HUMAN CHORIONIC GONADOTROPIN AND MALIGNANT MESOTHELIOMA

STUART RICH, MD,* CARY A. PRESANT, MD,* JOHN MEYER, MD,? SUE C. STEVENS, PHD,? A N D DAVID CARR, BS*

A 56-year-old male presented with ascites and gynecomastia. Laparoscopy demonstrated peritoneal tumors which were biopsied. Conventional histology and electron microscopy revealed the tumor to be a malignant mesothelioma. The ascitic fluid and tumor cell lysate, but not serum, contained hCG by specific assay, and the immunoreactive hCG had characteristics similar to purified hCG in filtration on Sephadex G-100. Malignant mesothelioma is a tumor which may be associated with elevated hCG concentrations. Demonstration of hCG in ascitic fluid should suggest the presence of neoplasm.

Cancer 43:1457-1462, 1979.

ESOTHELIOMA, once thought to be a very M rare tumor, is now being diagnosed with increasing f r e q ~ e n c y . ~ , ' ~ Polypeptide hormone production by mesothelioma cells has not previously been documented, although hypoglycemia has been observed in patients with mesothelioma." We report here a case of malignant mesothelioma associated with human chorionic gonadotropin in ascitic fluid and in tumor cells, but not in serum.

CASE REPORT A 56-year-old black male came to The Jewish

Hospital of St. Louis in December 1976, complain- ing of 6 weeks of increasing abdominal girth, de- creasing urine volume, and breast enlargement. There was no history of jaundice, liver disease, alcoholism or abdominal pain. The patient was receiving no medication and had been in excellent health. He stated that his occupation inluded han- dling of asbestos fibers for 6 years beginning 25 years prior to the onset of symptoms.

Physical examination revealed a cachectic patient with bilateral gynecomastia without discharge or tenderness. The abdomen was markedly distended by ascites. There was no adenopathy or organo- megaly. The penis and testicles were normal-sized, and the genital hair distribution was masculine.

From the Departments of *Medicine and ?Pathology, Washington University School of Medicine at The Jewish Hospital of St. Louis, St. Louis, Missouri.

Supported in part by the Multimodality Oncology Project, T h e Jewish Hospital of St. Louis, St. Louis, Missouri.

Address for reprints: Cary A. Presant, MD, Depart- ment of Medicine, T h e Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, MO 63110.

Accepted for publication May 22, 1978.

The serum lactate dehydrogenase was 27 I IU/l , serum glutamic oxalacetic transaminase 27 I U l l , and serum bilirubin 0.5 mg/dl. Radioimmunoassay for alpha fetoprotein (Clin-Chem Laboratories, Boston, MA) revealed less than 1 pg/ml. Total serum thyroxine was 13.0 pg/dl, and free serum thyroxine was 1.65 ng/dl. Serum thyroid stimulat- ing hormone was 4.9 pIU/ml. Serum estradiol (E2) was 109.0 pglml. Serum testosterone was 330 ng/dl. Serum human chorionic somatrotropin was less than 1.0 pglml. Chest roentgenograms were normal and there were no pleural masses. Bone and liver scans were normal. A paracentesis yielded straw- colored fluid with a total protein of 5.0 g/dl and a lactate dehydrogenase of 620 lUI1. The fluid contained 3650 erythrocytes and 290 nucleated cells per mm3. Cytological examination revealed the nucleated cells to be neoplastic, consistent with malignant mesothelioma. Laparoscopy revealed nodular, polyploid lesions diffusely covering pari- etal and visceral peritoneal surfaces. Biopsies were obtained which confirmed the diagnosis of malig- nant mesothelioma (see below).

The patient initially received 15 mCi of 32P intra- peritoneally. When the ascites reaccumulated rapidly in February 1977, the patient was begun on intravenous injections of adriamycin 60 mg/m2, cyclophosphamide 600 mg/mz, plus methotrexate 25 mg/m2 every 3 weeks. Although the patient's strength increased, palliative paracenteses were required approximately once per month. After 4 months of chemotherapy, a supraclavicular lymph node appeared and the volume of ascites increased. A second intraperitoneal injection of 32P produced a transient improvement, but the patient expired in August 1977.

LABORATORY INVESTIGATIONS Tissue obtained at biopsy was examined by

light microscopy. The tumor was papillary

0008-543X/79/0400/1457 $0.80 0 American Cancer Society

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1458 CANCER April 1979 Vol. 43

FIG.. 1. Photomicro- graph of mesothelioma showing papillary epi- thelial growth pattern with delicate supporting stroma. Nuclei are small and lack anaplastic fea- tures (H & E. X260).

FIG. 2. Electron micrograph showing several rnesothehma cells surrounded by a basal lamina on the left. At the lower right is a lumen into which microvilli protrude. Deposits of glycogen and a few lipid vacuoles are present, particularly in cell at upper left. Profiles of tubular endoplasrnic reticulum are present (Uranyl acetate, lead citrate, x 12,500).

No. 4 HCG A N D MALIGNANT MESOTHELIOMA . Rich et al. 1459

FIG. 3. Electron micrograph at higher power showing macula occludens adjacent to lumen and a desmosome on the left. Tubular profiles of rough endoplasmic reticulum, mitochondria, and aggregates of cytoplasmic microfibrils are present. A Golgi apparatus is visible between the nucleus and the luminal surface of the cell on the lower left (Uranyl acetate, lead citrate, X 18,900).

with minimal nuclear pleomorphism and del- icate supportive connective tissue growing on the peritoneal surface (Fig. 1). No glands were formed, and no mucin was present. Diastase- controlled periodic acid Schiff stains demon- strated moderate amounts of intracytoplasmic glycogen. Examination by transmission elec- tron microscopy (Figs. 2, 3) demonstrated several characteristic features of epithelial mesotheliomas: basal laminas, desmosomes, microvilli, and intracellular fibril^.'^^'^,^^ In further agreement with ultrastructural obser- vations on mesotheliomas, mitochondria were abundant and showed a tendency toward peri- nuclear grouping, Golgi lamellae and vesicles were well developed, only scattered lamellar or tubular profiles of rough endoplasmic re- ticulum were present, and glycogen particles were present focally.

Tissues were incubated with tritiated thy- midine and processed by autoradiography to give an estimate of cellular kinetic^.'^.'^ The S-

phase fraction of the neoplasm was 12.4% (Fig. 4), which indicates a high rate of DNA synthesis.

Two samples of ascitic fluid, 3.6 1 and 4.0 1, were obtained at separate times prior to ther- apy and processed identically. They contained 2.2 x lo8 and 5.0 x lo8 neoplastic cells. Fluid was centrifuged at 1000 x g for 10 minutes at 4 C. The supernatant fluids were assayed for hCG by quantitative radioimmuno- assay for the P subunits of hCG (P-hCG, BioRIA, Montreal, Canada). This radioim- munoassay shows no cross reactivity with fol- licle stimulating hormone, luteinizing hor- mone, growth hormone, or thyroid stimulat- ing hormone, and is sensitive to 0.15 mIU P-hCG.5 The first supernatant fluid contained 7.6 mIU/ml (9.7 ng/ml) or 3.93 mIU per mg protein determined by the method of Lowry et a1.12 The second contained 12.0 mIU/ml (15.3 ng/ml) or 6.3 mIU/mg protein. Assays for P-hCG in serum samples obtained

1460 CANCER April 1979 Vol. 43

FIG. 4. Autoradiograph of mesothelioma after incubation with tritiated thymidine for 2 hr. S-phase nuclei (engaged in DNA synthesis at time of incubation) are identified by overlying dark silver grains, and their large number indicates ii rapid rate of replication by the neoplastir tnesothelial cells ( H 8c E, X425).

simultaneously revealed less than 0.4 mIU/ml on each occasion (which was a normal result for males by this method). In contrast, ascitic fluid from a patient with hepatic cirrhosis con- tained no detectable P-hCG.

The cell pellet obtained from the first para- centesis and centrifugation was then subjected to discontinuous density gradient centrifuga- tion on Ficoll-Hypaque as previously described to separate any red blood cells from neoplastic cells.14 After addition of 10 ml 0 . 1 5 ~ NaCl to the neoplastic cell pellet, cells were lysed by sonication for 60 seconds and centrifuged at 20,000 x g for 30 minutes at 4 C. The supernatant cell lysate solution was then as- sayed for p-hCG, and contained 7.7 mIU p - hCG/mg protein.

T o characterize further the nature of the ectopic protein, the protein contained in the ascitic fluid was subjected to gel filtration on a Sephadex GlOO column which had previ- ously been standardized with partially-purified normal hCG (Sigma Chemical Co., St. Louis, MO) and “asialo-hCG.” The ectopic protein

displayed a filtration pattern similar to that of normal hCG (Fig. 5 ) . The pattern of the partially purified normal hCG consisted of 2 peaks, possibly representing both intact hCG and free p chains. The ascitic fluid hCG ac- tivity migrated in both regions, indicating that both intact hCG and possibly also free p chains were present in the ascitic fluid. There was no evidence of protein migrating with “asialo- hCG.”

DISCUSSION

Chorionic gonadotropin production, though characteristic of trophoblastic neo- plasms, has recently been associated with vari- ous nontrophoblastic tumors. Increased hCG concentrations in serum have been docu- mented in patients with carcinomas of the lung, stomach, pancreas, breast, ovary, pros- tate, liver and kidney, and leukemia, malig- nant melanoma, malignant lymphoma, islet cell tumors, and some soft tissue 8 3 9 3 1 3 * 1 9 In the current case, increased hCG con-

No. 4 HCG AND MALIGNANT MESOTHELIOMA . Rich et al.

500- FIG. 5. Gel filtration ofhCG and neoplastic ascitic fluid super- natant. Partially purified hCG (6700 mIC‘img protein) was ob- tained from Sigma Chemical Co. (St. Louis, MO), and 0.24 nig hCG w a s incubated with 150 units V . E cholmr neurarninidase (Calbio- L

chem, San Diego, CA) at 37 C for 90 minutes tn produce “asialo- hCG.” T h e reaction was ter- 2 minated by cooling to 4 C. The E solutions were dialyLed against 0 . l 5 ~ iYaCl prioi- t o gel filtration. 0 T w o ml containing 0.5 mg par- u tially purified hCG (O), 2 nil -C containing 0.5 tng “asialo-hCG” ’ (B), or 2 ml of neoplastic ascitic fluid (concentrate from ultra- filtration) (0) were applied to a Sephadex Gl00column (1.8 X 20 cni at 4 C) which was equilibrated

C- 400-

300-

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0-

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fractions were cchlected and as- sayed for p-IiCG.

centrations were observed repeatedly in ascitic fluid of a patient with malignant mesothelioma.

Trophoblastic differentiation has been rec- ognized by light microscopy in a few examples of extragonadal, nongerm cell malignancies,’ and has been associated with gonadotropin secretion,I3 but it was not observed in any of the 113 neoplasms associated with P-hCG studied by Braunstein and associates.2 The syncytial trophoblastic cell, which is the pre- dominant site of gonadotropin production in the normal placenta and in choriocarcinoma, has prominent dilated cisternas of rough endo- plasmic reticulum that occupy a large portion of its cytoplasmic volume. The cytotropho- blastic cell, in which only isolated droplets of gonadotropin can be detected, has a scanty, tubular, rough endoplasmic reticulum.

The cells of the mesothelioma that we studied resembled the cytotrophoblast in hav- ing only tubular rough endoplasmic reticulum similar to that observed in other mesotheli- omas.I0 We were not able to discern any pe- culiarities of structure, either by light or elec- tron microscopy, that would seem associated with secretion of chorionic gonadotropin.

The association of hCG and malignant mes- othelioma in this patient was indicated by demonstration of hCG in the ascitic fluid and in the lysate of neoplastic cells. Since the filtra- tion characteristics were the same as those of partially purified normal hCG, it is likely that the protein was mostly intact hCG, rather than

ELUTION VOLUME (ml)

just P-hCG or a-hCG subunits of asialo-hCG, all of which have slower filtration through Sephadex GIOO.” It is also unlikely that the assayed hCG activity was some nonspecific protein effect on the assay, since such effects would not be expected to have filtration characteristics similar to purified human hCG on Sephadex G100.

Our observations in this patient suggest that tumor cell synthesis and secretion of hCG can occur in malignant mesothelioma. However, it is also possible (but unlikely) that our demon- stration of hCG in tumor cell lysate represented uptake and storage of hCG, and not synthesis.

The absence of assayable hCG in the serum is notable. Preferential sequestration of the protein in the peritoneum is unlikely. Al- though clearance of hCG subunits3 or asialo- glycoproteins’ from serum has been shown to be more rapid than that of intact hCG, the filtration characteristics of the ectopic protein in our patient suggest neither explanation. The finding of hCG in ascitic fluid despite its absence in serum has potential clinical applications as a diagnostic indication of neoplastic etiology of the ascites. Indeed, one specimen of ascitic fluid from a patient with hepatic cirrhosis lacked detectable quantities of P-hCG. A prospective trial is necessary to document the frequency of P-hCG occurring in patients with ascites of both neoplastic and benign etiologies.

Kahn et al.9 noted elevated plasma levels of

1462 CANCER April 1979 Vol. 43

hCG or of its alpha or beta subunits in 17 of 27 patients with islet cell carcinomas, but not in 43 patients with benign islet cell tumors, which suggests that high rates of production of hCG or its subunits are associated with rapid growth. In a variety of benign tumors studied in our laboratory, we have not observed S- phase fractions in excess of 4.7%, and values in excess of 10% are associated with rapidly growing neoplasms that include colorectal and gastric carcinomas, poorly differentiated breast carcinoma, and histiocytic and Burkitt

lymphoma^.'^^'^ Further studies are indicated to determine whether synthesis of hCG or its subunits may be related to the growth rate or histologic features of mesotheliomas.

In summary, we have reported a patient with peritoneal mesothelioma which was as- sociated with ectopic production of hCG. Further studies are indicated to define the frequency of elevated P-hCG levels as a marker for this tumor, and the frequency of high P-hCG levels in effusions or ascites of neo- plastic etiologies.

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