polyamines as markers of response and disease activity in … · polyamines as tumor markers...

[CANCERRESEARCH37, 214-221, January 1977]

SUMMARY

Onehundred twenty-four patientswith hematological andsolid neoplasmshad pretreatment urinary polyamine determinations. Putrescine, spermidine, and spermine were allsignificantly increased as compared to normals (p < 0.001).Polyamine levels were directly related to disease activityand tumor burden. In patients with multiple myeloma, putrescine levels were significantly correlated with clinicaldisease activity as well as the in vitro labeling index ofmarrow plasma cells. Spermidine values reflected tumorcell burden. Serial studies in 56 patients indicated thatgreater than twofold rise in urinary spermidine during treatment was highly correlated with cell kill and subsequentclinical response (p < 0.001). Serum polyamine levels in 17patients were found to be comparable to urinary values. Ourdata indicate that polyamine determinations can potentiallybe clinically useful, i.e., baseline values as indicators oftumor cell mass and growth fraction, and increases in spermidine during treatment as an excellent marker of tumorcell kill.

INTRODUCTION

Polyamines are low-molecular-weight, highly charged organic cations that are ubiquitous in nature. They are intimately involved in cell growth, with possible importance inthe regulation of RNA-dependentprotein synthesis (1, 5).Intracellular levels of polyamines, particularly spermidine[NH2(CH2)4NH(CH2)NH2]and putrescine [NH2(CH2)4NH2],increase early and dramatically with growth in both normaland neoplastic cells (26). The most marked synthesis andaccumulation of polyamines occurs in rapidly growing tissues (29).

The report of increased excretion of putrescine, spermidine, and spermine (NH2(CH2)3NH(CH2)4]in the urine of patients with cancer was published in 1971 (25, 30). Since thattime there have been several reports of elevated polyaminelevels in patients with cancer(3, 7, 10, 11, 13, 15, 18, 21, 34â€”

1 Supported in part by Contract NIH-NCI-C-73-7313 from the Division of

Cancer Treatment, National Cancer Institute; American Cancer Society Institutional Grant IN-I 10; and Grants CA-14102, CA-17094, and CA-14783 fromthe National Cancer Institute.

2 Scholar of the Leukemia Society of America. To whom requests for

reprints should be addressed.3 RecIpient of Research Career Development Award CA-00072 from the

National Cancer Institute.Received May 21. 1976; accepted October 14, 1976.

37), as well as reports of decreased polyamine levels aftersuccessful treatment of various types of cancer (7, 10, 11,13, 16, 21, 30, 34, 36, 37). The effect of therapy uponpolyamine levels has been further evaluated in animalmodel systems. Regression of mammary carcinoma in response to hormone ablation (28) and response of a rapidlygrowing hepatoma to chemotherapy (5-fluorouracil) (32)and irradiation (31) are associated with dramatic increasesin extracellular polyamine levels in proportion to the cell killoccurring with treatment. In a preliminary study of a groupof patients who had polyamine levels determined before,during, and after chemotherapy (27), we obtained similarresults and developed a model for the interpretation ofchanges in extracellular polyamine levels. The essence ofthis model is that extracellular spermidine levels are proportional to spontaneous cell loss or cell kill with treatment,whereasextracellularputrescinelevelsaremore relatedtothe growth fraction (27).

In this study, we have expanded our original preliminaryobservations in patients with both hematological and solidneoplasms. To evaluate the usefulness of serial determinations of extracellular polyamine levels, we have measuredpolyamine levels in the urine (and in some cases, serum) ofpatients before, during, and after specific courses of cancerchemotherapy, and have attempted to relate the pretreatment baseline levelsand changeswith treatment to the cellkill with treatment and to the overall survival and prognosisin individual patients.

MATERIALSAND METHODS

One hundred twenty-four patients with hematological orsolid neoplasms who were candidates for cytotoxic chemotherapy were studied. All patients were considered to bewithout other factors that might significantly affect polyamine levels at the initiation of the study period (e.g.,recentsurgery or other cancer therapy, or intercurrent diseasessuch as infection, collagen vascular diseases, or chronicobstructive lung disease) and had objective evidence ofdisease that could be evaluated with respect to clinicalstage and response to chemotherapy. The patients' agesranged from 17 to 70 years (median, 51 years). All patientshad urinary polyamine determinations and some had, inaddition, serum polyamine determinations, in relation toparticular courses of chemotherapy. Changes in polyaminelevelswith surgeryor radiation therapy were also monitoredin a few patients. We (30) and others (37) have previouslydetermined urinary polyamines in a large number of normal

214 CANCER RESEARCH VOL. 37

Polyamines as Markers of Response and Disease Activity inCancer 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 ofArizona College of Medicine, Tucson, Arizona 85724

on June 15, 2021. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Polyamines as Tumor Markers

adults. However, for this study, 16 control subjects in apparent good health (age range, 18 to 60 years) were studiedconcurrently to confirm our normal range for each urinarypolyamine. In addition, a small group of patients with diseases other than cancer were studied to illustrate the potential for abnormal levels in a variety of circumstances.

ClInicalEvaluationand Treatment.Standardcriteriaforstaging of disease were used (4, 8). In addition, the activityof the malignant process was assessed, with the majorcriterion being the presence or absence of clinical evidenceof disease progression within 2 months of the time of observation. The clinical parameters of progression of diseaseused in this study were as follows: (a) for multiple myeloma:new bone lesions, hypercalcemia, rising serum or urinemonoclonal immunoglobulin (M-component), or increase inbone marrow plasma cells or extraskeletal myeloma; (b) forleukemia: rising marrow and/or peripheral blast counts orblast transformation in chronic myelogenous leukemia; and(c) for lymphomas and solid neoplasms: rapidly increasingmeasurable disease. Response to chemotherapy wasjudged on the basis of objective evidence of cell kill usingthe standard criteria of the Southwest Oncology Group foreach disease type. It should be noted that patients in whomcell kill was followed by significant tumor regrowth beforethe next course of therapy were all included in the partialresponse category. Patients in whom there was no evidenceof tumor kill at any time during or after treatment wereincluded in the no-response category.

Most of the patients in this study were treated on aninpatient basis with intensive combination chemotherapy; asmall number were treated with simpler intermittent schedules on an outpatient basis. The major drugs used weredoxorubicin hydrochloride (adriamycin), cyclophosphamide, cytosine arabinoside, 5-(3,3-dimethyl-1 -triazeno)imidazole carboxamide, vincristine, bleomycin , and prednisone. No drugs known to interfere directly with polyaminesynthesis and metabolism [e.g., methylglyoxalbis(guanylhydrazone) (13)] were used.

Measurementof Polyamines.Polyamineswere assayedby the amino acid analyzer technique as previously published, with the use of a Durrum Model D-500 analyzer(Durrum Instrument Corporation, Palo Alto, Calif.) (33).

Standards,Reagents,and Buffers.Putrescine,cadaverme, spermidine, and spermine, in the form of hydrochloridesalts, were obtained from Calbiochem (San Diego, Calif.)and recrystallized 3 times from ethanol before use. The 14C-labeled polyamines used to determine recovery rates wereobtained from New England Nuclear (Boston, Mass.). NmSol, a ready-to-use ninhydrin solution, and thiodiglycolwere purchasedfrom PierceChemicalCompany(Rockford,III.). Liquified phenol was purchased from Matheson Scientific (Elk Grove Village, lIl.).The 3 buffers used were: BufferA (0.2 mole of Na@per liter, pH 6.16), the loading buffer forurine and serum samples; Buffer B (2.4 moles of Na@perliter, 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 3rdbuffer used for serum samples. The method for the preparation of these buffers is described elsewhere (16).

UrineAssayProcedure.Twenty-four-hrurinespecimenswere collected in plastic bottles containing 10ml of concentrated HCI and refrigerated between collections. An aliquot

from each collection was filtered through a Millipore filter(0.22-sm pore size) and a 2-mi sample was hydrolyzed for14 to 16 hr at 110Â°in 6 N HCI, and then centrifuged, transferred, and evaporated. The residue was reconstituted in250 @dof 0.1 NHCI, and 50 @Iwere loaded on the amino acidanalyzer. Details of the elution program are published elsewhere (33). When serial 24-hr urine collections were obtamed as a baseline prior to treatment, expression of thepolyamine levels per unit creatinine (pg/mg creatinine) resuited in very consistent daily excretory patterns. Expression of the data in this way also corrected for incompletecollection. When the time-span of collection was uncertainor the standard creatinine clearance was reduced by 50% ormore, samples were discarded. It was found that total unnary creatinine per day was very consistent in individualpatients and not significantly affected by systemic chemotherapy. It should be noted that, in looking at the pre- toposttreatment ratios in individual patients, the creatininevalues cancel out.

SerumAssayProcedure.Serawerecollectedand immediately stored at â€”76Â°until analysis. A 3- to 5-mI sample ofserum was treated with an equal volume of cold 10% tnichloroacetic acid, and the proteins were allowed to precipitatefor 30 mm in an ice bath. The sample was centrifuged, thesupernatant decanted, the pellet resuspended in 1 ml of10% tnichloroacetic acid and recentnifuged, and the supernatant added to the previous wash. This procedure resultedin 95 to 100% recovery in the supernatant of free and conjugated polyamines. The tnichloroacetic acid was removedwith ether washes, and the sample was hydrolyzed for 14 to16 hr at 100Â°in 6 N HCI. After hydrolysis, the sample wascentrifuged, transferred, and evaporated. The residue wasreconstituted in 150 pi of 0.1 N HCI and a 75-pi aliquot wasloaded on the analyzer. Sample size and relative peak areafor putrescine, cadavenine, diaminopropane, spermidine,and spermine were linearly related over the range of 0.05 to20 nmoles.

TritlatedThymidine-labelingIndexStudies.Labelingindex studies were performed in patients with multiple myeloma as an additional parameter of disease activity. Hepaninized bone marrow cell suspensions (0.5 x 10@to 1.0 x106cells/mI of cell suspension) were incubated for 1 hr at 37Â°with high specific activity (40 to 60 Ci/mmole) tnitiated thymidine (dose, 5.0 @Ci/mlof cell suspension). Cytocentnifuge smears were then made on gelatin-coated slides andfixed with methanol. The tnitiated thymidine-labeling indexwas then determined using our previously published highspeed scintillation autoradiography method (9) with controlcomparison to the standard method (15). The amount ofPPO used in the scintillator solution was 5 g/500 ml ofdioxane.

RESULTS

PretreatmentLevels of Urinary Polyaminesin CancerPatients. One hundredtwenty-fourpatientshad at leastpretreatment baseline urinary polyamine levels determined.Table 1 shows the mean Â±standard deviation, median, andrange for the urinary excretion of putrescine, spermidine,and spermine in patients with hematological or solid neo

215JANUARY1977



Polyamineswereassayedin 24-hr urinecollectionsby meansof an amino acidanalyzertechnique(see â€œMaterialsandMethodsâ€•).PutrescineSpermidineSpermmne(@.tg/mg

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

ofNo. ofNo.ofsubMean Â±sub Mean Â±sub

jectsS.D. MDNaRangejectsS.D. MDNRangejectsMean Â±S.D.MONRangeHematologi684.4 Â±0.66b 4.20.45-38683.7 Â±0.79â€• 2.70.58â€”25620.8 Â±0.32'5 1.00.25-8.0cal

tumorsSolidtumors553.7 Â±Ã˜@39b 3.80.29-10562.7 Â±54b 1.81.09-8.1560.6 Â±0.28k0550.12-3.7Normals162.1

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

Examplesof polyamine elevationsin diseasesother thancancerEachvalue indicates 1 patient. The listed disease was thoughttobe

the major factor in polyamineelevation.Putrescineâ€•

Spermidineâ€•Spermine'@(jig/mgcre- (pg/mg cre- (pg/mg cre

Disease atinine) atinine)atinine)Perniciousanemia 3.4810.81Hemolytic

anemia 26.35 8.551.0811.8212.081.87Rheumatoid

arthritis 5.64 3.81tracePolymyositis16.45 25.470.946.33

3.39traceChronicobstructive 3.85 2.751.19lung

diseaseActivepulmonary tu- 23.184.91berculosisLiverabscess

10.601.70Psoriasis5.71 3.10

B. G. M. Dune et al.

Table1Pretreatmentlevels of polyaminesin the urine of patients with cancer and normal control subjects

a MDN, median.

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

Table 2plasms, and in normal control subjects. (Urinary cadaveninelevels were measured in all patients with cancer; however,since there was no obvious correlation with tumor status orresponse and since some of these levels could have beeninfluenced by any bacterial contamination of urine samples,the data are not presented.) The range of values is extendedin all tumor categories, but the mean and median values foreach polyamine in cancer patients are significantly elevated, compared with those for the controls (p < 0.001).Putrescine, spermidine, and spermine values were allhigher in the patients with hematological tumors than inpatients with solid tumors.

Striking examples of values found for patients with diseasesother than cancer are shown in Table 2. Significantelevations in both putrescine and spermidine were found inassociation with pernicious anemia, hemolytic anemia(Coombs' positive), rheumatoid arthritis, polymyositis,chronic obstructive lung disease,alcoholic cirrhosis, acuteand chronic infections, and psoniasis.

Urinary Spermidineand PutrescineExcretionand Response to Chemotherapy. For 56 patients, complete sequential polyamine data were available, and it was possibleto objectively evaluate clinical response to a given course ofchemotherapy. Chart 1 exemplifies the pre- and posttreatment putrescine and spermidine levels and Table 3 summanizes the post- to pretreatment ratios of putrescine andspermidine in 22 patients with no response to chemotherapy, 16 patients with a partial response, and 18 patients witha complete response. Of these patients, 36 had hematological tumors and 20 had solid tumors. Note that, in the patients who had no response to chemotherapy as judged bysubsequent clinical evidence, the posttreatment-to-pretreatment spermidine ratio was 1.2. Those patients witheither a partial or complete response had a mean increasein this ratio of 3.7 and 3.6, respectively. Although putrescineconcentration usually increasedafter chemotherapy, elevation was not correlated with response. Thus, the nonrespenders had elevations in putrescine excretion withoutmajor increases in spermidmne, resulting in a low value forthe posttreatment-to-pretreatment ratio for spermidine overthe posttreatment-to-pretreatmentratio for putrescine. Notethat this ratio (0.4 for nonresponders, 1.2 for partial responders, and 1.4 for complete responders) is perhaps themost helpful in distinguishing the 3 categories.

In Table 4, the pre- and postchemotherapy spermidinelevels as well as the posttreatment-to-pretreatment ratios

a Range, 0.4 to 2.1.

b Range, 0.4 to 2.1.

C Range, 0.02 to 0.3.

0 10 20 30

I (OAP/L-ASP)

DAYSChart 1. Serial polyammnelevels in a patient with diffuse histiocytic lym

phoma who had a complete response to chemotherapy (putrescins, â€¢;cadaverins, 0; spermidine, A; and spermine, tb). Treatment is indicated by abar on theaxis [0, oncovin(vincristins);A, cytosinearabinoside;P, prednisons; L-ASP, L-asparaginase].

are shown for individual patients in relation to diseasecategory and response to chemotherapy. Note that in noneof the disease categories of nonresponding patients (including patients with multiple myeloma. leukemia, lym

216 CANCERRESEARCHVOL. 37



Response to chemotherapy and urinary spermidine and puexcretion in 56 patients studiedseriallytrescinePosttreat-

Posttreatmentâ€•/pre- menV'/pretreatmenttreatmentputrescine

spermidineNo.of ratioratioB/AType

of response cases (A)(B)ratioNone

22 2.7 Â±3,5â€• 1.2 Â±0.50.4Partial16 3.0 Â±3.1 3.7 Â±2.1r1.2Complete18 2.5 Â±1.2 3.6 Â±1.3c1.4Combined,

hema- 36 2.6 Â±2.2 3.0 Â±2.0@'1.2tologicaltumorsCombined,

solid 20 2.8 Â±3.4 2.0 Â±1.31.4tumors


Table 3 with active, as compared with more indolent disease states,as defined in â€œMaterialsand Methods.â€•Table 5 lists the datafor patients with multiple myeloma in whom the labelingindex (in vitro growth fraction) of the bone marrow plasmacells was available for comparison. It can be seen that theputrescine values were significantly (p at least

Effect of chemotherapyon urinaryspermidinelevels ofcancer patients bytype of cancer andresponse tochemotherapyPretreatmentPosttreatmentaPosttreatmsnt/spsrmidinespsrmidinspretreatment(pg/mg

creati (@&g/mgcreati spermidine raDiseaseResponsePatientSexnine)nine)tioMeanÂ± S.D.

Multiple mysloma None 1.14 Â±0.59

B. G. M. Durieetal.

Table 4

23456789

1011121314151617181920212223242526272829

None 30Complete 31

3233343536

M 2.92M 4.84M 0.79M 2.44M 3.54M 4.69M 1.22M 1.86F 2.20M 1.38M 1.95M 1.62M 2.11M 1.50M 1.00M 6.94M 0.63F 1.26M 26.59F 2.69M 1.70F 3.34M 3.33M 3.78M 2.51F 3.66F 1.48F 9.40F 2.42M 6.01M 2.10M 8.50M 12.27F 3.56M 1.90M 2.50

2.003.151.204.263.398.991.921.910.302.916.249.0611.90

4.753.11

10.713.5411.2163.9915.31

5.317.896.74

24.447.959.697.48

34.006.968.286.43

39.5033.7212.365.608.20

0.680.651.521.750.961.921.571.030.142.103.205.595.643.173.111.545.628.902.415.693.122.362.026.473.172.655.053.622.881.383.064.652.753.472.953.28

Partial 4.13Â±1.77

3.08Â±0.12

1,54â€•4.30Â±2.54

1@38â€•3.36 Â±0.68

1.24 Â±0.36

2.66 Â±0.58

3,73 Â±1.68

CompleteLeukemiaNonePartialCompleteLymphomaSolid

tumorsBreastcarcinoma0.89Breastcarcinoma1.48Melanoma1.42Melanoma1.47Melanoma0.63Melanoma1.32Melanoma1.50Melanoma0.76Prostate

carcinoma1.55Prostatecarcinoma0.96Coloncarcinoma1.66Breastcarcinoma2.49Fibrosarcoma2.55Fibrosarcoma1.95Liposarcoma3.55Bladder

carcinoma2.77Breastcarcinoma2.52Breastcarcinoma5.29Testicularcarcinoma5.04Testicular

carcinoma2.05

None 37383940414243444546474849505152

Complete 53545556

F 1.49F 1.65M 1.63M 1.35F 8.11M 2.50F 3.76M 1.40M 1.75M 2.23M 2.86F 0.92M 3.30M 3.59F 1.63F 1.27M 3.23F 1.19M 4.11M 2.10

1.322.442.311.985.133.305.651.072.712.154.752.298.417.015.793.528.136.29

20.724.30

Partial

a Posttreatmsnt indicates within 72 hr of initiation of chemotherapy.

b Single value.

original observation that polyamines are elevated in theextracellular fluid (urine, serum, and cerebrospinal fluid) ofpatients with cancer (3, 7, 10, 11, 14, 16, 18, 21, 28, 32, 34,36, 37). However, it has not been clearly established in whatway single or serial determinations may be used to improvethe management of individual patients or groups of pa

tients. The current report documents potential clinical usesfor polyamine determinations. The increase in urinary spermidine with successful therapy can be used as a marker ofcell kill. Pretreatment (baseline) polyamine values can beused to evaluate a number of tumor characteristics including tumor size, growth rate, and prognosis.




DiseaseNo.

ofpa

tientsPutrescine(pg/mg creat

mine)Spsrmidine(pg/mg creat

mine)Acute

myelogenousleu 121 1.4 Â±6.5b9.7 Â±7.1kemiaSmoldering

leukemia43.0 Â±1.01 .8 Â±0.6Chronic

myelogenousleukemiaBlast

phase227.6 Â±14.625.8 Â±12.5Chronicphase45.6 Â±1.76.8 Â±6.5Disseminated

softtissuesarcomasRapidly

progressive218 Â±9.69.9 Â±2.6Slowlyprogressive43.2 Â±1.02.1 Â±0.7Metastatic

colon carcinomaRapidly

progressive512.7 Â±6.78.0 Â±6.4Slowlyprogressive64.0 Â±1.52.1 Â±1.6

Baseline urinary polyamine values and disease activityâ€• in multiplemyelomaNo.

ofpa

. ,Labeling index (%)Putrescine (pg/mg creatinine)Spermidine (@.tg/mgcreatinine)Mean putrescine/spermiMean Â±Mean Â±MeanÂ±tientsS.D.

MDN' RangeS.D. MON RangeS.D. MON RangedineratioHigh

cell mass attime of diagnosisâ€•60.8

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

\\@ < 0.01

/3.2eIntermediateor 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 1.30.2-2.612.6cell

mass at timeofdiagnosisâ€•Refractory

patientsin relapse

Patients stable in5 1018

Â±12.0 16.0 5.0â€”38.0

\@

B. G. M. Dune et a!.

clinically obvious conditions such as bowel or myocardialinfarction and surgical procedures. However, most noncancer processes that can result in substantial polyamine elevations evolve over a period of several days to weeks (e.g.,bacterial abscess or viral hepatitis) and should not lead toconfusion. Active bone marrow recovery can result in putrescine and spermidine elevations, and careful attentionmust be given to the magnitude and time course of thesechanges. The data of Rosenoff et a!. (24) and Miller et a!.(19) indicate rather prompt recovery of DNA synthesis (12 to36 hr) after single doses of alkylating agents. Thus, elevations in polyamines at the end of treatment or betweenclosely spaced pulses of chemotherapy must be looked atwith the view that very early marrow recovery could be acontributing factor. However, our studies thus far have mdicated that extracellular spermidine elevations occur duringtherapy rather than at the end of treatment. In addition,putrescine is the major polyamine associated with a rapidregrowth process, whereas tumor cell kill results in predominant elevation in spermidine. In a number of the patients with multiple myeloma and leukemia, marrow-labeling indices and serum and urine lysozyme determinations[marker of granulocyte turnover (12)] have been available toexclude marrow recovery during this period (the leukemiadata are not presented in this report). In this regard, polyamine levels could be used to evaluate marrow recovery andaccurately schedule the next course of therapy. In the interpretation of polyamine levels, it is clearly important to evaluate all possible factors in a given patient that could becontributing to urine and/or plasma elevations.

As indicated in Tables 5 and 6, baseline polyamine determinations can provide useful information about tumor sizeand growth rate. The polyamine data can be used to corroborate other clinical and laboratory findings with respect tostage of disease as well as evaluation of remission or relapse (e.g., Ref. 21). The putrescine level can be used toprovide information similar to that of the in vitro labelingindex or growth fraction (6, 20) but without the need forserial biopsy of the tumor (which may be inaccessible).Since the labeling index is dependent upon the cell generation time, polyamine determinations may provide a moreaccurate reflection of ongoing cell growth. Polyamine determinations could serve as a basis for treatment selection,using kinetic principles.

However, these baseline values must be interpreted withmore caution than the dramatically increased values associated with cancer therapy. The maximum values in patientswith cancer are about 30 times greater than those of normalsubjects but, unfortunately, are still within the same rangeas values (exemplified in Table 2) found in some patientswith noncancer pathologies. Attention to this nonspecifictype of elevation in polyamines is particularly important forany planned use of polyamines in cancer diagnosis, differential diagnosis, or screening. Sanford eta!. (34) and Faireta!. (10)found polyaminedeterminations helpful in the diagnosis of urological cancer (prostate, bladder, and kidney).There were correlations with disease stage and tumorgrowth. However, differentiation from nonurological diseases was not included. Nonetheless, Dreyfuss et a!. (7),concluded that by looking at the pattern of elevation ofspermidine, putrescine, and spermine, distinction between

neoplastic and other cell growth or death processes waspossible. It is emphasized that clinical processes leading tononspecific polyammne elevations are usually obvious onroutine physical examination or standard laboratory testing.

The major portion of our study is based upon 24-hr urinepolyammnedeterminations. These samples are cumbersometo obtain and susceptible to incomplete collection. It wouldthus be more convenient if serum or plasma samples couldbe used to obtain comparable information, especially forpatients being followed on an outpatient basis. Our preliminary analyses illustrated in Chart 2 indicate that serum andurine values are comparable. However, levels of polyaminesin serum and plasma are lower than those in the urine, andthe difficulty in distinguishing between cancer and noncancer elevations is magnified. In addition, because of thepotential for rapid fluctuations in plasma levels, multiplesamples may be necessary to obtain meaningful data. Thus,timed (e.g., 2 to 6 hr) or spot urine collections or samplesfrom specific tissues of interest such as bone marrow (22)may be necessary or more useful. With kinetic modeling ofthe patterns of conjugation and excretion of polyamines(23), it may be possible to predict the best timing andsources for sampling.

For polyamine determinations to be available in a clinically useful way, a rapid simple assay system is necessary.An immunoassay, if feasible, would seem ideal. Recently, adirect radioimmunoassay for measuring spermine in serumhas been developed (4). Unfortunately, the antibody usedhad significant (22%) cross-reactivity with spermidine.Hopefully, a more specific immunoassay system can bedeveloped.

In conclusion, our data suggest considerable potentialclinical usefulness for polyamine determinations. However,more widespread clinical testing will be necessary to establish the full role of this type of information.

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1. Bachrach, U. Functions of Naturally Occurring Polyamines. New York:Academic Press,Inc.,1973.

2. Bachrach,U., and Ben-Joseph,M. TumorCells,Polyaminesand Polyamine Derivatives. In: D. H. Russell (ed), Polyammnes in Normal andNeoplastic Growth, pp. 15-26. New York: Raven Press, 1973.

3. Bartos,D.,Campbell,A.A., Bartos,F.,andGrettis,D. P.DirectDetermination of Polyamines in Human Serum by Radloimmunoassay. CancerAss., 35: 2056-2060, 1975.

4. CIassiflcatii@n and Staging of Cancer by Sits. A Preliminary Handbook.Chicago: American Joint Committee for Cancer Staging and End ResultsReporting, 1976.

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6. Crowther, D., Beard, M. E. J., Bateman,C. J. T., and Sewell, A. L.Factors Influencing Prognosis in Adults with Acute Myslogenous Leukasmia. Brit. J. Cancer, 32: 456â€”464,1975.

7, Dreyfuss, F., Chayen, A., Drsyfuss, G., Dvir, A., and Ratan, J. PolyamineExcretion in the Urine of Cancer Patients. Israel J. Med. Sci. , 11: 785â€”795, 1975.

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9. Dune,B. G. M., andSalmon,S. E. HighSpeedScintillationAutoradiography. 5cience, 190: 1093â€”1095,1975.

10. Fair,W. A., Wehnsr,N., and Brorsson,U. UrinaryPolyamineLevelsinthe 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. UrinaryPutrescine, Spermidine, and Spermine in Human Blood and Solid Cancars and in an Experimental Gastric Tumor of Rats. Cancer Ass. , 36:1320â€”1324,1976.

12. Hansen, N. E. Plasma Lysozyms: A Measure of Neutrophil Turnover. AnAnalytical Review. Ser. Hasmatol., 7: 7-13, 1974.




13. Heby, 0., and Russell, D. H. Changes in Polyamine Metabolism in TumorCells and Host Tissues during Tumor Growth and after Treatment withVarious Anticancer Agents. In: D. H. Russell (ed.), Polyamines in Normaland Neoplastic Growth, pp. 221-237. New York: Raven Press, 1973.

14. Lipton, A., Sheenan, L. M., and Kessler, G. F. Urinary Polyamine Levelsin Human Cancer. Cancer Brussels, 35: 464â€”473,1975.

15. Livingston, A. B., Ambus, U., George, S. L., Freirsich, E. J, and Hart, J.S. In Vitro Determinationof Thymldlne-3HLabeling Index in HumanSolid Tumors. Cancer Ass., 34:1376-1380, 1974.

16. Marton, L. J., Heby, 0., Levin, @/.A., Lubich, W. P., Crafts, D. C., andWilson, C. L. B. The Relationship of Polyamines in Cerebrospinal Fluidto the Presence of Central Nervous System Tumors. Cancer Ass. , 36:973-977,1976.

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18. Marton, L. J., Vaughn, J. G., Hawk, I. A., Levy, C. C., and Russell, D. H.Elevated Polyamins Levels in Serum and Urine of Cancer Patients:Detection by a Rapid Automated Technique Utilizing an Amino AcidAnalyzer. In: D. H. Russell (ed), Polyamines in Normal and NeoplasticGrowth, pp. 367-372. New York: Raven Press, 1973.

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36. Waalkes, T. P., Gehrke, C. W., Bleyer, W. A., Zumwalt, A. W., Olweny, C.L. M., Kuo, K. C., Lakings, 0. B., and Jacobs, S. A. Potential BiologicMarkers in Burkitt's Lymphoma. Cancer Chemotherapy Rept., 59: 721-727, 1975.

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JANUARY1977



1977;37:214-221. Cancer Res Brian G. M. Durie, Sydney E. Salmon and Diane H. Russell Cancer ChemotherapyPolyamines as Markers of Response and Disease Activity in

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