~
Cancer Immunol Immunother (1990) 31: 187 - 190
ancer mlnunology mmunotherapy
© Springer-Verlag 1990
A controlled trial of Bestatin in hydatidiform mole Pak-Chung Ho l, Ling-Chui Wong 1, John W. M. Lawton 2, and Ho-Kei Ma 1 1 Department of Obstetrics and Gynecology and 2 Department of Pathology, University of Hong Kong Received 90ctober 1989/Accepted 20 December 1989
Summary. A prospective randomized controlled trial was conducted to study whether Bestatin, an immunomodifier, can reduce the incidence of persistent gestational trophoblastic disease in patients with hydatidiform mole. A group of 21 patients (Bestatin group) received 30 m g Bestatin daily after evacuation o f the hydatidiform mole. A second group of 23 patients (control group) did not receive any drug. B l o o d was taken for white cell counts, differential counts, l y m p h o c y t e subset counts (CD2 +, CD4 +, CD8 + and B cells) and natural killer cell activity before evacuation of the hydatidiform moles. The tests were repeated every 4 weeks after evacuation until the serum ~ subunit of h u m a n chorionic gonadotropin ([3hCG) had returned to normal or until the patient had to receive chemotherapy because of persistent gestational trophoblastic disease. There was no significant difference in the age of the patients, the pre-evacuation serum [3hCG, or the gestational age between the two groups. Chemotherapy was needed by 6 patients in the Bestatin group (28.6%) and 3 patients in the control group (13%) because of persistent gestational trophoblastic disease. There was no significant difference in any of the immunological parameters between the two groups before or after evacuation. W e conclude that Bestatin at this dosage does not improve the immunological functions or clinical outcome in patients with hydatidiform mole.
Introduction Bestatin (C16H24N204) is an immunomodifier of small molecular mass. Treatment of cancer patients with Bestatin has been shown to increase the proportion o f T cells in the blood and the frequency o f lymphocytes bearing Fc recep-
Offprim requests to: P. C. Ho, Department of Obstetrics and Gynaecology, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong
tors for IgG was also normalized. The natural killer (NK) cell activity against two allogeneic target cell lines was increased but the lymphocyte proliferative responses to phytohemagglutinin and purified protein derivative of tuberculin were not influenced. There were no detectable side-effects even with prolonged treatment. Bestatin has been used with radiotherapy and/or chemotherapy in patients with various malignancies, and there is some suggestive evidence o f beneficial antitumor effects [1, 2, 8]. Immunological studies in patients with hydatidiform mole showed that those patients with impaired immunological functions are more likely to develop persistent gestational trophoblastic disease [4]. It was thought possible that if the immunological functions were improved by Bestatin therapy, the risk of development of persistent gesrational trophoblastic disease might be reduced. Therefore, we conducted a prosPective randomized controlled study on the use of Bestatin in patients with hydatidiform moles.
Materials and methods The study was a prospective randomized controlled trial. Patients were recruited from the University Gynecology Units at Queen Mary Hospital and Kwong Wah Hospital. When a patient was suspected to have hydatidiform mole, from the clinical features and ultrasound findings, she was interviewed and the nature of the trial was explained. If the patient agreed to take part in the trial she was randomized into the Bestatin group or the control group. The hydatidiform moles were evacuated with suction evacuation. If the tissues resembled hydatidifonn mole macroscopically, oral Bestatin was started the next day at a dosage of 30 mg/day if the patient was in the treatment group. In the control group, the patients were not given any drug. All the tissues evacuated were examined histologically. If the diagnosis of complete hydatidiform mole could not be confirmed histologically, the patient was excluded from the study. The patients were followed up weekly at our trophoblastic disease clinic. Blood was taken weekly for assay of the [3-subunit of human chorionic gonadotropin (~3hCG). The [3hCG was measured by radioimmunoassay as described by Vaitukaitis et al. [9]. The purified ~3hCGfor , radioiodination and the antibody against ~hCG were supplied by the National Institute of Health. The sensitivity of the assay was 5 IU/1. The inter-assay and intra-assay coefficients of variation were 4.5% and 4% respectively. Since the distribution of the ~3hCGlevels was skewed, we
188 performed logarithmic transformation before statistical analysis. The criteria for selection of pafients for chemotherapy have been descfibed elsewhere [6]. Blood was taken for assaying the white cell count, differential count, T cell count, B cell count, T cell subset counts and NK cell activity before suction evacuation. A complete blood picture, liver function tests, and renal function tests wem also performed. The tests were repeated every 4 weeks until the serum ~hCG level had returned to normal or unfil the patient required chemotherapy. The B cell counts, T cell counts and the T cell subset counts were performed with an indirect immunofluorescence technique described previously [5]. B cells were idenfified by the presence of surface immunoglobulins (sig+). T cells (CD2+), the helper T cells (CD4 +) and the suppressor/cytotoxic T cells (CD8 ÷) were idenfified with the monoclonal antibodies OKT11, OKT4 and OKT8 (Ortho Pharmaceuticals, Raritan, NY, USA) respectively. NK cell activity was assayed against the target cell line K562 [5]. The target cells, 106 cells in 0.2 ml RPMI medium, were labelled with 100 gCi chromium-51 (1 mCi/ml) by incubafion at 37°C for 1.5 h. The target cells were then washed twice in RPMI medium supplemented with 10% fetal calf serum, and the concentration was adjusted to 105]ml. The mononuclear cells were adjusted to a concentration of 8 x 106/ml and diluted serially twofold to 0.5 x 106/rel. Aliquots of 0.1 ml lymphocyte suspension at these five concentrations were dispensed into the wells of a round-bottomed micro-T/C tray (Nunc, Roskilde, Denmark), and 0.1 ml target cells was added to each well. Therefore, the target : effector cell ratios studied were 1 : 80, 1 : 40, 1:20, 1 : 10 and 1 : 5. Assays were done in quadruplicate. The spontaneous release was assayed by incubafing 0.1 ml RPMI medium with 0.1 ml target cells and the total releasable 51Cr assayed by addition of 0.1 ml Nonidet P40 (1%) to 0.1 ml target cells. The whole plate was incubated at 37 ° C in 5% carbon dioxide for 3.5 h. After incubation, the plate was centrifuged at 400 g for 10 min. Aliquots of the supernatant fluid (0.1 ml) were dispensed into the counting tubes and counted in a gamma counter. The specific cytotoxicity at the different target : effector ratios was calculated by the formula: Cytotoxicity(%) = 100 x
51Crreleasedby effectors- 51Crreleasein medium 5iCr releasedby detergent- 51Crreleasedin medium
Comparison of the means was performed by Student's t-test for paired and unpaired data. Fisher's exact test was used to analyze the difference in incidence of persistent trophoblastic disease. The protocol was approved by the ethical committee of the Faculty of Medicine, University of Hong Kong.
Results Altogether 44 patients were recruited into the study: 21 in the Bestatin group and 23 in the control group. The patients' characteristics are shown in Table 1. There were no significant differences between the two groups in re-
Table 1. Characterisfics of patientsa Characteristic
Bestatin group (n = 21)
Control group (n = 23)
Age (years)
27.5 +_ 6.8
29.4 _+8.0
Gestational maturity (weeks)
12.3 _+ 3.2
13.3 _+3.5
5.2 _+0.7
5.2 -+ 0.7
11.9 ± 5.0
10.6 ± 4.3
Log (pre-evacuation serum ~hCG level) (IU/1) Time taken for [3hCG to return to normal (weeks)
a Results are expressed as means -+ 1 SD; [3hCG, ~3 subunit of human chorionic gonadotropin
spect of the patients' mean age, the mean gestational age at evacuation and the mean pre-evacuation serum [3hCG level. Suction evacuation was the method of termination of the hydatidiform moles in all these patients. The results of the immunological tests are shown in Tables 2 - 5 . In neither group of patients was there any significant change in the total white cell count after evacuation of the uterus. In both groups the percentage and absolute counts of lymphocytes and the absolute counts of T cells (CD2+), B cells, helper T cells (CD4 +) and suppressor/cytotoxic T cells (CD8 ÷) rose after evacuation and were significantly higher than those before evacuation (Tables 2 - 4 ) . However, the percentages of CD2 ÷ cells, B cells, CD4 + cells, and CD8 ÷ cells and the CD4:CD8 ratio did not show any change. There was no significant difference in any of these parameters between the Bestatin group and the control group. The results of the NK cell assays are shown in Table 5. In neither group of patients was there a significant change in the NK cell activity after evacuation of the uterus. There was no significant difference in the NK cell activity, at any target: effector cell rat±o, between the Bestatin group and the control group, except for a small but significant difference at 12 weeks after evacuation, when the NK activity in the control group was in fact higher than in the Bestatin group at the target: effector cell rat±os of 1:5 and 1 : I0. The clinical outcome was similar in the two groups of patients. There was no significant difference in the time taken for the serum ~3hCG to return to normal (Table 1). There were 6 patients (28.6%) in the Bestatin group and 3 patients in the control group (13%) who required chemo-
Table 2. Total blood white cell counts and lymphocyte counts in the Bestatin and control groups (Results expressed as means -+ 1 SD) Cells
Group
Preevacuation
Post-evacuation 4 weeks
white cell count (109/1)
Bestatin Control
8.06 + 3.89 7.55 + 1.83
Lymphocytes (%)
Bestatin Control
17.90+ 6.94 24.74+ 17.93
Lymphocyte count (109/1)
Bestatin Control
1.30_+ 0.62 1.52 _+ 0.66
7.75 ± 1.68 7.44 _+2.65 27.05_+8.69**** 27.83_+ 8.93** 2.06_+0.75**** 1.97 +_0.56*
8 weeks 6.86 + 2.13 7.00 + 1.81
12 weeks 7.00 ± 1.38 6.78 _+ 1.71
26.75_+8.48**** 32.23+ 8.39****
32.50_+8.44**** 32.50_+ 6.81"***
1.79_+0.75" 2.23 ± 0.89***
2.24_+0.62**** 2.17 ± 0.62***
No significant differences were observed between the control group and the Bestatin group. Some post-evacuation values were significantly higher than the corresponding pre-evacuation values: * P
Cancer Immunol Immunother (1990) 31: 187 - 190
ancer mlnunology mmunotherapy
© Springer-Verlag 1990
A controlled trial of Bestatin in hydatidiform mole Pak-Chung Ho l, Ling-Chui Wong 1, John W. M. Lawton 2, and Ho-Kei Ma 1 1 Department of Obstetrics and Gynecology and 2 Department of Pathology, University of Hong Kong Received 90ctober 1989/Accepted 20 December 1989
Summary. A prospective randomized controlled trial was conducted to study whether Bestatin, an immunomodifier, can reduce the incidence of persistent gestational trophoblastic disease in patients with hydatidiform mole. A group of 21 patients (Bestatin group) received 30 m g Bestatin daily after evacuation o f the hydatidiform mole. A second group of 23 patients (control group) did not receive any drug. B l o o d was taken for white cell counts, differential counts, l y m p h o c y t e subset counts (CD2 +, CD4 +, CD8 + and B cells) and natural killer cell activity before evacuation of the hydatidiform moles. The tests were repeated every 4 weeks after evacuation until the serum ~ subunit of h u m a n chorionic gonadotropin ([3hCG) had returned to normal or until the patient had to receive chemotherapy because of persistent gestational trophoblastic disease. There was no significant difference in the age of the patients, the pre-evacuation serum [3hCG, or the gestational age between the two groups. Chemotherapy was needed by 6 patients in the Bestatin group (28.6%) and 3 patients in the control group (13%) because of persistent gestational trophoblastic disease. There was no significant difference in any of the immunological parameters between the two groups before or after evacuation. W e conclude that Bestatin at this dosage does not improve the immunological functions or clinical outcome in patients with hydatidiform mole.
Introduction Bestatin (C16H24N204) is an immunomodifier of small molecular mass. Treatment of cancer patients with Bestatin has been shown to increase the proportion o f T cells in the blood and the frequency o f lymphocytes bearing Fc recep-
Offprim requests to: P. C. Ho, Department of Obstetrics and Gynaecology, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong
tors for IgG was also normalized. The natural killer (NK) cell activity against two allogeneic target cell lines was increased but the lymphocyte proliferative responses to phytohemagglutinin and purified protein derivative of tuberculin were not influenced. There were no detectable side-effects even with prolonged treatment. Bestatin has been used with radiotherapy and/or chemotherapy in patients with various malignancies, and there is some suggestive evidence o f beneficial antitumor effects [1, 2, 8]. Immunological studies in patients with hydatidiform mole showed that those patients with impaired immunological functions are more likely to develop persistent gestational trophoblastic disease [4]. It was thought possible that if the immunological functions were improved by Bestatin therapy, the risk of development of persistent gesrational trophoblastic disease might be reduced. Therefore, we conducted a prosPective randomized controlled study on the use of Bestatin in patients with hydatidiform moles.
Materials and methods The study was a prospective randomized controlled trial. Patients were recruited from the University Gynecology Units at Queen Mary Hospital and Kwong Wah Hospital. When a patient was suspected to have hydatidiform mole, from the clinical features and ultrasound findings, she was interviewed and the nature of the trial was explained. If the patient agreed to take part in the trial she was randomized into the Bestatin group or the control group. The hydatidiform moles were evacuated with suction evacuation. If the tissues resembled hydatidifonn mole macroscopically, oral Bestatin was started the next day at a dosage of 30 mg/day if the patient was in the treatment group. In the control group, the patients were not given any drug. All the tissues evacuated were examined histologically. If the diagnosis of complete hydatidiform mole could not be confirmed histologically, the patient was excluded from the study. The patients were followed up weekly at our trophoblastic disease clinic. Blood was taken weekly for assay of the [3-subunit of human chorionic gonadotropin (~3hCG). The [3hCG was measured by radioimmunoassay as described by Vaitukaitis et al. [9]. The purified ~3hCGfor , radioiodination and the antibody against ~hCG were supplied by the National Institute of Health. The sensitivity of the assay was 5 IU/1. The inter-assay and intra-assay coefficients of variation were 4.5% and 4% respectively. Since the distribution of the ~3hCGlevels was skewed, we
188 performed logarithmic transformation before statistical analysis. The criteria for selection of pafients for chemotherapy have been descfibed elsewhere [6]. Blood was taken for assaying the white cell count, differential count, T cell count, B cell count, T cell subset counts and NK cell activity before suction evacuation. A complete blood picture, liver function tests, and renal function tests wem also performed. The tests were repeated every 4 weeks until the serum ~hCG level had returned to normal or unfil the patient required chemotherapy. The B cell counts, T cell counts and the T cell subset counts were performed with an indirect immunofluorescence technique described previously [5]. B cells were idenfified by the presence of surface immunoglobulins (sig+). T cells (CD2+), the helper T cells (CD4 +) and the suppressor/cytotoxic T cells (CD8 ÷) were idenfified with the monoclonal antibodies OKT11, OKT4 and OKT8 (Ortho Pharmaceuticals, Raritan, NY, USA) respectively. NK cell activity was assayed against the target cell line K562 [5]. The target cells, 106 cells in 0.2 ml RPMI medium, were labelled with 100 gCi chromium-51 (1 mCi/ml) by incubafion at 37°C for 1.5 h. The target cells were then washed twice in RPMI medium supplemented with 10% fetal calf serum, and the concentration was adjusted to 105]ml. The mononuclear cells were adjusted to a concentration of 8 x 106/ml and diluted serially twofold to 0.5 x 106/rel. Aliquots of 0.1 ml lymphocyte suspension at these five concentrations were dispensed into the wells of a round-bottomed micro-T/C tray (Nunc, Roskilde, Denmark), and 0.1 ml target cells was added to each well. Therefore, the target : effector cell ratios studied were 1 : 80, 1 : 40, 1:20, 1 : 10 and 1 : 5. Assays were done in quadruplicate. The spontaneous release was assayed by incubafing 0.1 ml RPMI medium with 0.1 ml target cells and the total releasable 51Cr assayed by addition of 0.1 ml Nonidet P40 (1%) to 0.1 ml target cells. The whole plate was incubated at 37 ° C in 5% carbon dioxide for 3.5 h. After incubation, the plate was centrifuged at 400 g for 10 min. Aliquots of the supernatant fluid (0.1 ml) were dispensed into the counting tubes and counted in a gamma counter. The specific cytotoxicity at the different target : effector ratios was calculated by the formula: Cytotoxicity(%) = 100 x
51Crreleasedby effectors- 51Crreleasein medium 5iCr releasedby detergent- 51Crreleasedin medium
Comparison of the means was performed by Student's t-test for paired and unpaired data. Fisher's exact test was used to analyze the difference in incidence of persistent trophoblastic disease. The protocol was approved by the ethical committee of the Faculty of Medicine, University of Hong Kong.
Results Altogether 44 patients were recruited into the study: 21 in the Bestatin group and 23 in the control group. The patients' characteristics are shown in Table 1. There were no significant differences between the two groups in re-
Table 1. Characterisfics of patientsa Characteristic
Bestatin group (n = 21)
Control group (n = 23)
Age (years)
27.5 +_ 6.8
29.4 _+8.0
Gestational maturity (weeks)
12.3 _+ 3.2
13.3 _+3.5
5.2 _+0.7
5.2 -+ 0.7
11.9 ± 5.0
10.6 ± 4.3
Log (pre-evacuation serum ~hCG level) (IU/1) Time taken for [3hCG to return to normal (weeks)
a Results are expressed as means -+ 1 SD; [3hCG, ~3 subunit of human chorionic gonadotropin
spect of the patients' mean age, the mean gestational age at evacuation and the mean pre-evacuation serum [3hCG level. Suction evacuation was the method of termination of the hydatidiform moles in all these patients. The results of the immunological tests are shown in Tables 2 - 5 . In neither group of patients was there any significant change in the total white cell count after evacuation of the uterus. In both groups the percentage and absolute counts of lymphocytes and the absolute counts of T cells (CD2+), B cells, helper T cells (CD4 +) and suppressor/cytotoxic T cells (CD8 ÷) rose after evacuation and were significantly higher than those before evacuation (Tables 2 - 4 ) . However, the percentages of CD2 ÷ cells, B cells, CD4 + cells, and CD8 ÷ cells and the CD4:CD8 ratio did not show any change. There was no significant difference in any of these parameters between the Bestatin group and the control group. The results of the NK cell assays are shown in Table 5. In neither group of patients was there a significant change in the NK cell activity after evacuation of the uterus. There was no significant difference in the NK cell activity, at any target: effector cell rat±o, between the Bestatin group and the control group, except for a small but significant difference at 12 weeks after evacuation, when the NK activity in the control group was in fact higher than in the Bestatin group at the target: effector cell rat±os of 1:5 and 1 : I0. The clinical outcome was similar in the two groups of patients. There was no significant difference in the time taken for the serum ~3hCG to return to normal (Table 1). There were 6 patients (28.6%) in the Bestatin group and 3 patients in the control group (13%) who required chemo-
Table 2. Total blood white cell counts and lymphocyte counts in the Bestatin and control groups (Results expressed as means -+ 1 SD) Cells
Group
Preevacuation
Post-evacuation 4 weeks
white cell count (109/1)
Bestatin Control
8.06 + 3.89 7.55 + 1.83
Lymphocytes (%)
Bestatin Control
17.90+ 6.94 24.74+ 17.93
Lymphocyte count (109/1)
Bestatin Control
1.30_+ 0.62 1.52 _+ 0.66
7.75 ± 1.68 7.44 _+2.65 27.05_+8.69**** 27.83_+ 8.93** 2.06_+0.75**** 1.97 +_0.56*
8 weeks 6.86 + 2.13 7.00 + 1.81
12 weeks 7.00 ± 1.38 6.78 _+ 1.71
26.75_+8.48**** 32.23+ 8.39****
32.50_+8.44**** 32.50_+ 6.81"***
1.79_+0.75" 2.23 ± 0.89***
2.24_+0.62**** 2.17 ± 0.62***
No significant differences were observed between the control group and the Bestatin group. Some post-evacuation values were significantly higher than the corresponding pre-evacuation values: * P
