Hypercalcemia Due to Parathyroid Hormone-Related Protein Produced by Primary Ovarian Clear Cell Adenocarcinoma: Case Report

Gynecologic Oncology 76, 218 –222 (2000) doi:10.1006/gyno.1999.5619, available online at http://www.idealibrary.com on

CASE REPORT Hypercalcemia Due to Parathyroid Hormone-Related Protein Produced by Primary Ovarian Clear Cell Adenocarcinoma: Case Report 1 Ryosuke Tsunematsu, M.D.,* Toshiaki Saito, M.D.,* ,2 Haruo Iguchi, M.D.,† Toshiro Fukuda, M.D.,‡ and Naoki Tsukamoto, M.D.* *Gynecology Service, †Department of Biochemistry, and ‡Department of Pathology, National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka 811-1395, Japan Received June 30, 1999

have shown parathyroid hormone-related protein (PTHrP) to be the major cause of the HHM [1]. Clear cell adenocarcinoma of the ovary has been recognized as one of the most common histologic subtypes associated with hypercalcemia [2]. However, the mechanisms of hypercalcemia in clear cell carcinoma have yet to be elucidated because this life-threatening complication usually develops in the terminal stage of the disease. We experienced a case of ovarian clear cell adenocarcinoma associated with HHM prior to the primary surgery and proved that ovarian clear cell adenocarcinoma has the potential to produce PTHrP at the primary site.

Objective. Among ovarian carcinomas, clear cell adenocarcinoma is one of the most common histologic subtypes associated with hypercalcemia. However, the mechanisms of hypercalcemia in clear cell adenocarcinoma are still unclear. In the following case report, we tried to determine the etiology of hypercalcemia and also to demonstrate the management of hypercalcemia diagnosed preoperatively. Case. A 49-year-old woman was diagnosed as having a malignant ovarian tumor with hypercalcemia caused by elevated serum parathyroid hormone-related protein (PTHrP) prior to her primary surgery. Treatment with disodium incadronate promptly normalized the serum calcium level. An immunohistochemical study demonstrated PTHrP expression in the primary ovarian lesion, but not in the metastatic lesion. A Northern blot analysis of the cancer cells from the ovarian tumor confirmed the presence of PTHrP mRNA. Conclusion. Humoral hypercalcemia of malignancy in this case has been conclusively shown to be due to the production of PTHrP at the primary ovarian tumor, based on both immunohistochemical and molecular analyses. © 2000 Academic Press Key Words: ovarian cancer; clear cell adenocarcinoma; hypercalcemia; parathyroid hormone-related protein; incadronate.

CASE REPORT

INTRODUCTION

In April 1998, a 49-year-old woman, gravida 2, para2, was referred to our hospital with a pelvic mass and a 6-month history of increasing abdominal distension. For a week prior to admission, she had been having loss of appetite, nausea, malaise, constipation, and thirst. Her past medical history was not remarkable. She had had regular periods until reaching menopause at 46 years of age. A physical examination demonstrated abdominal distension without fluctuation. There was no mucosal dryness. Her pulse rate was 102/min and blood pressure was 110/60 mm Hg. Her body temperature was 36.4°C and deep tendon reflexes were brisk. A pelvic examination revealed a cystic abdominal mass measuring 25 cm in diameter arising from the pelvic cavity. The uterus was hard to outline due to the above mass. A serum chemistry analysis revealed a serum calcium level of 13.0 mg/dl, 2.9 mg/dl phosphorus, 3.4 g/dl albumin, and 82 IU/L alkaline phosphatase. The serum level of PTHrP was 259 pmol/L (normal range 13.8 –55.3 pmol/L), which was determined by a RIA kit specific for the C-terminal portion of PTHrP (Daiichi RI Co. Ltd., Tokyo, Japan). The parathyroid hormone level was 0.3 pmol/L (⬍0.5 pmol/L). The remaining laboratory findings were unremarkable. The serum ␣-fetopro-

Hypercalcemia is one of the common paraneoplastic syndromes in gynecologic malignancies. Hypercalcemia accompanied by malignancy is categorized into two major groups: One is local osteolytic hypercalcemia (LOH), due to local osteolysis induced by the tumor metastasized to bone, while the other is humoral hypercalcemia of malignancy (HHM), due to systemic bone resorption caused by humoral factors produced by carcinoma cells. Over the past decade, various studies 1

This case study was partly supported by a Grant-in-Aid for Cancer Research (10-12) from the Ministry of Health and Welfare, Japan. 2 To whom correspondence should be addressed. Fax: 81-92-542-8503. E-mail: [email protected]. 0090-8258/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Sequential changes in the serum calcium and PTHrP levels. The upper horizontal band represents the normal range of serum calcium (8.4 –9.8 mg/dl). The lower horizontal band represents the normal range of PTHrP concentration (13.8 –55.3 pmol/L).

tein, carcinoembryonic antigen, CA-125, and CA19-9 levels were not elevated. A computed tomographic scan demonstrated a pelvic mass extending to the level of the umbilicus measuring 24 ⫻ 22 ⫻ 12 cm with well-defined margins and a monoloculated cystic, low-density central area. The uterus was normal in size and was pushed by the mass toward the sacrum. A small amount of ascites was recognized in the pelvis. No abnormal findings were recognized in the bone, liver, spleen, pancreas, kidneys, and retroperitoneal lymph nodes. Bone scintigraphy revealed no abnormal uptake. Plain X rays of the skull, vertebrae, chest, and pelvic bones did not show any evidence of bone destruction. She was diagnosed as having HHM induced by PTHrP, which was probably produced by the ovarian tumor. Immediately after the above diagnosis was made, 10 mg of disodium incadronate (Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan) was administered by 2-h intravenous infusion. Four days later, the serum calcium level decreased to a normal level (9.7 mg/dl) (Fig. 1). One week after the administration of disodium incadronate, she underwent a laparotomy. In the abdominal cavity, a small amount of ascites was found. The tumor originated from the right ovary, while the gross appearance of the uterus and left ovary was normal. Neither the pelvic lymph nodes nor the paraaortic lymph nodes were apparently swollen. Small metastatic nodules were found in the undersurface of the bilateral diaphragm. A histopathological diagnosis based on the frozen sections of the ovarian tumor was clear cell adenocarcinoma. A total abdominal hysterectomy, bilateral salpingo-oophorectomy, pelvic lymph node dissection, paraaortic lymph node biopsy, partial omentectomy, and appendectomy were performed. The washing peritoneal cytology was reported positive. The right ovarian tumor had a multiple bosselated yellowish

surface and showed no invasion of the capsule macroscopically. The cut surface revealed a mixture of solid parts and small- to medium-sized cystic spaces separated by fibrous septae. The histological examination showed that cuboidal carcinoma cells with a pale-staining cytoplasm grew in solid aggregates, lined tubules, and papillae. In the solid portions, the carcinoma cells with clear or vacuolated cytoplasm diffusely proliferated. In the papillary structure, hobnail cells and stromal hyalinization were frequently observed. These microscopic findings were compatible with the clear cell adenocarcinoma of the ovary (Fig. 2). Microscopic foci of metastasis were found in the left obturator lymph node. She was staged as FIGO stage IIIc. Following surgery, she was treated with six courses of chemotherapy: one course of 899 mg CBDCA, AUC 6 with 175 mg/m 2 Paclitaxel in a 3-h infusion and five subsequent courses of 70 mg/m 2 CDDP with 500 mg/m 2 cyclophosphamide. The CBDCA with Paclitaxel regimen could not be continued due to an episode of severe pseudomembranous enterocolitis. Her serum calcium and PTHrP remained in the normal range after surgery. She recurred intraperitoneally 3 months after the cessation of chemotherapy. However, her serum calcium and PTHrP did not elevate at recurrence. IMMUNOHISTOCHEMICAL ANALYSIS Formalin-fixed and paraffin-embedded sections of the primary lesions were dewaxed with xylene and dehydrated through a series of graded alcohol. After endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol, the sections were incubated with anti-human PTHrP polyclonal antibody Y201 (Yanaihara Institute Inc., Shizuoka, Japan) in 50 mmol/L phosphate-buffered saline, diluted 1:1000. The avidin– biotin–peroxidase complex (ABC) method was used with Ultratek HRP (Anti-Rabbit) kit (Scytek Laboratories, Logan, UT, USA). The final development of the sections was carried out with 3,3⬘-diaminobenzidine containing 0.03% hydrogen peroxide. The cytoplasm of carcinoma cells of the primary lesion showed definite positive staining (Fig. 3). However, the metastatic lesion of the left obturator lymph node demonstrated negative staining. This indicates that the cancer cells in the ovarian tumor are the source of the elevated serum PTHrP. NORTHERN BLOT ANALYSIS FOR mRNA OF PTHrP A Northern blot analysis for mRNA of PTHrP was performed on cultured cancer cells derived from the ovarian tumor. Briefly, a single-cell suspension of tumor cells was prepared from a tissue specimen of the ovarian tumor obtained at surgery using mechanical and enzymatic techniques. The cells were placed in a tissue culture flask and incubated at 37°C in a humidified atmosphere of 5% CO 2 in air. The tumor cells

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FIG. 2.

Microscopic appearance of the ovarian tumor (H & E stain; original magnification, ⫻40).

were maintained in an RPMI 1640 medium containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 ␮g/ml). After confluent growth of tumor cells was achieved, a Northern blot analysis was performed.

FIG. 3.

Total RNA was extracted from the tumor cells using the guanidium thiocyanate technique. Aliquots of total RNA were electrophoresed on a 1% agarose–formaldehyde gel, transferred to a nylon membrane, and immobilized with UV light.

Immunohistochemical finding of the primary lesion (⫻200). Tumor cells show strong and diffuse positivity for immunoreactive PTHrP.

CASE REPORT

FIG. 4. A Northern blot analysis for the cancer cells derived from the ovarian tumor. Case-Y, tumor cells from the present case; KP-1N, pancreatic cancer-derived cell line as a negative control; HARA-B, lung cancer-derived cell line as a positive control. A transcript of the PTHrP mRNA was seen at around 1.1 kb in case-Y, while multiple transcripts were seen in the positive control (HARA-B).

Blots were prehybridized and hybridized in 5⫻ Denhaldt’s reagent, 4⫻ SSC, 100 ␮g salmon sperm DNA, 50% formamide, 0.5% SDS, and 10% dextran sulfate. Full-length PTHrP cDNA was labeled with [␣- 32P]dCTP to a specific activity of 1.2 ⫻ 10 9 cpm/␮g DNA by the random priming technique and blots were hybridized with 1 ⫻ 10 6 cpm/ml 32P-labeled cDNA probe for 12 h at 48°C. The washing conditions were 2⫻ SSC– 0.5% SDS for 20 min at room temperature, 0.2⫻ SSC– 0.5% SDS for 20 min at 54°C, and 0.1⫻ SSC at room temperature. The labeled PTHrP mRNA on the membrane was detected with a Bio-Imaging Analyzer (BAS 1000, Fuji Film, Tokyo, Japan). As shown in Fig. 4, a Northern blot analysis revealed the presence of PTHrP mRNA in the tumor cells derived from the ovarian tumor of the present case. DISCUSSION Hypercalcemia is thought to occur in approximately 8 to 10% of all patients with malignant disease. However, the diagnosis is frequently delayed. In order to avoid a delay of appropriate management, it is vital to know the tumor types associated with hypercalcemia, the mechanism that generates the hypercalcemia, and the symptoms that will thus help lead to a prompt diagnosis and timely and appropriate intervention. Among ovarian carcinomas, small cell carcinoma and clear cell carcinoma are the most frequently reported subtypes associated with hypercalcemia. Young and colleagues recently described in detail the clinicopathological characteristics of 150 cases of ovarian small cell carcinoma of the hypercalcemic type [3]. Matias-Guiu and colleagues examined the role of PTHrP in seven cases of Young’s series using immunohistochemical methods, but found no correlation between immunoreactivity and serum calcium levels [4]. They recommended additional studies using probes for PTHrP messenger RNA to determine whether or not the cells have the ability to synthesize this hormone [4].

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There have thus far been no large studies analyzing clear cell adenocarcinoma of the ovary in relation to hypercalcemia. Several case reports have suggested that PTHrP is the cause of hypercalcemia in advanced or recurrent clear cell carcinoma. Fujino et al. described a case of clear cell carcinoma of the ovary that demonstrated hypercalcemia only at recurrence with an elevated serum PTHrP level [5]. They detected a significant amount of immunoreactive PTHrP in the metastatic lymph node using a radioimmunoassay. In addition, Kitazawa et al. recently reported a case of recurrent clear cell carcinoma of the ovary with pulmonary metastases, manifesting hypercalcemia during the last 3 months of the patient’s clinical course [6]. They demonstrated the localization of PTHrP immunohistochemically at both the primary and the metastatic sites. Furthermore, they revealed a transcript of PTHrP at pulmonary metastatic sites based on in situ hybridization and the reverse transcription-polymerase chain reaction method. However, they failed to show any PTHrP transcripts by a Northern blot analysis because the amount of RNA extracted from the frozen tumor tissue was insufficient to perform an analysis. This is the first reported case of clear cell adenocarcinoma demonstrating immunoreactivity for PTHrP and the expression of mRNA of PTHrP using a Northern blot analysis in the primary site. This case proved the potential of cancer cells in clear cell adenocarcinoma of the ovary to cause humoral hypercalcemia by producing the PTHrP. Interestingly, PTHrP was only detected immunohistochemically in cancer cells of the primary site. The heterogeneity of cancer cells with regard to PTHrP production is probably the cause of this phenomenon and also the reason for inconsistent hypercalcemic events during the clinical course of our patient. PTHrP is now widely accepted as the most common cause of HHM [1]. The cDNA was first cloned and purified in 1987 [7]. PTHrP has a strong homology in the amino-terminal region with PTH. PTHrP binds to PTH/PTHrP receptor in bone and kidney with equal affinity to parathyroid hormone and thus represents a persistent hypercalcemic effect. PTHrP has also been reported to function as a growth factor in an autocrine/ paracrine fashion in many normal tissues. In breast cancer, it is suggested that PTHrP either enhances the survival of tumor cells or promotes their ability to invade bone [8]. Based on such evidence, PTHrP producing tumors could have a more aggressive nature than other tumors that do not produce PTHrP. Both clear cell adenocarcinoma and small cell carcinoma have been recognized to be histologic subtypes with a poor prognosis among the various subtypes of ovarian cancer. In addition, both of these histologic subtypes have the same characteristics of a higher frequency with hypercalcemia than other subtypes of ovarian cancer. The aggressive nature of both of these cancers might be attributed to the potential ability of the tumor cells to produce PTHrP. There has been a drastic change in the treatment of hypercalcemia of malignancy in recent years after introduction of bisphosphonate [9]. Etidronate, clodronate, and pamidronate

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have been the most extensively tested bisphosphonates in patients with hypercalcemia. These agents were shown to decrease serum calcium levels in the majority of patients. Incadronate is a third-generation bisphosphonate that was licensed as a drug for malignancy associated with hypercalcemia in Japan [10]. Incadronate is thought be the most potent inhibitor of bone resorption among the available bisphosphonates. The effect of incadronate on serum calcium in patients with hypercalcemia tends to be remarkable. A 2-h infusion is usually sufficient and the effect lasts approximately 2 weeks. In our case, a prompt normalization of serum calcium levels was achieved by only one administration of incadronate prior to surgery. The most effective way to control hypercalcemia of malignancy is to eradicate or reduce the tumor burden by surgery and/or chemotherapy. We need to keep in mind that some ovarian cancers might be associated with hypercalcemia without bony metastasis and that a prompt diagnosis and a rapid initiation of treatment could prevent serious complications associated with this metabolic abnormality. REFERENCES 1. Wysolmerski JJ, Broadus AE: Hypercalcemia of malignancy: The central role of parathyroid hormone-related protein. Annu Rev Med 45:189 –200, 1994

2. Holtz G: Paraneoplasitc hypercalcemia in gynecologic malignancy. Obstet Gynecol Surv 35(3):129 –136, 1980 3. Young RH, Oliva E, Scully RE: Small cell carcinoma of the ovary, hypercalcemic type. A clinicopathological analysis of 150 cases. Am J Surg Pathol 18:1102–1116, 1994 4. Matias-Guiu X, Prat J, Young RH, Capen CC, Rosol TJ, Delellis RA, Scully RE: Human parathyroid hormone-related protein in ovarian small cell carcinoma. Cancer 73(7):1878 –1881, 1994 5. Fujino T, Watanabe T, Yamaguchi K, Nagasaki K, Onishi E, Iwamoto I, Dozono H, Nagata Y: The development of hypercalcemia in a patient with an ovarian tumor producing parathyroid hormone-related protein. Cancer 70(12):2845–2850, 1992 6. Kitazawa R, Kitazawa S, Matui T, Maeda S: In situ detection of parathyroid hormone-related protein in ovarian clear cell carcinoma. Hum Pathol 28(3):379 –381, 1997 7. Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Mosley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI: A parathyroid hormone-related protein implicated in malignant hypercalcemia: Cloning and expression. Science 237:894 – 896, 1987 8. Bundred NJ, Ratcliffe WA, Walker RA, Coley S, Morrison JM, Ratcliffe JG: Parathyroid hormone related protein and hypercalcemia in breast cancer. Br Med J 303:1506 –1509, 1991 9. Roger MJ, Watts DJ, Russell RGG: Overview of bisphosphonates. Cancer 80:1652–1660, 1997 10. Usui T, Oiso Y, Tomita A, Ogata E, Uchida T, Ikeda K, Watanabe T, Higuchi S: Pharmacokinetics of incadronate, a new bisphosphonate, in healthy volunteers and patients with malignancy-associated hypercalcemia. Int J Clin Pharmacol Ther 35:239 –244, 1997