Polyamines As Markers of Response and Disease Activity in Cancer Chemotherapy1

[CANCERRESEARCH37,214-221, January 1977] Polyamines as Markers of Response and Disease Activity in Cancer Chemotherapy1

Brian G. M. Dune,2 Sydney E. Salmon, and Diane H. Russell3 Section of Hematology and Oncology of the Department oflnternalMedlcinefB. G. M. D., S. E. S.Jand Department of Pharmacology (D. H. R.J, University of Arizona College of Medicine, Tucson, Arizona 85724

SUMMARY 37), as well as reports of decreased polyamine levels after successful treatment of various types of cancer (7, 10, 11, Onehundredtwenty-four patientswith hematological and 13, 16, 21, 30, 34, 36, 37). The effect of therapy upon solid neoplasmshad pretreatment urinary polyamine deter polyamine levels has been further evaluated in animal minations. Putrescine, spermidine, and spermine were all model systems. Regression of mammary carcinoma in re significantly increased as compared to normals (p < 0.001). sponse to hormone ablation (28) and response of a rapidly Polyamine levels were directly related to disease activity growing hepatoma to chemotherapy (5-fluorouracil) (32) and tumor burden. In patients with multiple myeloma, pu and irradiation (31) are associated with dramatic increases trescine levels were significantly correlated with clinical in extracellular polyamine levels in proportion to the cell kill disease activity as well as the in vitro labeling index of occurring with treatment. In a preliminary study of a group marrow plasma cells. Spermidine values reflected tumor of patients who had polyamine levels determined before, cell burden. Serial studies in 56 patients indicated that during, and after chemotherapy (27), we obtained similar greater than twofold rise in urinary spermidine during treat results and developed a model for the interpretation of ment was highly correlated with cell kill and subsequent changes in extracellular polyamine levels. The essence of clinical response (p < 0.001). Serum polyamine levels in 17 this model is that extracellular spermidine levels are propor patients were found to be comparable to urinary values. Our tional to spontaneous cell loss or cell kill with treatment, data indicate that polyamine determinations can potentially whereasextracellularputrescinelevelsaremore relatedto be clinically useful, i.e., baseline values as indicators of the growth fraction (27). tumor cell mass and growth fraction, and increases in sper In this study, we have expanded our original preliminary midine during treatment as an excellent marker of tumor observations in patients with both hematological and solid cell kill. neoplasms. To evaluate the usefulness of serial determina tions of extracellular polyamine levels, we have measured polyamine levels in the urine (and in some cases, serum) of INTRODUCTION patients before, during, and after specific courses of cancer chemotherapy, and have attempted to relate the pretreat Polyamines are low-molecular-weight, highly charged or ment baseline levelsand changeswith treatment to the cell ganic cations that are ubiquitous in nature. They are inti kill with treatment and to the overall survival and prognosis mately involved in cell growth, with possible importance in in individual patients. the regulation of RNA-dependentprotein synthesis (1, 5). Intracellular levels of polyamines, particularly spermidine [NH2(CH2)4NH(CH2)NH2]andputrescine [NH2(CH2)4NH2],in MATERIALSAND METHODS crease early and dramatically with growth in both normal and neoplastic cells (26). The most marked synthesis and One hundred twenty-four patients with hematological or accumulation of polyamines occurs in rapidly growing tis solid neoplasms who were candidates for cytotoxic chemo sues (29). therapy were studied. All patients were considered to be The report of increased excretion of putrescine, spermi without other factors that might significantly affect polya dine, and spermine (NH2(CH2)3NH(CH2)4]inthe urine of pa mine levels at the initiation of the study period (e.g.,recent tients with cancer was published in 1971 (25, 30). Since that surgery or other cancer therapy, or intercurrent diseases time there have been several reports of elevated polyamine such as infection, collagen vascular diseases, or chronic levels in patients with cancer(3, 7, 10, 11, 13, 15, 18, 21, 34â€” obstructive lung disease) and had objective evidence of disease that could be evaluated with respect to clinical stage and response to chemotherapy. The patients' ages

1 Supported in part by Contract NIH-NCI-C-73-7313 from the Division of ranged from 17 to 70 years (median, 51 years). All patients Cancer Treatment, National Cancer Institute; American Cancer Society Insti had urinary polyamine determinations and some had, in tutional Grant IN-I 10; and Grants CA-14102, CA-17094, and CA-14783 from the National Cancer Institute. addition, serum polyamine determinations, in relation to 2 Scholar of the Leukemia Society of America. To whom requests for particular courses of chemotherapy. Changes in polyamine reprints should be addressed. levelswith surgeryor radiation therapy were also monitored 3 RecIpient of Research Career Development Award CA-00072 from the National Cancer Institute. in a few patients. We (30) and others (37) have previously Received May 21. 1976; accepted October 14, 1976. determined urinary polyamines in a large number of normal

214 CANCER RESEARCH VOL. 37

adults. However, for this study, 16 control subjects in ap from each collection was filtered through a Millipore filter parent good health (age range, 18 to 60 years) were studied (0.22-sm pore size) and a 2-mi sample was hydrolyzed for concurrently to confirm our normal range for each urinary 14 to 16 hr at 110Â°in6 N HCI, and then centrifuged, trans polyamine. In addition, a small group of patients with dis ferred, and evaporated. The residue was reconstituted in eases other than cancer were studied to illustrate the poten 250 @dof0.1 NHCI, and 50 @Iwereloaded on the amino acid tial for abnormal levels in a variety of circumstances. analyzer. Details of the elution program are published else ClInicalEvaluationand Treatment.Standardcriteriafor where (33). When serial 24-hr urine collections were ob staging of disease were used (4, 8). In addition, the activity tamed as a baseline prior to treatment, expression of the of the malignant process was assessed, with the major polyamine levels per unit creatinine (pg/mg creatinine) re criterion being the presence or absence of clinical evidence suited in very consistent daily excretory patterns. Expres of disease progression within 2 months of the time of obser sion of the data in this way also corrected for incomplete vation. The clinical parameters of progression of disease collection. When the time-span of collection was uncertain used in this study were as follows: (a) for multiple myeloma: or the standard creatinine clearance was reduced by 50% or new bone lesions, hypercalcemia, rising serum or urine more, samples were discarded. It was found that total un monoclonal immunoglobulin (M-component), or increase in nary creatinine per day was very consistent in individual bone marrow plasma cells or extraskeletal myeloma; (b) for patients and not significantly affected by systemic chemo leukemia: rising marrow and/or peripheral blast counts or therapy. It should be noted that, in looking at the pre- to blast transformation in chronic myelogenous leukemia; and posttreatment ratios in individual patients, the creatinine (c) for lymphomas and solid neoplasms: rapidly increasing values cancel out. measurable disease. Response to chemotherapy was SerumAssayProcedure.Serawerecollectedandimme judged on the basis of objective evidence of cell kill using diately stored at â€”76Â°untilanalysis. A 3- to 5-mI sample of the standard criteria of the Southwest Oncology Group for serum was treated with an equal volume of cold 10% tnichlo each disease type. It should be noted that patients in whom roacetic acid, and the proteins were allowed to precipitate cell kill was followed by significant tumor regrowth before for 30 mm in an ice bath. The sample was centrifuged, the the next course of therapy were all included in the partial supernatant decanted, the pellet resuspended in 1 ml of response category. Patients in whom there was no evidence 10% tnichloroacetic acid and recentnifuged, and the super of tumor kill at any time during or after treatment were natant added to the previous wash. This procedure resulted included in the no-response category. in 95 to 100% recovery in the supernatant of free and conju Most of the patients in this study were treated on an gated polyamines. The tnichloroacetic acid was removed inpatient basis with intensive combination chemotherapy; a with ether washes, and the sample was hydrolyzed for 14 to small number were treated with simpler intermittent sched 16 hr at 100Â°in 6 N HCI. After hydrolysis, the sample was ules on an outpatient basis. The major drugs used were centrifuged, transferred, and evaporated. The residue was doxorubicin hydrochloride (adriamycin), cyclophospha reconstituted in 150 pi of 0.1 N HCI and a 75-pi aliquot was mide, cytosine arabinoside, 5-(3,3-dimethyl-1 -triazeno) loaded on the analyzer. Sample size and relative peak area imidazole carboxamide, vincristine, bleomycin , and predni for putrescine, cadavenine, diaminopropane, spermidine, sone. No drugs known to interfere directly with polyamine and spermine were linearly related over the range of 0.05 to synthesis and metabolism [e.g., methylglyoxal 20 nmoles. bis(guanylhydrazone) (13)] were used. TritlatedThymidine-labelingIndexStudies.Labelingin Measurementof Polyamines.Polyamineswereassayed dex studies were performed in patients with multiple mye by the amino acid analyzer technique as previously pub loma as an additional parameter of disease activity. Hepa lished, with the use of a Durrum Model D-500 analyzer ninized bone marrow cell suspensions (0.5 x 10@to 1.0 x (Durrum Instrument Corporation, Palo Alto, Calif.) (33). 106cells/mI of cell suspension) were incubated for 1 hr at 37Â° Standards,Reagents,and Buffers.Putrescine,cadaver with high specific activity (40 to 60 Ci/mmole) tnitiated thy me, spermidine, and spermine, in the form of hydrochloride midine (dose, 5.0 @Ci/mlof cell suspension). Cytocentni salts, were obtained from Calbiochem (San Diego, Calif.) fuge smears were then made on gelatin-coated slides and and recrystallized 3 times from ethanol before use. The 14C- fixed with methanol. The tnitiated thymidine-labeling index labeled polyamines used to determine recovery rates were was then determined using our previously published high obtained from New England Nuclear (Boston, Mass.). Nm speed scintillation autoradiography method (9) with control Sol, a ready-to-use ninhydrin solution, and thiodiglycol comparison to the standard method (15). The amount of were purchasedfrom PierceChemicalCompany(Rockford, PPO used in the scintillator solution was 5 g/500 ml of III.). Liquified phenol was purchased from Matheson Scien dioxane. tific (Elk Grove Village, lIl.).The 3 buffers used were: Buffer A (0.2 mole of Na@per liter, pH 6.16), the loading buffer for urine and serum samples; Buffer B (2.4 moles of Na@per RESULTS liter, pH 4.68), the 2nd buffer for urine and serum samples; and Buffer C (3.05 moles of Na@per liter, pH 4.68), the 3rd PretreatmentLevels of Urinary Polyaminesin Cancer buffer used for serum samples. The method for the prepara Patients. One hundredtwenty-fourpatientshad at least tion of these buffers is described elsewhere (16). pretreatment baseline urinary polyamine levels determined. UrineAssayProcedure.Twenty-four-hrurinespecimens Table 1 shows the mean Â±standard deviation, median, and were collected in plastic bottles containing 10ml of concen range for the urinary excretion of putrescine, spermidine, trated HCI and refrigerated between collections. An aliquot and spermine in patients with hematological or solid neo

JANUARY1977 215

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. B. G. M. Dune et al. Table1 Pretreatmentlevels of polyaminesin the urine of patients with cancer and normal control subjects PolyamineswereassayedinMethodsâ€•).PutrescineSpermidineSpermmne(@.tg/mg 24-hr urinecollectionsby meansof an amino acidanalyzertechnique(see â€œMaterialsand

creatinine)No. creatinine)(@.tg/mg creatinine)(pg/mg

ofsubofNo. ofNo. Mean Â±sub Mean Â±sub MONRangeHematologijectsS.D. MDNaRangejectsS.D. MDNRangejectsMean Â±S.D. .00.25-8.0cal 684.4 Â±0.66b 4.20.45-38683.7 Â±0.79â€•2.70.58â€”25620.8 Â±0.32'5 1 tumorsSolid 0550.12-3.7Normals162.1tumors553.7 Â±Ã˜@39b 3.80.29-10562.7 Â±54b 1.81.09-8.1560.6 Â±0.28k Â±0.62 1.00.40-2.1161 .2 Â±0.18 0.80.40-2.1160.04 Â±0.007 0.040.02-0.3

a MDN, median. b Difference from values for normal control subjects is significant (p < 0.001).

plasms, and in normal control subjects. (Urinary cadavenine Table 2 levels were measured in all patients with cancer; however, ExamplesofcancerEach polyamine elevationsin diseasesother than since there was no obvious correlation with tumor status or tobe value indicates 1 patient. The listed disease was thought response and since some of these levels could have been the major factor in polyamineelevation.Putrescineâ€• influenced by any bacterial contamination of urine samples, Spermine'@(jig/mgSpermidineâ€• the data are not presented.) The range of values is extended cre- (pg/mg cre- (pg/mg cre atinine)PerniciousDisease atinine) atinine) in all tumor categories, but the mean and median values for each polyamine in cancer patients are significantly ele 10.81Hemolyticanemia 3.48 anemia 26.351.0811.82 8.55 vated, compared with those for the controls (p < 0.001). 1.87Rheumatoid 12.08 Putrescine, spermidine, and spermine values were all tracePolymyositisarthritis 5.64 3.81 higher in the patients with hematological tumors than in 16.450.946.33 25.47 patients with solid tumors. traceChronic 3.39 1.19lung obstructive 3.85 2.75 Striking examples of values found for patients with dis diseaseActive easesother than cancer are shown in Table 2. Significant 4.91berculosisLiverabscesspulmonary tu- 23.18 elevations in both putrescine and spermidine were found in association with pernicious anemia, hemolytic anemia 1.70Psoriasis 10.60 (Coombs' positive), rheumatoid arthritis, polymyositis, 5.71 3.10 chronic obstructive lung disease,alcoholic cirrhosis, acute a Range, 0.4 to 2.1. and chronic infections, and psoniasis. b Range, 0.4 to 2.1. Urinary Spermidineand PutrescineExcretionand Re C Range, 0.02 to 0.3. sponse to Chemotherapy. For 56 patients, complete se quential polyamine data were available, and it was possible to objectively evaluate clinical response to a given course of chemotherapy. Chart 1 exemplifies the pre- and posttreat ment putrescine and spermidine levels and Table 3 summa nizes the post- to pretreatment ratios of putrescine and spermidine in 22 patients with no response to chemother apy, 16 patients with a partial response, and 18 patients with a complete response. Of these patients, 36 had hematologi cal tumors and 20 had solid tumors. Note that, in the pa tients who had no response to chemotherapy as judged by subsequent clinical evidence, the posttreatment-to-pre treatment spermidine ratio was 1.2. Those patients with either a partial or complete response had a mean increase

in this ratio of 3.7 and 3.6, respectively. Although putrescine 0 10 20 30 concentration usually increasedafter chemotherapy, eleva I (OAP/L-ASP) tion was not correlated with response. Thus, the nonre DAYS spenders had elevations in putrescine excretion without Chart 1. Serial polyammnelevels in a patient with diffuse histiocytic lym major increases in spermidmne, resulting in a low value for phoma who had a complete response to chemotherapy (putrescins, â€¢; the posttreatment-to-pretreatment ratio for spermidine over cadaverins, 0; spermidine, A; and spermine, tb). Treatment is indicated by a bar on theaxis [0, oncovin(vincristins);A, cytosinearabinoside;P, predni the posttreatment-to-pretreatmentratio for putrescine. Note sons; L-ASP, L-asparaginase]. that this ratio (0.4 for nonresponders, 1.2 for partial re sponders, and 1.4 for complete responders) is perhapsthe are shown for individual patients in relation to disease most helpful in distinguishing the 3 categories. category and response to chemotherapy. Note that in none In Table 4, the pre- and postchemotherapy spermidine of the disease categories of nonresponding patients (in levels as well as the posttreatment-to-pretreatment ratios cluding patients with multiple myeloma. leukemia, lym

216 CANCERRESEARCHVOL. 37

Table 3 with active, as compared with more indolent disease states, Response to chemotherapy and urinary spermidine and pu as defined in â€œMaterialsandMethods.â€•Table5 lists the data excretion in 56 patientsseriallytrescinePosttreat- studied for patients with multiple myeloma in whom the labeling Posttreat index (in vitro growth fraction) of the bone marrow plasma mentâ€•/pre- menV'/pre cells was available for comparison. It can be seen that the treatmenttreatmentputrescine putrescine values were significantly (p at least <0.01) spermidineNo. ratioB/AType of ratio higher in patients with high cell mass at the time of diagno (B)ratioNoneof response cases (A) sis and in patients with actively relapsing myeloma, corn 0.50.4Partial 22 2.7 Â±3,5â€• 1.2 Â± pared with patients with stable disease in complete remis 2.1r1.2Complete 16 3.0 Â±3.1 3.7 Â± sion. The putnescine-to-spermidine ratio was also signifi 1.3c1.4Combined, 18 2.5 Â±1.2 3.6 Â± cantly higher in patients with high-cell mass myeloma at the 2.0@'1.2tologicalhema- 36 2.6 Â±2.2 3.0 Â± time of diagnosis, compared with patients in stable com tumorsCombined, 1.31.4tumors solid 20 2.8 Â±3.4 2.0 Â± plete remission. The spermidine level was clearly related to cell mass in that it was statistically higher in patients with high as opposed to intermediate or low cell mass at the time a Posttreatment indicates highest value within 72 hr of initiating therapy. of initial diagnosis and in patients with active relapsing b Mean Â± 5.0. disease as opposed to patients with stable disease in corn C Difference from values for patients with no response is signifi plete remission (p at least <0.01). The relationship between cant (p < 0.0001). the urinary putrescine and in vitro labeling index was most d Difference from values for patients with solid tumors is signifi cant (p < 0.05). interesting. For patients with active relapsing disease and those in stable complete remission, there was a significant phoma, and solid tumors) does the posttreatment-to-pre correlation between the putrescine values and the labeling treatment ratio ever exceed 2 [mean ratio, 1.2 (Table 3)], index (r > 0.75). However, in the patients with high cell whereas the mean ratios for patients with partial and com mass at the time of presentation, the labeling index or plete responses are nearly 4.0. These data suggest that growth fraction was very low, whereas the putrescine values spermidine can be used as a highly reliable marker of re remained elevated. This may indicate the much longer sponse to chemotherapy. Fluctuations in spermine did not myeloma cell generation times at the plateau phase of correlate well with response to chemotherapy. growth so that in vitro â€œflashlabelingâ€•underestimated the The pattern of polyamine excretion in a patient with non actual cell growth and cell death reflected by the polyamine Hodgkin's lymphoma (diffuse histiocytic) in a leukemic levels. phase before, during, and after chemotherapy is shown in Table 6 lists the polyamine levels with respect to disease Chart 1. The initial rise in spermidine was associated with a activity in myelogenous leukemia, soft tissue sarcomas, and complete response to therapy, and the subsequent low colon carcinoma. Since a quantitative tumor marker was levels were in a period of remission. not available for these disease types (as it was for myeloma), Relationshipbetween Serum and Urinary Polyamine the relationship to cell mass could not be exactly estab LevelswithChemotherapy.Duringtheperiodofthisstudy, lished. However, the polyamine levels were clearly more methods for accurately measuring serum and plasma polya elevated in patients with active disease. mine levels were developed (33). In 17 of the patients in Effectsof DifferentTypesof Chemotherapy,Irradiation, cluded in the present study of urinary polyamines, sufficient and Surgery. The effects of the different drug combinations serial serum and plasma polyamine data were available to upon urinary polyamine levels were also evaluated. No spe allow comparison with urine values both before and after cific patterns were found in association with specific single chemotherapy. In general, changes in the serum and agents or drug combinations. Although several patients had plasma paralleled those noted in the urine, suggesting that a marked rise in urinary putrescine (3- to 6-fold) within 24 hr serum (or plasma) values for polyamines can be used to after i.v. administration of 1 to 2 mg of vincnistine, this rise monitor response to chemotherapy. also occurred after administration of noncycle-active single A representative plot of serial diurnal values for serum agents (e.g., adniamycin) and combinations. Thus, no defi putrescine and spermidine in a patient responding to chem nite conclusion could be reached with regard to possible otherapy is shown in Chart 2. Upon initiation of chemother synchronization (with subsequent rapid cell proliferation apy, there was a rapid increase in the spermidine concen and putrescine release) or cell recruitment. Both local irra tration with a delayed rise in the putrescine concentration. diation (for instance, to painful bone metastases) and major This pattern closely followed changes in the urine, with the surgery were also followed within 24 to 48 hr by marked exception that the putrescine level increased earlier in the elevations in polyamines, particularly putrescine. The post urine after the initiation of chemotherapy. This may indicate irradiation therapy changes were similar to those noted in that putrescine is more rapidly cleared from the serum than the rat hepatoma model (31). The postsurgical values need spermidine. Levels of both spermidine and putrescine in the further evaluation with regard to the effect of factors such serum and urine subsequently fell to within the normal as wound healing and the growth fraction of the residual range. Note that because dramatic changes may occur rap tumor. idly, timing of serum-plasma sample collection can be of critical importance in evaluating response to therapy. DISCUSSION Disease Activityand PolyamineLevels. Tables5 and 6 summarize the putrescine and spermidine levels in patients In the past 3 years, several workers have confirmed the

JANUARY1977 217

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. B. G. M. Durieetal. Table 4 Effect of chemotherapyon urinaryspermidinelevels ofcancerchemotherapyPretreatmentPosttreatmentaPosttreatmsnt/spsrmidinespsrmidinspretreatment(pg/mg patients bytype of cancer andresponse to

creati (@&g/mgcreati spermidine ra DiseaseResponsePatientSexnine)nine)tioMeanÂ± S.D. Multiple mysloma None M 2.92 2.00 0.68 1.14 Â±0.59 2 M 4.84 3.15 0.65 3 M 0.79 1.20 1.52 4 M 2.44 4.26 1.75 5 M 3.54 3.39 0.96 6 M 4.69 8.99 1.92 7 M 1.22 1.92 1.57 8 M 1.86 1.91 1.03 9 F 2.20 0.30 0.14 Partial 10 M 1.38 2.91 2.10 4.13Â±1.77 11 M 1.95 6.24 3.20 12 M 1.62 9.06 5.59 13 M 2.11 11.90 5.64 CompleteLeukemiaNonePartialCompleteLymphomaSolid14 M 1.50 4.75 3.17 3.08Â±0.12 15 M 1.00 3.11 3.11 16 M 6.94 10.71 1.54 1,54â€• 17 M 0.63 3.54 5.62 4.30Â±2.54 18 F 1.26 11.21 8.90 19 M 26.59 63.99 2.41 20 F 2.69 15.31 5.69 21 M 1.70 5.31 3.12 22 F 3.34 7.89 2.36 23 M 3.33 6.74 2.02 24 M 3.78 24.44 6.47 25 M 2.51 7.95 3.17 26 F 3.66 9.69 2.65 27 F 1.48 7.48 5.05 28 F 9.40 34.00 3.62 29 F 2.42 6.96 2.88 None 30 M 6.01 8.28 1.38 1@38â€• Complete 31 M 2.10 6.43 3.06 3.36 Â±0.68 32 M 8.50 39.50 4.65 33 M 12.27 33.72 2.75 34 F 3.56 12.36 3.47 35 M 1.90 5.60 2.95 36 M 2.50 8.20 3.28 tumorsBreast carcinoma0.89Breast None 37 F 1.49 1.32 1.24 Â±0.36 carcinoma1.48Melanoma1.42Melanoma1.47Melanoma0.63Melanoma1.32Melanoma1.50Melanoma0.76Prostate38 F 1.65 2.44 39 M 1.63 2.31 40 M 1.35 1.98 41 F 8.11 5.13 42 M 2.50 3.30 43 F 3.76 5.65 44 M 1.40 1.07 carcinoma1.55Prostate 45 M 1.75 2.71 carcinoma0.96Colon 46 M 2.23 2.15 carcinoma1.66Breast 47 M 2.86 4.75 carcinoma2.49Fibrosarcoma2.55Fibrosarcoma1.95Liposarcoma3.55BladderPartial 48 F 0.92 2.29 2.66 Â±0.58 49 M 3.30 8.41 50 M 3.59 7.01 51 F 1.63 5.79 carcinoma2.77Breast 52 F 1.27 3.52 carcinoma2.52Breast Complete 53 M 3.23 8.13 3,73 Â±1.68 carcinoma5.29Testicular 54 F 1.19 6.29 carcinoma5.04Testicular 55 M 4.11 20.72 carcinoma2.05 56 M 2.10 4.30

a Posttreatmsnt indicates within 72 hr of initiation of chemotherapy. b Single value. original observation that polyamines are elevated in the tients. The current report documents potential clinical uses extracellular fluid (urine, serum, and cerebrospinal fluid) of for polyamine determinations. The increase in urinary sper patients with cancer (3, 7, 10, 11, 14, 16, 18, 21, 28, 32, 34, midine with successful therapy can be used as a marker of 36, 37). However, it has not been clearly established in what cell kill. Pretreatment (baseline) polyamine values can be way single or serial determinations may be used to improve used to evaluate a number of tumor characteristics includ the management of individual patients or groups of pa ing tumor size, growth rate, and prognosis.

218 CANCERRESEARCHVOL. 37

The rise in urinary spermidine with successful treatment be especially useful in evaluating response. Fortunately, the was highly significant (Table 3). Knowledge of such very dramatic changes in extracellular polyamine levels fol changes should prove clinically useful even in the presence lowing cancer therapy are unlikely to be confused with of objective disease which can be carefully evaluated for other effects. For example, the patient in Chart 1 who had a evidence of response to therapy, for instance with X-ray or complete response to chemotherapy had a complicated bone marrow examination. For example, X-ray changes recovery phase with spiking fever, documented bacteremia, may take several weeks to become apparent, whereas the triple antibiotic therapy, and granulocyte transfusions, polyamine changes occur actually during the treatment pe none of which substantially altered the polyamine levels. nod. For occult or difficult-to-follow lesions, including re Acute elevations in polyamine levels have been noted with tnopenitoneal or gastrointestinal tumors, or bone and/or liver metastases, the changes in urinary spermidine should Table 6 Baselineurinary polyamine valuesand diseaseactivity' in EMBRYONAL CARCINOMA OF TESTiS hematological and solid cancers of pa (pg/mg creat (pg/mg creat mine)AcuteDiseaseNo. tientsPutrescinemine)Spsrmidine 7.1kemiaSmolderingmyelogenousleu 121 1.4 Â±6.5b9.7 Â±

0.6Chronic leukemia43.0 Â±1.01 .8 Â±

myelogenousleukemiaBlast

12.5Chronicphase227.6 Â±14.625.8 Â± 6.5Disseminatedphase45.6 Â±1.76.8 Â±

tissuesarcomasRapidlysoft

2.6Slowlyprogressive218 Â±9.69.9 Â± 0.7Metastaticprogressive43.2 Â±1.02.1 Â±

colon carci 2/26 2126V272/283/I 3/2 312 3@3 3/3 3@4 3/4 315 3/5 315 3@ @ PM PM @lNA PM M@ @4PM nomaRapidly I Chemotherapy I 6.4Slowlyprogressive512.7 Â±6.78.0 Â± progressive64.0 Â±1.52.1 Â±1.6 Chart 2. Serum putrescins and spermidine levels in a patient with em bryonal cell carcinoma of the testis who had a complete response to chemo a Seeâ€œMaterialsand Methodsâ€•forparametersof diseaseactiv therapy. In the pretreatment period there is no significant diurnal variation in ity. values, whereas with therapy dramatic increases occur within a 24-hr period. b Mean Â± S.D.

Table 5 myelomaNo.Baseline urinary polyamine values and disease activityâ€• in multiple ., Labeling index (%)Putrescine (pg/mg creatinine)Spermidine (@.tg/mgcreatinine)Mean pu of trescine/ Â±tientsS.D.pa Mean Â±Mean Â±Mean spermi ratioHigh MDN' RangeS.D. MON RangeS.D. MON Rangedine cell mass at Â±0.3c 0.5 0.5-1.06.2 Â±1â€¢7d 5.1 4.9-8.92.1 Â±0.8 1.5 1.2-2.9 time of diagnosisâ€•60.8 \ \@ < 0.01 /3.2eIntermediate 0.2-2.612.6cellor low53.8 Â±3.0 3.0 1.0-7.03.6 Â±3.5 2.0 1.9â€”10.01.4 Â±0.8 1.3 ofdiagnosisâ€•Refractorymass at time

patients Â±12.0 16.0 5.0â€”38.0 7.0 3.5-13.9 Â±0.9 3.5 0.7-2.5 in relapse \@<0.001 @p<0.001 @p<0.001

Patients1.7Vcomplete stable in5 1018 [email protected] 0.5 0.5-3.08.5Â±4.7â€•[email protected] 1.8 0.9-2.93.4 [email protected] 0.4-2.52.5 remis sion (75%tumorregression)

a See â€œMaterials and Methodsâ€• for parameters of disease activity. b See Ref. 8.

C. d. e Statistically significant difference between values (p < 0.01). f MDN, median.

JANUARY1977 219

clinically obvious conditions such as bowel or myocardial neoplastic and other cell growth or death processes was infarction and surgical procedures. However, most noncan possible. It is emphasized that clinical processes leading to cer processes that can result in substantial polyamine ele nonspecific polyammne elevations are usually obvious on vations evolve over a period of several days to weeks (e.g., routine physical examination or standard laboratory testing. bacterial abscess or viral hepatitis) and should not lead to The major portion of our study is based upon 24-hr urine confusion. Active bone marrow recovery can result in pu polyammnedeterminations. These samples are cumbersome trescine and spermidine elevations, and careful attention to obtain and susceptible to incomplete collection. It would must be given to the magnitude and time course of these thus be more convenient if serum or plasma samples could changes. The data of Rosenoff et a!. (24) and Miller et a!. be used to obtain comparable information, especially for (19) indicate rather prompt recovery of DNA synthesis (12 to patients being followed on an outpatient basis. Our prelimi 36 hr) after single doses of alkylating agents. Thus, eleva nary analyses illustrated in Chart 2 indicate that serum and tions in polyamines at the end of treatment or between urine values are comparable. However, levels of polyamines closely spaced pulses of chemotherapy must be looked at in serum and plasma are lower than those in the urine, and with the view that very early marrow recovery could be a the difficulty in distinguishing between cancer and noncan contributing factor. However, our studies thus far have mdi cer elevations is magnified. In addition, because of the cated that extracellular spermidine elevations occur during potential for rapid fluctuations in plasma levels, multiple therapy rather than at the end of treatment. In addition, samples may be necessary to obtain meaningful data. Thus, putrescine is the major polyamine associated with a rapid timed (e.g., 2 to 6 hr) or spot urine collections or samples regrowth process, whereas tumor cell kill results in pre from specific tissues of interest such as bone marrow (22) dominant elevation in spermidine. In a number of the pa may be necessary or more useful. With kinetic modeling of tients with multiple myeloma and leukemia, marrow-label the patterns of conjugation and excretion of polyamines ing indices and serum and urine lysozyme determinations (23), it may be possible to predict the best timing and [marker of granulocyte turnover (12)] have been available to sources for sampling. exclude marrow recovery during this period (the leukemia For polyamine determinations to be available in a clini data are not presented in this report). In this regard, polya cally useful way, a rapid simple assay system is necessary. mine levels could be used to evaluate marrow recovery and An immunoassay, if feasible, would seem ideal. Recently, a accurately schedule the next course of therapy. In the inter direct radioimmunoassay for measuring spermine in serum pretation of polyamine levels, it is clearly important to eval has been developed (4). Unfortunately, the antibody used uate all possible factors in a given patient that could be had significant (22%) cross-reactivity with spermidine. contributing to urine and/or plasma elevations. Hopefully, a more specific immunoassay system can be As indicated in Tables 5 and 6, baseline polyamine deter developed. minations can provide useful information about tumor size In conclusion, our data suggest considerable potential and growth rate. The polyamine data can be used to corrob clinical usefulness for polyamine determinations. However, orate other clinical and laboratory findings with respect to more widespread clinical testing will be necessary to estab stage of disease as well as evaluation of remission or re lish the full role of this type of information. lapse (e.g., Ref. 21). The putrescine level can be used to provide information similar to that of the in vitro labeling REFERENCES index or growth fraction (6, 20) but without the need for serial biopsy of the tumor (which may be inaccessible). 1. Bachrach, U. Functions of Naturally Occurring Polyamines. New York: Since the labeling index is dependent upon the cell genera Academic Press,Inc.,1973. tion time, polyamine determinations may provide a more 2. Bachrach,U., and Ben-Joseph,M.TumorCells,PolyaminesandPolya mine Derivatives. In: D. H. Russell (ed), Polyammnesin Normal and accurate reflection of ongoing cell growth. Polyamine de Neoplastic Growth, pp. 15-26. New York: Raven Press, 1973. terminations could serve as a basis for treatment selection, 3. Bartos,D.,Campbell,A.A., Bartos,F.,andGrettis,D. P.DirectDetermi nation of Polyamines in Human Serum by Radloimmunoassay. Cancer using kinetic principles. Ass., 35: 2056-2060, 1975. However, these baseline values must be interpreted with 4. CIassiflcatii@n and Staging of Cancer by Sits. A Preliminary Handbook. more caution than the dramatically increased values associ Chicago: American Joint Committee for Cancer Staging and End Results Reporting, 1976. ated with cancer therapy. The maximum values in patients 5. Cohen, S. S. Introduction to the Polyamines. New Jersey: Prentice-Hall, with cancer are about 30 times greater than those of normal 1971. subjects but, unfortunately, are still within the same range 6. Crowther, D., Beard, M. E. J., Bateman,C. J. T., and Sewell, A. L. Factors Influencing Prognosis in Adults with Acute Myslogenous Leu as values (exemplified in Table 2) found in some patients kasmia. Brit. J. Cancer, 32: 456â€”464,1975. with noncancer pathologies. Attention to this nonspecific 7, Dreyfuss, F., Chayen, A., Drsyfuss, G., Dvir, A., and Ratan, J. Polyamine Excretion in the Urine of Cancer Patients. Israel J. Med. Sci. , 11: 785â€” type of elevation in polyamines is particularly important for 795, 1975. any planned use of polyamines in cancer diagnosis, differ 8. Dune, B. G. M., and Salmon, S. E. A Clinical Staging System for Multiple ential diagnosis, or screening. Sanford eta!. (34) and Fairet Mysloma. Cancer, 36: 842-854, 1975. 9. Dune,B. G. M., andSalmon,S. E. HighSpeedScintillationAutoradiog a!. (10)found polyamine determinations helpful in the diag raphy. 5cience, 190: 1093â€”1095,1975. nosis of urological cancer (prostate, bladder, and kidney). 10. Fair,W. A., Wehnsr,N., and Brorsson,U. UrinaryPolyamineLevelsin There were correlations with disease stage and tumor the Diagnosis of Carcinoma of the Prostate. J. Urol., 114: 88â€”92,1975. I 1. Fujita, K., Nagatsu, T., Maruta, K., Ito, K., Senba, H., and Miki, K. Urinary growth. However, differentiation from nonurological dis Putrescine, Spermidine, and Spermine in Human Blood and Solid Can eases was not included. Nonetheless, Dreyfuss et a!. (7), cars and in an Experimental Gastric Tumor of Rats. Cancer Ass. , 36: 1320â€”1324,1976. concluded that by looking at the pattern of elevation of 12. Hansen, N. E. Plasma Lysozyms: A Measure of Neutrophil Turnover. An spermidine, putrescine, and spermine, distinction between Analytical Review. Ser. Hasmatol., 7: 7-13, 1974.

220 CANCERRESEARCHVOL. 37

13. Heby, 0., and Russell, D. H. Changes in Polyamine Metabolism in Tumor 26. Russell, D. H. Polyamines in Growth: Normal and Neoplastic. In: D. H. Cells and Host Tissues during Tumor Growth and after Treatment with Russell (ed.), Polyamines in Normal and Neoplastic Growth, pp. 1-13. Various Anticancer Agents. In: D. H. Russell (ed.), Polyamines in Normal New York: Raven Press, 1973. and Neoplastic Growth, pp. 221-237. New York: Raven Press, 1973. 27. Russell,D.H.,Dune,B. G. M., andSalmon,S. E. PolyammnesasPredic 14. Lipton, A., Sheenan, L. M., and Kessler, G. F. Urinary Polyamine Levels tors of Success and Failure in Cancer Chemotherapy. Lancst, 2: 797- in Human Cancer. Cancer Brussels, 35: 464â€”473,1975. 799, 1975. 15. Livingston, A. B., Ambus, U., George, S. L., Freirsich, E. J, and Hart, J. 28. Russell, D. H., Gullino, P. M., Marton, L. J., and LeGendre,S. M. S. In Vitro Determinationof Thymldlne-3HLabelingIndex in Human Polyamine Depletion of the MTW9 Mammary Tumor and Subsequent Solid Tumors. Cancer Ass., 34:1376-1380, 1974. Elevation of Spermidine in the Sara of Tumor-bearing Rats as a Bio 16. Marton, L. J., Heby, 0., Levin, @/.A., Lubich, W. P., Crafts, D. C., and chemical Marker of Tumor Regression. Cancer Ass., 34: 2378-2381, Wilson, C. L. B. The Relationship of Polyamines in Cerebrospinal Fluid 1974. to the Presence of Central Nervous System Tumors. Cancer Ass. , 36: 29. Russell, D. H., and Levy, C. C. Polyamins Accumulation and Biosyn 973-977,1976. thesis in a Mouse L1210 Leukemia. Cancer Ass., 31: 248-251 , 1971. 17. Marton, L. J., Heby, 0., Wilson, C. B., and Lee, P. L. Y. A Method for 30. Russell, D. H., Levy, C. C., Schlmpff, 5. C., and Hawk, I. A. Urinary the Determination of Polyamines in Cerebrospmnal Fluid. Federation Polyamines in Cancer Patients. Cancer Ass., 31: 1555â€”1558,1971. European Biochem. Soc. Letters, 46: 305-307, 1974. 31. Russell, D. H., Looney, W. B., Kovacs, C. J., Hopkins, H. A., Dattilo, J. 18. Marton, L. J., Vaughn, J. G., Hawk, I. A., Levy, C. C., and Russell, D. H. W,, and Morris, H. P. Changes in Serum Putrescine and Spermidine Elevated Polyamins Levels in Serum and Urine of Cancer Patients: Levels following Local Radiation to Hepatoma 3924A of the Rat. Cancer Detection by a Rapid Automated Technique Utilizing an Amino Acid Ass., 36: 420-423, 1976. Analyzer. In: D. H. Russell (ed), Polyamines in Normal and Neoplastic 32. Russell, D. H., Looney, W. B., Kovacs, C. J., Hopkins, H. A., Marton, L. Growth, pp. 367-372. New York: Raven Press, 1973. J., LeGendre, S. M., and Morris, H. P. Polyamine Depletion of Tumor 19. Miller, J. L., Hudspith, B. N., and Blackett, N. M. Reduced Lethality in Tissue and Subsequent Elevation of Spermidmnein the Sera of Rats with Mice Receiving a Combined Dose of Cyclophosphamids and Busulphan. 3924AHepatomasafter5-FluorouracilAdministration.CancerAss.,34: Brit. J. Cancer, 32: 193-198, 1975. 2382-2385, 1974. 20. Murphy, W. K., Livingston, A. B., Ruiz, V. G., Gercovich, F. G., George, 33. Russell, D. H., and Russell, S. D. Comparison of the Relative Usefulness S. L., Hart,J. S.,andFrsirsich,E.J. SerialLabelingIndexDetermination of Serum, Plasma, and Urine Levels of Polyamines as Biochemical as a Predictor of Response in Human Solid Tumors. Cancer Ass. , 35: Markers of Cancer. Clin. Chem., 21:860â€”863.1975. 1438-1444, 1975. 34, Sanford, E. J., Drago, J. A., Rohner, T. J., Kessler, G. F., Sheehan, L., 21. Nishioka, K., and Romsdahl, M. M. Elevation of Putrescine and Spermi and Lipton, A. Preliminary Evaluation of Urinary Polyamines in the Diag dine in Sara of Patients with Solid Tumors. Clin. Chim. Acta, 57: 155, nosis of GenitourmnaryTract Malignancy. J. Urol., 113: 218-221 , 1975. 1974. 35. Sahimpff, S. C., Levy, C. C., Hawk, I. A., and Russell, D. H. Polyamines: 22. Aennert,0., Miale,T., Shukla,J., Lawson,D., and Frias,J. Polyammns Potential Roles in the Diagnosis, Prognosis, and Therapy of Patients with Concentrations in Bone Marrow Aspirates of Children with Leukemia Cancer. In: D. H. Russell (ed.), Polyamines in Normal and Neoplastic and Other Malignancies. Blood, 47: 695â€”701,1976. Growth, pp. 395-403. New York: Raven Press, 1973. 23. Aosenblum,M. G., Dune, B. G. M., Salmon,S. E., Chang,S. Y., and 36. Waalkes, T. P., Gehrke, C. W., Bleyer, W. A., Zumwalt, A. W., Olweny, C. Russell, D. H. A Spermidine Conjugate in Body Fluids of Rats and L. M., Kuo, K. C., Lakings, 0. B., and Jacobs, S. A. Potential Biologic Humans. Proc. Am. Assoc. Cancer Ass., 17: 15, 1976. Markers in Burkitt's Lymphoma. Cancer Chemotherapy Rept., 59: 721- 24. Rosenoft, S. H., Bull, J. M., and Young, A. C. The Effect of Chemother 727, 1975. apy on the Kinetics and Proliferative Capacity of Normal and Tumorous 37. Waalkss, T. P., Gehrke, C. W., Tormey, D. C., Zumwalt, A. W., Husser, J. Tissues ln Vivo. Blood, 45: 107-118, 1975. N., Kuo, K. C., Lakings, D. B., Ahmann, D. L., and Moertel, C. G. Urinary 25. Russell, D. H. Increased Polyamine Concentrations in the Urine of Hu Excretion of Polyamines by Patients with Advanced Malignancy. Cancer man Cancer Patients. Nature, 233: 144-145, 1971. Chemotherapy Rept., 59: 1103-1 116, 1975.

JANUARY1977 221

Brian G. M. Durie, Sydney E. Salmon and Diane H. Russell

Cancer Res 1977;37:214-221.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/37/1/214

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/37/1/214. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.