Methods and kits for determining a risk to develop cancer, for evaluating an effectiveness and dosage of cancer therapy and for correlating between an activity of a DNA repair enzyme and a cancer

Methods and kits for (i) determining a risk of a subject to develop cancer; (ii) evaluating an effectiveness and dosage of cancer therapy administered to a cancer patient; and (iii) determining a presence of correlation or non-correlation between an activity of at least one DNA repair enzyme and at least one cancer, are disclosed.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the field of diagnosis and prognosis. More particularly, the present invention relates to methods of and kits for (i) determining a risk of a subject to develop cancer; (ii) evaluating an effectiveness and preferred dosage of cancer therapy administered to a cancer patient; and (iii) determining a presence of correlation or non-correlation between an activity of at least one DNA repair enzyme and at least one cancer.

The DNA in each cell of a body is constantly subjected to damage caused by both internal (e.g., reactive oxygen species) and external DNA damaging agents (e.g., sunlight, X- and γ-rays, smoke) (Friedberg, et al., 1995). Most lesions are eliminated from DNA by one of several pathways of DNA repair (Friedberg, et al., 1995, Hanawalt, 1994, Modrich, 1994, Sancar, 1994). When unrepaired DNA lesions are replicated, they cause mutations because of their miscoding nature (Echols and Goodman, 1991, Livneh, et al., 1993, Strauss, 1985). The occurrence of such mutations in critical genes, e.g., oncogenes and tumor suppressor genes, may lead to the development of cancer (Bishop, 1995, Vogelstein and Kinzler, 1993, Weinberg, 1989). Indeed, DNA repair has emerged in recent years as a critical factor in cancer pathogenesis, as a growing number of cancer predisposition syndromes have been shown to be caused by mutations in genes involved in DNA repair and the regulation of genome stability. These include Xeroderma Pigmentosum (Weeda, et al., 1993), Hereditary nonpolyposis colon cancer (Fishel, et al., 1993, Leach, et al., 1993, Modrich, 1994, Parsons, et al., 1993), Ataxia Telangiectasia (Savitsky, et al., 1995), Li-Fraumeni syndrome (Srivastava, et al., 1990), and the BRCA1 (Gowen, et al., 1998, Scully, et al., 1997) and BRCA2 genes (Connor, et al., 1997, Patel, et al., 1998, Sharan, et al., 1997). In these cases, which represent a minority of the cancer cases, gene mutations have caused malfunction, leading to a strong reduction in DNA repair.

A possible extension of the role of DNA repair in hereditary cancer, would be a role for DNA repair in sporadic cancer. Several studies suggested that inter-individual variability in DNA repair correlates with variation in cancer susceptibility, with low repair correlated to higher cancer risk (Athas, et al., 1991, Helzlsouer, et al., 1996, Jyothish, et al., 1998, Parshad, et al., 1996, Patel, et al., 1997, Sagher, et al., 1988, Wei, et al., 1996, Wei, et al., 1993, Wei, et al., 1994).

7,8-dihydro-8-oxoguanine (also termed 8-oxoguanine or 8-hydroxyguanine; dubbed 8-OxoG) is formed in DNA by two major pathways: (a) Modification of guanine in DNA by reactive oxygen species formed by intracellular metabolism, oxidative stress, cigarette smoke, or by radiation (Asami, et al., 1997, Gajewski, et al., 1990, Hutchinson, 1985, Leanderson and Tagesson, 1992). (b) Incorporation into DNA by DNA polymerases of 8-oxo-dGTP, which is formed by oxidation of intracellular dGTP (Maki and Sekiguchi, 1992). Once in DNA, 8-oxoG is replicated by DNA polymerases with the misinsertion of dAMP, causing characteristic GC to TA transversions (Shibutani, et al., 1991, Wood, et al., 1990). When the modified dGTP is used as a substrate by DNA polymerases, it is often misinserted opposite an A in the template, causing AT to CG transversions (Pavlov, et al., 1994).

The major route for removing 8-oxoG from DNA is base excision repair, initiated by 8-oxoguanine DNA N-glycosylase, product of the OGG1 gene (in humans termed also hOGG1; (Aburatani, et al., 1997, Arai, et al., 1997, Bjoras, et al., 1997, Radicella, et al., 1997, Roldan-Arjona, et al., 1997, Rosenquist, et al., 1997). The OGG1 gene was recently knocked-out in mice, such that the effects on carcinogenesis can now be examined in this organism (Klungland, et al., 1999, Minowa, et al., 2000). Expression of theE. colienzyme in Chinese hamster cells reduced 4-fold the mutagenicity of γ radiation (Laval, 1994), indicating that the repair of 8-oxoG is important in negating the mutagenic activity of γ radiation. The following observations associate OGG1 with cancer: (i) OGG1 was mapped to chromosome 3p25, a site frequently lost in human lung and kidney cancers (Arai, et al., 1997, Audebert, et al., 2000, Ishida, et al., 1999, Lu, et al., 1997, Wikman, et al., 2000). (ii) OGG1 was found to be mutated in 2 out of 25 lung tumors (Chevillard, et al., 1998), and in 4 out of 99 renal tumors (Audebert, et al., 2000). (iii) OGG1 was found to be mutated in a leukemic cell line (Hyun, et al., 2000) and in a gastric cell line (Shinmura, et al., 1998). (iv) Analysis of p53 mutations in human lung, breast, and kidney tumors revealed a substantial occurrence of GC to TA mutations, a mutation type produced by unrepaired 8-oxoG (Hollstein et al., 1996; Hernandez-Boussard, et al., 1999).

Since preventive measures which reduce the risk of developing cancer, such as, but not limited to, the use of anti-oxidants, diet, avoiding cigarette smoking, refraining from occupational exposure to cancer causing agents, are known and further since periodic testing and therefore early detection of cancer offers improved cure rates, there is a great need for, and it would be highly advantageous to have methods and kits for determining a risk of a subject to develop cancer.

Since the effectiveness of cancer therapy depends on the sensitivity of cells to genotoxic (mutageic) agents, there is a great need for, and it would be highly advantageous to have methods and kits for evaluating an effectiveness and preferred dosage of cancer therapy administered to a cancer patient.

There is also a great need for, and it would be highly advantageous to have methods and kits for determining a presence of correlation or non-correlation between an activity of at least one DNA repair enzyme and at least one cancer, so as to allow to determine a risk of a subject to develop cancer and to evaluate an effectiveness and preferred dosage of cancer therapy administered to a cancer patient.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided method of determining a risk (e.g., odds ratio, relative risk) of a subject to develop cancer, the method comprising determining a level of a parameter indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject, and, according to the level, determining the risk of the subject to develop the cancer.

According to another aspect of the present invention there is provided a method of determining a risk of a subject to develop cancer, the method comprising determining (a) a presence or absence of exposure to environmental conditions, such as smoking and occupational exposure to smoke or ionizing radiation, associated with increased risk of developing cancer; and (b) a level of a parameter indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject; and according to the presence or absence and the level, determining the risk of the subject to develop the cancer.

According to still another aspect of the present invention there is provided a method of determining a presence of correlation or non-correlation between an activity of at least one DNA repair/damage preventing enzyme and at least one cancer, the method comprising determining a level of a parameter indicative of a level of activity of at least one DNA repair/damage preventing enzyme in tissue derived from a plurality of cancer patients and a plurality of apparently normal individuals, and, according to the level determining the correlation or non-correlation between the activity of the at least one DNA repair/damage preventing enzyme and the at least one cancer.

According to further features in preferred embodiments of the invention described below, the parameter is selected from the group consisting of a protein level of said DNA repair/damage preventing enzyme, a level of a RNA encoding said DNA repair/damage preventing enzyme and a level of catalytic activity of said DNA repair/damage preventing enzyme.

According to still further features in the described preferred embodiments the cancer is selected from the group consisting of lung cancer, blood cancers, colorectal cancer, breast cancer, prostate cancer, ovary cancer and head and neck cancer.

According to still further features in the described preferred embodiments the tissue is selected from the group consisting of blood cells, scraped cells and biopsies.

According to still further features in the described preferred embodiments the DNA repair/damage preventing enzyme is selected from the group consisting of a DNA N-glycosylase, deoxyribose phosphate lyase and AP endonuclease.

According to still further features in the described preferred embodiments the DNA N-glycosylase is selected from the group consisting of Uracil DNA glycosylase, hSMUG1, hMBD4, Mismatch-specific thymine/uracil glycosylase, Methylpurine DNA glycosylase, hNTH1, Adenine-specific mismatch DNA glycosylase and 8-oxoguanine DNA glycosylase.

According to still further features in the described preferred embodiments the risk is expressed as a fold risk increase as is compared to a normal, apparently healthy, population, or a reference control group.

According to still further features in the described preferred embodiments the risk is expressed in enzyme specific activity units.

According to still further features in the described preferred embodiments the risk is expressed as a magnitude of a scale.

According to still further features in the described preferred embodiments determining the level of catalytic activity of the DNA repair/damage preventing enzyme is effected using a DNA substrate having at least one lesion therein.

According to still further features in the described preferred embodiments the at least one lesion is at a predetermined site in the DNA substrate.

According to still further features in the described preferred embodiments the substrate includes at least two different lesions of at least two types.

According to still further features in the described preferred embodiments the substrate includes a single lesion.

According to still further features in the described preferred embodiments the substrate includes at least two different lesions of a single type.

According to still further features in the described preferred embodiments the subject is known to be, or is about to be, exposed to environmental conditions associated with increased risk of developing cancer.

According to yet another aspect of the present invention there is provided a method of predicting the efficacy of a mutagenic anti-cancer treatment, such as chemotherapy and/or radiotherapy, in a subject, the method comprising determining a level of a factor indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject, and, according to the level, predicting the efficacy of the mutagenic anti-cancer treatment in the subject.

According to still another aspect of the present invention there is provided a method of selecting dosage of a mutagenic anti-cancer treatment, such as chemotherapy and/or radiotherapy, for treating a subject, the method comprising determining a level of a factor indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject, and, according to the level, selecting dosage of the mutagenic anti-cancer treatment for treating the subject.

According to an additional aspect of the present invention there is provided a kit for determining a level of activity of a DNA repair/damage preventing enzyme in a tissue of a subject, the kit comprising, a package including, contained in sealable containers, a DNA substrate having at least one lesion therein and a reaction buffer.

According to further features in preferred embodiments of the invention described below, the kit, further comprising test tubes for separating lymphocytes.

According to still further features in the described preferred embodiments the test tubes are prepackaged with an anti-coagulant.

According to still further features in the described preferred embodiments the kit further comprising a liquid having a specific gravity selected effective in separating lymphocytes from red blood cells via centrifugation.

According to still further features in the described preferred embodiments the kit further comprising a solution having osmolarity selected effective in lysing red blood cells.

According to still further features in the described preferred embodiments the kit further comprising a protein extraction buffer.

According to still further features in the described preferred embodiments the kit further comprising reagents for conducting protein determinations.

According to still further features in the described preferred embodiments the kit further comprising a purified DNA repair/damage preventing enzyme, which serves as a control for such activity.

The present invention successfully addresses the shortcomings of the presently known configurations by providing, methods, kits and reagents useful in determining a risk of a subject to develop cancer and for evaluating an effectiveness and individual dosage of cancer therapy administered to a cancer patient.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods and kits which can be used for (i) determining a risk (e.g., odds ratio, reklative risk) of a subject to develop cancer; (ii) evaluating an effectiveness and dosage of cancer therapy administered to a cancer patient; and (iii) determining a presence of correlation or non-correlation between an activity of at least one DNA repair/damage preventing enzyme and at least one cancer.

The principles and operation of a method and kit according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

While conceiving the present invention it was hypothesized that inter-individual variations in DNA repair/damage preventing activity modulate susceptibility of developing cancer.

While reducing the present invention to practice an experimental system which is easily adaptable to clinical use was developed, such that a defined DNA repair activity can now be used in determining cancer risk, and be utilized as a tool in cancer prevention, early detection and prognosis. Since the repertoire of DNA lesions is very large, at present experimental focus was given to an abundant and mutagenic DNA lesion, 8-oxoguanine (also termed 7,8-dihydro 8-oxoguanine or 8-hydroxyguanine; dubbed 8-oxoG). However, other mutagenic DNA lesions, such as, but not limited to, those listed in Table 3 below, can be similarly used to implement the methods of the invention, following suitable adaptation.

Thus, while reducing the present invention to practice, whether inter-individual variations in the activity of OGG, correlate with increased susceptibility to several types of cancers was studied. A lower repair activity might lead to an increased load of DNA lesions, and therefore to increased mutation rate, and earlier occurrence of cancer. Similarly, a lower repair activity renders cancer cells more susceptible to cancer therapy, which is genotoxic by nature. It should be noted that different types of DNA repair may be critical in different types of cancer. The present invention is exemplified, in a non-limiting fashion, with respect to the removal from DNA of a specific type of mutagenic lesion, 8-oxoG, by the activities of one or more DNA N-glycosylase repair enzymes, present in protein extracts from peripheral blood lymphocytes.

The present invention is herein exemplified with respect to the use of the level of the DNA repair enzymatic activity of DNA N-glycosylase(s) directed toward 7,8 dihydroxy 8-oxoguanine (8-oxoguanine DNA N-glycosylase activity; OGG), as a risk factor for lung cancer, lymphomas, and colorectal cancer. The enzymatic activity is measured in a protein extract extracted from peripheral blood lymphocytes and is referred to herein interchangeably as the OGGA nicking assay, OGGA assay or OGGA test.

Using the OGGA test, a case-control study was conducted on 309 individuals: 123 healthy individuals, and a total of 186 cancer patients as follows: 102 lung cancer (NSCLC) patients, 31 breast cancer patients, 18 lymphoma patients, 19 CLL patients, and 16 colorectal cancer patients. The following results were found.

The mean OGGA in healthy individuals of ages <50 (7.6±0.9; N=34) was slightly higher than in healthy individuals of ages ?50 (7.0±1.0; N=89). The difference is statistically significant (P=0.02).

The mean OGGA in healthy men (7.3±1.0; N=53) was similar to healthy women (7.1±1.0; N=70), the difference was not statistically significant (P=0.36).

The mean OGGA in smokers (7.3±1.00; N=35) was similar to that of non-smokers (7.1±1.0; N=88; P=0.46), indicating that the smoking status had a negligible effect on OGGA.

The mean OGGA in lung cancer patients (6.0±1.5; N=102) was significantly lower than in healthy individuals (7.2±1.0; N=123), with P=0.0001.

A strong association was found between low OGGA and lung cancer with odds ratio varying from 3.9 (95% CI 1.7-8.6, P=0.0009), to 9.0 (95% CI 3.2-25.0, P=0.0001), depending on the definition of the cutoff level (≦7.3 and ≦5.6, respectively). This indicates that a low OGGA value is a risk factor in lung cancer.

The mean OGGA in lymphoma patients (6.2±1.8; N=18) was significantly lower than in healthy individuals (7.2±1.0; N=123), with P=0.0001.

Low repair is defined as OGGA value ?5.5 units/μg protein, representing <4% of the healthy individuals. Normal repair is defined as OGGA>5.5 units/μg protein. After adjustment for age, lymphoma patients were 15 times more likely than the healthy controls to have a low OGGA value (Odds Ratio 15.2; 95% confidence interval, 3.7-62.5). This provides evidence that a low OGGA value is a risk factor in lymphoma.

The data shows that OGGA was low in 2 out of 16 (12%) colorectal cancer patients (compared to 5/123 i.e., 4.1% among healthy individuals), indicating that low OGGA is a risk factor in colorectal cancer.

OGGA distribution was normal in breast cancer patients, indicating that OGGA is not a risk factor in this type of cancer.

It will be appreciated that the OGGA and similar tests for other DNA repair activities can be used for screening individuals for purposes of prevention, early diagnosis and prognosis of cancers. These uses will be described in more detail below.

The following provides examples:

(i) Screening for smokers who have low OGGA in order to prevent lung cancer.

Although 85% of lung cancer patients are smokers, the great majority of smokers deal well with carcinogenic effects of smoking, and does not develop lung cancer. Even among heavy smokers, approximately 90% do not develop the disease (Mattson et al, 1987; Minna et al, 2002). The results presented herein clarifies the fact that the combination of smoking and low OGGA causes a dramatic increase in susceptibility to lung cancer. For example, the estimated risk of 30-years old smokers, with an OGGA value of 3.0, is 221-fold higher than the reference (30-years old non-smokers with an OGGA value of 7.0). For comparison, the estimated risk of 30-years old non-smokers, with an OGGA value of 3.0 is only 12-fold higher than the reference. The simplest explanation for this finding is that smokers with Low OGGA in peripheral blood lymphocytes have a lower OGGA also in their lungs. Having a low repair to start with, smoking causes further overloading of DNA damage, therefore leading to a high cancer risk. This is a classical example in which the risk of developing cancer is a combination of genetic factors (level of DNA repair) and external factors (cigarette smoking). Such individuals may be persuaded to quit smoking. Such a screen will be effective as a preventive means against lung cancer, and will lead eventually to a decrease in the incidence of lung cancer.

A considerable amount of people work in places which deal with radiation or with smoke. These include radiology departments in hospitals, nuclear industry, nuclear reactors, army personal dealing with nuclear weapons, etc. These people can be tested for OGGA, as a mandatory test, for their own safety. Individuals with Low OGGA might have an increased probability of developing cancer in such places, since ionizing radiation and smoke each produce 8-oxoG. Such individuals will be advised to seek an alternative working environment.

(iii) Using the OGGA value as a prognostic marker for cancer therapy.

Cancer therapy relies heavily on chemicals and radiation. These agents act, in most cases, by inflicting massive DNA damage, which leads to selective killing of the rapidly dividing cancer cells. The problem with such therapeutic agents is that they damage, or kill, also non-cancer cells. Knowing the level of OGGA in a cancer patient, may be used as a marker to estimate the prognosis of a particular therapeutic treatment.

(iv) Screening for susceptibility to lymphoma or colorectal cancers.

OGGA can be used to screen individuals for susceptibility to lymphomas or colorectal cancer.

(v) Early detection of cancer.

Individuals with low OGGA (e.g., smokers with low OGGA who would not quit smoking) can be advised to undergo periodical follow-ups, in order to enable early detection of lung cancer.

Thus, according to one aspect of the present invention there is provided a method of determining a risk of a subject to develop cancer. The method according to this aspect of the present invention is effected by determining a level of a factor indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject, and, according to the level, determining the risk of the subject to develop the cancer.

As used herein throughout the term “indicative of” includes correlating to.

According to another aspect of the present invention there is provided a method of determining a risk of a subject to develop cancer. The method according to this aspect of the present invention is effected by determining a presence or absence of exposure to environmental conditions, such as smoking and occupational exposure to smoke or ionizing radiation, associated with increased risk of developing cancer; and determining a level of a factor indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject; and according to the presence or absence and the level, determining the risk of the subject to develop the cancer.

Anyone of several approaches may be exploited according to the present invention in determining a level of a factor indicative of a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject.

According to one embodiment a protein level of the DNA repair/damage preventing enzyme is determined, which is indicative of the level of activity of the DNA repair/damage preventing enzyme. Several alternative quantitative assays are available for determining protein levels. Each of which is based on the specific interactions between proteins and antibodies specific thereto. Table 1 below lists known antibodies recognizing different DNA repair/damage preventing enzyme.

Antibodies recognizing any specific protein can be readily elicited using methods well known in the art in which cells of an immune system are exposed in vivo or in vitro to at least one epitope of the protein of interest, preferably a plurality of epitopes thereof. Such antibodies can be polyclonal or monoclonal. Commercial antibody developing services are available throughout the world. Examples include: Antibody Solutions, Palo Alto, Calif., Washington Biotechnology Inc., Baltimore, Md.; TNB Laboratories Inc., St. John's, Newfoundland, Canada; and Genemed Synthesis Inc., South San Francisco, Calif.

Such antibodies can be used in a variety of well known antibody based detection assays, including, but not limited to, Western blot, ELISA, a protein chip assay and an antibody chip assay.

In Western blot, total protein preparation is electrophoresed typically under denaturing and optionally under reducing conditions through a gel, typically a polyacrylamide gel. Then, the proteins are blotted onto a membrane, which is thereafter blocked by a non-specific protein, such as milk proteins. An antibody specific to the protein of interest is then interacted with the blot. The antibody will quantitatively bind to the protein of interest. The binding between the antibody and the protein of interest can be monitored by either directly labeling the antibody, or, preferably using a labeled secondary antibody capable of recognizing the first.

In ELISA the antibody capable of binding the protein of interest is linked to an enzyme capable of catalyzing a colorimetric reaction, which serves for quantitative detection.

In a protein chip assay, the protein of interest, typically a plurality of different proteins of interest, are linked to a solid support in addressable positions, so as to form a matrix of proteins. An antibody or several antibodies specific to certain proteins, each being labeled by a distinguishable label, are interacted with the support in the presence of proteins derived from a biological sample. A protein recognized by an antibody and which is present in the biological sample will compete with its solid support bound counterpart, such that the level of binding of the antibody to the respective addressable location on the support, is determinable by such competition for binding.

In an antibody chip assay, antibodies are linked to a solid support in addressable positions, so as to form a matrix of antibodies each capable of binding a different protein. Proteins derived from a biological sample are labeled and the labeled proteins are interacted with the solid support. The level of binding to the solid support is determined, being indicative of the level of the protein in the sample.

According to another embodiment of the present invention the level of a RNA, such as mRNA, encoding the DNA repair/damage preventing enzyme is determined, which is also indicative of the level of activity of the DNA repair/damage preventing enzyme. Several alternative quantitative assays are available for determining RNA levels. Each of which is based on the specific interactions between complementary nucleic acids. Table 2 below lists the human genes encoding DNA repair/damage preventing enzyme.

Yet undescribed human genes of DNA repair/damage preventing enzyme can nowadays be readily isolated using in-silico searches, since the majority (nearly all) of the coding sequences of the human genome have been cloned and sequenced. Traditional methods of gene isolation can also be exploited as is further described in the list of references provided hereinbelow.

In an alternative embodiment, and as is further described in detail below and exemplified by the Examples section that follows, the factor which is determined is the catalytic activity per se of the DNA repair/damage preventing enzyme.

The present invention is useful in determining a risk of a subject to develop cancer, whereby any type of cancer is subject to risk determination by way of implementing the method of the invention. It is well known that all cancers arise from DNA mutations and that the progress of a specific cancer from a primary tumor to a metastatic tumor, reflects clonal selection of cancer cells that accumulate mutations as they develop and turn more cancerous (e.g., proliferate more rapidly, escape proliferation control, acquire autosignalling behavior, induce angiogenesis, etc.) and more metastatic. This process is subject to variations depending on the specific genes involved in the development and progression of different cancers. It is therefore expected that different in vivo DNA repair/damage preventing activities are required to prevent the formation of different cancers. Also, the level of exposure of body tissues to genotoxic agents such as smoke and radiation, differs. Since different types of genotoxic agents cause different types of DNA lesions, it is again expected that different in vivo DNA repair/damage preventing activities are required to prevent the formation of different cancers.

The results obtained while reducing the present invention to practice are in agreement with the above, as low OGGA was found to be associated with some, but not all cancers tested. However, assays similar to the OGGA assay described herein can be readily developed for correlating other cancers with one or more DNA lesions, some of which are listed in Table 3 below.

In effect, all known cancers can be evaluated by finding correlation or non-correlation between the occurrence thereof and the occurrence of low DNA repair/damage preventing activity of certain types. When positive correlation is identified, a predictive risk determination assay can be readily implemented.

Thus, according to an aspect of the present invention there is provided a method of determining a presence of correlation or non-correlation between an activity of at least one DNA repair/damage preventing enzyme and at least one cancer. The method according to this aspect of the invention is effected by determining a level of activity of at least one DNA repair/damage preventing enzyme in tissue derived from a plurality of cancer patients and a plurality of apparently normal individuals, and, according to the level determining the correlation or non-correlation between the activity of the at least one DNA repair/damage preventing enzyme and the at least one cancer. This aspect of the invention is exemplified herein with respect to a single DNA repair enzyme activity (8-oxoguanine DNA glycosylase) using a suitable substrate having a single lesion therein, for a plurality of cancers, for some correlation was found, whereas for other, non-correlation was found.

Thus, the methods of determining a risk of a subject to develop cancer described herein can be implemented for a variety of cancers, including, but not limited to, lung cancers, e.g., small-cells lung cancer and non-small cells lung cancer, blood cancers, e.g., lymphomas and leukemias, including, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and the like, colorectal cancer, breast cancer, prostate cancer, ovary cancer, malignant melanoma, stomach cancer, pancreas cancer, urinary cancer; uterus cancer, bone cancer, liver cancer, thyroid cancer, brain cancer; head and neck cancer, including, for example, salivary carcinoma and laryngeal carcinoma.

DNA repair/damage preventing activity can be measured in extracts of different body tissues or cells, which may be collected from a testee by known methods. Blood cells, scraped cells (e.g., mouth or skin scrapes) and biopsies are good examples as such tissues are routinely removed from subjects for diagnostics.

Several types of DNA repair/damage preventing activities can be assayed according to the present invention, e.g., DNA N-glycosylase, nucleotide pool sanitizing activity (dNTPase activity, e.g., 8-oxodGTPase), AP endonuclease and deoxyribose phosphate lyase (of DNA polymerase β).

An assay for determining the activity of a DNA N-glycosylase is described and exemplified herein with respect to 8-oxoguanine DNA glycosylase. In this respect it is convenient to monitor the nicking activity of DNA N-glycosylase towards DNA substrates including one or more lesion.

An assay for monitoring the activity of 8-oxodGTPase is, for example, as described by Mo et al. [Mo, J. -Y., Maki, H. and Sekiguchi, M. (1992) Proc. Natl. Acad. Sci. USA 89, 11021-11025]. Thus, 8-oxodGTPase activity can be assayed by measuring the hydrolysis of α-32P-labeled 8-oxodGTP to 8-oxodGMP. The reaction mixture (12.5 μl) contains 20 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 40 mM NaCl, 20 μM α-32P-labeled 8-oxodGTP, 80 μg/ml bovine serum albumin, 8 mM dithiothreitol, 10% glycerol, and a protein extract. The reaction is executed at 30° C. for 20 minutes. Thereafter, an aliquot (2 μl) from the reaction mixture is spotted onto a PEI-cellulose TLC plate, and the mixture is fractionated with a solution containing 1 M LiCl for 1 hour. The spots on the TLC plate are then visualized and quantified by phosphorimaging. The preparation of 8-oxodGTP is described in Mo et al., ibid.

An assay for monitoring the activity of AP endonuclease is, for example, as described by Wilson III, et al. [Wilson III, D. M., Takeshita, M., Grollman, A. P., Demple B. (1995) Incision activity of human apurinic endonuclease (Ape) at abasic site analogs in DNA. J. Biol. Chem. 270, 16002-16007]. The reaction mixture (10 μl) contains 50 mM Hepes-KOH pH 7.5, 50 mM KCl, 100 μg/ml bovine serum albumin, 10 mM MgCl2, 0.05% Triton X-100, 2 pmol of a the DNA substrate and a protein extract. Reactions are performed at 37° C. for 5-30 minutes, after which the reaction products are fractionated by urea-PAGE, to separate the intact and incised DNA strands. The activity is deduced from the extent of cleavage of the substrate. The preparation of the substrate is described in the same reference (Wilson III et al., ibid.).

An assay for monitoring the activity of deoxyribose phosphate lyase (dRPase) is, for example, as described by Prasad et al. [Prasad, R., Beard, W. A., Strauss, P. R. and Wilson, S. H. (1998) Human DNA polymerase β deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism. J. Biol. Chem. 273, 15263-15270]. Deoxyribose phosphate lyase (dRPase) activity can be assayed by following the removal of deoxyribose phosphate from a32P 3′ end-labeled duplex oligonucleotide containing a site-specific 5′-incised abasic site. The reaction mixture (10 μl) contains 50 mM Hepes pH 7.4, 2 mM dithiothreitol, 5 mM MgCl2, 20 nM32P-labeled duplex oligonucleotide with a site specific abasic site (pre-incised at the 5′ with AP endonuclease), and a protein extract. The reaction is carried out at 37° C. for 15 minutes. After the reaction is terminated, the product is stabilized by the addition of NaBH4to a final concentration of 340 mM, and incubated for 30 minutes at 0° C. The DNA is then ethanol precipitated and fractionated by urea-PAGE. The activity of the dRPase is deduced from the extent of formation of the shorter reaction product. The preparation of the DNA substrate is described in the same reference (Prasad et al., ibid.).

Table 3 below lists examples of DNA repair enzymes, the genes encoding same and the DNA lesion(s) they recognize:

The risk according to the present invention can be expressed in one of a plurality of ways. In one example the risk is expressed as a fold risk increase in developing cancer as is compared to a normal, apparently healthy, population, or a reference control group. In another example, the risk is expressed in enzyme specific activity units. In another example, a linear or logarithmic risk scale is generated for either the “fold risk increase” or the “activity units” and the risk is expressed as a magnitude of the scale.

According to still further features in the described preferred embodiments determining the level of activity of the DNA repair/damage preventing enzyme is effected using a DNA substrate having at least one lesion therein.

As is schematically exemplified byFIGS. 12a-b, a monomolecular (MMS,FIG. 12a) or plurimolecular (PMS,FIG. 12b) universal substrate can also be generated and used while implementing the methods and kits of the present invention. Such a universal substrate is used according to the present invention to simultaneously determine the activity of more than a single DNA repair/damage preventing enzyme. Thus, a universal substrate of the invention includes at least two (four are shown inFIGS. 12a-bidentified by L1-L4) different DNA lesions specifically recognized by at least two different DNA repair enzymes. Careful selection of the positions of the different DNA lesions along the universal substrate, can be used to ensure the generation of distinguishable (e.g., size distinguishable) reaction products (P1-P10 inFIG. 12a, P11-14 inFIG. 12b), being indicative of the activity of the different DNA repair enzymes. In order to ensure accuracy, the lesions are selected to be unique to the activities tested. The length of the universal substrate, especially for a monomolecular substrate, which preferably includes labels along its length, is selected such that reciprocal reaction products are substantially longer than all of the reaction products to be analyzed (P1-P10 inFIG. 12a). End labeling can be used in the case of a plurimolecular substrate to circumvent this problem altogether. Thus, the length of a substrate according to the present invention, without limitation, can range between 10 base pairs and several hundreds base pairs.

A substrate of the invention can thus have at least one lesion of at least one type or at least one lesion of at least two types (universal substrate), the lesions preferably being positioned at predetermined site(s) in the DNA substrate. The lesion(s) can be of any type, including, but not limited to, uracil, 5-fluorouracil, 5-hydroxyuracil, isodialuric acid, alloxan, uracil or thymine in U/TpG:5meCpG, uracil (U:G), 3,N4-ethenocytosine, (eC:G), T (T:G), 3-methyladenine, 7-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine, 1, N6-ethenoadenine, 1,N2-ethenoguanine, thymine glycol, cytosine glycol, dihydrouracil, formamidopyrimidine urea, adenine from A:G; A:8-oxoG; C:A, 2-hydroxyadenine, 2,5-amino-5-formamidopyrimidine, 7,8-dihydro-8-oxoguanine and abasic site.

A lesion can be introduced at a unique and defined location (site) in a DNA molecule using solid phase DNA synthesis, using in sequence the four conventional phosphoramidite building blocks used in the synthesis of oligodeoxynucleotides and additional at least one modified phosphoramidite building block, which when introduced into the DNA introduces a lesion therein, which lesion is recognizable by a DNA repair enzyme. In the alternative, a DNA molecule is exposed to a mutagenic agent (e.g., an oxidative agent or UV radiation) which forms one or more lesion of one or more types therein. Even when using this method, one can select a presubstrate which will result in a product (substrate of the invention) in which the lesions are non-randomly distributed, since the extent by which a specific lesion is formed in DNA is often dependent on the DNA sequence.

Other alternatives also exist. For example, one can oxidize a plasmid DNA with an oxidizing agent. This will form several lesions in the plasmid DNA. One can now use this plasmid DNA to assay a repair enzyme that acts on this DNA, without knowing precisely where the lesions are. The enzyme will produce a nick in the DNA, and this will convert the plasmid from the supercoiled closed form to the nicked (open circular) form. These two can be easily distinguished by gel electrophoresis or gradient centrifugation. In another example a piece of DNA is enzymatically synthesized in the presence of lesioned building blocks. Other alternatives are also known, such as chemical deamination, etc.

Thus, the substrate of the present invention can include at least two different lesions of at least two types, a single lesion, or at least two different lesions of a single type.

A cancer risk determination test according to the present invention is specifically advantageous for a subject which is known to be, or is about to be, exposed to environmental conditions associated with increased risk of developing cancer, such as smoking and occupational exposure to smoke, ionizing radiation and other carcinogens.

As is discussed hereinabove, the effectiveness of cancer therapy is due to its genotoxic effect affecting cancer cells more than normal cells. Thus, according to another aspect of the present invention there is provided a method of predicting the efficacy of a mutagenic anti-cancer treatment, such as chemotherapy and/or radiotherapy, in a subject. The method according to this aspect of the invention is effected by determining a level of activity of a DNA repair enzyme in a tissue of the subject, and, according to the level, predicting the efficacy of the mutagenic anti-cancer treatment in the subject.

Anti cancer therapy dosage can also be individually optimized in view of the teachings of the present invention. Thus, according to still another aspect of the present invention there is provided a method of selecting dosage of a mutagenic anti-cancer treatment, such as chemotherapy and/or radiotherapy, for treating a subject. The method according to this aspect of the invention is effected by determining a level of activity of a DNA repair/damage preventing enzyme in a tissue of the subject, and, according to the level, selecting dosage of the mutagenic anti-cancer treatment for treating the subject. In this case, the tissue is preferably a biopsy derived from the cancer itself.

According to an additional aspect of the present invention there is provided a kit for determining a level of activity of a DNA repair/damage preventing enzyme in a tissue of a subject. In its minimal configuration, the kit includes, a package including, contained in sealable containers, a DNA substrate having at least one lesion therein and a reaction buffer selected suitable for supporting DNA repair activity. Preferably, the kit also includes test tubes for separating lymphocytes. Preferably, the test tubes are prepackaged with an anti-coagulant, such as, but not limited to, heparin. Still preferably, the kit further includes a liquid having a specific gravity selected effective in separating lymphocytes from red blood cells via centrifugation, e.g., Ficoll contained in lymphocytes isolation tubes. Advantageously, the kit includes a solution having osmolarity selected effective in lysing red blood cells. In a preferred embodiment of the invention a protein extraction buffer is also included in the kit. Preferably, the kit further includes reagents for conducting protein determinations, e.g., reagents included in the BCA kit by Pierce. Still preferably, the kit includes a purified DNA repair enzyme, which serves as a control for such activity.

EXAMPLES

Materials and Experimental Methods

DNA substrates: The DNA substrate was prepared by annealing two complementary synthetic oligonucleotides, 32-bases long each. They were synthesized by the Synthesis Unit of the Biological Services Department at the Weizmann Institute of Science. The oligonucleotide containing 8-oxoG had the sequence 5′-CCGGTGCATGACACTGTOACC TATCCTCAGCG-3′ (SEQ ID NO:1) (O=8-oxoG). The 8-oxoG phosphoramidite building block was purchased from Glen Research. The oligonucleotide was32P-labeled using T4 polynucleotide kinase, and annealed to the oligonucleotide 5′-CGCTGAGGATAGGTCACAGTGTCATGCA CCGG-3′ (SEQ ID NO:2). The radiolabeled duplex was purified by PAGE on a native 10% gel. Its concentration was determined by the PicoGreen dsDNA quantitation assay (Molecular Probes).

Blood samples: Large blood samples were obtained from the blood bank in the Sheba Medical Center. Samples of 10 ml peripheral blood were obtained from healthy donors or from cancer patients. Those were collected after obtaining permission from the Institutional Helsinki Committee.

Isolation of peripheral lymphocytes: The blood samples were processed 18-24 hours after collection. A 100 μl aliquot from each sample of whole blood was analyzed using a Cobas Micros (Roche Diagnostic System) blood counter. Ten ml PBS (Dulbecco's phosphate buffered saline, Sigma) were added to the remaining blood portion, and peripheral blood lymphocytes were isolated by density gradient centrifugation of the diluted whole blood on a polysucrose-sodium metrizoate medium in UNI-SEP tube (NOVAmed, Jerusalem, Israel). Centrifugation was performed at 1,000×g for 30 minutes at 20° C.

Following centrifugation the lymphocyte band was removed and washed with PBS buffer. Elimination of red blood cells was done by lysis in 5 ml of 155 mM NH4Cl; 0.01 M KHCO3; 0.1 mM EDTA for 4 minutes at room-temperature. The lymphocytes were washed with PBS, and suspended in 1 ml PBS. The number of white blood cells in this suspension was determined using a Cobas Micros (Roche Diagnostic System) blood counter.

Samples containing 1-4×106cells were precipitated by centrifugation at 5,000 rpm, for 4 minutes at room temperature. The cells pellet was then resuspended to a concentration of 20,000 cells/μl in 50 mM Tris.HCl (pH 7.1), 1 mM EDTA, 0.5 mM DTT, 0.5 mM spermidine, 0.5 mM spermine, and a protease inhibitor cocktail (Sigma). The cells were incubated on ice for 30 minutes, after which they were frozen in liquid nitrogen. The frozen lymphocytes were stored at −80° C.

Preparation of a protein extract: The frozen lymphocytes were thawed at 30° C., after which their protein content was extracted with 220 mM KCl, for 30 minutes on ice. Cell debris was removed by centrifugation at 13,200 rpm for 15 minutes at 4° C. Glycerol was added to the protein extract to a final concentration of 10%, and the extract was frozen in liquid nitrogen. Protein concentration was determined by the BCA assay kit (Pierce) using bovine γ-globulin as a standard.

Standard analysis of OGG activity: The reaction mixture (20 μl) contained 50 mM Tris-HCl (pH 7.1), 1 mM EDTA, 115 mM KCl, 20 μg bovine γ-globulin, 2 pmol PolydA.polydT, 0.5 pmol substrate and 8-12 μg protein extract. The reaction was carried out at 37° C. for 30 minutes, after which it was stopped by the addition of 15 mM EDTA, 0.2% SDS. The proteins were degraded by incubation with proteinase K (20 μg) for one hour at 37° C., after which they were treated with 80 mM NaOH for 30 minutes at 37° C. The denatured DNA products were analyzed by electrophoresis on a 15% polyacrylamide gel containing 8 M urea, in 89 mM Tris-borate, 2.5 mM EDTA pH 8.0, at 1,500 V for 2 hours at 45-50° C. The distribution of radiolabeled DNA products was visualized and quantified using a Fuji BAS 2500 phosphorimager. One unit of OGG activity is defined herein to cleave 1 fmol of DNA substrate in 1 hour at 37° C., under the standard reaction conditions described herein. In the following, OGGA is presented as specific activity, i.e., activity units/1 μg of total protein extract.

Statistical analysis: A 3-way ANOVA was employed for healthy subject to compare mean OGGA values, with gender, age (?50, <50), and smoking status as fixed effects.

Student's t-test was used to compare the mean OGGA values, analyzed as a continuous variable, between adenocarcinoma and squamous cell carcinoma patients.

To neutralize possible effects on OGGA means originating from the difference in mean age between the cases and controls, OGGA means were compared using ANCOVA, with age (treated as a continuous variable) as a covariate. This analysis was possible since no significant interaction was found between age and health conditions.

Associations were calculated using Fisher's exact test, and Odds ratios (OR) were calculated from a 2×2 table. Adjusted ORs and CI values were calculated by fitting logistic regression models with adjustment for age, sex and smoking status for lung cancer; and adjustment for age only, for lymphoma. OGGA values were analyzed as a continuous variable or as a dichotomized variable at values corresponding to 4% (OGGA cutoff at 5.5), 5% (OGGA cutoff at 5.6), 10% (OGGA cutoff at 5.9), 15% (OGGA cutoff at 6.2), 25% (OGGA cutoff at 6.4) or 50% (OGGA cutoff at 7.3) of the control group. Age was analyzed as a continuous variable, whereas gender and smoking status were analyzed as dichotomic variables.

Odds Ratio (OR) were calculated by the formula (Kleinbaum, 1994)

O⁢⁢RX1⁢-⁢X0=ⅇ∑i=1k⁢⁢bi⁡(X1⁢i-X0⁢i)
using the biestimates from the logistic regression model where OGGA values were analyzed as a continuous variable (bOGGA=0.624; bage=0.1; bsmoker=2.9). For example, X0, the reference, was used to represent non-smoking, 30 years-old individuals with an OGGA value of 7.0, and Xiwas used to represent the tested subject group. Thus the formula for the current model is:
−0.624(OGGAi−7.0)+0.1(Agei−30)+2.9Si
ORi=e
where OGGAiand Ageiare the OGGA value and age of individual i, and Siis either 1 or zero, for a smoker or a non smoker, respectively.

All the statistical analyses were performed using SAS software (version 6.12; SAS Institute Inc., Cary, N.C.).

Experimental Results

The OGG activity (OGGA) DNA repair test: Base excision repair (BER) is initiated by a DNA N-glycosylase, that releases the damaged or unusual base from DNA, generating an abasic site. The latter is then repaired by an AP endonuclease (APE/HAP1) and/or the lyase activity of the glycosylase, as well as the deoxyribose phosphate lyase (dRPase) activity of DNA polymerase β. The resulting gap is filled-in by DNA polymerase β, forming a patch of 1-3 nucleotides, followed by ligation (Dianov, et al., 1992, Singhal, et al., 1995). A long patch pathway of BER was identified which requires also PCNA, and the FEN-1 flap endonuclease (Fortini, et al., 1998, Kim, et al., 1998). It was reported that 8-oxoG can be repaired in cell extracts also by nucleotide excision repair (Reardon, et al., 1997), however, the in vivo significance of this finding is not clear (Runger, et al., 1995). In addition, it was reported that there is transcription-coupled repair of 8-oxoG, and that it required the XPG, TFIIH and CSB (Le Page, et al., 2000), and the BRCA1 and BRCA2 proteins (Le Page, et al., 2000).

While reducing the present invention to practice, an assay was developed for OGG activity (the OGGA test), using as substrate a32P end-labeled synthetic oligonucleotide, 32-base pairs long, carrying a site-specific 8-oxoG. The source of the OGG activity was a protein extract prepared from human peripheral blood lymphocytes (PBL), obtained by Ficoll fractionation from 10 ml blood samples. A protein extract was prepared from the lymphocytes by freeze-thaw, followed by salt extraction. The removal of 8-oxoG from the oligonucleotide, by the OGG activity in the extract, generates an abasic site, which was rapidly incised either by the AP lyase activity of the enzyme, or by AP endonucleases present in the extract. Alkali treatment, which breaks abasic sites, was performed after the incubation with the extract in order to ensure complete cleavage of the abasic site, such that only OGG activity is measured in the test. Analysis by urea-PAGE followed by phosphorimaging was used to quantify the extent of nicking, indicated by the formation of a shorter radiolabeled DNA fragment, 17 nucleotides long (FIG. 1a). The OGG activity level (OGGA value) is measured as specific activity, i.e., units of OGG activity/1 μg of protein extract. One unit of OGG activity cleaves 1 fmol of DNA substrate in 1 hour at 37° C., under standard reaction conditions.

FIGS. 1b-cand2a-bshow a time course and a titration of OGG activity, respectively, in lymphocyte extracts. The activity was dependent on the presence of 8-oxoG in the DNA substrate. No activity was observed when the DNA contained a G instead of the 8-oxoG. This observed activity is mostly due to the OGG1 enzyme, which was shown to be responsible for most of OGG activity in extracts prepared from human cells (Monden, et al., 1999). The existence of OGG2, a second OGG enzyme was reported. However, its activity was much lower than OGG1 in whole cell extracts (Hazra, et al., 1998). In addition to OGG, APNG (alkylpurine DNA N-glycosylase), also termed Aag (alkyladenine DNA glycosylase), or MPG (N-methylpurine glycosylase), was reported to act on 8-oxoG (Bessho, et al., 1993) but this finding was challenged in Hang, et al. (1997). In vivo this protein has no significant role in removing 8-oxoG from DNA, at least in mice (Engelward, et al., 1997, Hang, et al., 1997). In order to establish whether MPG is involved in the removal of 8-oxoG from DNA by lymphocyte extracts, a competition experiment was performed with an unlabeled duplex oligonucleotide containing a site-specific hypoxanthine (a substrate of MPG but not for OGG1; (see, Engelward, et al, 1997, Hang, et al., 1997)). As can be seen inFIG. 3, this duplex oligonucleotide did not inhibit the incision of the 8-oxoG-containing DNA by the extract, suggesting that APNG is not involved in the incision reaction. A control experiment with an excess of unlabeled duplex oligonucleotide containing a G instead of 8-oxoG showed no inhibition, whereas a duplex oligonucleotide containing 8-oxoG-DNA did cause inhibition, as expected (FIGS. 3a-b). These competition experiments are an indication of the specificity of the OGGA test to 8-oxoG.

Reproducibility experiments showed that the assay is accurate and highly reproducible, with a coefficient of variation ?10%. An example of a reproducibility experiment is shown in Table 4.

TABLE 4Reproducibility of the OGGA testA Blood Sample:123456789101112OGGA (units/μg protein):6.86.96.46.56.77.46.96.56.66.75.96.9Average OGGA:6.7Standard variation:0.4Coefficient of variance:6%Twelve blood samples from a healthy donor (donor No. 54),10 ml each, were processed and assayed forOGGA. One unit of OGG activity incises 1 fmolGO-containing substrate in 60 minutes at37° C. under standard assay conditions.B Blood Sample:123456AveSDCVOGGA (units/μg protein)Experiment 1:6.77.26.76.87.16.76.90.23%Experiment 2:7.96.97.88.28.17.87.80.56%Experiment 3:6.97.07.17.37.97.27.20.45%Overall average OGGA:7.1Standard deviation (SD):0.5Coefficient of variance (CV):7%Six blood samples from a healthy donor (donor No. 50),10 ml each, were processed to prepare protein extracts.The table shows the results of three independent assaysperformed with these assays on three different days.

OGGA value in healthy individuals (control subjects): The OGGA test was performed on blood samples from 123 healthy individuals, and the distribution is shown inFIG. 4. The mean OGGA value was 7.2±1.0 units/μg protein (this will be also dubbed OGGA value of 7.2±1.0; Table 5).

The range of OGGA was 3.6-10.1 units/μg protein, representing a 2.8-fold range of OGG activity. This is a rather narrow distribution of activity, significantly narrower than previously reported (Asami, et al, 1996).

A 3-way ANOVA with gender, age (<50, ?50), and smoking status revealed that there was no significant difference in mean OGGA value between men (53 individuals; 7.3±1.0 units/μg protein) and women (70 individuals; 7.1±1.0 units/μg protein; P=0.36FIG. 5; Table 5), or between smokers (N=35; 7.3±1.0 units/μg protein) and non-smokers (N=88; 7.1±1.0 units/μg protein; P=0.46). This indicates that smoking does not affect the OGGA value in peripheral blood lymphocytes (FIG. 6; Table 5). This result differs from the result obtained by Asami et al. (1996), who reported that 8-oxoG repair activity was increased 1.6-fold in smokers. In contrast, there was a small (6.6%), but statistically significant decrease in mean OGGA values between the two age groups: Individuals under the age of 50 had a mean OGGA value of (7.6±0.9; N=34), whereas those 50 years or older had a mean OGGA value of (7.0±1.0; N=89; P=0.02;FIG. 7; Table 5). Taken together these results indicate little or no variation of the OGGA value with age, smoking status and gender.

OGGA is not reduced in patients with breast cancer, and is altered in chronic lymphocytic leukemia (CLL): The OGGA test was performed on blood samples from 31 breast cancer patients and 19 CLL patients. As can be seen in Table 6, the mean OGGA value was 7.3±1.4 units/μg protein in breast cancer patients, similar to that of control female subjects (7.1±1.0, P=0.29; Tables 5 and 6). Also, the distribution of OGGA values was similar (FIGS. 8a-d). These results indicate that OGGA is not a risk factor in breast cancer. The mean OGGA value of CLL patients was 7.9±1.5, higher than the control subjects (7.2±1.0, P=0.0007; Table 6). However, the distribution of OGGA values among CLL patients was similar to the control group (FIGS. 8c-d).

OGGA is a risk factor in lung cancer: The OGGA test was performed with blood samples from 102 patients who suffered from operable non-small cell lung cancer (NSCLC), and had not been subjected to either chemo- or radiotherapy at the time when the blood samples were taken. As can be seen inFIGS. 9a-band Table 6, the mean OGGA value was 6.0±1.5 units/μg protein, significantly lower than the mean value of controls (7.2±1.0, P=0.0001). Analyzing separately cases with adenocarcinoma or squamous cell carcinoma revealed a similar OGGA level in these two main sub-types of NSCLC: adenocarcinoma: 6.1±1.5, N=37; squamous cell carcinoma: 5.8±1.6 N=35; P=0.44 (the other 30 cases were either other sub-types or unclassified NSCLC). The comparison of the distributions of OGGA in controls and in cases highlights the difference between the two groups. As can be clearly seen inFIGS. 9a-b, there is a shift to lower OGGA values in cases as compared to controls. For example, only 4% of controls have OGGA values of ?5.5, whereas 38% of cases have OGGA values in this range. This includes values 2-3 fold lower than the mean OGGA values of the controls. The mean age of the control group (57±14) was significantly different from the cases group (68±10; P<0.0001). Therefore, logistic regression, adjusted for age, was used to analyze associations, and analysis of covariance was used to compare age-adjusted mean OGGA values.

To analyze the association between levels of OGGA and presence of lung cancer logistic regression was used, where the binary dependent variable was presence/absence of lung cancer, and with age as a continuous variable, and gender, smoking status and OGGA as dichotomic variables. The latter was dichotomized at values corresponding to 5% (OGGA cutoff at 5.6), 10% (OGGA cutoff at 5.9), 15% (OGGA cutoff at 6.2), 25% (OGGA cutoff at 6.4) or 50% (OGGA cutoff at 7.3) of the control group. As can be seen in Table 7, smoking is strongly associated with lung cancer, in agreement with its established role as a major risk factor in the disease. For example, in a model where OGGA was dichotomized at ?5.9 (corresponding to 10% of the controls), the Odds Ratio (OR) for smokers was 20.8 (95% CI 7.8-55.4, P=0.0001). In a model where the OGGA cutoff was defined as ?7.3 (corresponding to 50% of controls), the OR for smokers was 23.0 (95% CI 8.9-59.2, P=0.0001). The gender had no significant effect in any of the models, whereas increased age was associated with the presence of lung cancer. Notice that although the OR for age was relatively small in all models (1.1 95% CI 1.1-1.2, P=0.0001) it is statistically significant. The age was analyzed as a continuous variable, and the relatively small OR is given per one-year change. Therefore, its final effect when applied to a particular change of age might be much larger (see Table 8 below).

As can be seen from Table 7, a clear association was found between the level of OGGA and presence of lung cancer. Moreover, there is a dose-dependent effect, with higher OR obtained for lower OGGA. For example, OR values of 3.9, 5.2, 7.0 and 9.0 were obtained for cutoff OGGA values of 7.3, 6.4, 5.9 and 5.6, respectively (Table 7). These high OR values indicate a strong association between low OGGA and lung cancer. Moreover, the increase in OR with decreasing OGGA further strengthens the significance of the association. In addition, the high OR values argue against the possibility of a selection bias in the control group.

In case-control studies there is a possibility that the examined variable is a consequence of the disease, rather than being a risk factor. In the present case, the possibility that the lung tumor causes a decrease of OGGA in peripheral blood lymphocytes (PBL) was considered. The OGGA value may be affected, for example, by factors that the tumor secretes into the blood stream. The main treatment of NSCLC is surgical removal of the tumor. This offers a way to distinguish between a causative and a resultive model for the association of PBL OGGA and lung cancer. Once eliminated from the lung, the effect the tumor have (if any) on OGGA in lymphocytes should decay with time. No correlation between OGGA and the time period that passed between surgery and taking the blood sample (ranging from 4 months before surgery to over a year after surgery) was found, indicating that whether the samples were taken before or after surgery had no effect on the level of OGGA in PBL. In the current group of case subjects, most (67/102) samples were taken after surgery. These results, clearly indicate that reduced OGGA is indeed a risk factor in lung cancer.

The simplest biological explanation for the present finding is the following: Low OGGA in PBL reflects low OGGA in the lungs. Correlations between DNA repair activities in PBL and lung cells (Auckley et al., 2001) or gastric mucosa (Kyrtopoulos et al., 1990) were previously reported. The lower DNA repair capacity leads to a reduced ability to repair oxidative DNA damage, and as a result 8-oxoguanine accumulates and leads to an increased mutation rate, which causes a higher cancer risk. In smokers there is an overload of DNA damage in the lungs, and therefore a higher risk is expected. No interaction was found between OGGA values and smoking status, implying that each of the two is an independent risk factor for lung cancer. This means that low OGGA is a risk factor also in non-smokers. This is not surprising, since oxidative DNA damage is a common intracellular damage that occurs even without exposure to external agents (Lindahl, 1993).

As discussed before, OGGA is not reduced in patients with breast cancer. This suggests that the repair of 8-oxoG is a bottleneck in the case of lung cancer, and in some additional cancers, but not in others (e.g., breast cancer). This is consistent with the finding that hereditary defects in particular DNA repair genes cause predisposition to specific types of cancer. For example, defects in nucleotide excision repair were shown to cause skin cancer (Weeda et al., 1993) whereas defects in mismatch repair cause hereditary non-polyposis colon cancer (Modrich, 1994). To our knowledge the results presented herein, are the first demonstration that decreased activity of a specific base excision repair enzyme is associated with cancer.

A useful application of the results of this study would be a quantitative model, which will provide an estimation of the risk of lung cancer associated with a particular OGGA value, age and smoking status. For diseases that do not occur frequently, such as lung cancer, and assuming that the cases and the controls are reasonably representative of the population, the odds ratio can be used as estimated relative risk (Gordis, 1996). Thus, a model was formulated using logistic regression, with age and OGGA as continuous variables, and smoking status as a dichotomic variable (smokers or non-smokers). This yielded OR values for lung cancer and these were taken as an estimation of risk. The OR values were calculated by dividing the odds of each particular group (having a particular age, OGGA value and smoking status) by the odds of 30 years-old non-smokers with a normal OGGA value of 7.0 (the reference group; OR of 1.0). The OR values for a specific age, OGGA value and smoking status are listed in Table 8. For example, according to Table 8, the estimated risk for 30 years-old smokers with a low OGGA value of 4.0, is 118-fold higher than the reference. At the age of 40, the estimated risk will increase to 321-fold higher than the reference. This high estimated risk is primarily the combined result of smoking and low OGGA. Having a low repair activity to start with, smoking causes further overloading of DNA damage, therefore leading to a high cancer risk. This model is instrumental in clarifying the fact that the combination of smoking and low OGGA causes a dramatic increase in susceptibility to lung cancer. For example, 40 years-old non-smokers with an OGGA value of 4.0 have an estimated risk 18-fold higher than the reference, compared to an estimated risk of 321-fold higher than the reference of smokers with the same age and OGGA (Table 8).

The OGGA test can be used to screen smokers for reduced DNA repair capacity. These individuals can be persuaded to quit smoking based on their personal reduced ability to cope with DNA damage. Since smoking is the main contributor to the high relative estimated risk for lung cancer (Tables 7 and 8), quitting smoking is expected to significantly improve the chances of preventing lung cancer. Such an approach of personalized smoking cessation, based on personal susceptibility, may provide a successful and cost-effective strategy to prevent lung cancer, and may be extended to include additional DNA repair assays.

TABLE 8An odds ratio model for estimating the risk of lung cancer forspecific DNA repair OGGA values, age and smoking statusEstimated RiskOGGA(Odds Ratio*)Age, yvalueNon-smokersSmokers3071183062343053633047118303122214073494065924059172404183214033359950771345061425150526468504488735039016296072036560637681605701272604131237360324444297075599270610218527051903456704355645170366212040*The Table is based on logistic regression analysis of the case-control study, and therefore the numbers represent only estimated values of risk. The odds ratio is calculated relative to the odds ratio of 30 years-old non-smokers with an OGGA value of 7.0.

The data presented herein indicates that low OGGA is a risk factor for lung cancer also among non-smokers (Tables 7 and 8). What can non-smokers with low OGGA do to protect themselves? One possibility is to make sure that they are not exposed to external sources of oxidative DNA damage such as secondary smoking or ionizing radiation. The latter includes radiology departments in hospitals, nuclear industry, and nuclear reactors. However, oxidative DNA damage is caused also by internal agents; therefore, dietary anti-oxidants might have a protective effect. Large population studies found that oxidants had no protective effect against cancer (reviewed in Collins, 1999; Lippman and Spitz, 2001). However, these food additives might have a protective effect when taken by individuals with low capacity to repair oxidative DNA damage.

Low OGG activity is a risk factor in lymphoma: Analysis of 18 lymphoma patients showed a clear shift to lower OGG DNA repair values (FIGS. 10a-b; Table 6): The mean OGGA value was 6.2±1.8 units/μg protein, significantly lower than in healthy individuals (P=0.0001). Analysis of Normal and Low repair in healthy individuals and in lymphoma patients using logistic regression yielded an adjusted Odds Ratio of 15.2 (95% CI, 3.7-62.5). This means that after adjustment for age, lymphoma patients were 15 times more likely than the healthy controls to have a Low OGGA. This indicates that Low OGGA is a risk factor in lymphoma (Table 9).

OGG activity seems to be reduced in colorectal cancer patients: An analysis was performed with 16 colorectal cancer patients (FIGS. 11a-b). Two of the patients exhibited low OGG (12%). This data indicates that low OGG is a risk factor in colorectal cancer.

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(Additional References are Cited in the Text)

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