NO801018L - DIAGNOSTIC PROCEDURES. - Google Patents

Description

The present invention relates to a method for the diagnostic detection of malignant neoplasms.

There is a clear need for new methods for detecting cancer. In many cases, an early diagnosis of cancer will greatly increase the chances of causing complete remission of the disease.

Previous attempts to demonstrate the presence of tumor-specific components, such as hormones and antigens, in the blood of cancer patients have been described in the literature. However, such attempts have largely been without favorable results, and a practical, non-invasive diagnostic method, which is based on the content of a tumor-specific serum component, has so far stood as an unattainable goal.

Recently, DNA-binding proteins have been found in the serum of patients with neoplasia, systemic lupus erythematosus and other inflammatory diseases (see e.g. FEBS Letters 92(2) : 211-213 (1978); Europ. J. Biochem. 71 :1-8 (1976); Amer. J.. Med. 6_5: 437-445 (1978). However, no suitable examination method for such serum proteins, which will be necessary for a practical diagnostic procedure. Furthermore, it appears that the previously described serum proteins do not exhibit the degree of selectivity that. is desired by a screening method for cancer.

The present invention makes use of an inhibitor serum protein which is described in "The Pharmacologists" (Abs.) 2_0(3):238 (1978). However, the abstract gives no indication that the protein is present in different amounts in patients with cancer and in cancer-free patients. • The present invention thus provides a generally applicable method for diagnostic examination for the detection of malignant neoplasms in humans. More particularly, the invention provides a diagnostic method for detecting the presence of malignant neoplasms by examining human serum samples for the presence of a specific DNA-binding protein. The procedure is valuable for the early detection of malignant diseases and as an indicator of the development of the disease in a cancer patient who is undergoing treatment.

The present method is based on the discovery that a certain DNA-binding protein (hereafter called inhibitor protein X) is present in substantially reduced concentrations in the serum of patients with cancer compared to cancer-free patients. By providing a sensitive test for this serum protein, the present invention enables a rapid, non-invasive and accurate method for detecting the presence of cancer.

The presence of a particular DNA-binding protein in human serum was discovered by the present inventors while investigating the bleomycin-induced degradation of PM-2 DNA (Pseudomonas bacteriophage covalently closed circular DNA). Observation of the inhibition of this degradation by human serum led to studies on the nature of the inhibition and attempts to identify the inhibitor compound.

The present inventors' hypothesis, namely that the inhibition of the PM-2 DNA degradation reaction was due to a serum protein, was confirmed when treatment with pronase resulted in the loss of inhibitory activity. The treatment with DNAase 1 or pancreatic RNAase had no effect on the inhibitory activity of the serum. Sera from dogs and cats were not inhibitory, nor were human albumin and immunoglobulins.

The inhibitor protein was purified under non-denaturing conditions using molecular filters, dialysis and self-chromatography with Sephadex. Purification of the protein was significantly improved by binding the protein to DNA, isolating the DNA-protein complex from the unbound protein and then dissociating the protein from DNA by treatment with urea. Purification by SDS-polyacrylamide gel electrophoresis and isoelectric focusing is believed to result in more than 2000-fold purification. Isoelectric focusing gel electrophoresis suggests that the inhibitor is a protein with a molecular weight of approx. 64,000 daltons and a pl of 5.9. This protein is provisionally called inhibitor protein here

X.

Inhibitor protein X exerts its inhibitory effect by binding to DNA. DNA binding has been demonstrated by agarose gel electrophoresis, isolation of the DNA-protein complex, fluorescein quenching and circular dichroism studies.

When investigating the nature of inhibitor protein X, it unexpectedly turned out that there was a significant difference in the content of this protein in the serum of patients with cancer compared to the serum of cancer-free patients. Based on this important invention, a sensitive and rapid test for the presence of inhibitor protein X in human serum was sought as part of the present invention.

PM-2 DNA has previously been used in a spectrophotofluorometric assay for the determination of bleomycin biochemical activity (Cancer Res. 38:3322-3326 (1978)). Bleomycin's effect. mechanism appears to be related to its ability to degrade DNA, and the reduction in binding of ethidium bromide (2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide) to PM-2 DNA induced by bleomycin, as determined by fluorescence spectrometry, can therefore be used when examining bleomycin activity.

As noted above, inhibitor protein X has been shown to act as an inhibitor of bleomycin-induced degradation of PM-2 DNA. As a result, the fluorometric sample previously used to measure bleomycin activity can also be used to examine the concentration of inhibitor protein X in human serum. The content of inhibitor protein X can then be used to predict the likelihood of cancer in patients being examined.

The present invention thus prescribes a method for detecting cancer in humans where: (a) a blood sample is taken from a patient who is to be examined for cancer,

and the procedure is characterized by the fact that one

(b) determines the concentration of inhibitor protein X in the sample from step (a), and (c) compares the concentration determined in step (b) with the norm concentration of inhibitor protein X in the serum of cancer-free patients, whereby a significant reduction in the concentration of inhibitor protein X in relation to the norm concentration indicates the likelihood of cancer.

The special survey method: which is used in stages.

(b) for determining the serum concentration of inhibitor protein X is not essential and the present invention lies

in its broadest aspect in the discovery that inhibitor protein X concentration (however determined) can be used as a diagnostic tool for a wide range of human cancers.

One screening method that has been shown to be advantageous is the PM-2 DNA fluorescence assay described in "Cancer Res." 38: 3322-3326 (1976). Application of this test is based on the inhibitory effect of inhibitor protein X on the well-known bleomycin-induced breakdown of the PM-2 DNA reaction. When this test is used, it is appropriate to express the concentration of inhibitor protein X as an IC^q value, where ICb,.U..is defined as the concentration of inhibitor protein X required to inhibit 50% of bleomycin induced degradation of PM-2 DNA. This IC^Q value can then be compared with norm IC^^ values which are linked to the serum of cancer-free patients, and a significant rise in the IC^Q value in relation to the norm value will indicate the probability of cancer .

In the following, the PM-2 DNA fluorescence test for inhibitor protein X is described in more detail: First, a mixture of a serum sample (e.g. ■ eg a 50 µl sample) from a patient to be examined and a solution of bleomycin, PM-2 DNA and 2-mercaptoethanol in pH. 9.5 puffs. Typically, a 50 µl serum sample is mixed with 450 µl of a solution containing 35 nM bleomycin, 8.3 µM PM-2 DNA and 25 mM 2-mercaptoethanol in pH 9.5 sodium borate buffer (0.015M NaCl: 0.05M sodium borate). , '

The above mixture is then incubated at 37°C for 30 minutes

A solution of ethidium bromide in pH 12.1 denaturation buffer is then prepared by adding 0.1 ml of an ethidium bromide solution (22 µg ethidium bromide per ml of 12.1 denaturation buffer) to 0.9 ml of a pH 12.1 denaturation buffer. A suitable denaturing buffer f is comprises 0.09M Na^PO^; : 0.01M EDTA : 0.01M NaCl, adjusted to pH value 12.1 with 0.15M NaOH. To 1 ml of this ethidium bromide solution is added a portion, flex. 100 µl, of the incubated serum mixture prepared as indicated above.

The fluorescence of the ethidium bromide:PM-2 DNA mixture is determined with a spectrophotofluorometer at 530 nm input and 590 nm emission. Fluorescence greater than background is caused by the binding of ethidium bromide to PM-2 DNA, so that a change in fluorescence relative to a control reaction (identical to test sample except that it does not contain bleomycin) allows the degree of bleomycin-induced degradation of PM -2 DNA can be quantified. From the fluorescence values, the percentage inhibition of PM-2 DNA degradation caused by the presence of inhibitor protein X in the serum can be easily determined.

The concentration of inhibitor protein X in the serum sample is then determined by the conventional Lowry method (folin-phenol reagent) as described in "J. Biol. Chem." 193:265-275

(.19 51).

By plotting the percentage inhibition of PM-2 DNA degradation against the logarithm of the concentration of inhibitor protein X can. The IC^g value for the particular serum sample is easily determined..

When first IC,. The Q-value for a given patient has been calculated, this value can be used directly to predict the probability of cancer in such a patient. Extensive studies on healthy subjects

(cancer-free) patients and patients with many different malignant neoplasms have shown that inhibitor protein X is found in significantly lower amounts in the serum of patients with cancer. The IC|-Q value is therefore found in cancer patients to be significantly elevated compared to the norm for healthy individuals. This clear differentiation in the ICj-g values provides a simple and accurate basis for detecting the presence of cancer and is the core of the present invention.

The exact IC^Q value obtained from blood samples of cancer-free patients will vary to some extent with the particular individual. In patients who have previously been tested, the mean IC^^ value for cancer-free patients has been found to be 90.2-11.0 pg/ml (significant at the 0.001 level). The highest recorded ICj-g value for a healthy patient has been 120 yg/ml.

In cancer patients, the obtained IC[-Q values varied with the type of cancer involved, but in all cases the value has so far been significantly higher than in cancer-free individuals. The lowest IC5Q value to date for a cancer patient has been 270 vig/ml. However, an IC,-_ value above approx. 200, however, a high probability of. that the patient has a malignant neoplasm of one type or another.

The method according to the present invention has been successfully used for the detection of a large number of different types of cancer, and it is therefore assumed to be generally applicable for cancer diagnosis. As specific examples of proven tumor types. can be mentioned testicular carcinoma, lymphoma, leukaemia, adenocarcinoma of the breast, small cell adenocarcinoma of the lung, large cell adenocarcinoma of the lung, adenocarcinoma of the colon and melanoma, . to name just a few. The method also appears to be specific for cancer diagnosis, as cancer-free patients with various types of non-neoplastic diseases have not had the elevated IC^Q values that accompany the presence of cancer.

The above-described fluorescence test for inhibitor protein X according to the present invention is based on one inherent property of this substance, namely the inhibition of bleomycin-induced degradation of PM-2 DNA. The serum concentration of the protein determined using this method has been correlated with the presence or absence of malignant disease. The availability of a purified inhibitor protein X makes it possible that a wide range of methods known within biochemistry and clinical chemistry can used for its measurement. Prominent among these are immunological methods such as radioimmunoassay, fluoroimmunoassay, enzyme immunoassay (e.g. ELISA), spin immunoassay, chemiluminescence immunoassay, fluorescence polarization immunoassay, nephelometric assay, gel diffusion methods and classical methods, such as hemagglutination and complement fixation. Furthermore, other functional properties of inhibitor-protein.X can be exploited for its measurement in biological fluids. Catalytic phenomena in connection with the molecule can be measured by spectrophotometric or spectrofluorometric, kinetic or endpoint tests. Furthermore, separation techniques can be applied to the liquid in question and a wide range of detection methods can be used for quantification. ring of the protein, including spectral or densitometric methods and immunological methods. Then it by the previous ones

methods specific measurement of the concentration of inhibitor protein X is obtained, it follows that the serum concentration of the protein, determined by such methods, correlates with the presence or absence of malignant disease.

Inhibitor protein X is purified according to the following methods.

Partial purification - Method A

Inhibitor protein X is partially purified by "Sephadex" column chromatography. 1 ml of human serum is added to a "Sephadex G-75" column of 88 cm x 2 cm and was then eluted in a sodium phosphate buffer of 0.07 M and Ph 6.8 at a flow rate of 6-8 ml per minute. hour at room temperature. The inhibitor contained in the depletion volume collected was lyophilized resuspended in 1 ml of 1^0, dialyzed against 20 volumes of H2O for 24 hours at .4 C and then applied to a "Sephadex G-2 00" column, which was equilibrated in the same puffer.

Elution of the "Sephadex G-200" column resulted in three. peaks that absorbed at 280 nm. Vertex III which constitutes:; 7.7% of the total eluted protein contained inhibitor protein X. This was treated as described above and added again to a "Sephadex G-200" column. The elution was carried out with the phosphate buffer described above. 3 ml fractions from each column were collected with a fraction collector (Gilson Medical Electronics, Inc., Middleton, Wis.) and the elution patterns were obtained by absorbance at 280 nm with a spectrophotometer. Protein concentrations were determined using the Lowry method.

Cleaning - Method B

Purification of the inhibitor protein was achieved by molecular filtration with "Amicbn" filters, dialysis and "Sephadex" gel column chromatography. The human sera, 1-5 ml, were centrifuged in "Amicon Conical" filter tubes (Amicon, Lexington, Mass.) at 5000

g for 10 minutes at 4°C. The retentate that remained in the filter core after centrifugation was resuspended in deionized water to the original serum volume. Portions of the retentate and filtrate were tested for inhibitory activity. The active retentate was dialyzed in cellulose tubes against 20 parts by volume of deionized water at 4°C for 24 hours. The retentate and dialysate were lyophilized and resuspended in deionized water. The results

of the investigations of the two fractions showed that the inhibitory activity was in the retentate. The retentate, 1 ml, was added to a 60 x 5 cm column of "Sephadex G-50" and eluted with a sodium phosphate buffer (0.07 M, pH 6.8) at a flow rate of 30 . ml per hour at 4°C. The column eluate>5 ml per glass, became collected with a "Buchler Fractomette Alpha 200" fraction collector (Searle, Fort Lee, N.J.). The 280 nm absorbance for each fraction was measured with a spectrophotometer and the glasses are mixed according to the absorbance peaks. The mixed fractions were lyophilized, dialyzed and tested for inhibitory activity as described above. The "active material" which was in the discharge volume fraction was fed to "Sephadex G-75 and G-100" columns and eluted in the same way as the "G-50" column. The inhibitory activity was found in the depletion volume fractions for both columns. The "active" material collected in the "Sephadex G-200" column discharge volume fraction was eluted with sodium phosphate buffer (0.07M, pH 6.8) at room temperature at a flow rate of 6-8 ml per minute. hour. The fractions were collected, 3 ml per glass, with a "Gilson" type microfraction collector (Gilsdn, Middleton, Wis,). The column eluate was fractionated on the basis of the 280 nm absorption profile with mixing of the glasses for each absorption peak. Each fraction i was then lyophilized, dialyzed and tested for inhibitory activity as described above. The inhibitory activity was found in fraction III. The material in fraction III was again added to the "Sephadex G-200" column and eluted under the same conditions. Fraction III from the "Sephadex G-200" column and containing inhibitor protein X represented a purification of more than 1000 times.

The PM-2 DNA fluorescence examination method for inhibitor protein X is described in more detail below:

Determination of IC^ for inhibitor protein X

The determination of the IC,.Q value (the protein concentration required to give 50% inhibition of PM-2 DNA degradation) for inhibitor protein X in human serum takes place in two steps: 1. The protein concentration in the serum is determined by the Lowry method. 2. The PM-2 DNA fluorescence sample for bleomycin is used to obtain a dose-response curve for the serum protein inhibitor. The IC,-Q values are derived from plots of percentage inhibition of DNA degradation against the protein concentrations on onlog-probit graphs. Procedures;

1. The Lowry reaction

A. Preparation of base solutions:

Solution A. 2% Na2C03 in 0.1N NaOH

Solution B. 1% CuS04, 5H2O in H2O

Solution C. 2% Na<+>K<+>tartrate in H2O

Solution D. 2N folin-ciocalteau-phenol reagent

B. Preparation of reagent solutions:

Reagent solution 1. 1 volume of base solution B

mixed with 1 volume of base solution C.

Reagent solution 2. 50 volumes of base solution A

mixed with 1 volume of reagent solution 1.

Reagent solution 3.;. 1 volume of base solution D

mixed with 1 volume of H20.

C. Reactions:

1. Serum is diluted 1:10 with H20.

2. 10 µl of diluted serum is transferred to a 13 x 100 mm test tube and 290 µl of H20 is added. 3. 1 ml of reagent solution 2 is added, the whole is mixed thoroughly and incubated for 10 minutes at room temperature. 4. Add 100 µl of reagent solution 3, mix thoroughly and incubate for 30 minutes at room temperature. 5. A spectrophotometer is used to measure absorbance at 750 nm. 6.. The protein concentration is determined from the BSA standard curve. D. Bovine serum albumin (BSA) standard curve:

1. Serial dilutions of BSA are prepared, e.g.

25, 50, 75, 100, 150 ug/ml, in a final volume of 0.3 ml. 2. The reactions specified in section D are carried out beginning with step 3. namely addition of reagent solution 2. 3. Absorbance at 750 nm is plotted against BSA concentrations.

II. Protein inhibitor assay

A. Serial dilutions of the serum protein are made on the basis of the serum protein concentrations determined by the Lowry reaction. Dilutions are prepared in a constant volume of sodium borate buffer (0.05M, pH 9.5).

B. Samples of serum protein in volumes of 50 µl are added to 10 µl of bleomycin (34.5 nanomoles) contained in a 13 x 100 mm test tube. C. 390 µl of sodium borate buffer (0.05 M, pH 9.5) containing 25 mM 2-mercaptoethanol is added. D. 50 µl PM-2 DNA (8.3 x 10-6M in 0.15M NaCl) is added.

E. It is incubated for 30 minutes, at 37°C.

F. Triplicate portions of. 100 µl are transferred to 13 x 100 mm test tubes containing 0.9 ml Na^PO^ (0/09M, pH 12.1) containing 0.01M EDTA and 0.01MNaCl.. The whole is mixed thoroughly.

G. 100 μl of ethidium bromide (55.7 x 10-6M) is added in

Na 3 PO 4 buffer, pH 12.1. The whole thing is thoroughly mixed.

H.. Control reagents:

1.. Sodium borate (0.5M, pH 9.5) containing 2-mercaptoethanol without bleomycin or proteins.

2. Protein without bleomycin or PM-2 DNA.

3. Protein without bleomycin.

4. Sodium borate (0.05M, pH 9.5) containing 2-mercaptoethanol blank. I. Spectrophotofluorometry (Aminco-Bowman): The sample is transferred to a 1 cm quartz cuvette and the fluorescence is measured at 530 nm input and 590 emission.

Claims (3)

1. Procedure, for the detection of cancer in humans whereby one (a) takes a blood sample from a patient to be examined for cancer, characterized in that one (b) determining the concentration, of inhibitor protein X in the sample, and (c) compares the concentration determined in step (b) with the norm concentration of inhibitor protein X in the serum of cancer-free patients, in the case of a significant reduction of the concentration of inhibitor protein X in relation to the norm concentration indicates the probability of cancer . 2. Method according to claim 1, characterized in that one in step (b) determining the concentration of inhibitor protein X in the sample as the concentration required to inhibit 50% of bleomycin-induced degradation of PM-2 DNA and in step Ce) comparing the IC^Q determined in step (b) - value with the norm ICj-Q value in serum from cancer-free patients, whereby a significant increase in the ICj-Q value from the norm value indicates the probability of cancer 3. Method according to claim 2, characterized in that the determination of the ICo .-u-. value in step (b) is carried out in the steps that one (1) prepare a mixture of the blood sample with one solution of bleomycin, PM-2.DNA and 2-me.rcaptoethanol in pH 9.5 buffer, (2) incubating the mixture from step (1) for 30 minutes at a temperature of 37°C, (3) adding a portion of the mixture from step (2) to a mixture of ethidium bromide in a pH 12.1 denaturing buffer, (4) determine the fluorescence of the ethidium bromide : PM-DNA mixture from step (3) with a spectrofluorometer at 530 nm input and. 590 nm emission, (5) . determine the percentage inhibition of bleomycin-induced PM-2 DNA degradation based on changes in the fluorescence of the sample relative to a control sample that does not contain bleomycin, (6) determines the concentration of inhibitor protein X by the Lowry method, and (7) from the values obtained in steps (5) and (6) determine the concentration of inhibitor protein X required to inhibit 50% of bleomycin-induced PM-2 DNA degradation.