[CANCER RESEARCH 37, 646-650, March 1977]
`
`Free DNA in the Serum of Cancer Patients and the Effect of
`Therapy
`
`s. A. Leon, B. Shapiro, D. M. Sklaroff, and M. J. Varos
`Departments of Nuclear Medicine and Radiation Therapy, Division of Radiology, Albert Einstein Medical Center, Philadelphia , Pennsylvania
`
`19141
`
`SUMMARY
`
`INTRODUCTION
`
`A radioimmunoassay for ng quantities of DNA was devel(cid:173)
`oped . [' 25I)Iododeoxyuridine-labeled DNA was used as the
`antigen, and the serum of a lupus erythematosus patient
`served as the source of antibody . The level of free DNA in
`the serum of 173 patients with various types of cancer and
`in 55 healthy individuals was determined by this radioimmu(cid:173)
`noassay. DNA concentration in the normal controls had a
`range of Oto 100 ng/ml with a mean of 13 ± 3 ng/ml (S .E.) .
`For comparison purposes , the range of O to 50 ng/ml was
`designated as normal, and 93% of controls were found in
`this range. In the cancer patients , the DNA concentration
`ranged from zero to µ.g levels with a mean of 180 ± 38 ng/
`ml. Fifty% of the patients' values were found in the range of
`Oto 50 ng/ml ; the other 50% were between 50 and 5000 ng/
`ml. No correlation could be seen between DNA levels and
`the size or location of the primary tumor. Significantly
`higher DNA levels, however, were found in the serum of
`patients with metastatic disease (mean of 209 ± 39 ng/ml),
`as compared to non metastatic 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 the
`patients, whereas in glioma, breast , colon, and rectal tu(cid:173)
`mors, the DNA levels decreased only . in 16 to 33% of the
`patients. Generally , the decrease in DNA concentration in
`the serum correlated with improved clinical condition, such
`as decrease of tumor size and reduction of pain . Con(cid:173)
`versely , when DNA levels either increased or remained un(cid:173)
`changed, a lack of response to the treatment was noted. Of
`17 patients who died within a year, 13 showed DNA levels
`that remained high or unchanged, whereas only 4 showed
`lower levels during treatment . Persistent high or increasing
`DNA levels in the circulation, therefore, may signal a relapse
`and are probably a poor prognostic sign .
`The relatively high percentage (50%) of cancer patients
`with apparently normal DNA levels would suggest that this
`test may have low diagnostic value . It should be pointed
`out, however, that all these patients represent a selected
`group considered for radiation therapy, usually after sur(cid:173)
`gery and/or chemotherapy . It is possible that a better corre(cid:173)
`lation between DNA levels and cancer will be obtained prior
`to the initiation of treatment . On the other hand, DNA in the
`serum may be an important tool for the evaluation of ther(cid:173)
`apy or the comparison of different regimens.
`
`Received July 14, 1976; accepted November 22, 1976.
`
`646
`
`Tissue and cell injury take place under both nor
`pathological conditions . It is expected , therefore th:t and
`cellular material such as DNA may be released inttra.
`th
`circulation . Indeed , free DNA has been detected ins:
`e
`and other fluids such as synovial fluid (8) . For examr~m
`circulating DNA can be found in disorders such as syste~e,
`lupus erythematosus, rheumatoid art_hritis, pulmonary e~~ 1
`bolism or infarct, and cancer (5, 10) . In addition , therapeu(cid:173)
`tic procedures may also release DNA as after high-dose
`corticosteroid treatment or surgery (5 , 8) . Healthy controls
`on the other hand, usually showed lower levels of free DNA
`in the serum . The results for both disease and control
`groups are highly variable, depending on the methods used
`for detection of DNA. In some instances, indirect methods
`such as hemagglutinin inhibition (10), complement fixation
`(3), and double diffusion in agarose (8) showed µ.g levels of
`DNA. In other cases , using counterimmunoelectrophoresis,
`only fractions of a µ.g were detected (5, 15). Considerable
`controversy exists concerning the normal level of DNA in
`healthy controls, with reported values ranging from barely
`detectable levels (3, 5) to µ.g per ml (10). Similarly, a wide
`range of levels has been reported for a variety of disorders,
`including leukemia, solid tumors , and chronic inflammatory
`diseases such as systemic lupus erythematosus and rheu(cid:173)
`matoid arthritis (10) . In addition to the release of DNA, the
`common trait characterizing these diseases is lymph9id
`hyperplasia or sustained immunological stress. Most of the
`results agree in one respect : generally, the levels of DNA
`are higher than in healthy controls.
`The development of a direct competitive binding test for
`ng quantities of DNA (12, 13) in our laboratory provided a
`sensitive probe for the investigation of DNA release. The
`method is based on the binding between 125I-labeled DNA as
`the antigen and the serum of a systemic lupus erythemato·
`sus patient containing high-avidity antibody . Extraction and
`purification of the test sample are not necessary, and t~e
`determination can be performed directly on the patients
`serum . No interference has been found from either RNA or
`low-molecular-weight nucleotides (12) . The small amount 0
`:
`nonspecific binding by the basic component of complemen
`(C1 q) can be eliminated by heat inactivation (1 ). The confo;(cid:173)
`mation of DNA in the seru·m has been reported as sing:
`stranded by some investigators (10) and as double strande
`by others (2, 7, 8) . The antiserum used m ours u Y
`.
`.
`t d contains
`NA,
`antibodies against both double- and single-stranded D
`with a relative avidity of 9.6 and 5.6 x 105
`liters/m~~~
`respectively (11 ). This antiserum, therefore , Is used for
`
`CANCER RESEARCH VOL.
`
`37
`
`00001
`
`EX1043
`
`

`

`determination of total DNA and cannot discriminate be(cid:173)
`tween the 2 forms o_f DNA if present in a mixture.
`The purpose of this study was to determine the incidence
`of elevated DNA levels in cancer patients and wheth
`.
`tion therapy affects the DNA level.
`er rad1a-
`
`MATERIALS AND METHODS
`
`Labeling and Preparation of DNA. DNA was labeled b
`growing Eschertch1a
`in
`the
`y
`coli
`]. d d
`presence
`·d ·
`of
`['"I 10 _o eoxyun m_e as described previously (12, 13) . After
`extraction and punflcation, the Millipore filtration step (12)
`was omitted. and the DNA was passed through
`th
`1
`albumin-Kieselguhr column (11) . With this m date . Y ated
`nonspecific binding of DNA by serum com o I ication , the
`duced to 2 to 3% .
`ponents was re-
`
`e
`"' C
`" z
`
`0
`
`Free DNA in Cancer Serum and the Effect of Therapy
`
`5000
`
`4000
`
`3000
`
`200
`
`100
`
`700
`
`600
`
`so
`
`400
`
`30
`
`200
`
`DNA Antiserum. The serum of a lupus erythematosus
`patient {L. W.) was used as the antibody in th
`d. ·
`era 101 mmu-
`noassay (11, 12).
`Radioimmunoassay. The details have been described
`previously (12)_. Each sample was tested without antibody,
`for the determination of nonspecific binding. These counts
`were s_ubtracted from the counts obtained in the presence
`of antibody . In the absence of cold DNA {no inhibition
`control), 45 to 52% of the counts were found in the precipi(cid:173)
`tate. Known amounts of native salmon sperm DNA were
`added to normal human serum in the range of o to 1 ooo ng/
`ml, and a standard curve was constructed in the legit trans(cid:173)
`form (12) .
`Patients and Sera. Blood samples were drawn from pa(cid:173)
`tients and normal, healthy controls by venipuncture . The
`blood was allowed to clot at room temperature within 1 hr,
`and the serum was separated and frozen until use. Twenty(cid:173)
`five µI were taken for the determination of DNA concentra(cid:173)
`tion .
`Only cancer patients considered for radiation therapy
`were taken for this study . Samples were drawn before the
`beginning of the treatment and on biweekly intervals during
`the treatment. All patients received radiation daily for a
`minimum of 2 weeks, and some received it for as long as 6
`weeks . All patients had confirmed histological diagnosis of
`cancer. The test was performed without knowledge of their
`clinical condition, the J)resence or absence of metastases,
`or the outcome of surgical procedures . Subsequently, this
`information was obtained and correlated with the results of
`the DNA tests.
`The normal controls consisted of healthy hospital person(cid:173)
`nel, matched by age (17 to 61 years) and sex (24 males, 31
`females) to the cancer patients .
`
`RESULTS
`
`Normal Values of DNA in the Serum. The concentration
`of DNA was below detectable levels (0 to 25 ng/ml) m 42 of
`the 55 sera tested {76%) . Another 9 sera contained 25 to 50
`ng/ml (160/.) Only 4 sera {7%) contained 50 to 100 ng/ml,
`1 (Chart 1) Thus, 93%
`/
`·
`and none were found above 100 ng m
`1 1
`0
`the range of 0 to 5 ng m
`d ·
`of the normal sera were faun m
`.
`0
`with a mean of 13 :!: 3 ng/ml. For comparison purposes ,
`
`0
`
`•
`
`Chart 1. Distribution of ONA 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 reproduc(cid:173)
`ibility was within 10 to 15% (12) . • . metastatic; e, nonmetastatic. Fem . genit . ,
`female genital; Colon-rect. , colon-rectum ; Other dig ., other digestive; Genit.(cid:173)
`urin ., genitourinary; Unknown pr., unknown primary; Miscel., miscellaneous.
`
`to 50 ng/ml ·was designated as the normal range . On re(cid:173)
`peated tests with subsequent samples from the 4 individuals
`with a DNA concentration of 50 to 100 ng/ ml in the serum , 2
`showed lower values in the Oto 50 ng/ml range . It is un(cid:173)
`known at present whether this represents normal fluctua(cid:173)
`tion in DNA concentration in the serum or whether it is due
`to a latent or subclinical pathological condition. No correla(cid:173)
`tion 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 clarify
`these points .
`DNA Concentration in the Serum of Cancer Patients
`before Therapy. The 173 patients were classified according
`to the site of the primary tumor. All types showed very wide
`ranges , from zero to as high as 5000 ng/ml. The mean fo r
`each group{:!: S.E.) and the p value relative to the mean of
`the normal controls were: (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 not
`significant. The mean for all cancer patients was 180 :!: 38
`ng/ml , p < 0.005. It can be seen that in some groups of
`tumors {head-neck, lung , breast , ovary, uterus, lymphosar(cid:173)
`coma) very high concentrations of DNA could be found , up
`to µ,g levels. In others {digestive tract, genitourinary) th
`distribution had narrower range {Chart 1 ). In the ce~tr:i
`nervous system {glioma of brain or cord), all the patients
`
`)
`
`MARCH 1977
`
`647
`
`00002
`
`

`

`S. A. Leon et al.
`
`showed high values, but the small number of samples (4)
`precludes further evaluation of this finding . With the possi(cid:173)
`ble exception of central nervous system tumors , all groups
`showed some values in the normal range of Oto 50 ng/ml.
`Interestingly, almost all patients with high values (above
`100 ng/ml) had metastatic disease. In the low range (0 to 50
`mg/ml), on the other hand , both types were found . No cor(cid:173)
`relation could be found between the DNA concentration in
`the serum and the size, location, or histological composi(cid:173)
`tion of the tumor. However, in patients with well-contained ,
`slow-growing tumors, DNA levels tended to be lower than
`in those with faster-growing , poorly differentiated cells .
`This correlation was seen clearly when the DNA levels in
`patients without evidence of metastases were compared to
`those with proven metastatic disease (Chart 2). The mean
`DNA 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, healthy
`individuals is also included in Chart 2 for comparison .
`DNA Concentration in the Serum after Radiation Ther(cid:173)
`apy. 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 was
`observed only in a few of the patients treated (16 to 33%)
`(Table 1) . Generally, when the treatment was beneficial , as
`determined by decrease in tumor size or reduction of pain, a
`
`60
`
`50
`
`4 0
`
`....
`Vl
`z
`~ ....
`~
`.._ 30
`0
`5
`CD
`::!:
`:::,
`z
`
`20
`
`10
`
`n
`
`o·.f> _,oo_.,cP .,&
`ryo,ooo..:.i.~q..
`
`_.,o ,cP.p>.,&
`o ryo-,&~ . .{'(,,q..
`
`n
`
`NO META STAS ES
`
`W ITH M ETASTAS E S
`DN A LE VEL
`I N SERUM
`( ng / m l )
`Chart 2 . ONA levels in metastatic and nonmetastatic patients, compared
`to normal controls. The highest concentration in each group ranged between
`500 and 5000 ng/ml.
`
`NOR MAL
`
`648
`
`decrease in serum DNA concentration also was b
`Conversely, when the treatment was unsucces;f served.
`levels tended to remain unchanged or even . ul, DNA
`.
`mcrea
`S
`ome representative examples for each type of
`Sed.
`are illustrated in Table 2 . The results for all types r~sponse
`tested are summarized in Table 3. Clearly ·In pat·o cancer
`•
`Ients
`·
`metastatic disease, the DNA levels are generally hi h With
`above) and tend to stay high during and after tr~a:r (see
`Relapses occurred more frequently in this group
`lthrnent.
`th·
`b
`·
`' a ough
`1s o servatIon has not ~een evaluated critically be'cau
`of the small number of patients . Of the 17 patients wh
`. se
`within 1 year, 13 showed DNA levels that increased Odied
`or re-
`h
`d d
`.
`m . d
`urmg the treatment , whereas the D
`ame unc ange
`decreased in only 4 cases .
`NA
`
`DISCUSSION
`
`The source of free DNA in solution in the serum is un(cid:173)
`known at present. In addition to viral DNA, other sources
`have been reported such as normal lymphocytes (2) and
`tumor cells (14). The release of DNA from lymphocytes in
`vitro may also take place in vivo, although evidence for such
`a phenomenon is not available . Autolysis of cells after the
`collection of blood samples and release of DNA can be ruled
`out on the basis of control experiments . Repeated freezing
`and thawing of the white cell layer suspended in normal
`serum did not result in detectable DNA concentration in the
`supernatant fraction (S . A. Leon and B. Shapiro, unpub(cid:173)
`lished results) . The fact that DNA levels in normal individ·
`uals 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 to
`the method used. The choice of (125I)iododeoxyuridine-la(cid:173)
`beled DNA, with its low affinity for normal globulin , as well
`as 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-molec(cid:173)
`ular-weight nucleotides and RNA do not interfere in this
`direct competitive binding radioimmunoassay, indicating
`that the antibody is highly specific for DNA (12). For these
`reasons , we conclude that normal serum contains very little
`free DNA , in the range of 0 to 50 ng/ml and , in a small
`number of sera, up to 100 ng/ml. Further study will be
`requ ired to establish whether fluctuation in DNA levels
`within this range is a normal physiological phenomenon or
`due to a latent or subclinical pathological condition .
`In cancer patients , higher concentrations of DNA in the
`serum may be expected if we assume that the source of this
`material is proliferating tumor cells (14) . Another possibility
`is 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 be
`required to distinguish between these possibilities.
`The persistence of DNA in the circulation ra ises a~other
`question, namely, the function of DNAse I and II in the
`serum (17). In malignant disease, lower activity of these
`enzymes has been reported . This may be due to the release
`of an inhibitor which may be present in both tumors (4) an~
`normal cells (thrombocytes) (6 , 9) . We are presently inveStl(cid:173)
`gating this question in our laboratory.
`The elevated concentration of DNA in the serum of cancer
`
`CANCER RESEARCH VOL 37
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`00003
`
`

`

`---
`
`Free DNA in Cancer Serum and the Effect of Therapy
`
`Table 1
`Radiation therapy and DNA levels in serum of cancer patients
`Pulmo~a_ry tumors included bronchogenic and alveolar adenocarcinoma; female reproductive tract: ovary, uterus
`and cervix, colorectal tumors were examined separately from other digestive types: esophagus, pancreas, and
`gallbladder. The genitourinary types included bladder, prostate, and testicular carcinomas . In the head and neck
`?roup, 8 tumors are represented : palate, epiglottis, gingiva, nasopharynx, tonsil , larynx, maxillary sinus , and submax(cid:173)
`!llary gland - The _central nervous system group included glioma of the brain and cord . In the miscellaneous group were
`included bone giant cell carcinoma , epithelioma , melanoma, kidney, and hepatocarcinoma. Samples during and after
`treatment were not available for some patients. Levels were considered increased or decreased only if the change was
`greater than 25%.
`
`Type
`1 . Pulmonary
`2. Breast
`3. Female genitals
`4. Colon-rectum
`5. Other digestive
`6. Genitourinary
`7. Head-neck
`8. Central nervous system
`9. Lymphoma
`10. Unknown primary
`11. Miscellaneous
`
`Total
`
`12. Normals
`
`Total no.
`of pa-
`tients
`33
`32
`19
`9
`10
`29
`17
`4
`7
`5
`8
`
`173
`
`55
`
`Before therapy
`
`No . of patients
`
`Over
`50 ng/
`ml
`20
`12
`12
`5
`6
`10
`8
`4
`4
`4
`2
`
`Over 50
`ng/ml
`(%)
`61
`38
`63
`56
`60
`34
`47
`100
`57
`80
`25
`
`87
`
`50
`
`0-50
`ng/ml
`13
`20
`7
`4
`4
`19
`9
`0
`3
`1
`6
`
`86
`
`51
`
`Effect of therapy (% no . of patients)
`
`Decreased
`75
`22
`90
`16
`50
`55
`55
`33
`66
`0
`0
`
`Increased
`25
`66
`10
`50
`0
`27
`33
`66
`33
`100
`0
`
`No
`change
`0
`11
`0
`33
`50
`18
`11
`0
`0
`0
`100
`
`Table 2
`DNA levels in individual patients
`The 1st number in each sequence represents the DNA level before radiotherapy . Subsequent
`values represent the levels after 2, 4, or more weeks of treatment .
`DNA (ng/ml)
`
`Patient
`Tumor
`Lung (M)'
`H. M.
`Lung (M)
`G.D.
`Lung (M)
`A. M.
`Lung (M)
`J . G.
`Lung (M)
`H. S.
`Lung
`V. B.
`Lung (M)
`F. S.
`Lung
`J. G. A.
`Breast (M)
`I. B.
`Breast (M)
`I. R.
`Breast
`R. J.
`Breast (M)
`B. M.
`Breast
`D. H.
`Breast (M)
`H.P.
`Cervix (M)
`A. T.
`Ovary (M)
`F.O .
`Ovary (M)
`D. Y.
`Uterus
`P. M .
`Rectum (M)
`J . F.
`Colon (M)
`A. G.
`Rectum
`J. G. L.
`Pancreas (M)
`H.G.
`Larynx
`C. B.
`Tonsil (M)
`J. A.
`Glioma
`J . M.
`Lymphosarcoma
`E. K.
`• M, metastatic.
`
`Increase
`
`140-1000-250
`30-190-160
`25-115-120
`
`25-120-120
`125-160-320-300
`40-80-100
`115-210-200
`
`150-1600
`
`260-330-360
`110-55-350
`
`No change
`
`Decrease
`400-360-110
`250-140-75
`280-95-30
`
`35-50-50
`80-60-80
`
`90-95-60
`
`75-40-75
`175-200-150
`
`145-60-120
`
`140-90-80
`170-100-50
`1800-0-0
`650-50-50
`
`340-40-40
`
`550-2000-110
`
`2000-90-340
`
`MARCH 1977
`
`649
`
`00004
`
`

`

`S. A. Leon et al.
`
`Table 3
`DNA levels after radiotherapy in metastatic and nonmetastatic
`patients
`DNA levels were compared before therapy and at 2 weekly inte_r(cid:173)
`vals during the treatment in 81 metastatic and 92 nonmetastatic
`patients.
`
`DNA level
`Increased
`No change
`Decreased
`
`Patients (%)
`
`Metastatic
`61
`13
`26
`
`Non metastatic
`58
`3
`39
`
`.
`with reduction in tumor size, pain , and other signs of re
`sion . We hope , therefore, that sequential measurern
`rnis(cid:173)
`DNA concentration may be a useful tool for monito ~nts Of
`effects of therapy . Preliminary studies indicate tha:'~9 the
`results are obtained with chemotherapy (R . Bornst!:~1lar
`Shapir~ , and S. A. Leon, Effect of Chemotherapy on OB.
`Levels in Cancer Patients, in preparation). This test rna NA
`y be
`useful, therefore, to compare and evaluate objectiv
`I
`effectiveness of different treatments or their comb·inety the
`a ions.
`
`ACKNOWLEDGMENTS
`
`patients seems to correlate with the presence of tumor and/
`or metastatic disease . In preliminary studies, the DNA levels
`in patients with malignant lesions of the gastrointestinal
`tract were significantly higher than in those with inflamma(cid:173)
`tory conditions (carcinoma of the pancreas versus pancrea(cid:173)
`titis; Hodgkin's gastric ulcer versus duodenal ulcer) (B .
`Shapiro, S. A. Leon , E. Cohn , and M. Desai , DNA in the
`Serum of Patients with Tumors of the Gastrointestinal Tract,
`in preparation) . The diagnostic value of this finding, either
`for screening of suspected patients or for predicting a re(cid:173)
`lapse, may be questionable at present . The reason for this
`conclusion is the relatively high number of cancer patients
`(50%) with DNA levels in the normal range (0 to 50 ng/ml) . It
`should be pointed out, however, that the patients reported
`in this study by no means represent a random sample of
`malignant diseases. These patients represent a selected
`group considered for radiation therapy, in many cases after
`surgery and/or chemotherapy . It is possible that a prospec(cid:173)
`tive study of patients suspected for cancer, before , during,
`and after treatment, will yield results with better correlation
`between DNA levels and neoplastic disease . We conclude
`for the present that high levels of DNA in the serum accom(cid:173)
`pany a pathological condition , but normal levels do not
`rule out cancer and, probably , other diseases .
`After radiation therapy, a surprisingly high percentage of
`the patients (about 40%) showed decreased or unchanged
`DNA levels . This was especially obvious in patients who
`remained in the normal range of 0 to 50 ng/ml. Although
`radiation should have induced cell death and release of
`DNA in the circulation, it is equally possible that the arrest
`of tumor cell proliferation also reduced DNA release , on the
`assumption that they are the source of this DNA. Another
`explanation may be that the release of DNAse inhibitor from
`the tumor cells was reduced . In either case (and possibly
`both), low~r levels of DNA in the serum would result . Al(cid:173)
`though the mechanism is not clear at present, our findings
`suggest that persistence of DNA in the circulation after
`therapy correlates with poor response to treatment. In these
`patients , X-ray visualization or palpation (when possible)
`did not show a decrease in tumor size ; persistence of pain
`and relapses were observed . Conversely , decrease in DNA
`levels during treatment seem to be a better prognostic sign ,
`
`We thank Cynthia Baumel for her technical assistance .
`
`REFERENCES
`
`1. Agnello , V., Winchester, R. J., and Kunkel, H. G. Precipitin Reaction
`the C1q Component of Complement with Aggregated y-globulin 8 0d
`1
`Immune Complexes in Gel Diffusion . Immunology, 19: 909-919, 1970an
`2. Anker, P. , Stroun, M., and Maurice. P. A. Spontaneous Release of DNA
`~uman Blood Lymphocy1es in Vitro . Cancer Res., 35: 2375-2382,
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