[CANCER RESEARCH 37, 646-650, March 1977]

INTRODUCTION

A radioimmunoassay for ng quantities of DNA was developed. [125ljlododeoxyuridine-labeled DNA was used as theantigen, and the serum of a lupus erythematosus patie'ntserved as the source of antibody. The level of free DNA inthe serum of 173 patients with various types of cancer andin 55 healthy individuals was determined by this radioimmunoassay. DNA concentration in the normal controls had arange of 0 to 100 ng/mI with a mean of 13 ±3 ng/ml (S.E.).For comparison purposes, the range of 0 to 50 ng/ml wasdesignated as normal, and 93% of controls were found inthis range. In the cancer patients, the DNA concentrationranged from zero to p.g levels with a mean of 180 ±38 ng/ml. Fifty % of the patients' values were found in the range of0 to 50 ng/ml; the other 50% were between 50 and 5000 ng/ml. No correlation could be seen between DNA levels andthe size or location of the primary tumor. Significantlyhigher DNA levels, however, were found in the serum ofpatients with metastatic disease (mean of 209 ±39 ng/mI),as compared to nonmetastatic patients (mean 100 ±30, p <0.02).

After radiation therapy in lymphoma, lung, ovary, uterus,and cervical tumors, the levels decreased in 66 to 90% of thepatients, whereas in glioma, breast, colon, and rectal tumors, the DNA levels decreased only in 16 to 33% of thepatients. Generally, the decrease in DNA concentration inthe serum correlated with improved clinical condition, suchas decrease of tumor size and reduction of pain. Conversely, when DNA levels either increased or remained unchanged, a lack of response to the treatment was noted. Of17 patients who died within a year, 13 showed DNA levelsthat remained high or unchanged, whereas only 4 showedlower levels during treatment. Persistent high or increasingDNA levels in the circulation, therefore, may signal a relapseand are probably a poor prognostic sign.

The relatively high percentage (50%) of cancer patientswith apparently normal DNA levels would suggest that thistest may have low diagnostic value. It should be pointedout, however, that all these patients represent a selectedgroup considered for radiation therapy, usually after surgery and/or chemotherapy. It is possible that a better correlation between DNA levels and cancer will be obtained priorto the initiation of treatment. On the other hand, DNA in theserum may be an important tool for the evaluation of therapy or the comparison of different regimens.

Received July 14, 1976; accepted November 22, 1976.

Tissue and cell injury take place under both normal andpathological conditions. It is expected, therefore, that intracellular material such as DNA may be released into thecirculation. Indeed, free DNA has been detected in serumand other fluids such as synovial fluid (8). For example,circulating DNA can be found in disorders such as systemiclupus erythematosus, rheumatoid arthritis, pulmonary embolism or infarct, and cancer (5, 10). In addition, therapeutic procedures may also release DNA as after high-dosecorticosteroid treatment or surgery (5, 8). Healthy controls,on the other hand, usually showed lower levels of free DNAin the serum. The results for both disease and controlgroups are highly variable, depending on the methods usedfor detection of DNA. In some instances, indirect methodssuch as hemagglutinin inhibition (10), complement fixation(3), and double diffusion in agarose (8) showed @tglevels ofDNA. In other cases, using counterimmunoelectrophoresis,only fractions of a j.@gwere detected (5, 15). Considerablecontroversy exists concerning the normal level of DNA inhealthy controls, with reported values ranging from barelydetectable levels (3, 5) to @tgper ml (10). Similarly, a widerange of levels has been reported for a variety of disorders,including leukemia, solid tumors, and chronic inflammatorydiseases such as systemic lupus erythematosus and rheumatoid arthritis (10). in addition to the release of DNA, thecommon trait characterizing these diseases is lymphoidhyperplasia or sustained immunological stress. Most of theresults agree in one respect: generally, the levels of DNAare higher than in healthy controls.

The development of a direct competitive binding test forng quantities of DNA (12, 13) in our laboratory provided asensitive probe for the investigation of DNA release. Themethod is based on the binding between 125l-labeledDNA asthe antigen and the serum of a systemic lupus erythematosus patient containing high-avidity antibody. Extraction andpurification of the test sample are not necessary, and thedetermination can be performed directly on the patient'sserum. No interference has been found from either RNA orlow-molecular-weight nucleotides (12). The small amount ofnonspecific binding by the basic component of complement(Clq)can beeliminated by heat inactivation (1). The conformation of DNA in the serum has been reported as singlestranded by some investigators (10) and as double strandedby others (2, 7, 8). The antiserum used in our study containsantibodies against both double- and single-stranded DNA,with a relative avidity of 9.6 and 5.6 x i0@ liters/mole,respectively (ii). This antiserum, therefore, is used for the

646 CANCERRESEARCHVOL. 37

Free DNA in the Serum of Cancer Patients and the Effect ofTherapy

S. A. Leon, B. Shapiro, D. M. Sklaroff, and M. J. Yaros

Departments of Nuclear Medicine and Radiation Therapy, Division of Radiology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141

SUMMARY

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Free DNA in Cancer Serum and the Effect of Therapy

determination of total DNA and cannot discriminate between the 2 forms of DNA if present in a mixture.

The purpose of this study was to determine the incidenceof elevated DNA levels in cancer patients and whether radiation therapy affects the DNA level.

MATERIALS AND METHODS

Labeling and Preparation of DNA. DNA was labeled bygrowing Escherichia co/i in the presence of[1251]iododeoxyuridmneas described previously (12, 13). Afterextraction and purification, the Millipore filtration step (12)was omitted, and the DNA was passed through a methylatedalbumin-Kieselguhr column (ii). With this modification, thenonspecific binding of DNA by serum components was reduced to 2 to 3%.

DNA Antiserum. The serum of a lupus erythematosuspatient (L. W.) was used as the antibody in the radioimmunoassay(ii,12).

Radloimmunoassay. The details have been describedpreviously (12). Each sample was tested without antibody,for the determination of nonspecific binding. These countswere subtracted from the counts obtained in the presenceof antibody. In the absence of cold DNA (no inhibitioncontrol), 45 to 52% of the counts were found in the precipitate. Known amounts of native salmon sperm DNA wereadded to normal human serum in the range of 0 to 1000 ng/ml, and a standard curve was constructed in the logit transform (12).

Patients and Sera. Blood samples were drawn from patients and normal, healthy controls by venipuncture. Theblood was allowed to clot at room temperature within 1 hr,and the serum was separated and frozen until use. Twentyfive j.d were taken for the determination of DNA concentration.

Only cancer patients considered for radiation therapywere taken for this study. Samples were drawn before thebeginning of the treatment and on biweekly intervals duringthe treatment. All patients received radiation daily for aminimum of 2 weeks, and some received it for as long as 6weeks. All patients had confirmed histological diagnosis ofcancer. The test was performed without knowledge of theirclinical condition, the presence or absence of metastases,or the outcome of surgical procedures. Subsequently, thisinformation was obtained and correlated with the results ofthe DNA tests.

The normal controls consisted of healthy hospital personnel, matched by age (17 to 61 years) and sex (24 males, 31females) to the cancer patients.

RESULTS

Normal Values of DNA in the Serum. The concentrationof DNA was below detectable levels (0 to 25 ng/ml) in 42 ofthe 55 sera tested (76%). Another 9 sera contained 25 to 50ng/ml (16%). Only 4 sera (7%) contained 50 to 100 ng/ml,and none were found above 100 ng/ml (Chart 1). Thus, 93%of the normal sera were found in the range of 0 to 50 ng/mlwith a mean of 13 ±3 ng/ml. For comparison purposes, 0

500(

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Chart 1. Distribution of DNA levels in the serum of tumor groups (total,173 patients) and 55 healthy individuals, measured by radioimmunoassay.The values represent the average of duplicate determinations. The reproducibility was within 10 to 15% (12). U, metastatic; •,nonmetastatic. Fem. genit.,female genital; Colon-rect. , colon-rectum; Other dig. , other digestive; Genit.urin. , genitourinary; Unknown pr. , unknown primary; Miscel. , miscellaneous.

to 50 ng/mI was designated as the normal range. On repeated tests with subsequent samples from the 4 individualswith a DNA concentration of 50 to 100 ng/mI in the serum, 2showed lower values in the 0 to 50 ng/ml range. It is unknown at present whether this represents normal fluctuation in DNA concentration in the serum or whether it is dueto a latent or subclinical pathological condition. No correlation could be found between the DNA value and the age,sex, and smoking habits of the test subjects. Hopefully,further testing with larger numbers of individuals will clarifythese points.

DNA Concentration In the Serum of Cancer Patientsbefore Therapy. The 173 patients were classified accordingto the site of the primary tumor. All types showed very wideranges, from zero to as high as 5000 ng/mI. The mean foreach group (±SE.) and thep value relative to the mean ofthe normal controlswere: (1) 164 ±44,p < 0.002; (2) 193 ±79,p <0.02;(3)182 ±95,p <0.05;(4)102 ±37,p <0.01;(5) 104 ±28, p < 0.002; (6) 41 ±11,p < 0.01 ; (7) 108 ±35, p< 0.005; (8) 286 ± 106, p < 0.01 ; (9) 493 ±299, p = 0.05;

(10) 245 ±189, p not significant; and (11) 64 ±39, p notsignificant. The mean for all cancer patients was 180 ±38ng/ml, p < 0.005. It can be seen that in some groups oftumors (head-neck, lung, breast, ovary, uterus, lymphosarcoma) very high concentrations of DNA could be found, upto @glevels. In others (digestive tract, genitourinary), thedistribution had narrower range (Chart 1). In the centralnervous system (glioma of brain or cord), all the patients

647MARCH 1977

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S. A. Leon et a!.

showed high values, but the small number of samples (4)precludes further evaluation of this finding. With the possible exception of central nervous system tumors, all groupsshowed some values in the normal range of 0 to 50 ng/mI.Interestingly, almost all patients with high values (above100 ng/ml) had metastatic disease. In the low range (0 to 50mg/mI), on the other hand, both types were found. No correlation could be found between the DNA concentration inthe serum and the size, location, or histological composition of the tumor. However, in patients with well-contained,slow-growing tumors, DNA levels tended to be lower thanin those with faster-growing, poorly differentiated cells.This correlation was seen clearly when the DNA levels inpatients without evidence of metastases were compared tothose with proven metastatic disease (Chart 2). The meanDNA concentration in the 1st group was 100 ±30 ng/ml,whereas the mean calculated for the 2nd group was 209 ±39 ng/ml (p < 0.02). The distribution of normal, healthyindividuals is also included in Chart 2 for comparison.

DNA Concentration in the Serum after Radiation Therapy. In some tumors, such as lung, ovary, uterus, cervix,and lymphoma, the majority of the patients (66 to 90%)responded with lower DNA levels after radiation. In others,such as breast, colon, rectum, and glioma, this effect wasobserved only in a few of the patients treated (16 to 33%)(Table 1). Generally, when the treatment was beneficial, asdetermined by decrease in tumor size or reduction of pain, a

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Chart 2. DNA levels in metastatic and nonmetastatic patients, comparedtonormalcontrols.Thehighestconcentrationineachgrouprangedbetween500 and 5000 ng/ml.

decrease in serum DNA concentration also was observed.Conversely, when the treatment was unsuccessful, DNAlevels tended to remain unchanged or even increased.Some representative examples for each type of responseare illustrated in Table 2. The results for all types of cancertested are summarized in Table 3. Clearly, in patients withmetastatic disease, the DNA levels are generally higher (seeabove) and tend to stay high during and after treatment.Relapses occurred more frequently in this group, altt@oughthis observation has not been evaluated critically becauseof the small number of patients. Of the 17 patients who diedwithin 1 year, 13 showed DNA levels that increased or remained unchanged during the treatment, whereas the DNAdecreased in only 4 cases.

DISCUSSION

The source of free DNA in solution in the serum is unknown at present. In addition to viral DNA, other sourceshave been reported such as normal lymphocytes (2) andtumor cells (14). The release of DNA from lymphocytes invitro may also take place in vivo, although evidence for sucha phenomenon is not available. Autolysis of cells after thecollection of blood samples and release of DNA can be ruledout on the basis of control experiments. Repeated freezingand thawing of the white cell layer suspended in normalserum did not result in detectable DNA concentration in thesupernatant fraction (S. A. Leon and B. Shapiro, unpublished results). The fact that DNA levels in normal individuals in our study are usually zero or very low (25 to 50 ng/ml), in contrast to other reports (3, 8, 10), is probably due tothe method used. The choice of [1251]iododeoxyuridmne-labeled DNA, with its low affinity for normal globulin, as wellas an antiserum containing high-avidity antibody for DNA,results in a very specific and sensitive determination of DNA(12). For example, we had shown previously that low-molecular-weight nucleotides and RNA do not interfere in thisdirect competitive binding radioimmunoassay, indicatingthat the antibody is highly specific for DNA (12). For thesereasons, we conclude that normal serum contains very littlefree DNA, in the range of 0 to 50 ng/ml and, in a smallnumber of sera, up to 100 ng/mI. Further study will berequired to establish whether fluctuation in DNA levelswithin this range is a normal physiological phenomenon ordue to a latent or subclinical pathological condition.

In cancer patients, higher concentrations of DNA in theserum may be expected if we assume that the source of thismaterial is proliferating tumor cells (14). Another possibilityis that the DNA is released from normal cells (lymphocytes)during their interaction with virus or tumor cells (16, 18).Isolation and characterization of the DNA in serum may berequired to distinguish between these possibilities.

The persistence of DNA in the circulation raises anotherquestion, namely, the function of DNAse I and II in theserum (17). In malignant disease, lower activity of theseenzymes has been reported. This may be due to the releaseof an inhibitor which may be present in both tumors (4) andnormal cells (thrombocytes) (6, 9). We are presently investigating this question in our laboratory.

The elevated concentration of DNA in the serum of cancer

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DNA LEVEL IN SERUM (ng/mI)

648 CANCERRESEARCHVOL. 37

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greater than 25%.Total

no.of pa

Before thorapyEffect

of therapy (% no. ofpatients)No.of patients

Over0-50 50 ng/Over

50ng/mlNoTypetientsng/mI

ml(%)Decreased Increasedchange1.Pulmonary3313206175 2502.Breast3220123822 66113.Female

genitals197 126390 1004.Colon-rectum9455616 50335.Other

digestive104 66050 0506.Genitourinary2919103455 27187.Head-neck17984755 33118.Central

nervoussystem40 410033 6609.Lymphoma7345766 33010.Unknown

primary51 4800 100011.Miscellaneous862250 0100Total17386

875012.Normals5551

47

Table2DNAlevels in individualpatientsThe

1st number in each sequence representsthe DNA level before radiotherapy.Subsequentvaluesrepresentthe levelsafter 2, 4, or more weeksof treatment.DNA

(ng/mI)Patient

Tumor Increase Decrease NochangeH.M. Lung (M)°400-360-110G.D. Lung (M)250-140-75A.M. Lung (M)280-95-30J.G. Lung (M)140-1000-250H.S. Lung (M)30-190-160V.B. Lung25-115-120F.S. Lung (M)35-50-50J.G. A. Lung80—60-80I.B. Breast (M)25-120-120I.R. Breast (M)125-160-320-300R.J. Breast40-80-100B.M. Breast (M)115-210-200D.H. Breast90-95-60H.P. Breast (M)140—90-80A.T.

Cervix(M)170-100-50F.0. Ovary (M)1800-0-0D.V. Ovary(M)650-50-50P.M. Uterus75-40-75J.F. Rectum(M) 175-200-150A.G. Colon (M)150-1600J.G. L. Rectum340-40-40H.G. Pancreas(M)145-60-120C.B. Larynx550—2000-110J.A. Tonsil (M)260-330-360J.M. Glioma110-55-350E.K. Lymphosarcoma2000-90-340a

M, metastatic.

Free DNA in Cancer Serum and the Effect of Therapy

Table 1

Radiation therapy and DNA levels in serum of cancer patients

Pulmonary tumors included bronchogenic and alveolar adenocarcinoma; female reproductive tract: ovary, uterusand cervix; colorectal tumors were examined separately from other digestive types: esophagus, pancreas, andgallbladder. The genitourinary types included bladder, prostate, and testicular carcinomas. In the head and neckgroup, 8 tumors are represented:palate,epiglottis, gingiva, nasopharynx,tonsil, larynx, maxillarysinus, and submaxillary gland. The central nervous system group included glioma ofthe brain and cord. In the miscellaneous group wereincluded bonegiant cell carcinoma,epithelioma, melanoma,kidney, and hepatocarcinoma.Samplesduring and aftertreatmentwere not availablefor somepatients. Levelswereconsidered increasedor decreasedonly if the changewas

MARCH1977 649

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DNAlevelswere comparedvals during the treatment in 81patients.before

therapy and at 2 weekly intermetastatic and 92nonmetastaticDNA

LevelPatients

(%)Metastatic

NonmetastaticIncreased

No changeDecreased61

5813 326 39

S. A. Leon et a!.

with reduction in tumor size, pain, and other signs of rem ission. We hope, therefore, that sequential measurements ofDNA concentration may be a useful tool for monitoring theeffects of therapy. Preliminary studies indicate that similarresults are obtained with chemotherapy (R. Bornstein, B.Shapiro, and S. A. Leon, Effect of Chemotherapy on DNALevels in Cancer Patients, in preparation). This test may beuseful, therefore, to compare and evaluate objectively theeffectiveness of different treatments or their combinations.

ACKNOWLEDGMENTS

We thank Cynthia Baumel for her technical assistance.

REFERENCES

1. Agnello, V., Winchester, R. J., and Kunkel, H. G. Precipitin Reactions ofthe Clq Component of Complement with Aggregated ‘y-globulinandImmune Complexes in Gel Diffusion. Immunology, 19: 909-919, 1970.

2. Anker, P., Stroun, M. , and Maurice, P. A. Spontaneous Release of DNAby Human Blood Lymphocytes in Vitro . Cancer Res., 35: 2375-2382,1975.

3. Barnett, E. V. Detection of Nuclear Antigens (DNA) in Normal and Pathologic Human Fluids by Quantitative Complement Fixation. ArthritisRheumat., 11: 407-417, 1968.

4. Cooper, E. J., Trautmann, M. L. , and Laskowski, M. Occurrence andDistribution of an Inhibitor for Deoxyribonuclease in Animal Tissues.Proc. Soc. Exptl. Biol. Med., 73: 219-222, 1950.

5. Davis, G. L., and Davis, J. S. Detection of Circulating DNA by Counterimmunoelectrophoresis (CIE). Arthritis Rheumat., 16: 52-58, 1973.

6. Frost, P. G., and Lachmann, P. J. The Relationship of Deoxyribonuclease Inhibitor Levels in Human Sera to the Occurrence of AntinuclearAntibodies. Clin. Exptl. Immunol., 3: 447-455, 1968.

7. Harbeck, R. J., Hoffmann, A. A., and Carr, R. I. Studies on the Nature ofCirculating DNA in SLE. J. Rheumatol., 2: 194-203, 1975.

8. Hughes, G. A. ‘I., Cohen, S. A., Lightfoot, A. W. , Meltzer, J. I., andChristian, C. L. The Release of DNA into Serum and Synovial Fluid.Arthritis Rheumat., 14, 259-266, 1971.

9. Hughes, G. R. V. , and Lachmann, P. J. Systemic Lupus Erythematosus.In: P. G. H. Gell, R. A. A. Coombs, and P. J. Lachmann (ads.), ClinicalAspects of Immunology, p. 1131. Oxford: Blackwell Scientific Publications, 1975.

10. Koffler, D. , Agnello, V. , Winchester, A. , and Kunkel, H. G. The Occurrence of Single-stranded DNA in the Serum of Patients with SLE andOther Diseases. J. Clin. Invest., 52: 198-204, 1973.

11. Leon, S. A., Green, A., Ehrlich, G. E., Poland, M., and Shapiro, B. AvidityofAntibodies in SLE: Relation to Severity of Renal Involvement. ArthritisRheumat. , in press.

12.Leon,S.A.,Green,A.,Yaros,M.J.,andShapiro,B.Radioimmunoassayfor Nanogram Quantities of DNA. J. lmmunol. Methods, 9: 157-164,1975.

13. Leon, S. A. , Shapiro, B., Kollmann, G., and Green, A. A Comparison ofMethods for Labelling DNA for Use in the Radioimmunoassay of DNAantibodies. J. Immunol. Methods, 5: 1-8, 1974.

14. Rosenberg, B. Possible Mechanisms for the Antitumor Activity of Platinum Coordination Complexes. Cancer Chemotherapy Rept. , 59: 589-598,1975.

15. Schur, P. H., DeAngelis, D., and Jackson, J. M. Immunological Detectionof Nucleic Acids and Antibodies to Nucleic Acids and Nuclear Antigensby Counterimmunoelectrophoresis. Clin. Exptl. Immunol., 17: 209—218,1974.

16. Tonietti, G., Oldstone, M. B. A. , and Dixon, F. J. The Effect of InducedChronic Viral Infections on the Immunologic Diseases of New ZealandMice. J. Exptl. Med., 132: 89—109,1970.

17. Wroblewski, F., and Bodansky, 0. Presence of Desoxyribonuclease Activity in Human Serum. Proc. Soc. Exptl. Biol. Med., 74: 443-445, 1950.

18. Yoshida, T. 0. High Incidence of Antinuclear Antibodies in the Sera ofNasopharyngeal Cancer Patients. In: W. Nakahara, K. Nishioka, T. Hirayama, and V. Ito (eds.), Recent Advances in Human Tumor Virology andImmunology, pp. 443-460. Baltimore: University Park Press, 1971.

Table 3DNA levelsafter radiotherapy in metastatic and nonmetastatic

patients

patients seems to correlate with the presence of tumor and/or metastatic disease. In preliminary studies, the DNA levelsin patients with malignant lesions of the gastrointestinaltract were significantly higher than in those with inflammatory conditions (carcinoma of the pancreas versus pancreatitis; Hodgkin's gastric ulcer versus duodenal ulcer) (B.Shapiro, S. A. Leon, E. Cohn, and M. Desai, DNA in theSerum of Patients with Tumors of the Gastrointestinal Tract,in preparation). The diagnostic value of this finding, eitherfor screening of suspected patients or for predicting a relapse, may be questionable at present. The reason for thisconclusion is the relatively high number of cancer patients(50%) with DNA levels in the normal range (0 to 50 ng/ml). Itshould be pointed out, however, that the patients reportedin this study by no means represent a random sample ofmalignant diseases. These patients represent a selectedgroup considered for radiation therapy, in many cases aftersurgery and/or chemotherapy. It is possible that a prospective study of patients suspected for cancer, before, during,and after treatment, will yield results with better correlationbetween DNA levels and neoplastic disease. We concludefor the present that high levels of DNA in the serum accompany a pathological condition, but normal levels do notrule out cancer and, probably, other diseases.

After radiation therapy, a surprisingly high percentage ofthe patients (about 40%) showed decreased or unchangedDNA levels. This was especially obvious in patients whoremained in the normal range of 0 to 50 ng/ml. Althoughradiation should have induced cell death and release ofDNA in the circulation, it is equally possible that the arrestof tumor cell proliferation also reduced DNA release, on theassumption that they are the source of this DNA. Anotherexplanation may be that the release of DNAse inhibitor fromthe tumor cells was reduced. In either case (and possiblyboth), lower levels of DNA in the serum would result. Although the mechanism is not clear at present, our findingssuggest that persistence of DNA in the circulation aftertherapy correlates with poor response to treatment. ln thesepatients, X-ray visualization or palpation (when possible)did not show a decrease in tumor size; persistence of painand relapses were observed. Conversely, decrease in DNAlevels during treatment seem to be a better prognostic sign,

I

650 CANCER RESEARCH VOL. 37

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1977;37:646-650. Cancer Res   S. A. Leon, B. Shapiro, D. M. Sklaroff, et al.   TherapyFree DNA in the Serum of Cancer Patients and the Effect of

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