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GYNECOLOGIC ONCOLOGY 17, 386-393 (1984) Trophoblastic Cell Sensitivity to 8-Day Chemotherapy in Nonmetastatic Gestational Trophoblastic Neoplasia ROBERT D. HILGERS, M.D., JAMES C. STANDEFER, PH.D., J. MICHAEL RUTLEDGE, PH.D., AND FRANCISCO AMPUERO, M.D. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology and Department of Pathology, University of New Mexico School of Medicine, and the College of Pharmacy, University of New Mexico Medical Center, Albuquerque, New Mexico 87131 Received February 17, 1983 Serial radioimmunoassay determinations of serum j3hCG and methotrexate were compared in two patients with nonmetastatic gestational trophoblastic neoplasia (NMGTN) treated with Goldstein’s modification of Bagshawe’s intermediate-dose methotrexate-citrovorum factor rescue-treatment program. Pretreatment phCG levels (mIU/ml) ranged within the outer limits of the 10’ log level. Following intravenous methotrexate, sharp serum peaks between 10m6 and lo-’ M were observed. Plasma disappearance was rapid with a 3 log drop noted within 24 hr to levels incapable of inhibiting DNA synthesis. phCG levels manifested a 1 to 1.5 log drop over the 8 days of chemotherapy and complete remission was noted within 5 to 6 weeks of the first dose of methotrexate. No significant clinical or laboratory toxicity was observed. Although cell culture studies show that 100% of cell death can be achieved with serum levels of lo-’ M in methotrexate-resistant choriocarcinoma, similar data do not exist for previously untreated trophoblastic neoplastic cells. These preliminary observations suggest that serum methotrexate levels are important for establishing sensitivity levels in a heterogeneous population of trophoblastic cells in NMGTN and that the total dose of methotrexate may be safely preselected on the basis of the pretreatment PhCG. Trophoblastic cell proliferation, characterized by a postmolar rise in human chorionic gonadotropin (hCG), in nonmetastatic gestational trophoblastic neoplasia (NMGTN) manifests a unique sensitivity to single-agent chemotherapy, i.e., methotrexate or Actinomycin D. Methotrexate (MTX), often the drug of choice, is administered in a standard daily dose of 0.4 mg/kg for 5 days which is repeated after toxicity clears and while hCG levels remain abnormally high. Such conventional therapy is frequently associated with moderate to severe toxicity involving he- matopoietic, hepatic, renal, and epithelial tissues [ 1,2]. Furthermore, an average hospital stay of 58 days (range 29-88) is required for methotrexate to induce a complete remission in patients with nonmetastatic gestational trophoblastic neoplasia PI. To avoid the toxic manifestations of methotrexate, Bagshawe and co-workers [3] utilized intermediate-dose methotrexate (30 mg/m’ q.o.d. x 4 days) with 386 0090-8258/84 $1 SO Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved

Trophoblastic cell sensitivity to 8-day chemotherapy in nonmetastatic gestational trophoblastic neoplasia

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Page 1: Trophoblastic cell sensitivity to 8-day chemotherapy in nonmetastatic gestational trophoblastic neoplasia

GYNECOLOGIC ONCOLOGY 17, 386-393 (1984)

Trophoblastic Cell Sensitivity to 8-Day Chemotherapy in Nonmetastatic Gestational Trophoblastic Neoplasia

ROBERT D. HILGERS, M.D., JAMES C. STANDEFER, PH.D., J. MICHAEL RUTLEDGE, PH.D., AND FRANCISCO AMPUERO, M.D.

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology and Department of Pathology, University of New Mexico School of Medicine, and the College of Pharmacy,

University of New Mexico Medical Center, Albuquerque, New Mexico 87131

Received February 17, 1983

Serial radioimmunoassay determinations of serum j3hCG and methotrexate were compared in two patients with nonmetastatic gestational trophoblastic neoplasia (NMGTN) treated with Goldstein’s modification of Bagshawe’s intermediate-dose methotrexate-citrovorum factor rescue-treatment program. Pretreatment phCG levels (mIU/ml) ranged within the outer limits of the 10’ log level. Following intravenous methotrexate, sharp serum peaks between 10m6 and lo-’ M were observed. Plasma disappearance was rapid with a 3 log drop noted within 24 hr to levels incapable of inhibiting DNA synthesis. phCG levels manifested a 1 to 1.5 log drop over the 8 days of chemotherapy and complete remission was noted within 5 to 6 weeks of the first dose of methotrexate. No significant clinical or laboratory toxicity was observed. Although cell culture studies show that 100% of cell death can be achieved with serum levels of lo-’ M in methotrexate-resistant choriocarcinoma, similar data do not exist for previously untreated trophoblastic neoplastic cells. These preliminary observations suggest that serum methotrexate levels are important for establishing sensitivity levels in a heterogeneous population of trophoblastic cells in NMGTN and that the total dose of methotrexate may be safely preselected on the basis of the pretreatment PhCG.

Trophoblastic cell proliferation, characterized by a postmolar rise in human chorionic gonadotropin (hCG), in nonmetastatic gestational trophoblastic neoplasia (NMGTN) manifests a unique sensitivity to single-agent chemotherapy, i.e., methotrexate or Actinomycin D. Methotrexate (MTX), often the drug of choice, is administered in a standard daily dose of 0.4 mg/kg for 5 days which is repeated after toxicity clears and while hCG levels remain abnormally high. Such conventional therapy is frequently associated with moderate to severe toxicity involving he- matopoietic, hepatic, renal, and epithelial tissues [ 1,2]. Furthermore, an average hospital stay of 58 days (range 29-88) is required for methotrexate to induce a complete remission in patients with nonmetastatic gestational trophoblastic neoplasia PI.

To avoid the toxic manifestations of methotrexate, Bagshawe and co-workers [3] utilized intermediate-dose methotrexate (30 mg/m’ q.o.d. x 4 days) with

386

0090-8258/84 $1 SO Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved

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CASE REPORTS 387

citrovorum factor given on the alternate days. This resulted in a response rate similar to conventionally administered methotrexate with the advantage of minimal morbidity from fewer courses of chemotherapy over a shorter time span. Goldstein’s [4] modification of Bagshawe’s &day chemotherapy program has resulted in 81% of patients with NMGTN requiring only one course of inpatient chemotherapy to reach complete remission [5]. Likewise, their patients were allowed to resume normal activities and were nontoxic in the weeks immediately following chemotherapy.

This report presents preliminary observations of serum MTX values and their effect on clinical response and serum beta subunit (j3hCG) values in two patients with postmolar nonmetastatic gestational trophoblastic neoplasia treated with the g-day, modified Bagshawe, intermediate-dose methotrexate-citrovorum factor program (IDMTX-CFR).

MATERIALS AND METHODS

Pretreatment, a leukocyte count >3000/mm3, granulocyte count >1500/mm3, platelet count ~100,000/mm3, normal serum creatinine (~1.5 mg%), and normal liver function tests (bilirubin < 1.5 mg% and SGOT ~40 W/liter), without a prior history of hepatic disease, were present. The original hydatidiform moles were graded according to the criteria of Hertig and Manse11 [6]. Physical examination, roentgenograms, ultrasonography, isotope, and CAT scans were utilized to rule out extrauterine and metastatic disease.

Each patient received 1 mg/kg on Days 1, 3, 5, and 7, and 0.1 mg/kg CF 30 hr after the administration of each dose of methotrexate (Fig. 1). Each drug was administered by intravenous bolus through an angiocath with a heparin lock adaptor which remained in place the entire course of therapy. Blood (4 cc) was withdrawn 0, 0.5, 8, 24, and 48 hr after each dose of MTX. Methotrexate levels were measured by radioimmunoassay, as described by Raso and Schreiber [7], utilizing reagents supplied by Diagnostic Biochemistry, Inc. (San Diego, Calif.). The assay has a sensitivity of approximately 10 pg of MTX. The antibody cross- reacts insignificantly with folic acid, N”-methotetrahydrofolic acid, folinic acid, and approximately 7% with 7-OH-methotrexate. The within assay variation is 6% and between assay variation is 12%. Serum phCG levels were measured simultaneously from the aliquots of blood used to determine serum MTX levels and at weekly intervals until values reached ~5 mIU/ml. A normal level of phCG concentration in serum in nonpregnant women is ~5 mIU/ml as measured in our clinical laboratory. phCG was assayed by a method specific for the beta

DRUGS MTX CFR MTX CFF MTX CFR MTX CFR I I I , II I

-- HOURS 0 24 40 72 96 120 144 166 192

DOSE : METHOTREXATE : 1 mQ/kQ IV

CITROVORUM FACTOR : 0.1 lllQ/kQ IV

FIG. 1. Schematic of treatment protocol. Intermediate dose methotrexate-citrovorum factor rescue.

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388 HILGERS ET AL.

subunit portion of hCG [8] with a sensitivity of 2 mIU/ml. Antiserum, obtained from the National Pituitary Agency through the National Institute of Arthritis, Metabolic, and Digestive Diseases, showed less than 5% of cross-reaction with human luteinizing hormone (hLH). Iodinated hCG was prepared according to the procedure of Greenwood et al. [9] and was rechromatographed on Sephadex G-150 prior to each assay. The hCG standard was obtained from the World Health Organization (National Institute for Biological Standards and Control, Holly Hill, Hempstead, London), as the Second International Standard (1963).

A complete remission was reached when three consecutive weekly @hCG serum concentrations stabilized at ~5 mIU/ml. After the first course of IDMTX- CFR, further chemotherapy was withheld as long as the serum phCG levels decreased in an orderly manner. Once complete remission was reached, PhCG titers were followed monthly for 6 months and bimonthly for 6 months. Complete history and physical examinations, as well as pregnancy prevention through the use of low-dose oral contraceptives, were utilized to follow the patients during the first year after complete remission.

CASE REPORTS

Patient 1. M.M., a 23-year-old G3 Pl ABl Caucasian female underwent suction curettage of a lo-week-size uterus (grade II hydatidiform mole) on November 30, 1981. Postevacuation serum PhCG levels fell and then spontaneously rose to within the lo3 mIU/ml range for close to 1 month (Fig. 2). Subsequently the patient was referred to the Gynecologic Oncology Service at the University of New Mexico Medical Center where physical examination and metastatic survey were normal and the serum phCG value was 1.7 x lo3 mIU/ml. On February 9, 1982, IDMTX was administered by intravenous bolus at a dose of 70 mg/day every other day for four doses and rescued with 7 mg of CF 30 hr following each dose of methotrexate. The @hCG titer fell to 1.3 x lo* mIU/ml at the completion of the g-day cycle and to ~5 mIU/ml 4.3 weeks following the first dose of MTX. The patient remains in complete remission 10 months following IDMTX-CFR.

Case 2. A 19-year-old Hispanic Gl PO AbO female underwent suction curettage of a 24-week-size uterus (grade III hydatidiform mole) on October 12, 1981. The preevacuation serum /3hCG was 6.3 x 16 r&J/ml and her immediate postoperative course was uneventful (Fig. 2). By the seventh postevacuation week the serum phCG level had dropped to 24 mIU/ml. Following this the patient was temporarily lost to follow-up. Four months after suction curettage, the patient returned for follow-up care and a right adnexal mass was palpated. Laparoscopy showed bilaterally enlarged cystic ovaries. The uterus was lo-weeks gestational size and intrauterine curettage produced a deciduous-appearing endometrium with extensive Arias-Stella changes present in the endometrial glands. Metastatic survey showed no evidence of extrauterine disease and the PhCG level was 1.3 x lo3 mIU/ml. On February 13, 1982, she received as an inpatient 60 mg IDMTX every other day for four doses and 6 mg of CF 30 hr after each dose of MTX. At the completion of the S-day schedule, the serum phCG level dropped to 1.7 x lo* mIU/ml. Three and one-half weeks following initiation of MTX the serum PhCG

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CASE REPORTS 389

lo8

lo5

10‘

10:

lo2

” E 2 10’

6 0 y loc

$ w lo5 cn

104

lo3

lo2

10’

loo

&%E?4GE IDMTX-CFR

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PATIENT 1

IDMTX-CFR ml

\ PATIENT 2

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FIG. 2. phCG profile of study patients.

level reached <5 mIU/ml. Serum PhCG levels remain normal 10 months after IDMTX-CFR and she is on an oral contraceptive for pregnancy control.

RESULTS

Serial serum MTX levels are shown in Fig. 3. Within 30 min of each bolus dose of MTX, the serum concentration of MTX increased rapidly to a maximum of > 1O-6 M. In Patient 1, maximum methotrexate serum levels ranged between 2.9 x 10e6 and 2.1 x IO-’ M with a median of 8.2 x 10e6 M. In Patient 2, maximum serum MTX concentrations ranged from 4 x 10m6 to 1.8 x 10m5 M with a median of 8.5 x lop6 M. A rapid, apparent two-phase, lOOO-fold decrease

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HILGERS ET AL.

P I t

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CASE REPORTS 391

in serum MTX occurred within the first 24 hr and was followed by a pronounced decrease in MTX elimination after administration of CF. Serum MTX concentrations showed little change in the 30 to 48-hr period with serum MTX levels reaching an average low value of 5.9 ? 1.1 x 1O-9 M.

PhCG levels declined gradually during the 8 days of chemotherapy. In Patient 1, pretreatment phCG concentrations fell from 1.7 x lo3 mIU/ml to 0.13 x lo3 mIU/ml and in Patient 2 from 1.3 x lo3 mIU/ml to 0.17 x lo3 mIU/ml. A 1.57 and 1.13-log difference was observed, respectively. After a total dose of 280 and 240 mg of methotrexate, respectively, complete remission occurred at 6.3 and 5.5 weeks after the initial dose of IDMTX. Five of eight doses (63%) of IDMTX resulted in an average increase of 156 mIU/ml phCG (an average increase of 15%) within 8 hr of IDMTX infusion. Three of the five increased values were noted within one-half hour of IDMTX administration.

Patient 1 exhibited no evidence of clinical or laboratory toxicity. In Patient 2, the leukocyte count dropped to 2700/mm3 and the granulocyte count to 1 161/mm3 on Day 6 of chemotherapy. Renal and hepatic studies remained normal and no stomatitis was observed.

DISCUSSION

The g-day IDMTX-CFR protocol was used to treat two patients with non- metastatic gestational neoplasia characterized by a rising PhCG concentration in serum following uterine evacuation of a hydatidiform mole. Complete remission occurred at 6.3 and 5.5 weeks following the first of four doses of methotrexate with an absence of clinically observable toxicity.

Although gestational trophoblastic neoplasms are uniquely sensitive to meth- otrexate, the maximum level of MTX required to achieve 100% cell death remains unknown [10,11]. In vitro studies [12-141 have shown that 100% inhibition of DNA synthesis will occur with methotrexate levels of ~10~~ M. These data, drawn from the GeWo choriocarcinoma grown in cell culture, are based on proliferation of neoplastic trophoblastic cells known to be clinically resistent to methotrexate. Data from cell culture systems derived from MTX-sensitive tro- phoblastic cells are not currently available for comparison. In this study, a complete response followed serial peak MTX levels between 10e6 and lo-’ M.

Hammond et al. [2] graphically demonstrated that the total dose of methotrexate required to achieve a complete remission in patients with nonmetastatic gestational trophoblastic neoplasia is directly related to the premethotrexate level of hCG in urine. An extrapolation of their data to this study suggests the initial phCG levels measured in our patients require a total single dose of 150-200 mg/m* methotrexate for tumor eradication. In NMGTN the total dose required for a complete remission to occur seems to be the same regardless of whether the patient is treated by the coventional 5-day schedule or by the g-day IDMTX- CFR protocol. The data of Hammond et al. [3] (2 mg/kg/5-day course X 2.4 courses = 4.8 mg/kg total dose of MTX) are similar to the findings of Goldstein et al. [15] (4 mg/kg/&day course x 1.1 courses = 4.4 mg/kg total dose of MTX). Since the total dose of methotrexate is equivalent by both schedules, it would seem that IDMTX-CFR has the advantage of minimal morbidity and

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392 HILGERS ET AL.

fewer courses over a shorter time span. Differences in the route of administration, i.e., intravenous versus intramuscular, and drug scheduling [5,16] require further clarification however.

Formerly, selecting the maximal dose of methotrexate to treat patients with NMGTN was determined by normal tissue tolerance which avoided severe toxicity. With CFR and MTX-CFR pharmacokinetics, new protocols may be designed enabling treatment based on a single dose of MTX necessary to achieve a complete remission. The initial dose may be tailored to the height of the pretreatment phCG serum concentration rather than based on a fixed value-to-tolerance. Peak serum methotrexate levels for sensitive neoplasms reach >10e6 M to achieve 100% inhibition of DNA synthesis [ 111. Knowledge of the duration of time during which maximum serum MTX levels must be maintained will be required to ensure that neoplastic trophoblastic cells in the S-phase of the cell cycle are killed within one course of chemotherapy. Furthermore, the slope of the PhCG response curve during or following therapy may provide data that predict which tumors require more than 1 course of therapy or develop MTX resistance. In our limited experience, the response of phCG titers to MTX is very rapid and a 10 to 50-fold decrease was noted within the time frame of the treatment period. Berkowitz and Goldstein [ 171 defined response as a l-log drop of hCG within 18 days following one course of chemotherapy.

Citrovorum factor or N5-formyl tetrahydrofolic acid bypasses the inhibition of the enzyme dihydrofolate reductase by methotrexate to allow synthesis of DNA precursors. The advantages of CFR following MTX therapy include a lack of significant blood count nadirs, a lack of prolonged intervals between courses of chemotherapy, minimal clinical toxicity, and the use of higher doses of methotrexate. Standards for calculating the dose of CF have not been established as they have been with high-dose methotrexate [ 18,191. The threshold plasma concentrations of methotrexate required to inhibit DNA synthesis in bone marrow has been estimated to be 1 x lo-’ M whereas gastrointestinal epithelium is inhibited at 5 x 1O-9 M [20]. Past experience with toxicity associated with the 5-day course of conventionally administered methotrexate indicates that toxicity may be due to the terminal plasma one-half life which occurs 24 or more hours after MTX. Thus, timing of CFR may be critical. The data would suggest that CFR may be given at 24 instead of 30 hr after each dose of methotrexate.

In summary, these preliminary observations suggest that a complete phar- macokinetic analysis of serum methotrexate will become important to our un- derstanding of the sensitivity of heterogeneous trophoblastic cell populations [21] in nonmetastatic trophoblastic neoplasia and that the initial dose of methotrexate may ultimately be selected on the basis of pretreatment serum concentrations of phCG rather than on the basis of normal tissue tolerance and toxicity.

REFERENCES 1. Berkowitz, R. S., Goldstein, D. P., Jones, M. A., et al. Methotrexate with citrovorum factor

rescue. Reduced chemotherapy toxicity in the management of gestational trophoblastic neoplasms, Cancer 45, 423-426 (1980).

2. Hammond, C. B., Hertz, R., Ross, G. T., et al. Primary chemotherapy for nonmetastatic gestational trophoblastic neoplasms, Amer. J. Obsret. Gynecol. 98, 71-78 (1967).

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3. Bagshawe, K. D., and Newlands, E. S. High dose methotrexate in patients with trophoblastic tumors, in High dose methotrexate pharmacology, toxicology and chemotherapy (P. Periti, Ed.), Chemotherapia Oncologica 4, Editrice Giuntina, Firenze, pp. 281-288 (1978).

4. Goldstein, D. P., Goldstein, P. R., Bottomley, P., et al. Methotrexate with citrovorum factor rescue for nonmetastatic gestational trophoblastic neoplasms, Obstet. Gynecol. 48, 321-323 (1976).

5. Berkowitz, R. S., Goldstein, D. P., and Bernstein, M. R. Methotrexate with citrovorum factor rescue as primary therapy for gestational trophoblastic disease, Obstet. Gynecol. 50, 2024- 2027 (1982).

6. Hertig, A. T., and Mansell, H. Tumors of the female sex organs, Part I: Hydatidiform mole and choriocarcinoma, Armed Forces Inst. Pathol. Washington, D.C. (1956).

7. Raso, V., and Schreiber, R. A rapid and specific radioimmunoassay for methotrexate, Cancer Res. 35, 1407-1410 (1975).

8. Vaitukaitis, J., Braunstein, G. D., and Ross, G. T. A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone, Amer. J. Obstet. Gynecol 113, 751-758 (1972).

9. Greenwood, F. C., Hunter, W. M., and Glover, J. S. The preparation of In1 labeled human growth hormone of high specific radioactivity, Biochem. J. 89, 114-123 (1963).

10. Bertino, J. R. “Rescue” techniques in cancer chemotherapy: Use of leucovorin and other rescue agents after methotrexate treatment, Semin. Oncol. 4, 203-216 (1977).

11. Chabner, B. A. Pharmacologic principles of cancer treatment, Saunders, Philadelphia, pp. 229- 255 (1982).

12. Hussa, R. O., and Patillo, R. A. Effects of methotrexate on established cell lines of human choriocarcinoma, Eur. J. Cancer 8, 523-529 (1972).

13. Patillo, R. A., Gey, G. O., Delfs, E., et al. The hormone synthesizing trophoblastic cells in vitro: A model for cancer research and placental hormone synthesis, Ann. N. Y. Acad. Sci. 172, 288-298 (1971).

14. Patillo, R. A. Growth characteristics and glycoprotein hormone properties of the BeWo trophoblastic cell line, in Transcript of the jfth Rochester trophoblast conference (C. J. Lund and J. W. Choate, Eds.), Univ. of Rochester, Rochester, pp. 378-390 (1969).

15. Goldstein, D. P.. Saracco, P., Osathanondh, R., et a/. Methotrexate with citrovorum factor rescue for gestational trophoblastic neoplasms, Obstet. Gynecol. 15, 93-96 (1978).

16. Smith, E. B., Weed, J. D., Tyrey, L., and Hammond, C. B. Treatment of nonmetastatic gestational trophoblastic disease: Results of methotrexate alone versus methotrexate-folinic acid, Amer. J. Obstet. Gynecol. 144, 88-92 (1962).

17. Berkowitz, R. S., and Goldstein, D. P. Methotrexate with citrovorum factor rescue for nonmetastatic gestational trophoblastic neoplasms, Obstet. Gynecol. 54, 725-728 (1979).

18. Bertino, J. R. Clinical use of methotrexate-with emphasis on use of high doses, Cancer Treat. Rep. 65, 131-135 (1981).

19. Jacobs, S. A., and Santicky, M. J. Phase I trial of high-dose methotrexate with modified citrovorum factor, Cancer Treat. Rep. 62, 397-399 (1978).

20. Chabner, B. A., and Young, R. C. Threshold methotrexate concentration for in vivo inhbition of DNA synthesis in normal and tumorous target tissues, J. Clin. Invest. 52, 1804-1811 (1973).

21. Friedman, S. J., and Skehan, P. Morphological differentiation of human choriocarcinoma cells induced by methotrexate, Cancer Res. 39, 1960-1967 (1979).