'Department of Paediatrics, International Paediatric Growth Research Centre, University of Goteborg, Goteborg, and tDepartment of Paediatrics, University of Lund, Lund, Sweden

(Received 31 May 1994; returned for revision 2 August 1994; finally revised 24 August 1994; accepted 12 September 1994)

Summary

OBJECTIVE Low dose cranlai irradiation In chlidren with acute lymphobiastic ieukaemla (ALL) has been reported to reduce GH secretion in puberty. A recent study also reported a disturbed periodicity of GH secretlon durlng puberty. We have focused on the different stages of puberty in studying these two parameters of GH secre- tlon and have also compared the effects of 18 YS 24 Gy radiation dose.

prevlousiy treated for ALL were compared wlth a control group of 208 healthy normally growing children. GH secretion was measured as 24-hour profiles. RESULTS In children treated for ALL, OH Secretion rate and OH peak amplitude were below the median values for controls, both before puberty and during all stages of puberty. The difference between patients and controls was most pronounced in late puberty. Radiation with 18 or 24 Gy gave similar results. However, time sequence anaiysls showed a slmllar periodicity of GH secretlon In both patient and control groups before, as well as during, puberty. Thus, before puberty a broad range of cycles per 24 hours was seen. These synchronized during puberty to a predominant OH peak frequency of one every 3-4 hours. CONCLUSIONS After low dose cranial lrradiatlon wlth 18 or 24 Gy, the total amount of GH secreted is reduced both before and during puberty. We could not confirm previous findings of impaired periodicity of GH secretion in these Children.

PATIENTS AND MEASUREMENTS Thirty-four children

Correspondence: Birgitta Lannering, Department of Paediatrics, Children's Hospital, S-416 85 Goteborg, Sweden. Fax: 46 31 21 54 86.

With improved survival rates for children with acute lymphoblastic leukaemia (ALL) paediatric oncologists have become aware that some of these children show decreased growth rates long after cessation of therapy. Prophylactic cranial irradiation has been shown to induce a secondary GH insufficiency in children treated for ALL (Shalet et al., 1979; Blatt et al., 1984). A Scandinavian study observed no effects on longitudinal growth before puberty, whereas during puberty a loss in height of approximately 1 SD occurred in girls after cranial irra- diation with 20-24 Gy (Moell et al., 1994). Studies of GH secretion in these patients, however, have yielded somewhat contradictory results depending on the method of assessment used (pharmacological or physiological provocation tests, or estimation of spontaneous GH secretion) and the criteria for the selection of patients (dosage and fractionation of radiation treatment). For the clinician the questions remain: What patients are at risk of G H insufficiency? What patients are at risk of short stature, and who will need substitution treatment with GH?

We have previously shown that spontaneous GH secretion is unaffected for up to 2 years after 18-24 Gy of cranial irradiation for ALL and that the temporarily decreased growth rates seen during ALL treatment are not caused by GH insufficiency (Marky et al., 1991). About I years after cranial irradiation with 24 Gy, however, spontaneous G H secretion was found to be impaired. This was seen in both prepubertal and pubertal children (Moell et al., 1989). Crowne et al. (1992) found that at about 6 years after cranial irradiation with 18 Gy, G H insufficiency was observed only during puberty, documented as a reduced rate of GH secretion. They also reported for the first time a disturbance of periodicity of G H secretion restricted to puberty in these children.

The aim of the present study was to investigate when spontaneous G H secretion becomes impaired following treatment for ALL with cranial irradiation of 18-24 Gy and to assess the magnitude of this impairment by comparison with GH secretion in healthy normally growing children at different stages of puberty. We also wished to confirm, if possible, in a larger group of children the abnormalities of periodicity found by Crowne et at. (1992).

153

154 6. Lannering et al. Clinical Endocrinology (1995) 42

Table 1 Girls: clinical characteristics at time of ALL diagnosis and GH investigation

At diagnosis At investigation

Age Cranial irradiation Height Patient (years) dose (Gy) SDS

Survival Height (years) SDS

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22

1.1 1.4 1.7 1.9 2.0 2.0 2.1 2.9 2.3 2.5 2.7 2-8 2.9 3.0 3.9 4.2 4.4 4.5 5.1 6.6 7.1

14.5

20 18 20 18 24 24 24 18 24 24 24 24 18 24 24 24 18 24 24 18 24 18

1 .o 0.5

-0.4 0.5

-0.1 1 .o 1.3 1.1

-2.0 -0.6 -0.9

0.5 1.4 1.5 0.8

-0.8 -05 -02 -0.3

1.8

0.3 -0.5

9 -1.6 9 -1.2 9 0.7 4 0.6

10 -1.3 7 0.0 6 -0.3 5 0.3 7 -2.5 8 -2.3 6 -0.4 9 -1-4 7 2.0 7 0.7 7 -0.4 6 -1.3 4 -0-3 6 -0.6 9 0.1 4 0.8 6 -2.0 5 0.0

26 30 38 20 40 32 25 39 22 28 26 32 39 41 33 37 27 33 38 49 40 63

Pubertal stage

1 I 2 1 3 2 1 1 2 4-5 1 3 1 2 2 1 1 2 3 3 4-5 4-5

Patlents and methods

Patients

Thirty-four children (12 boys and 22 girls) participated in the study. They had been diagnosed 4-10 years (mean 7 years) previously as having ALL. The mean age at diagnosis of ALL was 3.9 years. They were not obese and had normal thyroid function. At the time of investigation their ages ranged between 6 and 19.5 years, and they were in good health.

Five boys and nine girls were still prepubertal (stage 1) at the time of the investigation, while the remaining children were in different stages of puberty (Tables 1 and 2). Puberty was assessed according to Tanner regarding pubic hair and breast development stages (Tanner & Whitehouse, 1976) and according to Prader for testicular volume (Zachmann et al., 1974). Height was expressed as standard deviation scores (SDS), in comparison with Swedish reference values for healthy children (Karlberg et al., 1976).

Treatment

The 34 patients were treated at two oncology centres

(Gotebog and Lund) for 2 or 3 years with chemotherapy, according to the recommendations of the Swedish Child Leukaemia Group. CNS prophylaxis was cranial irradiation with 18 Gy to the whole brain in 17 children, 20 Gy in two children and 24 Gy in 15 children. The two children receiving 20 Gy were grouped with those receiving 18 Gy. Data on height and plasma GH concentrations (estimated as area under the curve) have previously been published for 13 of these children (Moe11 et al., 1988).

Control subjects

A total of 208 children (91 girls and 117 boys) were investigated at the Childrens Hospital, Goteborg. Their heights and the height velocities were all normal (within f SD of the mean reference values) and all the children were healthy, well nourished, and had normal thyroid, liver and kidney function. Their chronological ages ranged between 2 and 18 years. Of the 9 1 girls, 22 were prepubertal, 12 were at pubertal stage 2, 17 at stage 3,24 at stage 4, and 16 at stage 5. Of the 117 boys, 58 were prepubertal, 27 were at pubertal stage 2, 9 at stage 3, 11 at stage 4, and 12 at stage 5 (Albertsson-Wikland et al., 1994).

Clinical Endocrinology (1995) 42 GH secretion after cranial irradiation 155

Table 2 Boys: clinical characteristics at time of ALL diagnosis and GH investigation At diagnosis At GH investigation

Age RT Height Survival Height Weight Pubertal Patient (years) (Gy) SDS (years) SDS (kg) stage

23 24 25 26 27 28 29 30 31 32 33 34

2.1 24 2.1 18 2.2 18 3.7 18 5.9 18 2.3 24 2.0 18 5.0 18

10.3 18 12.0 18 2.5 18

12.0 18

-1.5 -0.2

1.5 -0.7

1.8 2.2 0.2 1.5 1.9

-1.0 0.0 0.0

5 4 8 4 4

10 10 9 6 4

13 4

- 1.5 0.0 0 8

-0.3 1.6

- 1.7 0.2

-0.3 1.2

-0.4 0.8

-0.0

21 21 33 22 37 35 52 65 69 48 55 72

1 1 1 1 1 2 2 2 4-5 4-5 4-5 4-5

The study was approved by the Ethical Committee of the Medical Faculty, University of Goteborg. Informed consent was obtained from all the children and their parents.

Study protocol

The children stayed in the hospital for at least 24 hours. They received a normal diet, with breakfast at 0800 h, lunch at 1200h, and dinner at 1700h and they were allowed normal activity and sleep. A heparinized needle (Carmeda AB, Stockholm, Sweden) was inserted during the first evening or morning. Blood specimens were collected starting at 0800-0900 h. A constant withdrawal pump (Swemed, Goteborg, Sweden) with a non-thrombogenic catheter (Carmeda) was used, as described previously (Albertsson-Wikland el al., 1989). The rate of withdrawal was 0.5-2ml/h, and the volume of the testing system 0.1- 0.2ml. The heparinized tubes were changed every 20 minutes for 24 hours, thus giving 72 samples. The heparinized tubes of blood were stored at room tempera- ture and centrifuged within 24 hours. After centrifugation, the plasma samples were frozen and stored until assayed for GH.

GH measurement

GH concentrations were measured with a commercially available assay (Pharmacia hGH kit, Uppsala, Sweden), using polyclonal antibodies. In our hands the intra-assay coefficients of variation were 10, 6, 4 and 3% at GH concentrations of 2, 5, 15 and 40mU/1. The interassay CV was 5% at a GH concentration of 10mU/1 and 3% at 40 mU/l. The WHO International Reference Preparation

for hGH (66/217) was used as the standard (Albertsson- Wikland et al., 1993).

Analysis of 24-hour GH profiles

Pulse detection. Curves were analysed by the Pulsar program (Merriam & Wachter, 1982), using the previously described setting for G H (Albertsson-Wikland et al., 1989). This program gives the calculated baseline, number of peaks, peak amplitudes, peak widths and the area under the curve. The areas under the curve were used to calculate the amount of GH secreted in 24 hours using a simple formula derived from the deconvolution technique as described previously (Albertsson-Wikland et al., 1989).

Fourier time-series analysis. The original hormone concen- tration time-series was made stationary by taking the first- order difference (Chatfield, 1989), and was smoothed using a three-point moving average (weights w - ~ = w + ~ = 0.25, wo = 0.5) in order to reduce the influence of high- frequency components (low-pass filter). The smoothed series were analysed as Fourier expansions (Chatfield, 1989), which is a way of searching for underlying rhythmical components using sine and cosine waves of different frequencies, with periods (frequencies) spanning the whole sampling period (24 hours for a 24-hour profile) down to half the sampling interval. The resultant amplitude (power) at each frequency is shown in the results, displaying the dominant and subdominant harmo- nics in the underlying waveform.

Peaks analysis. All peaks of G H detected by Pulsar from children belonging to the same sex and pubertal stage were

156 B. Lannering et a / . Clinical Endocrinology (1995) 42

31

i5 0.5/ 0

( b )

I 2 3 4 5

( a1 A

e ’

1 2 3 4 5

t

Pubertal sioqe Pubertal stage

Fig. 1 Individual values of GH secretion rate are shown for a, boys and b, girls with ALL irradiated with 0, 18 Gy or 0, 24 Gy. GH secretion rate of healthy, normally growing children at pubertal stages 1-5 is also given (75th, 50th and 25th percentile).

Flg. 2 Maximal GH level over a 24-hour period in a, boys and b, girls with ALL irradiated with 0, 18 Gy or 0 , 2 4 Gy for ALL, and normally growing children (75th, 50th and 25th percentile) at pubertal stages 1-5.

pooled. For each group the distribution of GH peak amplitudes was calculated in increments of 5 mU/L

Statistics

Group data are presented as means &SEM. Significance between means was calculated by analysis of variance (ANOVA) followed by Student-Newman-Keul’s multiple range test. The GH peak distributions were compared by chi-squared (x2) analysis.

Results

GH secretion

The estimated GH secretion rate in all the irradiated ALL children was below the median of that of controls matched for pubertal stage and sex (Fig. 1). The difference between patients and controls was more pronounced in late puberty than before puberty. GH secretion, expressed as the AUC above the calculated baseline (AUCb) and baseline GH levels, as calculated by the Pulsar program, was also lower in the irradiated children than in the controls (data not shown).

GH pulsatile pattern

GHpeaks. The spontaneous maximal GH level over the 24-hour period was below 20mU/1 in all but two of the prepubertal boys and in all the prepubertal girls (Fig. 2). The number of GH peaks over the 24-hour period was, however, within the normal range for both boys and girls.

Time-sequence analysis. In order to further analyse the possible impact of irradiation on the rhythmicity of the GH

profiles, we applied Fourier analysis. The rhythmicity represented by the dominating cycles per 24 hours was unaffected by irradiation with 18 or 24 Gy. This was seen both before and during puberty compared with the control group of healthy children. Before puberty a broad range of cycles per 24 hours was seen which synchronized during puberty to a predominating frequency of approximately one every 3 hours. The spectral power was significantly lower during puberty in children irradiated with 18 and 24 Gy, indicating lower peak amplitudes in the irradiated children. Lower spectral power was seen also in patients before puberty, especially in children who received 24 Gy (Fig. 3). The pooled autocorrelation functions from irradiated children did not differ from that of the control groups (data not shown).

Time from diagnosis of ALL. The children were investigated between 4 and 10 years after cranial irradiation. Within these 6 years, there was no correlation between the time from diagnosis and GH secretion (expressed as the rate of total secretion per 24 hours or as the secretion rate/kg body mass, U/kg 24h) or the maximum GH level during the 24-hour period (data not shown).

Age at diagnosis of ALL. There was no obvious influence of the age at time of diagnosis on the GH secretion rate (see above). However, the maximum GH level was directly related to the age at irradiation: the younger the child at diagnosis, the lower the maximum GH level.

Height. The height standard deviation scores (SDS) of the children both at the time of diagnosis of ALL and at the time of investigation are shown in Tables 1 and 2. The

Clinical Endocrinology (1995) 42 GH secretion after cranial irradiation 157

FIg. 3 Fourier analysis of 24-hour GH profiles in ALL boys and girls irradiated with i, 18 Gy and ii, 24 Gy compared with the control group of healthy children (broad line) a, before puberty and b, during puberty. The spectral power is shown as a function of the time period in cycles. The dominant period is approximately 3-4 hours in both irradiated patients and in the control group.

0.5

L

0.5

I

mean height SDS was +0.3 at diagnosis of ALL. Children who were still prepubertal at the time of investigation lost on average 0.2 SDS (range -1-7 to +O-6 SDS), whereas children who had entered puberty at the time of investiga- tion lost on average 1.0 SDS (range -3-1 to f O SDS).

Dlrcusrlon

For many years, prophylactic CNS irradiation for ALL has been an important therapeutic method to prevent relapse. With intensified chemotherapy protocols, however, the total dose of irradiation required has gradually decreased from 24 to 18 Gy and may sometimes be as low as 12 Gy. In the future it could be completely replaced by high-dose methotrexate. At present, however, the majority of long- term survivors of ALL have received 18 Gy of cranial irradiation and need to be followed to assess the long-term effects of this type of treatment.

10 I Time period ( h )

10

GH secretion

It has previously been shown that spontaneous 24-hour GH secretion is blunted in girls 5 years after cranial radiotherapy with 20-24 Gy (Moell et al., 1988). The present results indicate that this is also the case after 18 Gy of cranial irradiation both before and during puberty. This is in contrast to the results of Crowne et al. (1992), who observed abnormal 24-hour G H profiles after 18 Gy of cranial irradiation only in pubertal children, whereas GH secretion was unaffected in prepubertal children. The authors also reported a disturbed periodicity with increased number of high-frequency pulses that was restricted to the pubertal period in these children. These findings could not be confirmed in our study. However, our large control group displayed, as Crowne et al. (1992) showed, a distinct periodicity during puberty that was not seen before puberty, but this pattern was maintained in our irradiated

158 6. Lannering et al. Clinical Endocrinology (1995) 42

children. The differences regarding spectral power between controls and patients were in our study an effect of lower pulse amplitudes in the irradiated patients. A similar disturbance of periodicity was also reported by Ryalls et al. (1993) at 2.5 years median time after cranial radiation. At that time they did not find a decrease in GH amplitude as could be expected within that relatively short period of time. In fact we postulate, in view of our earlier study (Marky et al., 1991) in which GH secretion was unaffected 2 years after radiotherapy with 18 Gy, that physiological GH insuffi- ciency has its onset between 3 and 4 years after cranial irradiation with 18 Gy. At present, the somewhat contra- dictory findings of disturbed GH periodicity cannot easily be explained. Further studies may show that differences in treatment protocols play some role in this respect.

Height

In this cross-sectional study, the distribution of heights for the whole group of children, with a mean follow-up time of 7 years from cranial irradiation, fell within the normal range of the Swedish population (Karlberg et af., 1976). Only two children had a height below -2 SDS. Final heights, however, were not reached in the majority of patients and in a few children there was a marked loss in height compared with their expected target height. Height loss was more obvious during puberty (- 1 SDS) than before puberty (-0.2 SDS) even though the survival time since radiotherapy was similar in the two groups (7.3 vs 6 years). Height loss was previously reported in girls treated with 20-24 Gy of cranial irradiation (Marky et al., 1991) and the present study indicates that this occurs also after irradiation with 18 Gy and is equally pronounced in boys and girls.

The implication of these results for children cured of ALL is that cranial irradiation with 18 Gy carries no less risk of impairment of physiological GH secretion and growth than does radiation with 24 Gy. However, as this impairment is small before puberty, compared with healthy and normally growing children, the consequences may not be seen until puberty, when a height loss of - 1 SDS could be expected. A tendency to early puberty in some children may also contribute to this height loss (Leiper et af., 1988). Most patients will not be short statured as a result of this height loss, which corresponds to approximately 8 cm (Moell et al., 1994). However, children diagnosed with ALL at young ages and with low target heights may attain short stature.

The implication of these results is different for young adults cured for ALL in childhood than it is for children. After puberty and completed growth, GH secretion normally decreases and is maintained at a low level in

adulthood (Zadik et af., 1985; Ho et al., 1987). In previously irradiated young adults, GH secretion may have reached such low levels that effects of GH impairment more subtle than decreased height velocity could occur. There may be a risk of reduced physical and mental capacity (McGaulley, 1989), of cardiovascular complications (RosCn t Bengtsson, 1990), and of changes in body composition with more fat and less muscle tissue (Roskn et al., 1993). Osteoporosis is another long-term complication which has been reported to occur only in ALL patients who have received cranial irradiation (Gilsanz et al., 1990). This may therefore be one of the late sequelae of long-standing impaired but not totally depleted GH secretion.

Children with acute lymphoblastic leukaemia are followed for many years after cessation of treatment. It may be that GH secretion studies need to be performed much later in life to assess the effects of reduced GH levels on organs other than the growth plate.

Acknowledgements

We thank the children and their families who participated in this study. We would also like to thank Ms Chatarina Jansson, Gunnel Hellgren and Ms Margaretha Nolbris. The Pulsar program was kindly provided by Dr George Merriam.

This work was supported by grants from the Swedish Cancer Foundation, the Swedish Foundation for Cancer in Children, the Swedish Medical Research Council (6465, 7509) and the Assar Gabrielsson Foundation.

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