bone by atomic absorption spectroscopy. Clin Chem 1985; by xww95991

VIEWS: 37 PAGES: 6

									in bone by atomic absorption spectroscopy. Clin Chem 1985;               enamel by the carbon cup AA method. Analyst 1974;100:884-90.
31:1172-4.                                                               14. Blust R, Van der Linden A, Declein W. Microwave-aided
7. Giordano R, Costantini S, Verniflo I, Casetta B, Aidrighetti F.       dissolution of a biological matrix in AAS sample cups prior to
Comparative study for aluminum determination     in bone by atomic       graphite furnace analysis. Atom Spectrosc 1985;6:163-5.
absorption techniques and inductively     coupled plasma atomic          15. Slavin W. Letter to the editor. Spectrochim       Acta 1987;
emission spectroscopy. Microchem J 1984;30:435-47.                       42B:933-5.
8. Stevens BJ. Electrothermal atomic absorption determination of         16. Welz B. Abuse of the analyte addition technique in atomic
aluminum in tissues dissolved in tetramethyl ammonium hydrox-            absorption spectrometry. Z Anal Chem 1986;325:95-101.
ide. Clin Chem 1984;30:745-7.                                            17. Leung FY, Henderson AR. Determination          of aluminum    in
9. Julshamn K, Andersen K-J, Willassen Y, Braekkan OR. A                 serum and urine using matrix modification and the L’vov platform.
routine method for the determination of Al in human tissue               Atom Spectrosc 1983;4:1-4.
samples using standard addition and graphite furnace AAS. Anal           18. Manning DC, Slavin W, Carnrick GR. Investigation of alumi-
Biochem 1978;88:552-9.                                                   num interferences using the stabilized temperature platform fur-
10. LeGendre GR, Aifrey AC. Measuring picogram amounts of                nace. Spectrochim Acta 1982;37B:331-41.
aluminum in biological tissue by flameless atomic absorption             19. Slavin W, Carnrick GR, Manning DC, Pruszkowska E. Recent
analysis of a chelate. Clin Chem 1976;22:53-6.                           experiences with the stabilized temperature   furnace and Zeeman
11. Krishnan 55, Quittkat S, Crapper DR. AA analysis for trace of        background correction. Atom Spectrosc 1983;4:69-86.
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12. McDermott JR, Whitehill I. Determination of Al in biological         1986;1:391-5.
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13. Dolinsek F, Stupar J, Spenko M. Determination of Al in dental        additions determination.  Fresenius Z Anal Chem 1986;325:263-6.




               465-471(1991)
CLIN.CHEM. 37/3,


Urinary Fibronectin Fragments (a Potential Tumor Marker) Measured by
lmmunoenzymometric Assay with Domain-Specific Monoclonal Antibodies
Masahlko
       Katayama,’
                Fumitsugu
                        Hino,1yoko Kamihagi,1
                             K                    Seklguchi,2 TItanl,2 and lkunoshinKato1
                                           Kiyotoshi       Kohl

We found that urinary fibronectin (UFN) in cancer patients               lular matrix, and in plasma. FN plays a role in various
was almost all fragmented and consisted mainly of the                    cellular processes, including  cell-to-substrateadhesion,
cell-binding domain. We developed a two-site immurloenzy-                cell migration, and the regulation of cell morphology (1).
mometnc assay forUFN, usingtwo monoclonal antibodies                     Webb and Lin first reported in 1980 (2) that the amount
that both recognize this    domain offibronectin. The amount             offragmented FN in the urine of patients with prostatic
of UFN was expressed as arbitrary units per milligram of                                                         A
                                                                         cancer ishigher than in normal subjects.nother group
creatinine. Some 2% of the 623 healthysubjects had UFN                   reported that an FN-like protein,with a lower molecu-
above theclinical cutoff point (200 arb. units/mg creatinine),           lar mass than plasma FN, is increased in sera from
as did 14% of the 271 patients with nonmalignant diseases.               patients with malignant disease (3). Since then, mea-
In contrast, concentrations of UFN exceeded the cutoff point             surements of the concentration of FN in body fluids and
in 59% of the 589 patients with cancer. in 37 patients with              tissues  from patients with cancer have usually involved
gastrointestinai   cancer tested for UFN and forone or more              useofa polyclonal            toplasma FN. Inparticular,
                                                                                               antibody
of three established serum tumor markers, UFN was found                  many examinations have been done to evaluate the
in 25, significantly more often than the other markers. These            clinical usefulness of measurements of plasma FN (4-7).
results indicated that UFN is a marker that may be helpful in            However, FN concentrations in body fluids and tissues
evaluating   many kinds   ofcancer.                                      are not correlated with malignancy or the metastatic
                                                                         potential of cancer (4-6, 8). Plasma FN does not seem
AdditionalKeyphrases:cancer            cutoff value                      suitable as a tumor marker, because plasma FN                     is
                                                                         sensitive to clinical events unrelated to the malignancy
  The adhesive glycoprotein fibronectin (FN),3 Mr                        status(4-6).
440 000, is widely distributed cells, the extracel-
                              on    in                                     We found by immunoblotting that urinary FN (UFN)
                                                                         in cancer patientsisalmost allpresent as fragments of
                                                                                    s      a
                                                                         differentizes, nd that a polyclonal     antibodyraised
  1Biotechnology     Research   Laboratories, Takara Shuzo Co.,  Ltd.,
                                                                         against native plasma FN cannot react well with UFN.
Seta 3-4-1, Otsu, Shiga 520-21, Japan.
                for Comprehensive Medical Science, Fujita Health         Monoclonal antibodies can be used to detect the target
University School of Medicine, Toyoake, Aichi 470-11, Japan.             substance in such situations. We have already devel-
   3Nonstandard      abbreviations: FN, fibronectin; UFN, urinary        oped four monoclonal antibodies against three different
fibronectin; LEMA, immunoenzyinometric        assay; CEA, carcinoem-     domains (the N-terminal,   the C-terminal, and the cen-
bryonic antigen; CA19-9, carbohydrate   antigen 19-9; AFP, a-feto-
protein; Hep, heparin; and Fib, fibrin.                                  tral region) of the FN molecule (9).
   Received September 7, 1990; accepted December 19, 1990.                  Here we demonstrate that two monoclonal antibodies,

                          37,
466 CLINICALCHEMISTRY, Vol. No.3, 1991
FN1O and FN3O, which are reactive with the central                                                    patients were collected before surgery. Some of the
region of the FN molecule, the “cell-binding domain,”                                                 malignant diseases were staged at the time of diagnosis
are suitable for in vitro assays of FN fragments excreted                                             after the urine samples were collected. To evaluate the
in the urine of cancer patients. The antibodies were used                                             correlation     between   increased   UFN   and tumor   grade,
together in a monoclonal imniunoenzymatic        sandwich                                             we performed a detailed staging in 58 of the 164 pa-
for a specific and simple urine test for cancer.                                                      tients with stomach cancer who provided urine samples.
                                                                                                      The samples were frozen without preservatives at
Materials and Methods                                                                                          until
                                                                                                      -20 #{176}C analysis. Serum samples were also col-
                                                                                                      lected from 37 of the patients with gastrointestinal
    Monoclonal        antibodies.          Two murmne monoclonal                              anti-   cancer.
bodies, FN1O and FN3O, were derivedby fusionwith the                                                    Standard    material for UFN assay. We used recombi-
use of splenocytes rom a mouse immunized with puri-
                    f                                                                                 nant FN cell-binding fragment as the standards for the
fied human plasma FN (Collaborative Research Inc.,                                                    assay. Fragments were prepared by the expression of
Lexington, MA) and identified as being of the IgG1                                                    human FN complementary          DNAs in Escherichia coli.
subclass. The domain epitopes of these antibodies were                                                The construction and expression of the plasmid used
characterized as described elsewhere (9, 10). Locations                                               were described elsewhere (9).
of antibody-binding sites on FN are shown in Figure 1.                                                   Assay of creatinine in urine. A commercially available
   Immunoblotting   analysis. UFN was purified from 20                                                kit [“Creatinine Test Wako,” based on the method of
mL of a urine sample by use of FN3O monoclonal                                                        Jaffe (11)] was used according to the manufacturer’s
antibody immobilized on an agarose column. Urine                                                      instructions  to assay creatinine in urine.
samples for electrophoresis    were collected from one                                                   Cutoff value for UFN. UFN concentrations    were ex-
healthysubject and three patients with bladder, lung, or                                              pressed in terms of arbitrary units, 1 arb. unit being
stomach cancer. UFN eluted from the column and                                                        equivalent   to 1 ng of plasma FN. A value 200 units/mg
plasma FN were separated simultaneously by 10% so-                                                    of creatinine was considered to be positive for cancer,
dium dodecyl sulfate-polyacrylamidegel electrophore-                                                  based on the values found in 623 healthy subjects (200
sis and then transferred to a nitrocellulose sheet by                                                 units/mg      is the mean of these observed values + 2 SD).
electrophoresis. The bound antibodies were detected                                                     Sandwich        JEMA. Antibody  FN1O was purified by Pro-
with anti-mouse IgG antiserum labeled with horserad-                                                  tein A chromatography      and labeled with horseradish
ish peroxidase (EC 1.11.1.7; Bethesda Research Labora-                                                peroxidase   (Boehringer   GmbH,    Mannheim,    F.R.G.).
           G
tories, aithersburg, MD). All samples were reduced                                                    First, 96-well plates coated with   antibody FN30 were
with 2-mercaptoethanol. The nitrocellulose membrane                                                   blocked with bovine serum albumin solution. To each
was immunostained with FN1O monoclonalantibody as                                                     well we added 100 L of a solution of peroxidase-labeled
reported previously (10).                                                                             FN1O antibody plus 20 L of FN standard (0, 50, 100,
    Clinical specimens.  For evaluation of the immunoen-                                              200, 400, or 800 arb. units/mL) or a urine sample. The
zymometric assay (IEMA) of UFN, a total of 1483 urine                                                 plate was incubated for 0.5 h at room temperature and
samples from healthysubjects n = 623),
                                  (          patientswith                                             washed with de-ionized water. We then added 2.9
benign disease (271), and patients with cancer (589)                                                  mmollL 3,3’,5,5’-tetramethylbenzidine       2HC1 (Sigma
were obtainedfrom the NationalCancer Center Hosp-                                                     Chemical Co., St. Louis, MO) solution as substrate and
tial (Tokyo),   Showa University   Hospital(Tokyo),  Toho                                             left the mixture for 15 mm at room temperature, after
University    Hospital  (Tokyo), okkyo University
                                D                  Hospi-                                             which we stopped the enzyme reaction by adding 100 p.L
tal (Tochigi), and Shiga University Hospital (Shiga,                                                  of HC1, 1 mol/L. We measured the absorbance of each
Japan). All of the 589 patients with pathologically and                                               well at 450 nm with a Titertek Multiscan densitometer
histologically proven cancer, but with no previous treat-                                             (Flow Laboratories, McLean, VA). The amount of UFN
ment by chemotherapy and radiotherapy, were entered                                                   was expressed per milligram of creatinine.
in this study. All the urine samples from the cancer                                                     Other clinical markers. Besides UFN, we assayed the
                                                                                                      concentrations in serum of carcinoembryonic antigen
                                                                                                      (CEA), carbohydrate antigen 19-9 (CA19-9), and a-feto-
                                                                                                      protein (AFP) in 37 of the 589 patients with malignant
                           Ur)JLfflifflj                                                              tumors. Serum and urine samples were collected from
                         Monoclonal   antibodies          FN3O   FNIO                                 these 37 patients before surgery; their cancers were
                                                                                                      classified as stage III or IV. Serum and urinary markers,
      MeUrillllllH                                                                1icxDl              except for UFN, were measured with commercially
           L_)                                                    -J L)           J
                                                                                                      available kits: CEA-EIA kits were purchased from Ab-
Domsina:     top-li         llep-3                 Coil                   imp-2       Fib-2           bott Laboratories (North Chicago, IL), CA19-9 radioim-
            Fe.,
                                                                                                      munoassay    kits from Centocor (Malvern,    PA), and AFP
Fig. 1.       Domain structure of fibronectin and the binding epitopes of
the monoclonal antibodies FNIO and FN3O, which recognize the                                          radioimmunoassay       kits from Pharmacia (Uppsala, Swe-
cell-binding domain                                                                                   den).
Arrows indicate the epitopes of the antibodies.Hep-1/Fib-1,first hepann- and
fibrin-binding domain; Gel, collagen- and gelatin-binding domain; Hep-2,                              Results
second heparln-blnding domain; Cell, cell-binding domain (involved in cell
attachment);Hep-3,third hepann-bindingdomain; Fib-2,second fibnn-binding                                Precision   of assay and recovery. We evaluated the
domain                                                                                                precision of the assay by assaying two samples of urine

                                                                                                                                             37,No.3,1991 467
                                                                                                                       CLINICALCHEMISTRY, Vol.
10 times each in a continuous series (intra-assay)     or
                                                                                        Table 1. Mean Concentrations of UFN in Healthy
twice each in 10 consecutive assays (interassay). Intra-
                                                                                                   Subjects, by Sex and Age
assay CVs were 7.0% for a UFN concentration of 72.5
                                                                                                                                              UFN, arb. unIts/mg
arb. units/mL and 6.5% for 141.0 arb. units/mL; interas-                                                                                             creatlnine
say CVs were 6.0% for 70.4 arb. units/mL and 7.8% for                              Age,                                   No. of
143.1arb.umts/mL.                      r
                     Mean analyticalecovery      ofstan-                          years                  Sex            specimens             Mean                SD
dard UFN (200,400, or 800 arb.units/mL)added to                                   20-29          Men                            57             61.7           35.0
three urine samples in equivolumeratios     was 99.1%.                                           Women                          57             54.5           29.8
  Immunoblotting  analysis of UFN in cancer patients.                                            Total                         114             59.0           32.9
Three samples of UFN from patients with bladder, lung,                            30-39          Men                            15             79.1           61.7
                                                                                                 Women                          30             76.2           52.8
or    stomach cancer showed a positive reaction, as did
                                                                                                 Total                          45             76.8           56.5
plasma FN, in immunoblotting analysis with FN1O
                                                                                  40-49          Men                           29              76.0           55.1
(Figure  2). UFN in these three cancer patients had                                              Women                         20              97.0           79.3
multiple molecular masses in the range of 30 000 to
                                                                                                 Total                         49              84.6           66.9
180 000 Da. The Mr of the antigens detected ranged                                50-59          Men                           21              89.1           68.2
widely, but were generally in the region of 60 000 or                                            Women                         16              65.4           42.4
100 000. The amount of UFN in a healthy subject                                                  Total                         37              78.9           59.5
chosen without conscious bias was so small that no                                60-69          Men                            8             106.6           76.4
antigen  was   detected.  Nonfragmented   FN,   Mr                                               Women                          4             107.8           71.1
-220 000, was almost undetectable in the urine sam-                                              Total                      12                107.0           74.6
ples from the three cancer patients.                                              20-69          Total Men                 132                 73.8           54.5
     Use of the assay      to measure       UFN in cancer patients.                              Total Women               125                 69.0           52.1
The mean concentration               of UFN in the 623 healthy                                   Overall Total             257                 71.7           53.1
subjects      was 68.6 (SD 47.7) arb. units/mg                  creatinine.
The mean concentrations of UFN for different ages and                             were slightly  increased in the urine of the healthy
by sex for all 257 subjects tested for suspected disease at                       subjects ages 60-69 years (Table 1).The mean + 2 SD
Showa University      Hospital are shown in Table 1.                              concentration of UFN in the 623 healthy subjects was
Among    the healthy controls, UFN concentrations in                              164.0 arb. units/mg creatinine, and the mean + 3 SD
men and women were not significantly          different,but                       was 211.7 arb. units/mg     creatinine. Concentrations
                                                                                  200       arb. units/mg        creatinine,         i.e., greater     than       the
                                                                kDa               mean      +    2 SD but less than the mean + 3 SD, were
                                                                                  therefore considered to be high. With this as the cutoff,
                                                          220                     only 13 (2%) of the 623 healthy subjectsgave positive
                                                                                  results. The distribution of UFN values of the patients
                                                                                  and healthy subjects is shown in Figure 3. Concentra-
                                                                                  tions were high in 73 (14%) of the patients with nonma-
                                                                                  lignant       diseases and in 356 (59%) of the patients                     with
                                                     .    93                      malignant diseases.
                                                                                    We assayed UFN in urine from the 58 patients with
                                                          66                      stomach cancers at differentstages.UFN                             concentra-
                                                                                  tions were high in four (33%) of the 12 patients with
                                                                                  stage I stomach cancer,in one (50%) ofthe two patients
                                                                                  with stage ifistomach cancer, in 14 (74%) of the 19
                                                                                  patients with stage IV stomach cancer, in 23 (96%) of
                  --    :4             -    -       -45                           the 24 patients with recurrent stomach cancer, but not
                                                                                  in the one patientwith stage IIstomach cancer.Metas-
                                                                                  tases to distant siteswere detected in two of the 19
                                                                                  patients with stage IV cancer and in nine of the 24
                                                                                  patients with recurrent cancer. UFN concentrations
                                                                                  were extremely high in all of these patients with distant
                                                           31                     metastases.
                                                                                    Comparison     of UFN and serum markers. We assayed
                                                                                  concentrations    of UFN and serum markers in urine
          1       2        34                   5                                 samples from patients with malignancies     (Table 2).
Fig. 2. immunostaining of IJFN and plasma FN by a monoclonal
antibody specific to the cell-binding domain of FN                                UFN was found much more often than the serum
Purified UFN and plasma EN were electrophoresed separately on a 10%               markers    tested (CEA, CA19-9, and AFP).
sodiumdodecyl sulfate-polyacrylamide gel. Proteinswere transferred electro-
phoretically to nitrocellulose and immunostainedwith FN10 monoclonalanti-         Discussion
body. Lane 1, UFN in a healthy subject; lane 2, plasma EN; lane 3, UFN in s
patient with bladder cancer; lane 4, UFN in a patient with lung cancer; lane 5,     We showed here that UFN excretion was increased in
UFN in the patient with stomach cancer                                            59% of the patients with various cancers and conclude

468    CLINICAL CHEMISTRY, Vol. 37, No. 3, 1991
                                            ,IN -                                                     The amount of UFN is given here per milligram of
                             1902O    3)0           5)0       )0
                  q                                                                                creatinine. Previous studies suggest that the ratio of
                                                                                                   urinary protein per unit of creatinine concentration in
                                                                                9I164(57)          random (untimed) urines accurately reflects 24-h uri-
                                                                                                   nary protein excretion (12, 13). Serial urine samples
             O.                                                                             5)     from the same subject should have consistent values for
                                                                           .-   73/137(53)         the UFN concentration when the excretion of UFN is
                                                                                            (76)   expressed per milligram of creatinine.
                                                                                                     The tumor markers CEA, CA19-9, and AFP circulate

                                            :z.                                 27/31




                                                                                    7/ti    4)
                                                                                                   in increased   amounts
                                                                                                   testinal malignant
                                                                                                                                                with gastroin-
                                                                                                                              in sera of patients
                                                                                                                            disease. Our study shows that the
                                                                                                   clinical usefulness of these assays in the detection of
                                                               .                                   cancer isnot superiorto that of UFN. Increasesin UFN
                                                                                                   seem to be independent of renal function, because the
                        k-
                                                                                                   incidence of increased UFN concentrations was not
                                                                                 5,#{176}
                                                                                       #{176})     significantly different between patients with renal dis-
                                                                                15/25 (60)         eases and those with nonmalignant diseases. In addi-
   NiIMdI.
                                                                                 013        (6)    tion, we preliminarily   studied the correlation between
                                                          .

                         i.---.                                                 ‘          )       two urinary markers (albumin and f32-microglobulin)

                        .                                                                          establishedfor early assessment of lesions of glomerulus
                                                                                                   and UFN in some patients with kidney failure, as well
                                                                                17/83              as the correlation between four markers (blood urea


                        ii.
Fig. 3. Concentrations of UFN in individual patients with various
cancers, patients with benign diseases, and healthy subjects
                                                                                                   nitrogen, serum creatinine, urinary glucose, and un-
                                                                                                   nary protein) for renal function and UFN in some cancer
                                                                                                   patients. We observed no significant      correlation
                                                                                                   tween these six markers and UFN (unpublished).
                                                                                                   preliminary examination shows that increases in UFN
                                                                                                                                                          be-
                                                                                                                                                         This

The dashed line indicates the cutoff for UFN (200 arb. units/mg creatinine).                       in the patients are unrelated to renal function. We
The number of positive findings/number of urine samples tested and the
percentages,in parentheses, are given at the right                                                 detected no difference between UFN concentrations in
                                                                                                   cancer patients pretreated with chemotherapy or radio-
                                                                                                   therapy and those in untreated cancer patients (unpub-
 Table 2. Positive Findings of UFN and Serum Markers                                               lished), perhaps because chemotherapy or radiotherapy
                   In Cancer Patients                                                              does not independently cause increased UFN.
                                  No. positive/total no. tested (and %)                               Several studies have shown that FNs isolated from
 Type of cancer          UFN                    CEA                 CAl 9-9           AFP
                                                                                                   normal or oncofetal tissues are similar in many proper-
Stomach               13/21 (62)             4/18(22)               8/18(44)    1/19(5)            ties but differ slightly in their subunit size; FN of
Colorectal            10/13 (77)             9/12 (75)              4/12(33)    0/12(0)            oncofetal origin has some extra domains and glycosyla-
Esophagus              2/3 (67)              1/3 (33)               1/3 (33)    1/3 (33)           tion changes (14-17). However, the actualamount of
Total                 25/37 (68)            14/33 (42)             13/33 (39)   2/34(6)            matrix containing FN produced by tumor cells is often
                                                                                                   much less than normal. Cultured cells of metastatic
                                                                                                   carcinomas of rats and humans apparently do not ex-
that this assay of UFN would be useful for widespread                                              press high concentrations of cellular FN (18, 19); the
diagnostic screening of many kinds of cancer. We ob-                                               increased concentrations of UFN in cancer patients may
tamed positive rates ranging from 37% to 88% for                                                   not be the result of an increased expression of cellular
different kinds of cancer, and a mean of 14% for nonma-                                            FN by the tumor cells.
lignant diseases. However, positive results suggesting                                                The increase in UFN concentrations is closely associ-
the presence of malignancy when only nonmalignant                                                  ated with malignant      diseases, but the basis for the
disease was actually present would not be a problem,                                               apparent correlation is not clear. The diversity of mo-
because false-positive  results for healthy subjects were                                          lecular masses of UFN suggests that UFN arises from
rare (2%).                                                                                         FN fragmented     by several     FN-degrading    proteases.   A
   Distribution of UFN concentrations in 58 patients                                                         e
                                                                                                   possiblexplanation fortheincreases fUFN in cancer
                                                                                                                                           o
with stomach cancer could be classified into stages. Our                                           patients might be the relationship between proteases
results indicate substantial differences between the per-                                          that degrade FN and cell invasion (20,21). The ability of
centages of patients positive for UFN at stage I and                                               cells to invade the surrounding    connective   tissue, an
those at stage IV, so we expected a direct correlation                                             important factor in tumor metastasis, seems to be a
between thestageofthetumor and the concentration       of                                          multiple-step   process requiring degradation of compo-
UFN in cancer patients.     Moreover,theUFN test would                                             nents of the extracellular matrix by proteases on the cell
indeed be primarily     advantageous for patients with                                             surfaces (22). FN is a large extracellular    glycoprotein
resectable stomach cancer (such as stage I).                                                       that interacts in various ways with cell surfaces (1), and

                                                                                                                  CLINICAL CHEMISTRY, Vol. 37, No. 3, 1991 469
 ismore sensitiveto digestion by various neutral prote-                chromatography.           We describe here a reliable and repro-
 ases than is laminin or collagen, which are also compo-               ducible        IEMA   for measuring   the forms of human FN
 nents of the extracellular      matrix (21).                          excreted into urine. With this assay we measure frag-
    Analysis of the course of tryptic or thermolysic diges-            ments made up mainly           of the cell-binding     domain,
 tion of plasma FN in vitro has shown that the a and /3                fragments that originate from the central portion of one
 subunits of FN are first rapidly cleaved into 215- and                of the subunits of FN and account for about one-fifth of
  185-kDa fragments        when the Hep-lfFib-1           and Fib-2    the molecular mass of the native protein. The accuracy
 domains are released (10,23). The 185-kDa fragment is                 of the assay was improved when the monoclonal anti-
 further degraded into the cell-binding domain, a 75-kDa               bodies FN1O and FN3O, specific to the cell-binding
 fragment, upon the release of the Gel, Hep-2, and Hep-3               domain, were used instead of polyclonal             antibodies
 domains. The cell-binding         domain of FN, specifically          against intact plasma FN; moreover, a sandwich assay
 recognized by both the FN1O and FN3O antibodies,                      performed with a pair of monoclonal           antibodies had
 seems to resist protease attack, to judge from its being              better specificity and sensitivity than did assays involv-
 the main component of UFN. The terminal                    domains    ing only one monoclonal antibody (unpublished).
                                                                          The apparent heterogeneity        of UFN, together with
 released, except for the cell-binding           domain, bind to
                                                                       the possibility that different forms of the antigen have
 other matrix       components:     collagen, fibrin, and gly-
                                                                       different affinities for polyclonal antibodies, was discov-
 cosaminoglycans       (1, 10, 23). Thus, the released frag-
                                                                       ered during immunoblotting.        We use the recombinant
 ments probably remain in the extracellular           matrix in an
                                                                       FN cell-binding     domain (9), recognized by FN1O and
 insoluble state. We could detect the few N- and C-ter-
                                                                       FN3O monoclonal        antibodies,    which also recognize
 minal domain fragments of FN in urine samples from                    plasma FN, in the assay to overcome the problem of
 both healthy subjects and patients by using monoclonal                multiple   molecular    forms of native FN (29), which
 antibodies specific to the Hep-1/Fib-1,           Gel, and Fib-2      limits the use of the latter as a standard.
 domains (unpublished).
    Some oncogenically      transformed      cells have a dimin-          We describe here a new urinary tumor marker for
 ished ability to adhere to FN because of the decreased                many     kinds of cancer and find it superior to other
 expression of integrin, the FN receptor (24). Therefore,              markers in the simplicity,    speed, and noninvasiveness
the cell-binding     domain of FN or the degradation prod-             of its assay. These results suggest that the assay will be
ucts of this domain may tend to be released from the                   useful in the diagnosis of cancer, especially in screening
 surface of malignant       cells into the body fluid. These                                         c
                                                                       groupsundergoingphysicalheckups and in monitoring
processes of degradation        and release may explain why            cancer patients.
the UFN detected consists mainly of the cell-binding
domain of FN. In our preliminary                experiments,      we   References
transplanted     human tumor tissue into nude mice and                 1. Hynes RO, Yamada KM. Fibronectins:          multifunctional modu-
observed the gradual increase of UFN excretion from                    lar glycoproteins. J Cell Biol 1982;95:369-77.
these mice (unpublished         data). This study strongly in-         2. Webb KS, Lin GH. Urinary fibronectin: potential as a biomark-
                                                                       er in prostatic cancer. Invest Urol 1980;17:401-4.
dicates that changes in FN organization             in tumor cells     3. Parsons RG, Todd HD, Kowal R. Isolation and identification of
may account for the release of FN or its fragments into                a human serum fibronectin-like protein elevated during malignant
the blood.                                                             disease. Cancer Res 1979;39:4341-5.
    Several low-Mr tumor markers in urine have been                    4. Choate JJ, Mosher DF. Fibronectin concentration in plasma of
                                                                       patients with breast cancer, colon cancer, and acute leukemia.
investigated (25-28). UFN is different from these mark-                Cancer 1983;51:1142-7.
ers because it is generated from a large extracellular                 5. Boccardo F, Guarneri D, Zanardi S, Castellani P, Borsi L, Zardi
matrix protein through proteolysis. Perhaps fragmented                 L. Fibronectin concentration in the plasma of patients with malig-
                                                                       nant and benign breast disease. Cancer Lett 1986;33:317-23.
FN is initially    released from tumor sites into the blood,
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