[CANCER RESEARCH 39, 5010-501 5. December 19791 0008-5472/79/0039-0000$02.00 Putrescine as a Biochemical Marker of Malignant Brain Tumor& Sami I. Hank2 and Carl H. Sutton Departments of Neurology (S. I. H.], Neurological Surgery, and Pathology (C. H. SI, University of Miami School of Medicine, Miami, Florida 33101 ABSTRACT tumors. The samples represent a variety of tumor types and in some instances normal brain tissue. A preliminary summary of Putmescine, spermidine, and spermine levels were deter our findings has been reported previously (7). mined in normal brain and central nervous system-related tumor tissues obtained at operation from 50 patients. The MATERIALS AND METHODS biochemical data were correlated with morphological histo pathological descriptions of the same tissues. There was little Tissues were obtained during neurosurgical procedures for variation in putrescine levels in normal cerebral cortical tissue. the excision of intracranial and intraspinal mass lesions under Subcortical white matter had lower putrescine but higher sper general anesthesia. After excision, tissue samples were im midine content than those of the overlying cortex. Putmescine mediately divided into 2 portions. The smaller portion was levels were elevated in all astrocytomas assayed, and the quickly frozen and stored at —60°untilassayed for its putres magnitude of this elevation was proportional to the malignancy cine and polyamine content. The larger portion was processed of the tumor as determined by histopathological criteria. In for conventional histopathological evaluation. In many in contradistinction, putrescine content of ‘‘benign'‘tumorswas stances, several samples of tissues were obtained from the generally equal to or lower than that of the normal cerebral same patient, and each sample was coded and processed cortex. Spermidine and spemminelevels varied widely in the separately. tumors assayed and did not correlate with criteria of malig For biochemical assays, the frozen tissue samples were nancy. It is concluded that putmescinemay be a good biochem homogenized in 9 volumes of cold distilled water in motor ical marker of malignancy in central nervous system-related driven glass homogenizers. Immediately after homogenization, tumors. a small volume of the homogenate was taken for the putrescine assay. To the remainder, an appropriate volume of 6 M per INTRODUCTION chloric acid was added to a final acid concentration of 0.4 M. The portion taken for the putrescine assay was heated in a Polyamine biosynthesis and accumulation are closely asso water bath at 95°for 20 mm and centrifuged for 10 mm at ciated with cellular growth and proliferation (2, 22) be it phys 20,000 x g, and 10- to 40-@slahiquotsof the supemnatant iological, such as the response of the hepatic remnant to partial (representing 1 to 4 mg of tissue) were assayed for putrescine hepatectomy (17) and the prostate to the administration of by the enzymatic-isotopic technique (5). The acidified homog andmogens(12), or neoplastic (1). Recently, there has been enates were allowed to stand in an ice bath for 30 mm and increasing interest in the polyamines as biochemical markers were centrifuged for 10 mm at 20,000 x g, and 0.5- to 2-mI that can be clinically useful in the diagnosis and follow-up of ahiquotsof the supemnatantwere assayed for spermidine and cancer patients (3). This interest was generated mostly by the spermine as described previously by Kmemzneret a!. (10). The finding of high levels of putrescine and spermidine in the urine amounts of putrescine, spermidine, and spermine in ahiquotsof and sera of patients harboring malignant tumors (4, 13, 14). the tissue extracts assayed were within the linear range of the Also, high levels of putmescineand spermidine were found in respective assay procedure. Putrescine was assayed in triphi the cerebrospinal fluid of patients with malignant brain tumors cates with less than 7% interassay variation. Spermidine and (11).Thehighlevelsofthediamine,putmescine,andthetria spermine were assayed in duplicates with an interassay varia mine, spermidine, in the serum, urine, or cemebrospinalfluid of tion less than 5%. The recovery of known amounts of authentic tumor-bearing patients presumably reflect extracellular leak putrescine, spermidine, and spermine added to tissue homog age of these intracellular amines resulting from spontaneous enates was studied with each batch of determinations, and the cell necrosis (4, 11, 14). recovery rates were used in calculating the tissue amine con The need for basic information regarding the levels of pu tent. trescine, spemmidine,and sperrnine in human tumors and the Samples for histopathological evaluation were fixed in for correlation of these levels with known criteria of malignancy is maIm (10 g/1 00 ml) and processed in the usual manner for essential for adequate understanding of results describing the paraffin sectioning. Sections from all biopsy specimens were concentration of these arnines in body fluids. We report here stained routinely with hematoxylin and eosin. Those from ghial on the levels of putrescine, spermidine, and spermine in tissue tumors were also stained with phosphotungstic acid-hematox samples obtained from 50 patients undergoing neurosurgical yhinto confirm the presence of ghialfibers. Reticulum stain and procedures for the excision of central nervous system-related Masson trichrome stains were used whenever needed to iden tify mesodemmaltumors and to demonstrate areas of collage nous fibrous tissue, respectively. Histopathological descrip 1 Supported in part by a grant from United Way of Dade County, The Rita Cohen Memorial Fund, lnstituticinal Grant IN-Si S and Grant POT 74 from the tions and diagnoses were made on each sample of tissue by American Cancer Society, and the Diane and Scott Austin Brain Tumor Research C. H. Sutton without knowledge of the biochemical results. The Fund. grading of primary ghialbrain tumor was carried out according 2 To whom requests for reprints should be addressed. Received May 21, 1979; accepted September 17, 1979. to the criteria of Kemnohanand Sayre (8). In many instances, 5010 CANCERRESEARCHVOL. 39 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Po!yamines in Brain Tumors although more than one sample of tissue was obtained from same cerebral cortical samples were more variable (Table 1; the same patient, each sample was evaluated separately, and Charts 2 and 3). Normal subcortical white matter was found to the biochemical data derived from each sample were tabulated have lower (p < 0.01 ) putrescine levels and higher (p < 0.005) under the appropriate histopathological diagnosis made on that spermidine levels (Table 1). The levels of putrescine, spermi piece of tissue. As an example, several samples of tissue were dine, and spermine in cerebellam cortical samples were not obtained from a patient with a large ghioma. One sample, significantly different from those of the cerebral cortex (Table suspected at operation to contain tumor, was found to mepre 1). sent normal brain tissue; another sample of tissue was diag All astrocytomas had elevated levels of putrescine (Table 1; nosed as Grade Ill astrocytoma; while a third sample was Chart 1). Even the least malignant of these tumors had putres histopathologically described as Grade II astrocytoma. The cine levels higher than the range of values obtained from biochemical data obtained from the 3 samples were tabulated normal brain tissue. Putrescine levels in the supratentorial under 3 different headings, normal cerebral cortex, Grade III astrocytoma groups increased progressively in direct relation astrocytoma, and Grade II astrocytoma, respectively. When ship to the histopathological criteria of malignancy of these different tissue samples taken from one patient showed similar tumors (Table 1; Chart 1). In astrocytoma Grade II, Ill, and IV histopathological appearance, the biochemical data were av groups, where the number of observations allowed adequate eraged and their means were used for data analysis. There statistical analysis, putrescine levels were higher than normal was little variation in biochemical results obtained from histo cerebral cortical levels at p < 0.001 . Also, putrescine levels in pathologically similar samples obtained from the same patient. Grade IV astrocytomas were significantly higher than those of Tissue samples that showed moderate to marked histopatho Grade II or Grade Ill tumors ( p < 0.01 ), while putrescine levels logical heterogeneity were excluded. Similarly excluded were in Grade II and Grade III astrocytomas did not differ signifi samples of tissue showing significant edema or reactive cantly. Sperrnidine and spermine levels in the various astrocy changes. Samples from patients who had had radiation them tomas varied over a large range, which overlapped with values apy, chemotherapy, and/or heat therapy to their brain tumors obtained from normal brain tissue (Table 1; Charts 2 and 3). were also excluded. Only in Grade II astrocytomas were spermine levels signifi cantly higher than those of normal cerebral cortex ( p < 0.01). RESULTS Putrescine was minimally to moderately elevated in cerebellar astrocytomas and in the single sample of the spinal cord Levels of putrescine, spermidine, and spermmnein normal astrocytoma (Table 1). human brain tissue and in various tumor types of the nervous A variety of slowly growing and relatively benign intracranial system are detailed