METHODS FOR DETERMINING OXIDATIVE INJURY TO DNA

Abstract: The invention relates to a diagnostic and prognostic method for detecting breast cancer at an early stage of its development utilizing analysis of intact DNA specimens by vibrational spectroscopy to determine the lesion profile of the DNA. The method also identifies certain types of DNA lesions, evidenced in spectra profiles having increased infrared absorption, that are characteristic of neoplastic specimens. Tissues having DNA absorption values approaching those values associated with established neoplastic DNA values in these regions are at a heightened risk of impending metastasis or of developing neoplasia unless cellular conditions in the tissue are altered to reduce DNA modifications.

Description: METHODS FOR DETERMINING OXIDATIVE INJURY TO DNA TECHNICAL FIELD: The invention relates to the field of molecular epidemiology and more particularly to the field of DNA lesion analysis through vibrational spectroscopy of lesion profiles to determine the risk of manifesting neoplasia. BACKGROUND ART: For some time, researchers have been analyzing the pathological differences between normal tissue and diseased tissue to identify characteristic differences between the two. In this manner, a diagnostician could use the findings to screen tissue samples and determine whether the observed tissue was cancerous if not apparently so. Of particularly greater importance is the ability to examine a histologically normal specimen taken from a subject not exhibiting neoplasia and propose a risk factor for manifesting neoplasia or tumorigenesis. Such screening procedures are becoming increasingly more common as they allow preventative measures to be adopted prior to the onset of the disease. In the field of molecular epidemiology, it has been shown that an attack on DNA by a reactive oxygen species (ROS) such as by a hydroxyl radical ( # OH), causes modifications to the DNA nucleotide bases. By analyzing the lesion profile of DNA via modified bases, a relationship was found wherein an increased risk of manifesting neoplasia was associated with an increased concentration of oxidatively modified DNA bases when compared to normal or baseline levels. A more complete description of this technology can be found in United States patent application serial number 088,626 (the '626 application) which is incorporated herein by reference. Subsequent extended studies into the role and use of oxidatively modified nucleotide bases identified two product pathways resulting from an attack on cellular DNA by a ROS -- reductive pathways yielding ring opened base products and oxidative pathways yielding hydroxyl adduct base products. It was thus determined that upon attack of the DNA by a ROS, the DNA would mutate and form modified base products that favored reductively or oxidatively derived end products depending upon the cellular redox potential as it affected cellular DNA. form modified base products that favored reductively or oxidatively derived end products depending upon the cellular redox potential as it affected cellular DNA. For a more detailed description of this technology, see United States patent application serial number 055,818 (the '818 application) which is also incorporated herein by reference. These subsequent studies continued to rely on analysis of modified bases by using a Gas Chromatograph Mass Spectrometer with Selected Ion Monitoring (GC/MS-SIM) as a preferred means for identifying the quantity and type of the base lesions. This methodology, however, required substantial preparation of the cellular DNA prior to analysis and the structural composition was lost during analysis due to the nature of the apparatus. While the methodology relied upon in the '626 and '818 applications would give direct evidence of base damage to DNA, direct assessment of base fidelity might not always be possible or appropriate. If it could be established that another biomarker was present in proportion to the degree of base mutations resulting from ROS attack, then analysis of this new biomarker would provide information similar to that identified in the '626 and '818 applications. A well accepted analytical method for determining the atomic and/or molecular structural properties of a specimen is the use of infrared spectroscopy (IRS). IRS causes atoms and groups of atoms of organic compounds to vibrate faster about the covalent bonds that connect them. The IRS method beneficially does not destroy the specimen, and smaller amounts of the specimen are needed in order to conduct an analysis compared to the GC/MS-SIM apparatus. Previous and present researchers have used IRS to establish spectral profiles of whole tissue specimens. Profiles of cancerous and ostensibly "normal" tissue have been obtained and compared. These research efforts have disclosed that as between cancerous and "normal" tissues, cancerous tissues exhibit increased hydrogen bonding of the phosphodiester groups of nucleic acids, decreased hydrogen bonding of the C-OH groups of proteins, and a shift in the 1082 cm "1 absorption peak. See e.g. , Wong, Distinct infrared Spectroscopic Patterns of Human Basal Cell Carcinoma of the Skin, 53 Can. Res. 762-65 (02/15/93). These prior efforts, typified by Wong et al., however, have not concentrated on the cellular DNA. Instead, many researchers believe it to be improper to analyze anything but whole tissues or cells, since to do so would introduce artifacts. Consequently, the results drawn by researchers utilizing intact cells and or tissue to make conclusions regarding the spectra of the samples ostensibly associated with DNA is questionable in view of the presence of similarly constituted biomolecules present in the cell or tissue. Nevertheless, the fact that there exist cellular or tissue level vibrational spectroscopy differences between cancerous and "normal" tissues is of great importance. Noticeably lacking, however, is any discussion of prognostic applicability. DISCLOSURE OF INVENTION The present invention concerns methods for establishing the level of oxidative damage to DNA. By comparing the level of oxidative damage of cellular DNA to a baseline or reference level, the state of the tissue from which the DNA was obtained can be established and the identification of neoplasia or a risk factor for developing neoplasia can be determined. The invention relies on the fact that unique DNA lesion profiles exist between low risk or normal, medium risk or susceptible, and high risk or diseased states. In particular, the invention identifies a progressive lesion profile associated with all stages or neoplasia and indirectly identifies the degree of mutagenic damage due to formed DNA lesions. The DNA lesion profile identified by the inventor concerns the structural modifications to the sugar-phosphate backbone of DNA and deoxyribose moiety of DNA nucleosides or nucleotides. Research has now shown that a characteristic alteration in the vibrational spectral profile of cellular DNA occurs after an attack by a ROS on the DNA: certain ascertainable changes occur to the sugar-phosphate backbone of nucleotides and sugar linked molecule of nucleosides after a ROS attack on DNA, attacks that also causes quantifiable changes to the DNA bases. By assessing the degree of sugar-phosphate modifications or deoxyribose modifications, one can indirectly determine the degree of base modifications and thus assign a probability factor that the type of specimen analyzed will manifest neoplasia if in vivo conditions do not change. In particular, the spectral profile of DNA obtained from a tissue specimen having histological evidence of neoplasia indicates that a strong band of infrared (IR) absorption occurs at approximately 1 250 to 1220 cm "1 , and to a lesser extent at approximately 1385 to 1365 cm "1 and 141 5 to 1390 cm "1 . This strong absorption at around 1240 cm 1 is characteristic of the phosphodiester backbone of DNA exhibiting strong asymmetric vibrations upon excitation by infrared radiation. These vibrations are extremely muted in histologically normal tissues that have been removed from subjects not believed to be at risk of neoplasia. The absorption bands at around 1375 cm 1 and 1405 cm "1 are characteristic of deoxyribose exhibiting a strong vibration as well as vibrations associated with nucleic acids. The absorption band at around 1375 cm "1 is also muted in specimens obtained from histologically normal tissues that were obtained from subjects not believed to be at risk of neoplasia. However, IR spectra show that at around 1405 cm '1 , normal tissue strongly absorbs in this region. Consequently, a DNA profile of relevant spectra for cancerous and non cancerous specimens has been established. To provide prognostic applications for the invention, further research showed that histologically normal tissue DNA did not necessarily exhibit spectral data conforming to the known normal tissue DNA. At the relevant wave numbers described above, the tested DNA specimens were intermediate between the average values for cancerous and non cancerous DNA specimens. The intermediate nature of these specimens was confirmed by comparing the overall spectral profile of the putatively normal specimens against the spectral profile of known cancerous specimens. The results confirmed the propriety of using selected wave numbers as valid parameters for status assignment. Thus, it was established that DNA from apparently histologically normal tissue exhibited, to varying degrees, similarity in spectral profiles to that of known cancerous tissue, both at selected wave numbers and in overall profile. To further establish the effectiveness of the invention, numerous histologically normal specimens were analyzed according to the principles recited herein. The DNA spectral profile of each specimen was compared to a DNA spectral profile that was consistent with the spectra obtained from IDC specimens and given a correlation value which was equivalent to the risk factor for each analyzed tissue. The log of the correlation value was then compared to a the log of the ratio of reductively formed base lesions to oxidatively formed base lesions from those specimens, a method comprehensively described in the '818 patent application. The comparison results confirmed the propriety of using spectral vibrational analysis of DNA to determine neoplastic status and risk. Infrared profiles of DNA from normal and transformed tissues of the female breast have revealed that a distinct IR profile, particularly in the 1262 cm '1 and 810 cm "1 region, is linked to the metastatic state (e.g., as revealed by positive lymph nodes). The distinctive profile has also been found to be present in the microscopically normal (benign) tissue surrounding invasive ductal carcinomas (IDCs) with no evidence for metastasis on the basis of lymph mode or lymphatic- vascular space invasion. Also, a strong correlation exists between IDC with lymph involvement and the occurrence for the "metastatic IR profile" in adjacent tissues of the cancerous breast. Further, some "normal" breast tissues have exhibited early evidence of the metastatic profile, suggesting a definite risk for the ultimate development of metastatic tumors. A fine needle biopsy and the extraction of DNA from the normal, cancerous, and potentially cancerous breast tissue provide a diagnostic tool for predicting the occurrence or risk of breast cancer metastasis before the disease has spread to the lymph nodes and widely throughout the body. While the preferred means for determining the result of ROS attack on DNA and its related components was performed with a Fourier Transform Infrared Spectrometer, other suitable means exists that are well known in the art and include, but are not limited to the following: Ramen infrared spectroscopy, Nuclear Magnetic Resonance analysis, DNA melting curve analysis, circular dichroism, and the use of antibodies. BRIEF DESCRIPTION OF DRAWINGS Fig. 1 shows an average of deconvoluted spectra of DNA obtained from IDC tissue specimens; Fig. 2 shows the mean Euclidean correlation values of DNA obtained from 41 RMT specimens when compared to a library of DNA spectra obtained from IDC specimens; Fig. 3 shows an average of deconvoluted spectra of DNA obtained from RMT tissue specimens having no histological evidence of neoplasia; Fig. 4 shows a comparison of spectra in overlayed fashion between DNA obtained from RMT (solid line) and IDC (dashed line) specimens; Fig. 5 is substantially the same as Fig. 4 but the spectrum of analysis is from 1500 to 1000 cm "1 ; Fig. 6 shows a comparison of spectra in overlayed fashion between DNA obtained from low (dotted line), medium (dashed line), and high (solid line) correlation value RMT; Fig. 7 shows the deconvoluted DNA spectra of a biopsy specimen 1090 (solid line) in overlayed fashion with low (dotted line) and high (dashed line) correlation RMT and IDC (dash-dot line) tissue; Fig. 8 shows the deconvoluted DNA spectra of a biopsy specimen 941 (solid line) in overlayed fashion with low (dotted line) and high (dashed line) correlation RMT and IDC (dash-dot line) tissue; Fig. 9 shows a melting curve in absorbance vs. temperature for DNA obtained from specimens having a low correlation to the IDC spectral profile; Fig. 10 shows a melting curve in absorbance vs. temperature for DNA obtained from specimens having a medium correlation to the IDC spectral profile; Fig. 1 1 shows a melting curve in absorbance vs. temperature for DNA obtained from specimens having a high correlation to the IDC spectral profile; Fig. 12 is a comparison the log of the ratio between Fapy Adenine and 8- hydroxyadenine with the log of the mean Euclidean correlation value of low, medium, high RMT specimens and IDC specimens; and Fig. 13 is a graph showing the mean IR spectral profiles for examples of invasive ductile carcinoma for breast tissue specimens with and without positive lymph node indications of a metastatic state. BEST MODE FOR CARRYING OUT THE INVENTION As detailed above, the invention is characterized as methods for determining the neoplastic status of a specimen based upon the molecular structural profile of its DNA. In order to make this association, the inventor identified general and specific DNA spectral signatures that were consistent among diseased tissue specimens having histological evidence of neoplasia. The instrument chosen to obtain the spectral signatures was a Fourier Transform Infrared Spectrometer (FT-IR) manufactured by Perkin-Elmer, under the model of System 2000. The unit has a liquid nitrogen cooled wideband MCT detector. The first step was to determine the spectral profile of diseased tissue specimens. As with prior studies, breast tissue having pathologically established invasive ductile carcinoma (IDC) was chosen for study. Tissue from each subject from which a specimen would be obtained was immediately frozen in liquid nitrogen after removal from the subject and maintained at -180° C until removed for analysis. A specimen, usually 50 to 100 mg, from each tissue sample was obtained and the DNA extracted therefrom by traditional phenol-chloroform methods. Each DNA specimen was then tested for purity by UV analysis wherein a suitable sample was characterized as having a 260/280 nm ratio of 1 .6 or higher. Thereafter, each DNA specimen was freeze dried at -80 ° C and placed on a lyophilizer and further freeze dried to remove water that would otherwise adversely affect the FT-IR spectra. The DNA specimens were then sealed under vacuum until analysis. To conduct the FT-IR analysis, each DNA specimen was removed from the lyophilizer and placed on a BaF 2 window. Each of the specimens are subsequently flattened to as to permit optimal infrared absorption spectra. With the aid of a microscope targeting apparatus having a 100 micron aperture, which was linked to the FT-IR, the transmission beam of the FT-IR was focused on the cellular DNA and 256 spectrographic scans at various locations in the specimen were then conducted wherein the center burst of infrared radiation had been maximized. The spectral bandwidth for each scan was set for 4000 to 700 cm "1 , resolution for 4 cm "1 , J stop size 2.5 mm, and B stop size 21.5 mm. To store and process the spectral data in a useful fashion, significant spectral absorption peaks must be ascertained and labeled. To carry out this function, the software supplied by Perkin Elmer performed a deconvolution of the spectral data. Deconvolution of the data was carried out by computer and software provided by Perkin-Elmer wherein the width factor was set at 42 and the smoothing factor was set for 30. The deconvoluted spectral data is then linked with PE Grams 2000 software also supplied by the manufacturer and compared to a baseline between 1350 and 900 cm "1 to adjust the spectra and maximize absorption values. After processing the spectra with this software, the spectra from each specimen was analyzed and each significant absorption peak was assigned a Peak Search Unit (PSU). The PSUs for each specimen were then entered into the IDC library to establish the spectral profile for cancerous or diseased tissues. A total of nine (9) diseased or cancerous specimens were analyzed in this manner. The results of the efforts are shown as an average absorption spectra in Fig. 1 . Having then established a library of spectral data for IDC specimens, additional breast tissue samples from cancerous and non cancerous subjects were similarly subject to the FT-IR procedure described above and compared to the IDC library using an Euclidean search routine. The results of the comparisons to the IDC library yielded a Euclidean correlation value. A total of forty one (41 ) tissue samples were thus analyzed and compared. The results of this procedure are shown in Fig. 2. As can be seen, the Euclidean search identified three predominant groups of sample types: a low group consisting of 12 specimens and having a correlation value of between 0.21 and 0.36, a medium group consisting of 15 specimens and having a correlation value of between 0.45 and 0.80, and a high group consisting of 14 specimens having a correlation of between 0.82 and 0.88. IDC specimens were run as a control and averaged a correlation value of 1 .00, thus confirming the consistency of the procedure to the procedure used to establish the IDC library. The spectral results obtained from tissue having the lowest correlation with the IDC library were assigned as having a low risk of neoplasia, assuming that cellular conditions inherent in the tissue would not change. The spectra of the low correlation value DNA samples is shown in Fig. 3. By comparing the average spectral profile of the IDC library to the average spectral profile of low correlation specimens as is shown in Fig. 4, it can be seen that certain identifiable differences exist between the two. In particular, it was found that absorption bands between 1450 and 1390 cm '1 , 1385 and 1365 cm "1 , and 1250 and 1200 cm "1 were significantly different between the specimens of the two tissue types. To better illustrate these differences, Fig. 5 shows an overlay of Figs 1 and 3 from between about 1500 and 1000 cm '1 wherein the two spectra have been normalized as discussed below. The band occurring at around 1410 cm "1 is indicative of IR absorption of the 2'- deoxyribose moiety; the band occurring at around 1375 cm "1 is also indicative of IR absorption of the 2'-deoxyribose moiety; the band occurring at 1240 cm "1 is indicative of asymmetrical IR absorption the phosphodiester moiety present in all nucleotides. These spectral differences observed between the IDC and the low correlation specimens were in agreement with the proposition that ROS attack on DNA leads to base damage thereby creating oxidatively modified nucleotide bases. The modification of a DNA base will cause a certain change in the DNA spectra associated with the sugar-phosphate backbone. Spectral changes occur in part because of stearic alterations induced by the oxidative bases on the conformation of the sugar-phosphate backbone. Additionally, spectral properties will be modified by introduction of oxidized base structures. Multiple stable products can be formed, at least some of which result in strand breakage. The relevant chemical environment surrounding the carbon centered radical, which affects radical stabilization and reaction products, can be altered by the relative excess or deficiency of free electrons, i.e. that is the redox condition of the chemical environment surrounding the cell and DNA. The differences also showed that such attacks were not limited to the actual purine or pyrimidine bases but were also manifested in the sugar-phosphate backbone of the DNA helix. ROS attack on the nucleotide backbone residue in the form of carbon centered radicals potentially at each carbon of the deoxyribose moiety depends upon the nature of radical stabilization -- a factor of which is a function of the chemical environment surrounding the carbon centered radical. While identifying a new method for establishing the presence or absence of cancer, or more generally neoplasia, is desirable, it was a specific objective of the inventor to identify a DNA profile that would indicate that a tissue from which a specimen was obtained was "at an increased risk" of manifesting neoplasia. It was speculated that at risk tissue would have overall spectral profiles and particularly spectral peaks intermediate to those previously identified and associated with cancerous and non cancerous tissue DNA. This speculation was due in part to the results obtained when attempting to establish a correlation between reduction mammoplasty tissue (RMT), i.e. the normal tissue, which was considered putatively not at risk and the IDC library. As indicated previously, the RMT correlations did not show consistent correlation with the IDC library as did the cancerous tissue specimen correlations. As shown in Fig. 2, the correlations actually comprised two groups significantly divergent in correlation values, with a third group, the medium correlation group, having values intermediate the low and high correlation value groups. The spectra obtained from the medium correlation specimens were averaged and the DNA spectral profile compared to both the high correlation profile and the low correlation profile. By simultaneously analyzing the spectral profiles of the average of the high, medium and low correlation profile spectra, it can be seen that not only were the correlation values intermediate between the high and low correlation specimens, but the spectra at the wavelengths of interest were also intermediate the two specimen types. It should be noted that all comparison spectra described here in have been normalized. This has been carried out by identifying one or more reference peaks that do not or should not vary significantly between correlation groups under study. For purposes of research, the chosen reference peak(s) occurs around 1080 to 1050 cm 1 and represent either the phosphodiester symmetrical stretching or the C-O vibration occurring in the deoxyribose molecule. In either case, these absorption peaks do not vary significantly, either by absorption or wave number shift, between cancerous and known normal DNA specimens. Alternatively, the reference peak(s) could remain the same and indirectly normalized by ratios wherein each absorption band of interest is adjusted proportionally by the amount that would be necessary to normalize the reference peak. To further illustrate the prognostic utility of this methodology, Fig. 7 shows a DNA profile of a biopsy sample (sample 1090) removed from a subject during the removal of a suspected lump. Previous to this surgery, the subject had undergone surgery for removal of a tumor in the breast. The pathology report for biopsy 1090 indicated that there was no evidence of residual or in-situ carcinoma in the test specimen. Yet as shown in the figure, the DNA spectral profile is generally intermediate to the profile for the low correlation DNA specimens and the high correlation DNA specimens as well as the IDC reference specimens. Moreover, the spectral profile is nearly at all time intermediate to the low correlation and IDC DNA specimens. Thus, one would conclude that the tissue from which the specimen was obtained was at an elevated risk of further neoplastic development, likely cancer. Knowing that the tissue was obtained from a subject having already exhibited IDC, such a conclusion is not unexpected. This result was also consistent with research showing a five (5) fold greater increase in reoccurrence of cancer in residual tissue after removal of original cancer compared to subjects not having had cancer. A similar illustration is presented in Fig. 8. This time, however, the pathology report for the biopsy sample (sample 941 ) stated that the tissue sample was negative for atypia and malignancy. Yet the spectral profile indicated that at all relevant wave numbers, the tissue was at an increased risk of developing neoplasia or cancer because the absorption level was intermediate to the normal tissue profile and the extreme risk tissue profile. The biopsy tissue was obtained during a routine breast reduction surgery. While less selective, it is also possible to determine the neoplasia risk for a tissue specimen by. comparison of the deconvoluted spectral profiles for a DNA sample to a profile library of known status. This methodology was employed when analyzing the RMT DNA specimens, and as will be discussed below, proved to be exceptionally accurate when compared to analysis of modified base lesions. As further evidence of the impact of ROS attack on DNA and its role in neoplasia, the inventor subjected the various tissue groups (low, medium, and high correlation) to melting curve analysis. The data was obtained from a Perkin-Elmer Lambda 2 spectrophotometer operating at 260 nm with a temperature program varying from 25 to 90° C at a rate of 1 ° C per minute. The results of these tests are illustrated in Figs. 9, 10, and 1 1 . The sharp transition slope (from about 78° C to about 87 ° C) of the melting curves shown in Fig. 9 are indicative of intact duplex DNA having a high molecular weight. The specimens analyzed in this Fig. were obtained from low correlation value RMT samples. In Fig. 10, which represents analysis of medium correlation value RMT, a noticeable decrease in the transition slope is apparent. In Fig. 1 1 , the transition slope for high correlation value samples is almost unidentifiable in certain specimens. The high frequency of specimens demonstrating such submelting behavior is characteristic of DNA containing significant strand breakage. The high incidence of strand breakage in the IDC and transitional RMT DNA indicates that inherent structural differences exist in the nucleotide backbone structure of these DNA classes. These results are fully consistent with the alterations shown by the FT-IR spectra shown previously. Thus, substantial strand breakage occurs in cancer derived specimens as opposed to the formation of stable products not associated with strand breakage but capable of modifying the FT-IR spectra of the nucleotide backbone in the normal RMT tissues. As detailed above, the present findings are supported by the inventor's work with oxidatively modified nucleotide bases as analyzed by means of a GC/MS-SIM. Figure 12 demonstrates the correlation between the two methodologies. Through statistical analysis, the inventor found that a model for determining tissue status (cancerous and non cancerous) and predicting the likelihood of neoplasia had a sensitivity of 91 % and a specificity of 96%. When the methods described herein are compared against the GC/MS-SIM derived model, a direct variation of both parameters was observed, indicating a close correlation of results between the two methodologies. Thus, the present model is not only internally consistent, but has been verified using another technique for diagnostic and prognostic applications, as well as confirming that the observed differences in spectral profiles are related to ROS attacks on DNA and are at least partially related to the degree of DNA base modifications. To evaluate the procedure outlined above for diagnostic and prognostic use in detection and treatment of human breast cancer, tests were conducted on human breast tissue from subjects with and without lymph node manifestation of metastasis. Infrared profiles of DNA from normal and transformed tissues of the female breast have revealed that a distinct I R profile, particularly in the 1262 cm '1 and 810 cm "1 region, is linked to the metastatic state (e.g., as revealed by positive lymph nodes). The distinctive profile is present in the microscopically normal (benign) tissue surrounding invasive ductal carcinomas (IDCs) with no evidence for metastasis on the basis of lymph mode or lymphatic-vascular space invasion. Also, a strong correlation exists between IDC with lymph involvement and the occurrence for the "metastatic IR profile" in adjacent tissues of the cancerous breast. Further, some "normal" breast tissues have exhibited early evidence of the metastatic profile, suggesting a definite risk for the ultimate development of metastatic tumors. A fine needle biopsy and the extraction of DNA from the normal, cancerous, and potentially cancerous breast tissue provide a diagnostic tool for predicting the occurrence or risk of breast cancer metastasis before the disease has spread to the lymph nodes and widely throughout the body. In Fig. 13, mean spectral profiles of breast tissue having invasive ductal carcinomas is shown, based upon one group of eighteen subjects whose lymph nodes did not exhibit positive symptoms of metastasis and fourteen subjects with positive symptoms of metastasis in lymph glands. Two distinct and characteristic peaks at about 1262 cm "1 and 810 cm '1 are shown for IR spectral profiles in both sets of subjects. Healthy tissue did not exhibit these peaks. The foregoing methods for analyzing DNA removed from a tissue sample are not exclusive. The invention has identified several relevant properties associated with a DNA profile and consequently, other means known to persons skilled in the art are contemplated. For example, Ramen spectroscopy can be substituted for the FT-IR procedure with little modification. With knowledge that the sugar-phosphate backbone is physically altered, use of Nuclear Magnetic Resonance can also be employed as well as circular dichroism and mono or polyclonal antibodies. In addition, knowledge that strand breakage is more prevalent in medium and high risk samples permits the use of DNA melting curve analysis to determine DNA structure and tissue risk. The examples cited above used breast tissue. Because DNA is inherent in all living organisms, the methods of the invention are not limited to the use of breast tissue samples. Tissue samples from other organs are equally susceptible to ROS attack and the methods described herein are equally applicable. In addition, the type of specimen is not limited to tissue. Fluids are equally suitable samples. In addition, DNA need not be isolated in order to carry out the invention. , For example, it is possible to examine DNA directly using the FT-IR device described above. By specifically limiting the IR scan to cellular nuclei, spectral profiles of high concentration will be generated. Such a method will enable streamlined procedures for analyzing DNA spectral profiles. Isolation of DNA was used by the inventor to establish the methodology, not to limit the various applications apparent thereafter. As described in the '626 application, use of the present invention will enable analysis of environmental hazards. By analyzing the DNA spectral profile of an organism prior to its introduction to an environment of unknown genotoxicity and comparing that profile to a DNA spectral profile obtained from the organism after exposure to the environment, an assessment of the genotoxicity of the environment can be made.

Claims: What is claimed: 1 . A method for diagnosis of a cancer of precancerous condition at a stage before onset of a metastatic state comprising the steps of: obtaining a biopsy specimen from a patient; preparing an infrared profile of DNA from said specimen; reviewing said profile for distinctive characteristics at about 1262 cm 1 and 810 cm 1 ; said distinctive characteristics indicating the presence of a cancerous or precancerous state even before onset of cancerous invasion into lymphphatic and vascular space. 2. The method of claim 1 wherein said tissue is human breast tissue. 3. A method for establishing the cancerous or pre-cancerous state of a specimen removed from human breast tissue to determine the status of the tissue wherein the status relates to a probability of developing neoplasia comprising the steps of: a) isolating a tissue or fluid sample from a test subject; b) removing a specimen from the sample; c) analyzing the DNA lesion profile of the specimen to establish an IR spectral profile; d) examining the spectral profile for distinctive absorbance peaks at about 1262 cm "1 and 810 cm '1 , said distinctive absorbance peaks being indicative of a cancerous or pre-cancerous state. 4. A method for establishing the DNA profile of a specimen removed from a tissue to determine the status of the tissue wherein the status relates to a probability of developing neoplasia comprising the steps of: a) isolating a tissue or fluid sample from a first test subject; b) removing a first specimen from the sample; c) analyzing the DNA lesion profile of the first specimen; d) comparing the DNA lesion profile of the first specimen to a baseline profile of DNA from a tissue or fluid specimen obtained from one or more samples similar to the type obtained from the first test subject and having established neoplasia wherein the tissue obtained from the first test subject is at a progressively greater risk of manifesting neoplasia as the DNA profile progressively approximates the profile of the baseline.

Inventors: MALINS DONALD C, MALINS, DONALD, C.
Applicants: MALINS DONALD C, PACIFIC NORTHWEST RESEARCH FOU, MALINS, DONALD, C., PACIFIC NORTHWEST RESEARCH FOUNDATION
Application year: 1995
Main classification: G01N33
IPC codes: A61B17, C12Q1, G01N33