PT99981A - PROCESS FOR THE DIAGNOSTIC DETECTION AND LOCATION OF CANCER IN PATIENTS BY NUCLEAR MAGNETIC RESONANCE OF A LIQUID SAMPLE - Google Patents

Description

3

Reference to Related Patent Applications The present invention is a continuation-in-part application of the pending pending U.S. application Serial No. 418,182, filed as a continuation-in-part of USSN 325,773 filed October 6, 1989, filed as a continuation from the now abandoned USSN application 262.073, filed as a continuation of USSN 188,752, now abandoned, filed as a continuation of USSN 036.943, now abandoned, filed as a divisional application of ERIC T. Fossei's US Series No. 833,840, filed on 26 February 1986, now abandoned.

Declaration on government-supported research

Federal

The funds for the work described here were provided by the Federal Government in the form of a grant from the Department of Health and Human Services. The Government may have certain rights to this invention.

Field of the Invention The present invention relates to a method for detecting and locating cancer in a living patient. 4

// t \ y, ·. BACKGROUND ART Methods utilizing Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) techniques to assist in the clinical diagnosis of cancer are well known in the prior art.

Damadian was the first to propose a medical application for NMR. He suggested that it be used to detect malignant tumors in tissues. See R. Damadian, " Tumor Detection by Nuclear Magnetic Resonance ", Science 171: 1151-1153 (1971). U.S. Patent 3,789,832 to Damadian covers an apparatus and method for the application of nuclear magnetic resonance to surgically removed specimens to measure Ti and T2 relating to proton relaxation times, the values of which, compared to tissue values healthy, were taken as an average for the diagnosis of cancer. U.S. Patents 4,411,270 and 4,354,499, issued to Damadian, cover an apparatus and method for the detection of cancer with NMR imaging and scanning of specimens throughout the body. Several other investigators have also reported that nuclear magnetic resonance (T ^) relaxation times for the protons in tumor-bearing organs are higher than the corresponding T1 values for the aqueous structure in organs of healthy animals. See Frey et al, J. Natl. Cancer, Inc., 49, 903 (1972); Inch et al, J. Natl. ϊ.η £ ΐΐ. 52, 252 (1974); Iijima et al, Physiol. Chem. and Physics, 5, 431 (1973); and Hazlewood et al, J. Natl. Cancer Inst. , 52, 1849 (1974).

Nowadays, despite some uncertainty regarding mechanical details, it is known that biophysical changes occurring in malignant cells often alter the proton nuclear magnetic resonance signal. See D.G. Taylor et al., &Quot; A review of the Magnetic Resonance Response of Biological-gical Tissue and Its Applicability to the Diagnosis of Cancer by NMR Radiology ", Computed Tomography, 5: 122-133 (1981). These changes form the physical basis for the detection of tumors by the representation of proton nuclear magnetic resonance images. See R. Zimmerman et al., Cerebral NMR: Diagnostic Evoluation of Brain Tumors by Partial Saturation

(1985) and K. Ohtomo, " Hepatic Tumors: Differentiation by Transverse Relaxation Time (T) of Magnetic Resonance Imaging ", Radiolo-gy 155: 421-423 (1985).

Studies of proton NMR in extracted, as well as plasma and serum tumors from experimental animals and patients have often revealed differences in the relaxation parameters T ^, T ^ and T * (T = a combination of T ^ from the intrinsic relaxation and relaxation induced by inhomogeneities of the magnetic field (as a function of malignant tumor (s)) .These findings were described by the following: L. Mclachian, " Cancer-induced Decreases in Human Plasma Proton NMR Relaxation Rates ", Phys. Med. Biol. 25: 309-315 (1980): F. Smith et al., Nuclear Magnetic Resonance Imaging of the Pancreas, Radiology 142: 677-680 (1982); P. Beall et al, " The Systemic Effect of Elevated Tissue and Serum Realation Times for Water in Animals, and Humans with Cancers ", NMR Eteic Principles and Progress, P. Diehl et al, Eds., 19: 39-57 (1987), R. Floyd, " Time Course of Time Water Proton Spin Lattice Relaxation in M-ice DEveloping Tumor Ascites ", Cancer Res. 34:91 (1974); C. Hazlewood et al. &Quot; Relationship Between Hydration and Proton Nuclear Magnetic Resonance Relaxation Times of Tumor Bearing and Non-Bearing Mice: Implication for Course Detection ", J. Natl. Cancer Inst., 52: 1849-1853 (1974); and R. Klimek et al., " A Discussion of Nuclear Magnetic Resonance (NMR) Relaxation Time of Tumors in Terms of Their In-terpretation as Self-Organizing Dissipative Structures and of Their Study of NMR Zeugmatopgraphic Imaging ", GineSol Pol. 52 : 493-502 (1981).

However, due to the extensive overlap of groups and small differences between the means of the groups, these methodologies are not clinically useful.

While a large part of the aforementioned prior art suggests the use of NMR to analyze tissue, it is also known that body fluids are subjected to such analysis, as described by Beall et al., Supra. 7

ra aqueous " In others it was known as water) of the spectrum

Prior art NMR methods cited for de-tecting malignant tumors are based on the interpretation of the composite NMR signal from all protons in tissue samples or blood derivatives. This composite signal is dominated by water protons, obscuring the - NMR signal of other constituents of the proton-containing sample. Certainly the prior art believed that the apparent correlation between the malignant tumor (s) and the observed changes in the NMR parameters was due to changes in the structure-refering Frey et al., Supra. In a preferred embodiment of the present invention, it is found that NMR components having a significant predictive value can be disguised by other materials in the sample By eliminating the aqueous signal, The previously disguised spectrum of these components has been disclosed. Patent pending application of Eric T. Fossei entitled " Process for the Detection of Cancer, by Use of Nuclear Magnetic Resonance ", US Serial No. 303,586, filed January 27, 1989, the disclosure of which is hereby incorporated by reference, the above-mentioned findings have been incorporated into a safe method for diagnosing cancer in a living patient.

According to this invention, a sample of a patient's body fluid is subjected to the Nuclear Magnetic Resonance Spectrum. A resonance line generated by a non-aqueous, sample component is selected by measuring the full width of this line of resonance at a given height, for example, half of its height. The full width thus measured proved to be a statistically safe measure of the presence or absence of cancer in a patient. 8

The above described plasma water free proton NMR test discriminates between people with untreated and other cancers with an accuracy greater than 90%. As such, the test was widely hailed as one of the decade's most important inventions. No previous non-invasive cancer test reached such a level of accuracy, however, false positive results were obtained.

In co-pending application of Eric T. Fossei, entitled " Process for the Detection of Cancer using NMR of (carbon 13), USSN 295,746, the teachings of which are hereby incorporated by reference, it has been shown that the main source of positive results false is high plasma triglyceride levels. Said invention teaches a non-invasive method and apparatus with improved accuracy over the previous non-invasive methods for determining the presence of cancer in a living patient.

According to this invention, the triglyceride level is measured in patients with a positive result in the proton NMR scanning test. A normal triglyceride level confirms the diagnosis of cancer; however, fluid samples from patients with elevated triglyceride levels are subjected to C-13 NMR. An abnormal result in this test confirms the diagnosis of cancer, while a normal result indicates that the previous diagnosis was a false positive diagnosis.

In the copending patent application of Eric T. Fossei entitled " Apparatus and Method for the Detection of Cancer Using Nuclear Magnetic Resonance " (" Apparatus and Method for De-tection Cancer Using Nuclear Magnetic Resonance ") USSN 418,182, filed October 6, 1989, whose teachings 9

incorporated herein by reference, it was shown that a diagnosis could be made by an automated process incorporating proton NMR and C-13 NMR spectroscopy.

According to this invention, an apparatus was provided which automated the process for diagnosing cancer using NMR. The apparatus has a spectrometer component capable of taking non-aqueous proton NMR and C-13 NMR readings from a fluid sample. Additionally, the apparatus has a computer for processing proton and C-13 readings, and for obtaining a numerical value corresponding to those readings. The apparatus further comprises a memory for storing a set of standard normal values and comparing said stored values with the values obtained from the NMR. The computer program that directs your function, analyzes the data and creates a diagnosis with a high degree of precision. Diagnosis is only the first step in the treatment of cancer. It has to be followed by the identification of particular malignities (of malignant signs). There is in the prior art nothing which describes a method using a method of obtaining NMR spectra, interpreting those spectra, drawing a diagnosis, and then, when diagnosing positive cancers, locating cancer by viewing the images magnetic resonance imaging (MRI). It is highly desirable to couple an MRI apparatus for an initial diagnosis and MRI visualization of the whole body, leading to a specific and extremely safe diagnosis of cancer. 10

SUMMARY OF THE INVENTION

According to the present invention, a method has been developed for detecting and localizing cancer through the use of a nuclear magnetic resonance apparatus and a magnetic resonance imaging system. The method and apparatus are capable of generating a diagnosis using proton NMR spectroscopy, factoring levels and, if

In the case of a true positive reading in either of the triglyceride or C-13 phases, the patient is then submitted to whole-body MRI for identify the malignant tumor or tumors.

In preferred embodiments, the sample fluid is blood, spiral fluid or plasma from the bone marrow; the blood is especially advantageous. The component of interest is lipid, preferably the methyl and methylene groups of the lipoprotein lipids.

Thus, it is an aim of the present invention to provide an accurate method of diagnosis using an NMR spectroscopy method for detecting the presence of cancer in a living patient, and when there is a true positive diagnosis of cancer, immediately using a scan per view magnetic resonance imaging to identify and locate the malignant tumor (s).

Other objects and advantages of the present invention will become apparent from the descriptions of the drawings and the following description.

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Brief Description of the Drawing Fig. 1 represents a typical 360 MHZ NMR spectrum for the non-aqueous components (without water) of a plasma sample from a healthy control obtained according to the first step of the present invention ; Fig. 2 represents an NMR spectrum for the same plasma sample, from which the spectrum of Fig. 1 was obtained, using the same equipment and pulse rate, but without water suppression; Fig. Fig. 3 is an enlarged view of the methyl and methylene regions from the sample reading of Fig. 1; 4 is an enlarged view of the methyl and methylene region of an NMR spectrum for a plasma sample of a patient with an untreated malignant tumor;

Figures 5A and 5B depict C-13 NMR spectra of a plasma sample from the olefinic region for a normal control and an untreated cancer patient respectively obtained according to the first step of the present invention;

Figures 6A and 6B represent views of C-13 NMR spectra between 10 ppm and 90 ppm, with the particular inclusion of the region included between 48 ppm and 80 ppm, of the plasma samples shown in Figures 5A and 5B for normal control and a patient with untreated cancer, obtained according to the first step of the present invention; η //

Figure 7 schematically shows the first phase of the apparatus of the present invention; Fig. 8 shows the results of a study using the method of the present invention; Fig. 9 shows a flowchart diagrammatically plotting the operation of the invention; 10A shows a flowchart for filling, an operation performed by the first stage of the apparatus which ensures reproducible results from readings to which water has been suppressed. Fig. 10B shows a sample program for filling corresponding to the proton spectrum phase of the flow chart in Fig. 10A. Fig. 10C shows a sample program for filling corresponding to a C-13 spectrum; Fig. 10D shows a sub-program of phase 8, RJ FQSET, of the filling program shown in Fig. 10C; Fig. 10E shows a sub-program of phase 2, RJ TUNE, of the program shown in FIG. 10B; and Fig. 10F shows a sub-program in Fig. 10B.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the beginning, the invention is described in its broader general aspects followed by a more detailed description. The present invention is a method for detecting the presence and location of cancer in a living patient. According to the present invention, a patient's body fluid sample is subjected to proton nuclear magnetic resonance spectroscopy to generate a nuclear magnetic resonance spectrum. Since the components of the NMR spectrum, which have a significant predictor value, can be hidden by other materials in the sample, dissimulation is eliminated to produce an informative nuclear magnetic resonance spectrum. A resonance line generated by a non-aqueous compound of the sample is selected, a predetermined width, preferably the total width, of the resonance line being measured at a predetermined height, e.g., half its height, in order to provide a safe measure for viewing the cancer in the patient. The above theory or process is described in co-pending U.S. Serial No. 303,586, the disclosures of which are hereby incorporated by reference.

In practice, a component of the NMR apparatus makes the measurement, or measurements, if more than one resonance line is selected, and compares the mean value with a stored value, which is an indicator of the normal value; i.e., to a cancer-free person.

A positive reading may indicate the presence of cancer in the patient, or it may be a false positive reading. It has been found that an important source of false positive readings are people with elevated plasma triglyceride levels. Of this 14

If the measured value of the resonance line is higher than the stored normal value, the program will diagnose that the patient is free of cancer. However, if the measured value is less than the stored value, the apparatus will direct the apparatus in order to obtain a measure of the patient's triglyceride level. In order to establish a difference between true and false positive readings, samples with high triglyceride levels will be subjected to C-13 NMR spectroscopy. This will result in a diagnosis. False positive results, due to hypertriglyceridemia and, conversely, the presence of cancer in the patient, can be safely determined from certain characteristics of the C-13 spectra.

If a positive cancer detection is obtained, either a positive proton NMR spectroscopy reading and a normal triglyceride level, either proton positive NMR spectroscopy, a high triglyceride level and a positive C-13 result, the patient is then subjected to magnetic resonance (MRI) spectra of the whole body to determine the location of the malignant tumor.

According to one embodiment of the present invention, proton NMR spectroscopy is performed initially on the sample to be tested. The water-free proton NMR spectra obtained in human blood plasma is dominated by the lipoprotein lipid resonances of the plasma. Without water suppression, these non-aqueous resonances are virtually erased by water. The means of the signals, allows the observation of the resonances of some parts (some clusters) associated with the components of the body fluid no. in high magnetic fields, even in the presence of water resonance. The ability of modern NMR spectrometers to almost completely suppress the proton resonance of water facilitates this reading. The non-aqueous Proton Nuclear Magnetic Resonance Spectrum of plasma is essentially that of plasma lipoproteins and some low molecular weight molecules. Protons of plasma proteins are obscured since they comprise a broad patch of unresolved resonances. The sharpest resonances of the protons of the more mobile lipoproteins are superimposed on this broad bottom. The NMR spectrometer of the present invention operates on any body fluid containing lipids. Whole blood, serum, or plasma may be used. Although the assay can be performed on any lipid-containing body fluid, work has so far focused on blood plasma. In the blood, lipids, including cholesterol, triglycerides and phospholipids are present in the form of lipoprotein. The cancer assay will typically be performed in vitro, preferably in serum or plasma. The fluid selected from a person to be swept to find cancer is exposed to a filmed magnetic energy and a radio frequency energy to generate a nuclear magnetic resonance signal which is then processed by the Nuclear Magnetic Resonance (NMR) spectrometer, which obtains a value for a selected parameter, eg W 1/2, for the methyl and / or methylene lipid protons. A relatively wide range of proton frequencies, e.g. 60MHz or higher, may be employed; at 360 MHz or higher is a preferred frequency. If costs are not a preferred factor, then 500 MHz may be the preferred frequency. 16

Figure 1 shows a non-aqueous proton spectrum of healthy control, and Figure 2 shows a proton spectrum of the same sample without water suppression. The truncated resonance line of water is marked with (A) in Figure 2. The resonance lines between 0.5 and 2 ppm (parts per million resonance frequency) are derived from the methyl and methylene groups of the lipoprotein lipids. A broader view of this region of the proton spectrum is shown in Figure 3 for a normal control and in Figure 4 for a patient with untreated malignant tumors. Thus, in its preferred embodiments, the present invention uses one of a number of conventional water suppression techniques, i.e., techniques for suppression of the aqueous proton NMR signal. Numerous techniques have been devised to suppress the proton NMR signal from water in other contexts. These can be divided roughly into two categories: (1) those whose aim is not to stimulate the aqueous proton signal, eg rapid scan correlation spectroscopy and the selective excitation technique, and (2) those that cause the aqueous proton magnetization is extremely small at the time the observed radio frequency pulsation analyzer (RJ) is applied, eg the inversion and saturation recovery technique. These and other solvent suppression techniques are described by PJ Hore in " Solvent Suppression in Fourier Transform Nuclear Magnetic Resonance ", Journal of Magnetic Resonance 55: 283-300 (1983) and the footnote references contained therein. water suppression is preferred when using a conventional NMR apparatus because of its inability to distinguish between the solvent proton signal and that of the pool or species of interest, a sufficiently sensitive apparatus would eliminate the need for total water suppression. or

Half the width of the resonance line at a predetermined height, preferably mid-height, e.g. in the methyl and methylene groups, associated with the plasma lipoprotein lipids, constitutes the variable of interest. The total width at half-height W1 / 2 (line width) of an NMR resonance line is inversely proportional to the apparent spin-spin (T2 *) relaxation time, ie W 1/2 --i --- ' m # 2 The value detected for the selected parameter is compared with the corresponding parameter for healthy controls, this comparison being preferably done by computer within the NMR apparatus. In a preferred embodiment, the mean values for the methyl and methylene protons are determined, averaging 33 Hz or less at a proton frequency of 360 MHz (8.45 T) per 400 MHz (9.40 T) as a indication of malignant tumor.

If a positive reading of the non-aqueous proton NMR spectra of a patient's plasma is obtained, a second level of exams is performed to confirm the diagnosis. First, a conventional test, usually termed triglyceride analysis, is performed to determine the patient's triglyceride level. If the triglyceride level is normal, the positive reading of non - aqueous proton NMR spectroscopy is a true positive reading and indicates the presence of cancer in the patient, in which case the patient would then be subjected to MRI scan of the whole body, to determine the location of the malignant tumor (s). 18

If, however, the triglyceride level is above the normal level, the NMR spectrometer apparatus is directed, preferably by computer means, to obtain a spectrum of.

C-13 NMR of the patient's plasma sample, which is already available due to the previous proton NMR spectrum, in order to differentiate between true and false positive results. False positive results due to hypertriglyceridemia can be safely distinguished from true positive results by substantial differences in the characteristics of C-13 spectra. Thus, the plasma sample already obtained from the suspect patient to be diagnosed is exposed to a magnetization and radio frequency magnetic energy to generate a C-13 nuclear magnetic resonance spectrum.

Initially, the olefin region, 120-140 ppm, of the spectrum is examined. Two peaks will appear, one at about 128-129 ppm and another at approximately 130-131 ppm, at a distance of about 2ppm. The ratio of the general region resonance of 128 ppm to that of 130 ppm is cancer determinative. In plasma readings from normal controls and people with non-cancerous disease, the height ratio of these two resonances (128/130 ppm) is 0.9 or greater, ie the peak resonance at 128 ppm is approximately equal to or of a height higher than the 130 ppm peak. The peak heights are measured by computer from the center of the reference line noise to the top of the peak. In plasma readings of patients with untreated cancer, the ratio of peak heights is less than 0.86, or the peak resonance at 130 ppm is higher in height by at least 5% from the peak at 128 ppm. It should be noted that in patients with hypertriglyceridemia, the ratio of resonance height (128/130) is the same as normal control values. In this way, the

NMR spectrometer apparatus will calculate the proportion of peak heights already measured, and if the ratio is greater than a stored value, it will diagnose the patient as healthy, but if not (otherwise) it will provide in cancer diagnosis. In a preferred embodiment, the stored (recorded) value is 0.9. A diagnosis of cancer will mean that the patient is then subjected to MRI scanning, as described above.

Figures 5A and 5B show the olefin regions of spectra drawn at 125.76 MHz with a wide-band proton decoupling from a normal control patient and a patient with untreated cancer. In Figure 5A, the ratio is 1.14 in the normal control patient and in Figure 5B the ratio is 0.70 in the untreated cancer patient. In patients with hypertriglyceridemia who have been studied, the proportion varies between 1.05 and 1.68.

Changes in the olefin region of the spectra of untreated cancer patients can be explained by increases or decreases in the polyunsaturated fatty acid chains in the lipids. The levels of oleic and linoleic acid are particularly indicative. Oleic acid is a monounsaturated fatty acid produced by the human body. Linoleic acid is a diunsaturated fatty acid and is not produced by the human body but obtained by consumption. Dietary fatty acids include polyunsaturated acids, such as linoleic acid. A peak resonance in the general region of 128-129 ppm evidenced only linoleic acid in the pacante. A peak resonance in the general region of 130-131 ppm evidences oleic acid and linoleic acid in the patient. 20

It has been found that the height of these resonance peaks in relation to each other are affected by certain patient conditions. For example, people with elevated triglyceride levels generally have a high proportion of linoleic acid levels for oleic. It has been found that patients with untreated cancer have low levels of linoleic acid in their bodies, presumably because the cancer causes the oxidation of polyunsaturated fatty acids, including linoleic acid. This is consistent with the hypothesis that lipids are oxidized by free hydroxyl radicals in cancer patients, since polyunsaturated fatty acids are more susceptible to oxidation.

Thus, if the patient has elevated triglycerides and untreated cancer, the peak resonance at 130 ppm will be higher, reflecting the decrease in linoleic acid at both peaks. If, however, the peak at 128 ppm not less than 130 ppm at more than 7%, no depression occurs or insignificant depression of linoleic acid levels occurs, the positive result being obtained from the proton NMR spectra confirmed as a false positive result. In that case, the NMR spectrometer apparatus provides a diagnosis that no cancer is present.

In addition, the region of spectra between 48 ppm and 80 ppm is far more complex in plasma with untreated cancer than in normal control plasma or hypertriglyceridemia plasma.

By " more complex " it is understood that there are more resonance peaks in the region. A peak of resonance is counted by the program if its height is at least 50% greater than that of the background noise during a normal test period. As those skilled in the art will know, the more spaced the data collected the better the resolution. Figures 6A and 6B show this region for normal control plasma and for untreated cancer plasma, respectively. These spectra were obtained at 125.76 MHz using a 5 mm test tube and 14 hours of accumulations. C-13 spectra with appropriate information can also be obtained, at 90.5 MHz in 10 mm tubes or in longer tubes.

Naturally, changes to the parameters of the assay process will be apparent to any person skilled in the art.

These parameters include sample tube size, pulse width, pulse repetition rate, and exponential multiplication of free induction decline by different factors. For example, it is obvious to any person skilled in the art that the higher the sample tested, the faster the spectra of suitable quality will be obtained. Other changes in the conditions referred to herein will be apparent to any person skilled in the art. C-13 spectroscopy or MRI scanning may initially be performed on a patient as a method of diagnosing the presence of cancer without first obtaining a proton NMR spectrum as described above. Although it generally takes 30 seconds to obtain proton NMR spectroscopy, a C-13 spectrum can take from one to fifteen hours, so it is very costly and costly. Thus, according to a preferred embodiment, C-13 spectroscopy is used to verify the positive results obtained from proton NMR spectra and statistically and clinically illuminates significant differences in a plasma C-13 spectrum between the true and false positive results of the test.

of proton non-aqueous NMR spectroscopy. MRI scanning of the whole body is equally expensive. The use of C-13 NMR spectroscopy prior to MRI scanning may prevent the use of another piece of machinery as well as unnecessarily subject patients to false positive readings to other medical procedures.

In preferred embodiments, a magnetometer with a constant field strength magnet is used and the NMR signal is transformed by the Fourier method, where the NMR parameters of interest constitute the entire line width at half height , for the proton resonances of the methyl and methylene groups and the C-13 resonances of linoleic acid.

As noted in U.S. patent application Serial No. 8,818,840, proper sample preparation and execution is essential for carrying out a successful measurement of a plasma sample. Blood is collected in tubes containing 70 microliters of a 15% Na2 EDTA solution. Blood is maintained at 4 ° C until centrifugation. Plasma was separated and stored at 42C until NMR analysis. Plasma samples have never been frozen since freezing destroys the lipid structural integrity of lipoproteins. Samples showing any visible signs of hemolysis were excluded.

All spectra were obtained at 20-22 ° C and with magnetic field strengths of 360 MHz or higher. Samples were individually filled by computer in the free proton induction decline area to full width at half the height of the water resonance to be 4 Hz or less.

Of course, careful filling is an assumed component of good laboratory NMR technique. 23

It can be seen that from those experimental conditions, the temperature and the fill are not as critical with C-13 NMR spectra-copy because no line width measurement has been made.

Of course, the field strength used will determine the time required to obtain a spectrum. In addition, accurate diagnosis requires careful review of the patient's medical records.

Figure 9 shows a flowchart of the process operation in which a sample of body fluid is obtained and subjected to NMR spectroscopy to give a non-aqueous H-1 spectrum. The apparatus then selects and measures the resonance lines and discovers a median line width comparing to the 33 Hz value, a predetermined normal value. If the average line width is greater than (or equal to A?) 33 Hz, the device will produce a negative diagnosis; otherwise, you will get a measure of the triglyceride level in the patient. If the triglyceride level is below 190 mg / dl then it will provide a positive diagnosis for the cancer and the patient will undergo MRI scans of the whole body. If, however, the triglyceride level is greater than 190 mg / dl, then the apparatus will obtain a C-13 spectrum from the sample.

It will then analyze the C-13 spectrum by measuring the peak ratio at 128 ppm for the peak at 130 ppm and counting the number of peaks at a mean of 48 ppm at 80 ppm. If the ratio is 0.9 or higher, the device will produce a negative diagnosis; however, if the ratio is less than 0.9 or if there is an abnormally high peak number, in the range of 48 ppm to 80 ppm, then the apparatus will diagnose that the patient is cancer and, as noted above, the patient is then

scanned by RMI to locate malignant tumors. The above diagnostic procedure, which comprises evaluating triglyceride levels and C-13 NMR copy spectra, is described in the pending co-application of Eric T. Fossei, "Process for the Detection of Cancer Using Nuclear Magne- Tic Ressonance (C-13) " ('Process for Detection of Cancer Using (C-13) Nuclear Magnetic Resonance', U.S. Ser.

No. 325,773, filed March 20, 1989, the teachings of which are hereby incorporated by reference.

NMR spectrometer 2 was prepared. In order to make it necessary

Referring to Figure 7, there is shown a nuclear magnetic resonance imaging which, in the preferred form, is capable of performing both the C-13 and the spectroscopic and is preferably but not of the type that suppresses the NMR signal of the water to produce reproducible Hl or C-13 spectra, it should fill in spaces. The filling process is shown in the form of a flowchart in Fig. 10A in which a sample is placed inside the machine, the parameters are realized relative to the temperature is stabilized, are called the pa-fill, the receiver advantage is adjusted , the filling is found to be the frequency of the water in non-aqueous HI spectra, the receiver advantage, is adjusted in the decline area of the suppression of the suppressed proton frequency to the non-aqueous spectra, the data is acquired, it is written a process (report) and the process (report) is processed. Fig. 10B shows a program (written using BRUKER's DISNMR software program) used to automatically perform the processes depicted in Fig. 10A. 25

10C shows a sample program (type program) for filling, corresponding to a -13 carbon spectrum. Fig 10D shows a sub-program of phase 8, RJ FQSET, of the fill program shown in Fig. 10C. Fig. 10E shows a sub-program of phase 2 RJ TUNE of the program shown in Fig. and Fig. 10F shows a sub-program of phase 6, RJ FQSET, of the program shown in Fig. 10B. The spectrometer 2 is adapted for examination of a sample 4, which in this example is human blood plasma, in a test tube 6. The above process is described in co-pending application of Eric T. Fossei under the title : "Apparatus and Method for Detecting Cancer Using Nuclear Magnetic Resonance", US Serial No. 418,182, filed October 6, 1989, is hereby incorporated by reference. , the teachings of which are reproduced by reference herein.

According to the invention, the spectrometer 2 contains means 8 for selecting at least one and preferably a plurality of Nuclear Magnetic Resonance (NMR) lines in the NMR spectrum of sample 4 and measuring the line width of the line or selected lines. Preferably, the line width is measured at half the height of the line, but this need not be so, and the line width can be measured at any predetermined fraction of the height of the line in question. It is preferable that the measurement be half the height of the line, since this is a Standard measurement performed in the field of NMR spectroscopy. Various commercially available computer programs can be used to automatically measure all line widths at half height. The medium 8 of the spectrometer 2 of the invention also measures peaks selected for the examination of the C-13 NMR spectra. 0 is - 26

pectrometer 2 is also of conventional construction and includes, in addition to all of its other structure, a means 10 for storing a value or range of values. According to the invention, the spectrometer 2 also includes means 12 for comparing a line width which is either directly measured or is derived from a plurality of said direct measurements with a value or a range of values representing the value or range of values that can be expected from normal patients, i. and. , patients who are cancer free. Means 12 are also used to classify measured or derived line widths, peak heights and peak numbers, as normal (i.e., cancer free), or abnormal (i.e., cancerous), based on the stored information. This can be done by comparing subtraction, or any other appropriate mathematical operation.

According to a preferred embodiment, the selection and measuring means 8 is preset to measure the line widths of the methyl and methylene groups of the lipoprotein lipids and the peak heights and the number of peaks in the spectra of NMR of C-13. This may include suppression of the aqueous signal from the NMR spectrum of the sample 4 or may alternatively be made directly when the spectrometer 2 is sufficiently sensitive to do such an operation.

According to a preferred embodiment, the line widths of the methyl and methylene groups are evaluated by the measuring means 8 to produce a composite line width which is the mathematical average of the two. This composite line width is compared to 33 Hz, the preferred stored value in the medium 10, by the sorting means 12. When the comparison reveals that the composite line width is less than 33 Hz, this indicates an abnormal sample 4 (iecancerous ). 27 9

EXAMPLE

In this Example, the method of the present invention was applied to a group of 135 patients who underwent a breast biopsy with palpable and non-palpable breast lesions. For the prospective breast study, blood was collected and kept at 4 ° C until centrifugation. The blood was collected in tubes " vacutainer " non-siliconized solutions containing 70 microliters of a 14% Na2 EDTA solution. The plasma was separated and stored at 42 ° C until NMR analysis was performed. Plasma samples were never frozen, since freezing destroys the structural integrity of lipid lipoproteins. Samples showing any visible signs of hemolysis were excluded.

Plasma triglyceride concentrations (Damon Clinical Laboratories, Westwood, Massachusetts) were measured on all fresh plasma samples. All spectra were obtained at 212 ° C using an NMR spectrometer of the type described in the present invention, operating at 360 MHz for (H-1) proton and 90.5 MHz for carbon (C-13). Additional C-13 spectra were obtained on a Bruker AM 11.8 J spectrometer operating at 125.7 MHz. All studies were carried out on 5 mm 0D sample (Wilmad, Vineland, New Jersey: 507 PP or 528 PP). Each sample, containing 0.6 ml of plasma, was individually filled in the area of the decrease in free proton induction (FID), up to full width at half height (FWHH). of the aqueous resonance is 4 Hz or less. Internal quality control was found in the EDTA resonance line width. 28

If all was well with the sample preparation and filling, the line width (FW Η H) of the EDTA resonances (without exponential enlargement) had to be 2Hz or less, often being between 1.0-1.5 Hz In order to achieve these values, most HI probes need to be de-tuned in order to avoid damping the radiation. The probe was de-tuned until the radio frequency pulse reached 902 in 20 msec. In the 8.45 T spectrometer this resulted in the probe being detonated at about 2 MHz. The sample was rotated during filling of the fill coils Z and during data acquisition. Our H-1 spectra were acquired using pre-saturation to suppress water and an inversion-recovery sequence to override any lactate-methyl protons present. The pre-saturation heart rate was 4.0 sec. with a delay of about 8.0 sec. between the pulses of 180e and 902. The signal mean of eight FIDSs was then converted by the Fourier method followed by multiplication by an exponential resulting in the line widening by 2Hz. The portion of the spectrum of 0.5 at 1.6 ppm was divided into phases so that the baseline level at the edges of the diagram was the same. This resulted in defective phasing of other (non-burnt) portions of the spectra.

The C-13 spectra were obtained at the 8.45 signal and at the 11.5 T signal with a broadband proton decoupling averaging between 2,000 and 28,000 FIDs, depending on the signal-to-noise level and the desired resolution. The sample was identical to the samples for the H-1 spectra except that 100 microliters of D-O were added to close the field. It was found that a minimum of 2,000 FIDs were required to produce reliable resonance intensities. The exponential multiplication equivalent to a 25 Hz line amplification 29 29 # 4 Ul ξβ, was used in the spectra obtained at 8.45 T.

In this study we prospectively obtained plasma from a series of women submitted to breast biopsy. Samples were withdrawn before the biopsy, analyzed by NMR, and the results correlated with the pathology reports. Two groups of patients were included in this study: 63 patients with palpable lesions and 72 patients with non-palpable lesions, discovered through mammography, who required localization through a sound recording device. The results of the evaluation by H-1 NMR spectroscopy are shown in Fig. 8. In both groups, benign lesions were distinctly distinct from malignant lesions (p = 0.0001) based on proton values. For these values, the triangles indicate patients who also had high triglyceride values. Open symbols indicate samples in which the results of C-13 collide with the proton results. Thus, for the open symbols, the sample would be changed from the benign column to the malignant column, or vice versa.

The patients in the study were a group of different healthy, outpatient women called for evaluation because of an abnormality on a routine breast examination or a scan mammogram.

In this group, sensitivity and specificity were 93% and 95%, respectively. The predictive value of a positive test was 84% and the predictive value of a negative test was 98%. Reclassified patients based on C-13 data increased sensitivity and specificity to 97% and 98% respectively and the predictive value of a positive test to 93%. These data suggest that the H-1 NMR line width, confirmed by 30

a ratio of C-13, could be used as a diagnostic aid in patients with breast lesions.

There were five apparent false positive results and two false negative results. Elevated triglyceride levels (265 mg / dl and 206 mg / dl) were associated with two of the five false positive results. The C-13 ratio was negative in three false positive results, two of which also had elevated triglycerides; and was positive in all but one case, with malignant breast biopsy. The false negative result for C-13 was also negative for H-1. While patients with elevated triglyceride levels may need to be evaluated for C-13, not all patients with elevated triglycerides had tight line widths. The invention may be carried out according to other specified forms without departing from the spirit or the essential characteristics thereof.

I

The present embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all such changes as may lie within the meaning and scope or equivalence of the claims shall be construed as being covered by the claims.

Claims (1)

31 31 The. A method for diagnosing the existence and location of cancer in a living, relatively non-invasive patient, characterized in that it comprises the following operational steps consisting of a) obtaining a body fluid sample from a patient; b) preparing the fluid sample for the nuclear magnetic resonance (NMR) test; c) obtaining a proton NMR spectral order, from which the undesirable signals of the body fluid sample were suppressed; i) measuring a predetermined width at a predetermined height of a lipoprotein resonance line in said spectrum; (ii) classify the predetermined width or within the category of normal widths or within the category of abnormal widths by comparison with a pre-established standard, for which the abnormal widths indicate the existence of cancer; d) measuring the triglyceride level of the body fluid sample used when the body fluid sample is classified as having abnormal widths; (i) classify the triglyceride level thus measured in a category of normal levels or in a category above the normal level, for which a normal triglyceride level and an abnormal spectrum indicate the presence of a cranberry; e) make the notification of a diagnosis of positive or negative cancer; f) subjecting the patient with the positive diagnosis of cancer to a nuclear magnetic resonance examination throughout the body; g) interpreting the scan image to determine the presence or absence of tissue mass; and (h) notifying the presence or absence of masses. 2a. A method according to claim 1, wherein said body fluid is blood. 3a. A method according to claim 1, characterized in that said blood sample is prepared by separating the red blood cells therefrom. 4a. A process as claimed in claim 1, wherein the undesired signal deleted is water. 5a. Method according to claim 1, characterized in that the predetermined width at a predetermined height is the predetermined width is the total width at half height. 6a. A method according to claim 1, characterized in that said proton resonance is greater than 60 MHz. The method of claim 1, wherein said proton resonance is equal to or greater than 360 MHz. 8a. Method according to claim 1, characterized in that said width measuring operation is carried out at a predetermined height by means of a computer. 9a. Method according to claim 1, characterized in that said predetermined width classification is carried out by means of a computer. 10a. A method for diagnosing the existence and location of cancer in a living, relatively non-invasive patient, characterized in that it comprises the following operational steps consisting of a) obtaining a body fluid sample from a patient; b) preparing the fluid sample for the nuclear magnetic resonance (NMR) test; c) obtaining a proton NMR spectral order, from which the undesirable signals of the body fluid sample were suppressed; i) measuring a predetermined width at a predetermined height of a lipoprotein resonance line in said spectrum; (ii) classify the predetermined width or within the category of normal widths or within the category of abnormal widths by comparison with a pre-established standard, for which the abnormal widths indicate the existence of cancer; d) notify the predetermined width as normal or abnormal; e) measuring the triglyceride level of the body fluid sample used when the body fluid sample is classified as having full abnormal widths; (i) classify the level of triglycerides as thus measured in an allegory of normal levels or in a category of levels above normal; f) notify the level of triglycerides as normal? g) obtaining a C-13 nuclear magnetic resonance spectrum from the body fluid sample used when said body fluid is classified as having above-normal levels of triglycerides; i) classify the nuclear magnetic resonance spectrum and C-13 within the normal spectrum category or within the abnormal spectrum category, the abnormal spectrum indicating the presence of a cancer; h) consider the nuclear magnetic resonance spectrum of C-13 as indicative of the presence of cancer; i) subjecting the patient with a positive diagnosis of cancer to examination of the whole body by means of MRI; j) interpreting the screening image to determine the presence or absence of masses of cancer cells; and k) notify the presence or absence of mass of cancerous cells. 11a. A method according to claim 10, wherein said body fluid is blood. 12a. A method according to claim 10, wherein said blood sample is prepared by separating the red blood cells therefrom. 13a. Process according to claim 10, characterized in that the undesired signal deleted is that of water. 14a. Method according to claim 10, characterized in that the predetermined width at a predetermined height is the total width at half height. 15a. The method of claim 10, wherein said proton resonance is greater than 60 MHz. 16a. The method of claim 15, wherein said proton resonance is equal to or greater than 360 MHz-17a. A method according to claim 15, wherein the ratio of the peaks measured between C-13 nuclear magnetic resonance at 128 ppm and at 130 ppm constitutes a diagnosis of the presence of cancer. 18a. The method according to claim 15, wherein the ratio of the peaks measured between C-13 nuclear magnetic resonance at 48 ppm and at 80 ppm is determinant with respect to the presence of cancer. 19a. Method according to claim 10, characterized in that said measurement operation is carried out at a predetermined height by means of a computer. 20a. Method according to claim 10, characterized in that the predetermined width classification is carried out by means of a computer. 21a. A process according to claim 10, characterized in that said classification of the triglyceride level is carried out by means of a computer. 22a. Method according to claim 10, characterized in that the classification of the nuclear magnetic spectrum is carried out by means of a computer. Lisbon, January 6, 1992 0 Official Agent of Industrial Property Agent of Proprcdado Industrie! Rua Marquês da Fronteira, No. 127 - 2 "1000 LISBOA TbIs.3377373-3877453