DE2729893A1 - Detection of human cancer - by analysing for cancer-specific virus-associated protein(s) using iodine-125 isotope - Google Patents

Abstract

Method comprises taking a sample (pref. blood plasma) from the patient and analysing it for a virus-associated protein which is specific to the cancer in question. Also claimed are certain cancer-specific virus-associated proteins in pure form, and reagents for the immunoassay of such proteins. The method is esp. applicable to breast cancer, can be used for diagnosis or for monitoring the effectiveness of surgery, radiation treatment and/or chemotherapy. Specifically claimed virus-associated proteins specific to breast cancer are an RNA-dependent DNA-polymerase (reverse transcriptase) and a viral tumour-associated protein both of which are cross-reactive with Mason-Pfizer Monkey virus (MPMV) antibodies.

Description

Procedure for the detection of cancer and means for

Carrying out the method The invention relates to a method for the detection of cancer, especially in humans, as well as a means for carrying out of the procedure.

Studies on mouse tumor models showed the possibility using plasma concentrations of viral protein to estimate presence and the status of solid tumors.

The viral protein used for these studies was a 52,000 Dalton glycoprotein Cgp 52) isolated from mouse mammary tumor virus (ITV) using affinity chromatography.

The availability of purified gp 52 by the above procedure enabled the development of a radioimmunological determination on this protein, which was sensitive to plasma levels as low as 0.1 ng / 100) 11. This is done on the following reference by Ritzi et al., Virology, 75, (1976) p. 188 and Ritzi et al., Proc. Natl. Acad. Sci. USA,, No. 11, (1976) p. 4190.

It was found that the relationship between the mammary tumors and the plasma levels of gp 52 are as follows: (a) Tumor-bearing, male, or female mice showed markedly increased (100-1000 ng / ml) levels of gp 52 as free soluble protein in plasma and the mean concentration decreased with the mean Tumor size too; (b) The presence of another malignant disease (leukemia) did not show any change in the levels of gp 52 in the plasma; (c) through Transplantation of mammary tumor tissue located outside the mammary glands is also used detected by high plasma levels of gp 52; (d) low (2-10 ng / ml) plasma levels of gp 52 are found in tumor-free mice, regardless of whether they are from Strains caused by high or low incidences of spontaneous mammary tumors are marked; (e) Tumor-free lactating females show the normal low levels of plasma gp 52 despite the fact that their milk has a Contains an average value of 23,000 ng / ml of this protein; and (f) elimination time of gp 52 in circulation in tumor-infected animals is sufficiently short (Half-life of 4-6 hours) to indicate the need for ongoing Close replenishment to maintain the observed high levels.

The 1TV model provides the basis for establishing the feasibility the use of a viral protein "marker" in the plasma to determine presence and the status of breast cancer in humans. The use of a viral protein marker respectively. Viral protein marker differs from the methods of determination, such as for example, they are described in U.S. Patent 3,999,944, which is based on Evidence the antigen-induced leukocyte adhesion inhibition caused by tumor-specific cells are based on mediated immunity.

It has now been found that the existence and status of cancer in the Humans can be proven by doing an examination for virus related Proteins in plasma samples. Appropriate virus-related proteins include the enzyme RNA-dependent DNA polymerase (reverse transcriptase) or a extracellular tumor-associated protein that originates from viruses. The enzyme described above and the protein associated with the tumor are immunological cross-reactive with antibodies to Mason-Pfizer monkey viurs (I1PMV), thus creating an appropriate Source of reagents for the process of the invention is supplied.

The invention therefore relates to a method for detecting presence and the status of cancer in humans by screening for certain tumor-specific ones virus-related proteins in plasma samples and for the method useful new reagents. Such a procedure is therefore useful in diagnosing Programs for an initial review for early detection of the disease therapy to determine the status of the disease after surgical interventions, Radiation treatments and / or chemotherapeutic treatments and in the Forecast as with evidence of the possibility of recurrence or evidence of metastases beneficial.

Virus-related proteins used as markers for the Detection of cancer can be used, particularly breast cancer Viral enzymes such as RIJA-dependent DNA polymerase (reverse transcriptase) or alternatively a tumor-associated protein that originates from viruses.

A first embodiment of the invention therefore relates to detection of human breast cancer through the use of RNA-dependent DNA polymerase as a plasma marker.

It was previously known in the art that particles of breast cancer possess many of the characteristics of RNA tumor viruses in humans. In addition to the expected size (600 S) and density (1.16 g / ml), these include Features the presence of an outer membrane and an inner membrane, which envelops a "core" containing a DNA polymerase and a high molecular weight RNA (70 S), which has a demonstrable homology to the RNA of the tumor tumor viruses (MMTV) and from Mason-Pfizer simian virus (NPfiV).

The purification and characterization of the DNA polymerase from the Particle of breast cancer in humans has now been reached and forms a part of the present invention.

The key properties of this enzyme are very similar to those derreversenTranscriptasen found in MMTV and MPMW. Like those viral enzymes the purified human breast cancer DNA polymerase shows the following three Characteristics, which together the known reverse transcriptases from viruses of normal Differentiate DNA polymerases from cells: a) a strong preference for oligo (dT): poly (rA) versus oligo (dT): poly (dA) as a template for the synthesis of poly (dT); b) the assumption of highly specific oligo (dG): poly (rCm) as template for the formation of poly (dG); c) the ability to use an RNA (ate) from viruses as a template for production an exact complementary DNA copy.

The similarity of the human enzyme to the reverse transcriptases of MMTV and IIPMV extends even further to the presence of molecular weight of 70,000 Daltou and the preference for Mg ++ over Mn ++. So far has been a Enzyme with these properties not found in normal breast tissues or in benign ones Detected breast tumors.

The isolation of DNA polymerase from human breast cancer was made by homogenizing breast cancer tissue and layers across discontinuous Sucrose gradient reached. The density range at 1.16-1.19 g / ml was put together diluted and centrifuged. Suspensions of the pellets were made by gel filtration fractionated over polyacrylamide agarose. The factions involved in determining on DNA polymerase found to be active were pooled and placed over one Column chromatographed with phosphoric cellulose. Elution was done with a linear Gradients of 0.1 M to 0.5 M phosphate buffer from pH 7.2.

The main peak of polymerase activity was pooled, and the enzyme was concentrated by dialysis.

The purified DNA polymerase from human breast cancer can be used for Develop a diagnostic determination on human breast cancer on multiple Way to be used. They can be used to develop specific antibodies against DNA polymerase used by human breast cancer by the purified Enzyme preferably in an emulsion of complete Freund's adjuvant in a suitable host animal such as a rabbit, a guinea pig, a goat Horse, etc. is injected for a period of time and then blood is drawn from the host animal (usually after reinforcement injections have been given to achieve the desired To obtain antisera.

In addition, the purified enzyme can be used as a substrate for radioododing can be used to obtain the g125J7 enzyme. A suitable way of working for radioiodination involves treatment of the enzyme with g125J7-3- (4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester (Bolton-Hunter reagent) followed by purification on a G-100 column.

The aforementioned antibody and the labeled enzyme can be used in a radioimmunological determination based on DNA polymerase human Breast cancer can be used on human plasma specimens. Appropriate working methods for radioimmunological determination are known in the art. So is for example an analogous procedure that can be used by Ritzi et al. in Virology 22 (1976) p. 188. In such a way of working, a buffered Sample treated with the antisera, i.e. rabbit anti-DNA polymerase, and then after incubation for about 45 minutes at 37 ° C., the rl 25J7-marl: ated Enzyme added. The sample is then incubated for a further 2 hours at 370C, and the bound radioactivity is removed from the free radioactivity by adding from normal IgG C rabbits) and a sufficient amount of secondary antibody (Goat anti-rabbit IgG) to achieve optimal precipitation of the IgG (rabbit) severed.

The concentration of DNA polymerase in the sample can be determined by a Comparison of the determined count rates of radioactivity in the bound and / or free fractions against a standard curve, which using different, known amounts of DNA polymerase in the same determination procedure was obtained, to be determined-.

In another way of working, the enzyme concentration in the Plasma samples by isolating the enzyme and measuring the enzymatic activity to be determined. Isolation can be carried out in a simple manner by treating the plasma sample with ammonium sulfate to precipitate the enzyme and subsequent affinity chromatography the reconstituted precipitate on a column with agarose pearls (Sepharose pearls), to which a synthetic template of the enzyme was covalently attached will. Suitable stencils for this purpose include polyriboadenylate (poly (rA) or poly (2 '-0-methylcytidylate) (poly (rCm). The elution of the enzyme from the column is applied using a gradient from 0.01 M KCl to 1.0 M KCl in 0.01 M phosphate, buffered to pH = 7.2.

The DNA polymerase activity of the isolated enzyme can under use the same determinative procedures that are observed when following purification of the enzyme from breast cancer tissue discussed above.

Another embodiment of the method according to the invention is based on finding a human tumor associated protein that is his Originates in viruses. Such a protein can be found in substantial concentrations the intercellular spaces of samples from human breast cancer tissue and further detected as circulating in the plasma of breast cancer patients will. The protein associated with the tumor appears to be superfluous protein, either by the virus after it infects the breast cells, or by of the cell itself under the control of the virus. Another embodiment the invention therefore consists in determining the presence of this tumor-associated Protein in plasma samples as a diagnostic or prognostic test for cancer, e.g. breast cancer.

Isolation of the tumor-associated protein from homogenized Cancer tissue can be identified by a combination of affinity chromatography and column chromatography be performed. The column used for affinity chromatography comprises a commercial filling, namely concavalin coupled to an agarose (Sepharose 4B) A, hereinafter referred to as "Con-A-Sepharose". The elution of the protein from the Column for affinity chromatography is performed using buffered X -Methyl-D-mannoside solution carried out. An additional purification of the protein is carried out by chromatography on DEAE cellulose. The previously described Procedures are those of Ritzi et al., Virology 22 (1976) p. 188 for purification The working methods used by gp 52 from MMTV are directly analogous and they are in detail described there.

The purified tumor-associated protein derived from viruses can be used in the same way as before for DNA polymerase to develop a radioimmunological Determination that is beneficial in the detection of breast cancer can be exploited.

In this way, the protein can be formed in host animals in a known way of antibodies that target the tumor-associated protein that its Originates in viruses, are specific. In addition, the purified, tumor-associated Protein radiolabeled, preferably with r125J7 Bolton-Hunter reagent radioiodinated to remove the labeled, i.e.

125J tumor-associated protein to act as a marker is used in such a radioimmunological determination. The one with this one Embodiment of the invention applied method of radioimmunological determination is not very critical and any conventional method can be used. The method used for the determination is preferably that for the radioimmunological one Determination of DNA polymerase used and previously described blocking double antibody procedure.

According to a further feature of the invention it has been found that for Mason - Pfizer Monkey Virus (MPMV) specific antibodies caused a cross reaction reasonably high levels with both human breast cancer DNA polymerase and also with tumor-associated protein from virus-induced human Have breast cancer. Therefore it is possible to use such antibodies and radiolabelled, preferably 125J-MPMV in radioimmunological determinations for human breast cancer to apply according to the invention. Because MPMV can be grown in tissue culture and is therefore easily available in large quantities, this constitutes a particularly advantageous one Is a source of reagents for large-scale practice of the invention.

In a further embodiment of the invention, a suitable Enzyme covalently bound to the desired antibody. The complex of antibody-enzyme is still enzymatic active, and when he is using tissue samples that contain the appropriate antigen, the antibody combines herewith. A substrate of the enzyme is then added, which releases a colored precipitate at the site of the activity. With a specific Embodiment was peroxidase to antibodies against Mason-Pfizer virus proteins coupled. The appearance and distribution of the colored product does not allow only identifying the presence of antigens, but actually their localization in the malignant cells of the tissue samples.

Another method of applying this principle is quantitative Assessment of the same antigens in body fluids, e.g. plasma. The way of working is as follows: 1) first, the antibody is placed on a solid surface, e.g. Polystyrene, stacked; 2) it gets a known volume of plasma from a patient placed in the test tube, and including any relevant, in the sample Antigens present on the surface of the coated antibody is made possible; 3) the sample is then removed and the test tube washed; 4) the complex off Antibody enzyme is added and incubated for binding. Everyone not absorbed antibody bound to enzyme is then washed out; 5) it becomes the chromogenic Substrate added which gives either the coloration or the fluorescence required can be measured to estimate the amount of tumor antigen present. Preferred enzymes that can be used include alkaline phosphatase and β-galactosidase, both of which have excellent chromogenic substrates.

More details on how to work with the Implementation of this embodiment of the invention are described in the prior art, see, for example, U.S. Patents 4,002,532 and 4,016,043.

The various provisions that the embodiments of the invention Procedure form can be used to detect the presence of breast cancer in humans can be used. A statistically significant number will be used to apply them from blood plasma samples from patients with clinically determined breast cancer, from normal Patients and from patients with either benign or non-breast cancer tumors according to the direct DNA polymerase activity method, the DNA polymerase immuno-determination method or the tumor-associated protein immunoassay method. the The concentration of the marker protein found in these determinations is im Case of the breast cancer plasma samples compared to the normal samples or the tumor samples from non-breast cancer patients. Reputation this way it is possible to use an arbitrary limit line between the concentration values Marker protein to draw with each determination method, which of the presence of breast cancer, and the values corresponding to normal states or non-breast cancer states correspond. An unknown plasma sample can then indicate the possibility for breast cancer at the examined person are classified by the sample in accordance is examined with one of the methods according to the invention and it is determined whether the Marker protein is present in an excess concentration to the control value.

Alternatively, a patient's own levels of marker protein serve as internal control. So can a patient diagnosed with breast cancer before and after the start of the therapy. A noticeable drop in the content of marker protein would be an indication of a favorable prognosis of the Treatment. A subsequent substantial increase in marker protein concentration levels on the other hand it would be an indication of a possible recurrence or of metastases the disease so that it would be possible for the supervising doctor to apply the therapy to start again at an early stage.

In addition, the effectiveness of such therapy could be through the method according to the invention can be monitored.

The invention is illustrated in more detail by means of the following examples.

Example 1 Subcellular Fractionation of Breast Tumor Tissue Depending of the amounts of available material, between 9 and 30 g of tumor were thawed, thinly sliced, in four volumes of 5, S sucrose (w / v) TNE (0.01 M Tris-HCl, pH 8.0, 0.15 M Nach, 3 mM EDTA) and suspended in a Silverson homogenizer mixed. The homogenate was at 4000 x g and then at 10,000 x g for removal the nuclei or mitochondria are centrifuged.

The post-mitochondrial supernatant fluid became trypsin added to a final concentration of 0.5 mg / ml. After an incubation at 20 ° C for 10 min, the proteolytic activity was increased by adding two Polypeptides, timabean trypsin inhibitor (simple excess) and trasylol (100 KIU / ml) inhibited. The sample was drawn over discontinuous sucrose gradients layered, composed of 6 ml of 50% sucrose-TNE and 8 ml of 25% sucrose-UNE was. Following centrifugation at 25,000 rpm for 90 min at 40 ° C. in a Spinco SW-27 rotor collected the material at the 25/50% interface, diluted with TNE and layered over linear 20-50 ß sucrose-TNE gradients. The samples were centrifuged for 16 hours as before, and the different ones Areas of density were collected. The density range of 1.16-1.19g / ml in which the ZA tumor virus localized was pooled, diluted and as before centrifuged for 90 min. The resulting pellets were approximately 0.6 ml resuspended by 0.1 M Tris-HCl, pH 8.0.

Six sets of tumors were enzyme-based in the manner described processed. Four of these (A, B, C and D) gave sufficient enzyme for characterization. A preparation CA), a metastatic liver tumor came from a single patient, all other preparations were pooled from a number of different individuals, Polyacrylamide agarose gel filtration The resuspended pellet was at OOC for 15 min by adding KCl (to 0.4 M), DTT (dithiothreitol to 0.01 M) and a nonionic surfactant like Triton X-100 (to 0.6%) solubilized and disrupted. The samples of approximately 0.9 ml was applied to a 0.9 x 50 cm column with a polyacrylamide agarose gel (Ultrogel AcA44) abandoned with 0.3 M potassium phosphate, pH = 8.0 in buffer A (2 mM DTT, 1 mM EDTA, 0.02% Triton X-100 and 10 ß glycerol) was equilibrated. Elution was carried out with 0.3 M phosphate buffer A at one flow rate of about 2 ml / h. There were 0.5 ml fractions for DNA polymerase and final transferase activity, as previously described, examined.

PhosDhozellulosechromatograshie The top fractions from the ultrogel column were combined (3 ml) and Trasylol was added to a concentration of 100 KIU / ml was added. The sample was against 0.01 M potassium phosphate, pH = 7.2, dialyzed in buffer A until the phosphate concentration was less than 0.02 M, then it was applied to a phosphocellulose column of 0.9 x 10 cm (Whatman P-11), which with equilibrated with the same buffer was abandoned.

The column was washed with 30 ml of the 0.01 M phosphate buffer, and the enzyme activity was measured with a 120 ml linear gradient from 0.01 M to 0.5 M potassium phosphate buffer A, pH = 7.2, at a flow rate of 14 ml / h eluted. Fractions (1.2 ml) were assessed for both DNA polymerase and final transferase activities examined. The top of the polymerase activity was put together, and Trasylol was added up to 100 KIU / ml. This fraction of the enzyme (called PC enzyme) was by dialysis at OOC against an osmotically active, synthetic polymer of high molecular weight such as Aquacid 11-A concentrated.

Centrifugation with glycerine gradients For the estimation of the molecular weight the concentrated PC-Enzam was diluted three times with 0.1 M potassium phosphate, pH 8.0 and layered over a linear 10-30% glycerol gradient containing 0.1 M potassium phosphate, pH 8.0, 2 mM DTT and 0.02 ffi Triton X-100. Centrifugation took place at 48,000 rpm for 12 h at 10c in a Spinco SW-50.1 rotor.

Fractions were picked from the bottom and checked for reverse transcriptase activity with oligo (dG): poly (rC) examined as a template. Bovine serum albumin served as a sedimentation marker in a parallel gradient experiment.

DNA polymerase determinations The determination mixtures for polymerase activity with templates made of synthetic polymer contained (in 100 µl): 5 M o1 Tris-HCL, pH = 8.0, 0.5 mol MgCl21 0.1 mol DTT and the following combinations of polymer and dNTPs-0.4 µg oligo (dG): poly (rC) or oligo (dG): poly (rCm), 0.02 µmol dCTP and 1.0 nmoles [3H] -dGTP (4000 Ipm / pmoles); 0.4 g Oligo (dT): Poly (rA) or Ologo (dT): Poly (dA), 0.02 µmole dATP and 1.0 mmole [3H] -dTTP (4000 Ipm / pmole). For reactions with oligo (dG): poly (rcm) 0.02 µmol MnCl2 was used instead of Mgcl2. Ipm = pulses per minute.

Determinations using AMV RNA contained (in 100 5 µmol Tris-HCl, pH = 8.0, 0.8 µmol MgCl2, 0.1 µmol DTT, 10 µg actinomycin D (Sigma Corp.), 5 µg distamycin A (Calbiochem), 2 µg AMV 7OS 'RNA, 0.1 µg oligo (dT) 12 ~ 18, 0.1 FMol each of dATP, dGTP, dTTP and 5 nmoles [3H] -dCTP (1.5 x 10 4 Ipm / pmol).

All reaction mixtures were incubated at 360C for 15 to 30 min, and the incubation was stopped by adding 0.5 ml of cold 0.067 M sodium pyrophosphate - 1 M sodium phosphate solution, pH = 7.2 and then 0.5 ml of cold 80% TCA solution completed. The acid-insoluble radioactivity was collected on membrane filters collected and measured in a scintillation counter.

The final deoxynucleotidyl transferase activity was increased by the polymerization of r3H7-dGTP measured in the absence of a template of complementary polymer. The reactions were carried out as previously described with the exception that the Polymer-dNTP combination by oligo (dG) 10-18 plus 0.02 ZMol dCTP and 1.0 nmol g3H7-dGTP has been replaced.

All synthetic oligo- and polynucleotides, tritiated dGTP, dTTP and dCTP were commercially available substances. AMV 70S RNA was purified from the Virus according to the method described by Weiss et al., Virology 22 (1976) p. 242 isolated.

Hybridization: s reactions The working methods for the hybridization reactions and their analysis with 5 nuclease were described by Weiss et al., log.citO. The conditions for equilibrium density centrifugation, as described by Axel et al., Nature 235 (1972) 5. 32 described, were by adding 0.02 of a sodium N-lauroyl sarcosinate and 20g each of E.coli DNA and RNA modified to the gradient.

Results The values given below are based on independent Enzyme isolations from four different tumor clusters (labeled A to D). The behavior during fractionation and the properties of breast tumor polymerase did not vary significantly from one preparation to another.

The polymerase preparation A was derived from metastatic damage in isolated from the liver of a patient with breast cancer, 9 g of the tumor were homogenized, and the particulate material that accumulated at a density of 1.16-1.19 g / ml, became collected as previously described. The pellet obtained was made by adding Triton X-100 solubilized and then passed through a column of polyacrylamide agarose gel (Ultrogen ACA-44) fractionated. Each column fraction was assayed for: 1) DNA polymerase with oligo (dG) 12 18: poly (rC) as template and 2) end transferase using of Oligo (dG) 12-18 as a base material (primer). Three tips (two larger ones and one smaller peak) of DNA polymerase activity were noted and it was evident that the first, larger tip also contained the end transferase (terminal T.). The two larger peaks contained (see Table I) 90 μ of the polymerase activity given up and 3 ß of the protein as per the fluorescamine procedure of Udenfriend et al., Science 178 (1972) p.871. Table I Purification of DNA polymerase from a human breast (sample A) total total specific protein activity activity (mg) (pmol) (pmol / mg) 1. Virus density range 8.3 168 20 2. Polyacrylamide agarose 0.25 148 5.6 x 10² 3. Phosphocellulose 0.02 96 4.8 x 10³ All reactions contained Olige (dG): Poly (rC) as a template and were used for 30 incubated min at 36 ° C. The total activity was expressed as pmoles of polymerized [3 H] dCMP calculated.

To the reality of the previously observed separation of polymerase activities to check, the two main peaks were merged to reduce the Dialyzed phosphate buffer concentration and then passed through a phosphocellulose column (PC) chromatographed with a linear phosphate gradient from 9.01 M to 0.5 M. The polymerase activities again resolved into two major peaks, one eluted at 0.08 M phosphate and the other at 0.18 M. It should be noted that the second major polymerase peak does not contain any final transferase activity. The latter divides in two peaks, one is with the first DNA polymerase activity at 0.08M Phosphate linked, and another eluted by itself at 0.23 M phosphate.

The second peak (0.18 M phosphate) of polymerase activity found in The PC column observed is in normal tissues (3 samples from breast tissue and 3 samples from the spleen) or in benign fibroadenomas of the breast (3 collected Amount of 3-4 fibroadenomas each) not found, making them the merely breast cancer tissue own DNA polymerase is. The fractions that make up the top 2 of the PC column contained 65% of the abandoned DNA polymerase activity and 8% of the protein. These Fractions were pooled and concentrated as previously described to obtain the To give breast tumor polymerase. In Table I are the yields of activity and protein compiled at each of the three stages of a typical purification.

Example 2 Evidence that Breast Cancer Polymerase is a Reverse Transcriptase is.

There are several useful criteria which reverse transcriptases distinguish RNA tumor viruses from normal mammalian DNA polymerases. The reverse Virus transcriptases show a preference for oligo (dT): poly (rA) over Oligo (dT): Poly (dA) and they continue to take Oligo (dG): Poly (rC) and Oligo (dG): Poly (rCm) as excellent templates for the synthesis of poly (dG). Another and stronger diagnostic feature is the ability of a reverse transcriptase, a heteropolymer To use RNA to direct the synthesis of an exactly complementary DNA, like by suitable back hybridization of the cDNA product to that used in the synthesis Stencil is shown.

The response of four breast cancer polymerases to the synthetic ones Polyribonucleotides are summarized in Table II below. The results show a pattern of activities that is completely in line with that with Reverse transcriptases isolated from RNA tumor viruses from authentic animals was obtained. So in all cases Oligo (dT): Poly (rA) is the Oligo (dT): Poly (dA) superior to the synthesis of poly (dT). Furthermore, both oligo (dG) ;: poly (rC) as well as Oligo (dC): Poly (rCn) excellent templates for the formation of poly (d). It should be noted that in addition to the four breast tumor enzyme preparations described herein numerous others (more than 50) received from additional patients the further investigations in the same way with different degrees of purity were examined using different methods of fractionation all of which showed the pattern described in Table II.

Table II Synthetic Polynucleotides as Templates for Breast Tumor Polymerase Template [³H] dNTP pmol [³H] dNMP, polymerized Prep.A Prep.B Prep.C Prep.D Oligo (dT): Poly (rA) T 0.472 0.445 0.221 0.495 Oligo (dT): Poly (dA) T 0.044 0.021 0.062 0.071 Oligo (dG): Poly (rC) G 0.534 0.444 0.180 0.532 Oligo (dG): Poly (rCm) G 0.502 0.410 N.T. N.T.

Oligo (dG) G 0.010 0.008 N.T. N.T.

The polymerase reactions were carried out using the template provided, such as previously described, carried out.

The incubation was carried out at 36 ° C. for 15 min (preparation C) or for 30 min (Preparations A, B and D). Polymerase preparation A was obtained from a single tumor (9g) isolated, the polymerase preparation B from a collected amount of 3 tumors (14g), the polymerase preparation C from a collected amount of 4 tumors (32g) and the polymerase preparation D from a pooled amount of 5 tumors (61g).

The definition of the function of a reverse transcriptase requires the Evidence that you are using a heteropolymeric RNA to make a DNA copy can. The response (Table III) of breast cancer polymerase to Avian RNA myeloblastosis (AMV) is that expected from the synthesis of a heteropolymeric DNA Speak to. Omit any or all of the required three unchecked Deoxyriboside trisohosphates lead to the same practical disappearance of the synthetic ones Activity as occurs when the RNA template is omitted. Tabel III Deoxynucleoside triphosphate and RNA requirements of breast tumor DNA polymerase pmol [3 H] dCMP, polymerizes reaction A B 1. Complete 0.200 0.170 2. -dATP 0.013 0.011 3rd -dGTP 0.025 0.022 4th -dTTP 0.012 0.005 5th -dATP, dGTP, dTTP 0.008 0.011 6. -RNA 0.026 0.016 The polymerase preparations A and B were dependent on the RNA DNA synthesis studied using AMV RNA as a template. During the reactions, for 30 incubated min at 36 ° C under the conditions given in Example 1.

The most informative test of a presumed reaction of a reverse Transcriptase comes from an examination of the match of the DNA copy. this requires the isolation of the r3K7 DNA product and its verification in an annealing reaction with the RNA template used in the synthesis.

For this purpose, a 2 ml reaction was carried out for 15 min with the DNA polymerase preparation A and AMV-RNA performed as a template, resulting in the synthesis of approximately 3 ng of t3H7 DNA at 2 x 107 Ipm / g. The r3H7DNA product was prepared according to standard procedures purified and recovered, and then annealed with AMV and RLV 70S RNAs. The product was determined by separation using a Cs2S04 gradient and the resistance examined against S1 nuclease. Both methods gave less than 5% response (annealing) with unrelated RLV-RNA and between 80 and 85 ffi hybridization of AMV-RNA, the template or template used to align the synthesis. These results therefore suggest that ~ ~ DNA is a one-stranded DNA Is the complement of AMV RNA. A more informative review of the [³H] DNA product is provided by a kinetic study of the annealing reaction. It was a comparison of the annealing kinetics on AMV-RNA of two r3K7 DNA products was carried out, one being synthesized by MV reverse transcriptase aligning MV RNA and the other by means of human breast cancer polymerase, using the same template had been synthesized. It was found, that the annealing kinetics to AMV-RNA of the two DNA products are indistinguishable was. Hence, the human enzyme is equally effective in reverse in either case Reverse transcription of AMV RNA as the homologous one purified from the avian virus Transcriptase.

Example 3 Other Properties of Breast Cancer Polymerase Changes in the temperature and the pH were on their influences on the activities of various preparations of breast cancer polymerase using oligo (dG): poly (rC) and Oligo (dT): Poly (rA) examined as templates or matrices. The maximum amount of polymerization speed occurred at 370C in 5 mM MgOl2 in a pH range from 7.0 to 8.5.

The need for divalent ions for reverse transcriptases has a certain interest, as they are used to divide the virus of origin into different ones Serving groups. So, all 6 contain strands of MMTV from a variety of sources a reverse transcriptase that has a strong preference for Mg ++ compared to Mn ++ shows. The same applies to the Mason-Pfizer monkey virus, the bromodeoxyuridine-induced Guinea pig virus and the bovine leukemia virus.

In contrast, reverse transcriptases from mouse leukemia and sarcoma viruses are much more effective in the presence of Mn ++. For the human breast cancer enzyme optimally supplies Mn ++ only about one seventh of that with Mg ++ achievable activity. It is clear that, just as between mouse mammary tumor and Leukemia viruses the human breast cancer enzyme requires a divalent ion that is most similar to the reverse transcriptases of the mammary tumor virus.

The molecular size of the breast cancer enzyme was determined using sedimentation estimated by a linear (10-30%) glycerol gradient. The enzyme was through Examination of fractions with AMV-RNA localized as a template or template. The enzyme activity sediments between 5S and 6S, slightly faster than the bovine serum albumin marker, thereby placing the molecular weight at about 70,000 Daltons. This result also agrees with the relative elution positions of the same proteins Ultrogel match. A number of enzyme preparations from various breast tumor sources resulted in identical sedimentation values.

Example 4 Materials and Methods Viruses: Nason-Pfizer Monkey Virus was in a suspension culture of normal, human lymphocyte cells, line NO-37, grown and concentrated as previously reported by Schlom and Spiegelman, Proc.Nat.

Acad. Sci. USA 68 (1971) p. 1613. The virus was further by centrifugation through an 8 ml column of 20% glycerol in TNE buffer (0.01 M Tris-HCl, pH = 8.3, 0.15 M.-AOL, 0.002 EDTA) on a pad made of 100% Glycerin purified at 93,000 x g for 60 min at 400. The virus pellet was in TNE buffer picked up and in a continuous 20-50; Sucrose gradients in TNE Centrifuged 98,000 x g to equilibrium for 16 h. The particles between Densities of 1.14-1.19 g / ml were collected, diluted and at 98,000 x g centrifuged for 45 min at 40C. This pellet was used immediately for purification the DNA polymerase used.

Avian myeloblastosis (AMV, BAI strain-A) viruses, simian sarcom virus Strain-1 (SSV-1), Friend leukemia virus (FLV), feline leukemia virus (FeLV) and Rauscher leukemia virus (RLV) were also used in this example. All virus concentrates were cleaned according to the description given above.

Production of RNA-instructed DNA polymerase from malignant tumors the human breast reverse transcriptase from malignant tumor of the human 3rust was, as before, by Ohno et al., Proc. Natl. Acad. Sci.

USA 74 (1977) 5,764 described from purified by isopycnic separation Particles prepared. After disruption by incubation in 0.2% Triton X-100 for For 15 min at OOC, some of the samples were tested for endogenous polymerase activity such as smoke on oligo (dG): poly (rC) and oligo (dT): poly (rA) directed synthesis of poly (dG) resp. Poly (rA) examined. The torn viruses were on density areas on a Polyacrylamide Agarose column (Ultrogel AcA44, SIEB, Co.) chromatographed and the eluted enzyme tip was applied to a phosphocellulose column (Whatman p11) given up. The enzyme was eluted with a 0.01-0.5 M gradient of potassium phosphate and would be from Ohno et al. loc. cit. described, concentrated.

Predaration of viral areas of human leukemia and HodRkin "s Spleen The spleen of patients with chronic lymphocytic leukemia (CLL), chronic, myelogenous leukemia (cD4L) and Hodgkin's lymphoma were used as sources for the viral density range preparations used, and the polymerase activities were analyzed by endogenous kinetics, as described by Witkins et al., Proc. Natl. Acad. Sci. USA 72 (1975) p. 4133.

PreDaration of MPMV polymerase That prepared from viruses as before MPMV pellet was resuspended on 0.05 M Tris-lICl, pH 9.2, 0.001 M EDTA and 2 M KCl, sonicated and then centrifuged at 98,000 x g for 120 min. The pellet was used to produce purified DNA polymerase by means of column chromatography, as previously reported by Witkins et al., loc. cit. described, used, prenaration of Antiserum Antiserum against MPMV DNA was induced in New Zealand white rabbits. Three immunization cycles were required to achieve the desired anti-polymerase IgG titre to obtain. In each cycle the enzyme was incorporated (1 x 103 pmoles per min TMP) emulsified with an equal volume of Freund's adjuvant and mixed into the two injected rear foot pad. This was followed by two additional, similar inoculations, given at the same sites at two-week intervals.

The sera were determined by chromatography on Sephadex G-200, 0.1 M Tris-HCl, pH = 8.0, chromatographed. The rabbit gamma globulins were serologically tested Identified immunodiffusion with goat anti-rabbit IgG antiserum. The relevant Fractions were concentrated by precipitation with ammonium sulfate (50% saturation) and dialyzed against 0.1 M Tris-HCl, pH 8.0. The protein concentration of the IgG fraction was measured according to Lowry's procedure.

Determinations of DNA polymerase mixtures for the determination of the polymerase activity with synthetic polymer matrices or templates contained (in 100 µl): 5 µmol Tris-HCl, pH 8.0, 0.5 µmol MgCl2, 0.1 / mol DTT and the following combinations on polymer and dNTPs: 0.4 µg oligo (dG): poly (rC) or oligo (dG): poly (rCm), 0.02? Mol dCTP and 1.0 nmol [³H] dGTP (4000 Ipm / pmol), 0.4 µg oligo (dT): poly (rA) or oligo (dT): poly (dA), 0.02 µmole dATP and 1.0 nmole [3 H] dTTP (4000 Ipm / pmole). In the reactions with oligo (dG): poly (rCm), MnCl2 (0.02 µmol) replaced the MgCl2.

The determinations using endogenous RNA contained (in 100 µl): 5 µmol Tris-HCl, pH = 8.0, 0.8 µmol MgCl2, 0.1 µmol DTT, 10.2 g actinomycin D, 5 µg distamycin A, 0.1 µg oligo (dT) 12-18; 0.1 µmol each of dATP, dGTP, dTTP, and 5 nmoles [3 H] dCTP (1.5 x 10 4 Ipm / pmoles).

All reactions were carried out at 360C for 15-30 min as indicated incubated, and they were by adding 0.5 ml of cold 0.067 M sodium pyrophosphate solution, 1 M sodium phosphate solution, pH = 7.2 and then from 0.5 m. Cold 80% TCA solution canceled. The acid-insoluble radioactivity was collected on membrane filters collected and counted in a scintillation counter.

The final deoxynucleotide transferase activity was determined by polymerization of [³H] dGTP carried out in the absence of a complementary polymer template, the latter was replaced by 0.4 µg oligo (dG) 10-18.

The Influence of Antibody on DNA Polymerase Activities. Reaction Mixtures for the neutralization of DNA polymerase activity (total volume 55 po) contained in addition to 2511g bovine serum albumin (BSA) and DNA polymerase the specified Amount (25-150 / µg) of purified IgG fraction.

The buffer used was 0.01 M Tris-HCl, pH 8.0, 0.15 M potassium chloride. After 15 minutes of incubation at 40C, a polymerase determination was made using from Oligo (dG): Poly (rC), performed as previously described. In certain cases the effect of the antibody on the activity was checked by the endogenous RNA.

Detection of antibody-enzyme complexes in glycerol gradients concentrated enzyme fractions (reverse transcriptase from MPMV or from malignant human breast tumors) were triple with 0.1 M potassium phosphate, pH = 8.0, which 0.5 mg / ml of bovine serum albumin, diluted, and the amounts indicated of purified IgG fractions were added. After an incubation during For 15 min at 40 ° C., the samples were set over a 10-30% glycerol gradient on 0.1 M potassium phosphate, pH 8.0, 0.002 M dithiothreitol and 0.02% Triton X-100 layered. The samples were run at 48,000 rpm for 12 hours in a Spinco SW50-1 rotor sedimented at 10C. The fractions were dropped from the bottom of the glasses and the enzyme activity was determined as described above certainly.

Results Comparison of the effects of anti-MPMV DNA polymerase IgG on a variety of polymerases The effects of anti-MPMV DNA polymerase on a number of viral DNA polymerases and the corresponding enzyme of particles of human breast cancer were compared.

In these experiments isopycnically grouped particles, cleaned according to the information in materials and methods as Source for the DNA polymerase and oligo (dG): Poly (rC) as template or template used. The antibody inhibits MPMV DNA polymerase by more than 80, and reached a 26% inhibition of DNA polymerase associated with particles from human breast cancer is associated. In contrast, there is no detectable effect on the DNA polymerases observed from any of the other animal tumor viruses including viruses of Avian myeloblastosis (AMV), Rauscher and Friend mouse leukemia viruses (RLV and FLV), feline leukemia virus (FeLV), a simian sarcoma virus (SSV-1), or mouse mammary tumor virus (F! TV).

Specificity of the inhibition by the anti-MPMV polymerase IgG Das The next question examined focused on whether those with the enzyme from breast cancer particles observed inhibition was limited to this tumor disease. As already on known in the art, represents the spleen of patients with mesenchymal cancers constitutes a suitable source of particulate enzyme, and therefore it has been made for the immunological Comparison used. The particle fractions were prepared and the activities the endogenous polymerase were determined as previously described, and for human breast tumor diseases and for spleen diseases due to mesenchyme Neoplasms. At least 5 cases of each type of neoplastic tissue have been made and typical results are shown in Table IV below.

Table IV Effect of anti-MPMV polymerase IgG on activity of endogenous reverse transcriptase from particles of tissue from base tumor in humans normal IgG BSA anti-MPMV IgG RNase cells or tissue 100 µg / 0.1 ml 100 µg / 0.1 ml 100 µg / 0.1 ml 8 µg / 0.1 ml Ipm Ipm% Ipm% Ipm% breast cancer, ex. 1 1862 1748 93.8 765 41.1 331 17.7 Breast cancer, ex. 2 3682 3429 93.1 138 3.7 195 5.3 Breast Cancer Ex. 3 2340 2118 90.5 283 12.1 321 13.7 CML Spleen 3086 3110 100.7 2806 90.9 168 5.4 CLL spleen 1540 1640 106.5 1621 105.2 126 8.2 Hodgkin spleen 1261 1205 95.5 1018 80.7 186 14.8 The reaction mixtures (50 µ #) contained 50 µg of either anti-MPMV polymerase IgG, or normal KLanine IgG, or bovine serum albumin (BSA) and particles torn out in the surfactant. Incubation for Allowing the complex to form with antibody was carried out for 15 min at 0 ° C. The activity of the endogenous enzyme was then correspondingly at 37 ° C for 15 min carried out according to the description given in "Methods". The sensitivity to RNase (80 µg / ml) was also tested.

There are no significant inhibitions when using the particle enzymes which were derived from leukemias and the lymphoma spleen preparations. However the enzymes of breast cancer particles were inhibited from 59 ffi to over 95 ffi. It it should be pointed out that these endogenous reactions are caused by the. Anti-MPMV polymerase IgG are more affected than by the synthetic stencils or matrices directed synthesis. As expected, all are described in Table IV DNA polymerase activities sensitive to RNase and to its presence resistant to actinomycin D (100 µg / ml) and distamycin (50 / µg / ml), this being for RNA-directed DNA polymerase are distinctive features.

The data shown in Table IV were obtained with the endogenous reactions obtained from surfactant / detergent disrupted particles, which had been isolated from the indicated neoplastic tissues. For completeness a similar comparison was made using the corresponding, purified by Oligo (dG): Poly (rC) directed enzymes carried out. Try using this Methods provided the response to anti-MPMV polymerase IgG from the purified, reverse ranscriptases from particles produced from breast cancer and from chronic, myelogenic, leukemic spleen. Over the entire tested concentration range of IgG will. does not significantly affect leukemic reverse transcriptase. In contrast, the anti-MPMV polymerase IgG suppresses the activity of the reverse Breast cancer transcriptase at all concentrations tested, with a 37% Inhibition is achieved at 150 µg per reaction mixture.

In Table V below is another aspect of specificity the interaction by studying the response of DNA polymerases from normal Cells shown to the anti-MPMV polymerase IgG.

Table V Influence of nti-MPMV-IgG on polymerases from cells and Breast Tumor Particles Normal IgG Anti-MPMV Polymerase IgG Source (75 mg / 0.1 ml) (75 µg / 0.1 ml) (enzyme) Ipm Ipm% of control breast tumor 1628 662 40.7 (reverse transcriptase) Breast tumor 2166 2196 101.4 (DNA-Polymerase-γ) HeLa cells 1865 2011 107.8 (DNA-Polymerase-γ) HeLa cells 1658 1819 109.7 (DNA polymerase-ß) The indicated DNA polymerases were determined according to the description given in the "Methods" section. The enzyme activity in the presence of normal Kanische IgG was taken as a control. It seo on it noted that Table IV compares normal IgG to BSA. Each 100 µl des Analysis mixture contained 0.4 mg oligo (dT): poly (rA), and incubation was carried out for 30 min at 37 ° C.

Neither the preparation of DNA polymerase-γ, no matter whether isolated from breast tumor or from the strain of HeLa cells, was detectably inhibited, the same was true for DNA polymerase-ß. At the same salary, the oppressed Anti-MPMV polymerase IgG reduced breast cancer reverse transcriptase by 40 ».

Similar experiments were carried out with normal DNA.-polymerase-α carried out, here again no inhibition was found the anti-MPMV polymerase IgG.

Evidence of sedimentation of complexes between breast cancer DNA polymerse and anti-polymerase IgG. It seems desirable to establish whether another Indication of the existence of physical complexes between reverse breast cancer transcriptase and the anti-MPMV polyterase IgG could be obtained. First would be such a Note a direct support of only from the simple suppression of the enzyme activity deduced conclusions and, secondly, such attempts could identify the fundamentals that of the apparent inability of antiwV DNA polymerase IgG to achieve a complete neutralization of reverse breast cancer transcriptase activity underlie. Basically, two mechanisms can be used to explain incompleteness the inhibition can be proposed. One mechanism would suggest that the enzyme preparation is heterogeneous and that only one subponulation inactive complexes with the added one IgG forms. The other mechanism would assume that the population of enzyme molecules in this case is homogeneous, and that all form complexes which, however, form a fraction the original activity. These two ways are easier Way by a sedimentation analysis of the enzyme activity before and after the reaction distinguishable with the corresponding IgG.

To monitor the effectiveness of this experiment, a positive was found Control experiment carried out with the homologous system consisting of IÇPMV DNA polymerase and their antibody passed. A negative control was also carried out, using normal (pre-immunized) IgG from the same rabbit. One 250 rX reaction, which contained enzyme and 150 μg of the indicated IgG, was performed at 40C for 15 min as described in the "Materials and Methods" section.

The mixture was then layered on a 10-30% gradient and centrifuged at 48,000 rpm for 12 hours at 10 ° C.

Fractions were then collected from the bottom and checked for reverse transcriptase and examined for the presence of IgG by immunodiffusion. It was found, that incubation with normal IgG the position (glass 13) of the peak of the MPMV reverse transcriptase activity with regard to the external labeling of bovine serum albumin (BSA) not changed. That Enzyme still sediments at a rate equivalent to one molecular weight of 70,000 Daltons. The IgG in the glasses was in the same gradient 10, 11 and 12 localized. Incubation of the polymerase with 150 Tg of anti-MPMV polymerase showed an 80% loss of enzyme activity and a markedly different sedimentation pattern the rest of the activity. In the original position near BSA there is no enzyme detectable, the entire enzyme appears as complexes, which more rapidly than free Sediment enzyme or IgG. The IgG is now immunodiffused in the fractions 13, 14 and 15 as well as in fractions 6 to 10 detected which the peak polymerase activity.

A very similar situation is found in experiments with purified, reverse Transcriptase obtained from particles from human breast cancer. The incubation with normal IgG leaves the human enzyme in its original position (glass 15) Inside a glass the BSA label, and the IgG was determined by immunodiffusion found in jars 12-14. However, that results Reaction with Anti-MPMV polymerase IgG suffer from a loss of activity and postpone it Residue after lower glasses as rapidly moving complexes that are in the Find fractions 6 to 10 in which IgG is also detected by immunodiffusion can be. IgG is still in the original position (fractions 12 to 14) found.

From the results described above, it can be seen that neither the MPMV reverse transcriptase nor that isolated from human breast cancer particles reverse transcriptase contains a significant proportion of molecules that lead to Are incapable of complexing with anti-MPMV polymerase IgG. The fact that the enzyme complexes IgG showing some activity is not a new phenomenon. Indeed in some reported cases, such complexes are fully active.

Discussion The experiments described here show that anti-MPMV DNA IgG, if it is in excess, complex it completely with the reverse transcriptase can and partially reverse transcriptase, isolated from particles from human Breast cancer. The specificity of the inhibition is determined by the inability of the same antibody supports the activities of normal DNA polymerases from cells or from a variety of reverse transcriptases from animal tumor viruses (e.g. AMV, RLV, FLV, FeLV, SSV-1 and MMTV). Still complex normal IgG obtained from the same rabbit before immunization, neither with the reverse transcriptases from MPMV nor from human breast cancer it still inhibits them.

In addition to this interest concerning the cause, the immunological one has Cross-reactivity between the reverse transcriptases of MPMV and of particles from human breast cancer has another important meaning. She delivers that Basis for testing for human breast cancer through clinically advantageous working methods. MPIC can be used in tissue cultures in reasonable yields for the purification of the herein contained enzyme and other protein components. these can in turn can be used again to generate antisera that have a use as specific detection reagents in immunofluorescence staining and immunoperoxidase staining of frozen sections as diagnostic tools for clinical pathology own. Of further interest is the application of this development to immunodetermination methods for the systematic detection of immunologically related protein in plasma and body fluids from patients with other than breast cancers, to lung cancer, stomach cancer, rectal and colon cancer, fallopian tube cancer, brain cancer, Bone cancer, cancer of the lymph glands, skin cancer (carcinomas and helanomas) of squamous cells and basal cells and the like. Each of these cancers has its own distinctive protein associated with the particles.

- patent claims -

Claims (21)

Patent claims method for the detection of breast cancer in humans, Noted that a sample of the patient is on for breast cancer specific protein associated with viruses is examined. 2. The method according to claim 1, characterized in that g e k e n n z e i c h -n e t that the presence of this breast cancer specific virus associated Protein in higher concentrations than the control values is a diagnostic clue for breast cancer in this patient is. 3. The method according to claim 1, characterized in that g e k e n n z e i c h -n e t that plasma samples from the patient before and after initiation of therapy and that the provision for monitoring the effectiveness of this Therapy is used. 4. The method according to claim 1, characterized in that g e k e n n z e i c h -n e t that the virus-linked proteins specific for breast cancer are human Breast cancer DNA polymerase and that the sample is a blood plasma sample. 5. The method according to claim 4, characterized in that g e k e n n z e i c h -n e t that the DNA polymerase by isolating the enzyme from the plasma sample and measuring the enzymatic activity is determined. 6. The method according to claim 5, characterized in that g e k e n n z e i c h -n e t that the DNA polymerase is removed from the plasma sample by precipitation with ammonium sulfate and subsequent affinity chromatography of the reconstituted precipitate through a column, which contains Sepharose pearls, to which a synthetic template or template for the enzyme has been covalently bound, is isolated. 7. The method according to claim 6, characterized in that g e k e n n z e i c h -n e t that as a synthetic template or template polyriboadenylate or poly (2'-O-methylcytidylate) is used. 8. The method according to claim 4, characterized in that g e k e n n z e i c h -n e t that the DIiA polymerase is determined by a radioimmunological determination. 9. The method according to claim 8, characterized in that g e k e n n z e i c h -n e t that in radioimmunological determination for human breast cancer DNA polymerase specific antibodies and 125J human breast cancer DNA polymerase are used will. 10. The method according to claim 8, characterized in that g e k e n n z e i c h -n e t that in the radioimmunological determination for Mason-Pfizer monkey virus specific Antibody that is cross-reactive with human breast cancer DNA polymerase, and 125J Mason-Pfizer simian virus can be used. 11. The method according to claim 1, characterized in that g e k e n n z e i c h -n e t that the breast cancer-specific, virus-linked protein is associated with tumor is viral origin-associated protein and that the sample is a blood plasma sample is. 12. The method according to claim 11, characterized in that g e k e n n z e i c h -n e t that the protein associated with tumor of viral origin by a radioimmunological Determination is determined. 13. The method according to claim 12, characterized in that g e k e n n z e i c h -n e t that in the radioimmunological determination, specific antibodies for protein, associated with tumor of viral origin, and 125J protein associated with tumor associated with virus origin. 14. The method according to claim 12, characterized in that g e k e n n z e i c h -n e t that in radioimmunological determination. specific antibodies for Mason-Pfizer monkey virus, which cross-reacts with the protein associated with the tumor, and 125J Mason-Pfizer monkey virus be used. 15. Human breast cancer DNA polymerase, which essentially is free from other virus components and breast tissue components. 16. 125 j-human breast cancer DNA polymerase. 17. Protein linked to human breast cancer of virus origin is essentially free of other viral components and breast tissue components. 18. 125J protein associated with human breast cancer of viral origin connected is. 19. Method for the detection of cancer in humans, thereby g e k NOTICE that a sample from the patient for virus-related protein, that is specific to that cancer is being investigated. 20. Antibodies specific to human breast cancer DNA polymerase. 21.) Antibody which is specific to protein associated with human Viral origin breast cancer is associated.