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Vol. 82, No. 4, February 21, 1990

The syndrome of inappropriate antidiuretic hormone (SIADH) secretion

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(14) HINTZ RL: Plasma forms of somatomedin and the binding protein phenomenon. Clin Endocrinol Metab 13:31-42, 1984

commonly occurs in patients with small cell lung cancer (1-10). Experimental evidence {11-16) has suggested that Expression of the Atrial this syndrome is caused by the ectopic Natriuretic Factor Gene in production and secretion of arginir.e Small Cell Lung Cancer vasopressin (AVP). However, although measurement of plasma AVP in paTumors and Tumor Cell Lines tients with small cell lung cancer and SIADH may reveal normal AVP levels David P. Bliss, Jr., James F. (75), it has not been demonstrated in all Battey, R. Ilona Linnoila, tumors from such patients (15). When Michael J. Birrer, Adi F. tumor studies have failed to reveal AVP Gaidar, Bruce E. Johnson* production, mechanisms involving abnormal posterior pituitary secretion of AVP have been postulated (17). To circumvent difficulties with imHyponatremia in patients with small munohistochemical analysis of AVP in cell lung cancer can be caused by tumor specimens and the plasma AVP tumor production of arginine vasopeptide measurement, we studied AVP pressin (AVP) and result in the synmRNA expression in tumors and tudrome of inappropriate antidiuretic mor cell lines from patients with small hormone. In evaluating the expression cell lung cancer and either SIADH of AVP mRNA from tumor and tuor normal serum sodium values. In mor cell line specimens from five patients with small cell lung cancer and the initial experiments, not all patients hyponatremia (presumed to have the with SIADH expressed AVP mRNA, syndrome of inappropriate antidiuretic which corroborated the occasional abhormone), we found that the tumors sence of AVP peptide production preand tumor cell lines from two of these viously described. Small cell lung canfive patients expressed AVP mRNA. cer tumors or tumor cell lines, or both, The RNA samples from the three pa- are known to synthesize various peptients with undetectable AVP mRNA tides, such as adrenocorticotrophic horexpressed abundant atrial natriuretic factor (ANF) mRNA. Analysis of specimens from three patients with small cell lung cancer and normal serum sodium levels revealed no detectable Received October 20, 1989; revised November AVP mRNA expression, and samples 9, 1989; accepted November 15, 1989. from only one of these three patients' D. P. Bliss, Jr., J. F. Battey, R. I. Linnoila, specimens expressed detectable ANF M. J. Birrer, A. F. Gazdar, B. E. Johnson, mRNA. The AVP and ANF peptide lev- NCI-Navy Medical Oncology Branch, National els in lysate preparations of the tumor Cancer Institute and National Naval Medical cell lines from four of these patients Center, Bethesda, MD. Present address: D. P. Bliss, Jr., Department were tested by radioimmunoassay and of Surgery, Washington University School of confirmed the gene expression data. Medicine, St. Louis, MO. These studies demonstrate ectopic proM. J. Birrer, Department of Medicine, Uniduction of ANF mRNA in small cell formed Services University of the Health Scilung cancer specimens from patients ences, Bethesda, MD. We thank Ms. S. Jensen and Mr. J. Way for with this cancer and the syndrome technical assistance; Dr. H. Oie, Mr. E. Russell, of inappropriate antidiuretic hormone. and Ms. S. Stephenson for establishment of the These findings will be of particular small cell lung cancer cell lines used in this study; interest if future studies demonstrate and Drs. J. Minna, S. Wells, M. Kuehl, S. Sethat ectopic ANF production can cause gal, N. Seibel, and J. Brennan and Messrs. K. sodium abnormalities in patients with Nakahara and J. Way for helpful suggestions. We are also grateful to Drs. R. Scarborough and S. small cell lung cancer. [J Natl Cancer Hilliker of California Biotechnology, Inc., MounInst 82:305-310, 1990] tain View, CA, for providing the ANF probe phNFpUClA.713. * Correspondence to: Bruce E. Johnson, M.D., NCI-Navy Medical Oncology Branch, National Naval Medical Center, Bldg. 8, Rm. 5101, Bethesda, MD 20814.

REPORTS

305

Patients and Methods Patients, Tumors, and Tumor Cell Lines The study group consisted of eight patients with extensive-stage, small cell lung cancer proven by histologic examination. They were treated at the National Cancer Institute-Navy Medical Oncology Branch of the Institute and Bethesda Naval Hospital, Bethesda, MD, or Veterans Administration Medical Oncology Branch, Washington, DC, from April 1981 through January 1986. The classifications for staging and response to chemotherapy are as defined {26). Survival was measured from the initiation of chemotherapy. Five patients were selected who were hyponatremic before chemotherapy was initiated, had the diagnosis of SIADH, and from whom tumors or tumor cell lines, or both, were available. The laboratory criteria used to establish SIADH were serum sodium values less than 130 mmol/L, a low serum osmolality of less than 280 mOsm/kg, and a high concurrent urine osmolality of greater than 500 mOsm/kg {10). Patients with SIADH were identified before medical treatment because morphine and chemotherapeutic drugs, such as cyclophosphamide and vincristine, can impair free water excretion 306

by various mechanisms {27). No patients who had hyponatremia and brain metastases, intrinsic renal insufficiency, or hepatic failure were included in the study. Three other patients with accessible tumor and tumor cell lines, who exhibited serum sodium levels above 130 mmol/L during their disease course and who did not have evidence for SIADH, were used as a control group. All eight patients were deceased at the time of this analysis. The study of patient materials was performed according to approved protocols by the respective institutional review boards. Metastatic tumor samples were obtained at postmortem examination and stored at —70 °C. Tumor cell lines were established from biopsy tumor specimens in seven patients according to techniques we and our associates described {28,29). RNA (Northern) Blot Hybridization Tumor specimens were minced with a razor blade in 4 M guanidium isothiocyanate buffer and further homogenized in a polytron obtained from Kinematica GmbH, Lucerne, Switzerland. Tumor cell lines were grown as described {30). Tumor or tumor cell line total cellular RNA was prepared by the guanidium isothiocyanate and cesium chloride gradient method {31). Rat atria and brain total cellular RNA were prepared from one 7-week-old male Sprague-Dawley rat (National Institutes of Health, Bethesda, MD) after the animal was anesthetized with ether and decapitated. Ten micrograms of total cellular RNA was fractionated on 1% agarose and 0.66 M formaldehyde gels and transferred to nitrocellulose {31). RNA samples showed no significant degradation, as determined by a comparison of the ethidium-bromide staining intensities of the 28S and 18SribosomalRNA species. The AVP probe was a first-exon, 345-base pair Sau3A-Rsal fragment {32). The ANF probe (phNFpUCl A.713) was a 713-base pair insert cDNA fragment cloned into the £coRI site of a pUC-9 plasmid. DNA fragments derived from AVP and ANF cDNA clones were radiolabeled by nick translation with [a