CN1063360A - The method of detection and localization of cancer - Google Patents

Abstract

The present invention discloses a kind of with nuclear magnetic resonance (NMR) spectrum with magnetic resonance radiography (MRI) detects and the method for localization of cancer, specifically, mensuration be class METH and/or methylene proton the NMR parameter and itself and value of healthy patients accordingly compared.

Description

The method of detection and localization of cancer

The present invention relates to method for cancer in a kind of detection and the position patient body.

In the prior art, utilizing nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) technical assistance is well-known in the method for the clinical diagnosis of cancer.

Damadian is that first proposes nuclear magnetic resonance the people who medically uses.He advises with the malignant tumour in the magnetic resonance detection tissue.See R.Damadian, " Tumor Detection by Nuclear Magnetic Resonance, " Science 171: 1151-1153(1971), authorize the United States Patent (USP) 3 of Damadian, 789,832 comprise that the sample that nuclear magnetic resonance is applied to surgical removal is to measure proton relaxation time T ₁And T ₂Instrument and method, with measured T ₁And T ₂Make comparisons as a kind of means of cancer diagnosis with the numerical value of health tissues.The United States Patent (USP) 4,411,270 and 4,354,499 of authorizing Damadian comprises with the NMR imaging of whole body sample and the instrument and the method for scanning detection cancer.

Many other researchists have also reported the corresponding T of water-bound in having the animal organ of tumour ₁Long.See Frey etal.J.Natl.Cancer Inst.49，903（1972）;Inch etal.J.Natl Cancer Inst.52，353（1974）;Iijima et al physiol Chem.and physics 5，431（1973）;and Hazlewood et al J Natl Cancer Inst，52，1849（1974）。

Now, well-known although its machine-processed details are not still had final conclusion, the biophysics that takes place in malignant cell changes and usually changes proton N MR signal.See D.G.Taylor et al. " A review of the Magnetic Resonance Response of Biological Tissue and Its Applicability to the Diagnosis of Cancer by; NMR Radiology; " Computed Tomography, 5: 122-133(1981).Such variation has constituted the physical basis that detects tumour with proton N MR imaging.See R.Zimmerman et al " Cerebral NMR:Diagnostic Evaluation of Brain Tumors by Partial Saturation Technique with Resistive NMR; " Neuroradiology 27: 9-15(1985) and K.Oktomo, " Hepatic Tumors:Differentiation by Transverse Relaxation Time(T ₂) of Magnetic Resonance Imaging, " Radiology 155: 421-423(1985).

To the tumour of in animal used as test and patient body, downcutting, and to the proton N MR of blood plasma and serum research has demonstrated relaxation parameter T ₁, T ₂, and T ₂ ^*(T ₂ ^*Be intrinsic loose T ₂The loose combination that causes with heterogeneity) different and different with malignant tumour by magnetic field.Such discovery has following report:

L.McLachlan，“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 Relaxation Times for water in Animals and Hamans with Cancers”NMR Basic Principles and progress，P.Diehl et al.Eds.，19∶39-57（1981）：

R.Floyd，“Time Course of Tissue water proton spin latice Relaxation in Mice Devloping Ascites Tumor，”Cancer Res.34∶91（1974）;

C.Hazlenoocl et al.“Relationship Between Hy dration and proton Nuclear Magnetic Resonan-ce RelaxationTimes in Tissues of Tumor Bearing and Nontumor Bearing Mice;Implications for Cancer Detection，”J.Natl Cancer Inst.52∶1849-1853（1974）;and

R.Klimek et al.“A Discussion of Nuclear Magnetic Resonance（NMR）Relaxtion Time of Tumors in Terms of Their Interpretation as Self-organizng Dissipative Structures，and of Their Study of NMR Zeugmatopgrarhic Imaging，”Ginekol pol 52∶493-502（1981）。

Yet because the extensive gravity treatment of group and the fine difference between each class methods, they are inapplicable clinically.

Although above-mentioned prior art suggestion is mostly analyzed tissue with NMR, learn that also body fluid can be used for such analysis, as described in people such as Beall, sees above.

The explanation of the compound NMR signal that all protons produce in the NMR method of the detection malignant tumour of being quoted described in the prior art depends on the sample of being derived by tissue or blood.This composite signal is controlled by the proton of water, and it can be covered from other of sample and contain the NMR signal of proton composition.In fact, prior art thinks that the surperficial correlativity between the change of malignant tumour and viewed NMR parameter is because the change of water-bound, the people's such as Frey of the usefulness that sees above article.

In other application of proton N MR spectroscopy, the signal from solvent (as water) in the inhibition sample is known.It is found that some components that have in the NMR spectrum of remarkable pre-value can be covered by other composition in the sample.

Common pending application at Eric T Fossel (is entitled as " using the magnetic resonance detection method for cancer ", UssN303,586, the applying date is on January 27th, 1989, the content of this application is at this as a reference) in, above-mentioned discovery is merged into the reliable method of cancer in a kind of patient body of diagnosis.

According to this invention, measure the NMR (Nuclear Magnetic Resonance) spectrum of patient's humoral sample, select the resonance line that non-water constituent produced by sample, measure this resonance line at assigned altitute, for example in the full duration at its mid-height place.Proved measured full duration is whether to have believable measuring on the statistics of cancer in the patient body.

The above-mentioned test that blood plasma water is suppressed proton N MR can be distinguished suffers from cancer and untreated people and other people, and its accuracy is higher than 90%.Therefore, this test is subjected to praising widely as one of most important invention over 10 years, does not have a kind of Non-Invasive test that detects cancer to reach such precision in the past.Yet, obtained false positive results.

Common pending application at Eric T Fossel (is entitled as and " uses NMR( ¹³C) detect method for cancer ", ussN295,746, its content is at this as a reference) in, the main source that has proved false positive results is the plasma triglyceride of high-load.Non-invasive method and the instrument that whether has cancer in a kind of definite patient body talked about in this invention, and its degree of accuracy increased than former non-invasive method.

According to this invention, the result's that is positive when the proton N MR shaker test patient is carried out the mensuration of content of triglyceride.Normal content of triglyceride can be examined the diagnosis of cancer, but needs to measure through C-13NMR for the high patient's humoral sample of content of triglyceride.The abnormal results of this test can further confirm the diagnosis of cancer, and normal result shows that then former diagnosis is a false positive.

Common pending application at Eric T Fossel (is entitled as " with the instrument and the method for magnetic resonance detection cancer ", ussN418,182, the applying date is on October 6th, 1989, its content is at this as a reference) in, proved and can diagnose by a kind of automated process that proton N MR and C-13NMR spectrum are combined.

According to this invention, designed a kind of instrument that makes the method robotization that utilizes the NMR cancer diagnosis.This instrument has one can read the water inhibition proton N MR of humoral sample and the spectrometer parts of C-13NMR reading.In addition, this instrument has and is used to handle proton N MR and C-13NMR reading and obtains counter corresponding to the numerical value of those readings.This instrument also comprises and is used to store one group of normal value standard and compares the memory of those Stored Values with the numerical value that is obtained by NMR.Instruct the computer program of its function can analyze data, obtain diagnostic result with accuracy highly.

Diagnosis only is the first step of treatment of cancer, must carry out the evaluation of specific malignant tumour subsequently.Do not speak of a kind of utilization in the prior art and obtained NMR spectrum, and translated and release this spectrum, and carried out this and break, when cancer diagnosis, determined method for cancer then by using magnetic resonance imaging (MRI).But the nuclear magnetic resonance analyser that is used to obtain tentative diagnosis is urgently to expect with the coupling of the whole body MRI scanning of single-minded ground of clock like precision cancer diagnosis.

According to the present invention, developed a kind of method of utilizing nuclear magnetic resonance analyser and magnetic resonance imaging system detection and localization of cancer.This method and instrument can utilize proton N MR spectrum, and triglyceride levels is necessary, further sample is made the C-13NMR spectroscopy analysis and draws diagnostic result.If the true positives reading occurs, then patient is carried out whole body MRI to identify malignant tumour at triglyceride or C-13 step.

In preferred embodiments, sample liquid is a blood, spinal fluid, or marrow blood plasma; Especially be advisable with blood.Interesting component is a lipoidis, best methyl and methylene from the class lipoprotein lipid.

Therefore, the purpose of this invention is to provide a kind of NMR of utilization spectroscopic method and diagnose,, identify and locate the exact method of malignant tumour immediately with the MRI scanner in case obtain the true positives diagnostic result of cancer to detect in the patient body whether have cancer.

Other purpose of the present invention and advantage can be hereinafter to obviously finding out in accompanying drawing and the description of the invention.

Fig. 1 is the typical 360MHz NMR spectrum of nonaqueous component (water is suppressed) in the normal healthy controls plasma sample that obtains according to the phase one of the present invention;

Fig. 2 is the NMR spectrum of the plasma sample identical with Fig. 1 specimen in use, and except that anhydrous inhibition, used equipment all is identical with pulsed frequency;

Fig. 3 is the methyl of Fig. 1 sample reading and the enlarged drawing in methylene zone;

Fig. 4 suffers from malignant tumour and the methyl and the methylene regional enlarged drawing of the NMR spectrum of untreated patient's plasma sample;

Fig. 5 A and 5B are respectively by the normal control of phase one acquisition of the present invention and the alkene zone of untreated cancer patient's plasma sample C-13NMR spectrum;

Fig. 6 A and 6B are normal control and untreated cancer patient's plasma sample (being shown in 5A and 5B) the C-13NMR spectral region between 10ppm and 90ppm that obtain according to the phase one of the present invention, particularly including the diagram in 48ppm to 80ppm zone;

Fig. 7 shows the phase one of instrument of the present invention;

Fig. 8 has provided the result of study that obtains with method of the present invention;

Fig. 9 has provided operational flowchart of the present invention;

Figure 10 A has provided the process flow diagram of adjusting, and adjustment is to be undertaken by the phase one of instrument, and it can guarantee that water suppresses reading result's reappearance;

Figure 10 B has provided the sample program of adjusting, corresponding to the proton spectrum step of Figure 10 A process flow diagram;

Figure 10 C has provided the sample program of adjusting, corresponding to C-13 spectrum;

Figure 10 D has provided the step 8 of the adjustment program shown in Figure 10 C, the i.e. subroutine of RJFQSET;

Figure 10 E has provided the program step 2 shown in Figure 10 B, the i.e. subroutine of RJ TuNE;

Figure 10 F has provided the subroutine of Figure 10 B.

At the first bruss, the present invention has been done general introduction, be described in more detail below. The present invention is the method for cancer in detection and the position patient body. According to the present invention, utilize proton nuclear magnetic resonance spectroscopy to measure the NMR spectrum of patient's humoral sample. Can be covered by other material in the sample owing to have the component of the NMR spectrum of important expectation numerical value, so just can obtain the abundant NMR spectrum of information content through eliminating this covering. Select one by the resonance line of the nonaqueous component generation of sample and at the height of being scheduled to, for example measure at half place of its height, the preset width of this resonance line, preferably full duration thinks that the interior cancer of detection patient body provides a kind of and measures reliably. This theoretical or above program is at common pending application ussN303, State in 586, the content of this patent application at this as a reference.

In fact, if select one or more resonance line, then a certain parts of available core magnetic resonance device are measured, and the storage values of the mean value that obtains and expression normal value (being non-cancer) is made comparisons.

Positive reading can show the existence of cancer in the patient body, otherwise it may be false positive readings. The main source of having found false positive readings is the high people of plasma triglyceride content. Therefore, if the resonance line numerical value that records greater than the normal value that stores, then this program will diagnose patient not have cancer. Yet, if the value that records less than storage values, this program draws indicating instrument the measured value of patient's triglyceride levels.

In order to distinguish true and false positive reading, those samples that triglyceride levels is higher are made the C-13NMR spectroscopy analysis. To draw like this a diagnostic result. Can determine reliably because the false positive results that causes of hypertriglyceridemia and on the contrary the existence of the interior cancer of patient body according to some feature of C-13NMR spectrum.

If with positive proton N MR spectroscopy reading and normal triglyceride levels or positive proton N MR spectroscopy, high triglyceride level and positive C-13 result obtain the positive test symbol of cancer, then also will make whole MRI to patient and describe to determine the position of malignant tumour.

In one embodiment of the invention, earlier testing sample is carried out proton N MR spectroscopy analysis.The water inhibition proton N MR spectrum that human plasma is recorded is controlled by plasma proteins fat.Under the situation that does not have water to suppress, these non-water resonances are covered by water in fact.Under the highfield, even in the presence of water resonance, can be observed the signal homogenizing of some partial resonance relevant with non-water body fluid component.Modern NMR spectrometer can almost completely suppress water proton resonance, thereby helps this reading.It is exactly that the water of plasma lipoprotein and minority low-molecular-weight molecule suppresses proton N MR spectrum basically that the water of blood plasma suppresses proton N MR spectrum, and why the plasma proteins proton is not differentiate the wide fuzzy of resonance because they comprise by coverage.The more sharp-pointed resonance of the lipoprotein proton that metabolic is stronger is attached on this broad background.

NMR spectrometer of the present invention starts with any body fluid that contains lipoid.Can use whole blood, serum or blood plasma, although available any such lipoid body fluid that contains is tested, present work has concentrated on blood plasma.In blood, lipoidis comprises that cholesterol, triglyceride and phosphatide exist with the form of lipoprotein.Detection to cancer generally is to carry out external, preferably with serum or blood plasma.

Will for detect body fluid exposure that cancer selects in magnetic field and radio-frequency (RF) energy to produce NMR signal, handle this signal with nuclear magnetic resonance spectrometer then, for lipoid methyl and/or methene proton, obtain selected parameter value, for example W1/2.Can use the proton frequency of relative broad range, for example 60MHz or higher; 360MHz or be optimized frequency more than the 360MHz.If do not mind expense, the preferred frequency of 500MHz.

The water that Fig. 1 has provided normal healthy controls suppresses proton spectrum, and Fig. 2 has provided at the proton spectrum that does not have same sample under the water inhibition situation.The shortening of water resonance line in Fig. 2, represent with A.0.5 the resonance line and the umber of per 1,000,000 resonant frequencies of 2ppm() is to be produced by the methyl of class lipoprotein lipid and methylene.The enlarged drawing of the regional proton spectrum of this of normal control is shown in Fig. 3, and this regional enlarged drawing of proton spectrum with malignant tumour untreated patient is shown in Fig. 4.Therefore, in its preferred embodiment, the present invention has adopted one of numerous conventional water inhibition technology,, suppresses the technology of water proton NMR signal that is.In other context, designed the technology of many inhibition water proton NMR signals.These technology can be divided into two classes substantially: (1) purpose does not lie in the technology that excites the water proton signal, for example, scan associated light spectrometry and selective excitation technology fast, (2) make the technology of water proton magnetization when using viewed radio frequency (rf) pulse, to become quite little, for example, backward recovery technology and saturation technique.These and other solvent suppresses technology and is described (seeing " Solvent Suppression in Fourier Transform Nuclear Magnetic Resonance, " Journal of Magnetic Resonance 55: 283-300(1983) with the list of references that wherein indicates footnote) by P.J.Hore.Though should adopt water inhibition technology because it can not distinguish the solvent proton signal with the interesting part or the signal of kind when using conventional nuclear magnetic resonance analyser, enough delicate does not then need the water inhibition completely.

At predetermined height, the half eminence group relevant preferably with the plasma lipid proteolipin, for example the resonance line width of methyl and methylene is interesting variable.The NMR resonance line is in the full duration W1/2(of half eminence live width) and show the relaxation time (T that spins * ₂) be inversely proportional to,

Be w 1/2=1/ (T ₂ ^*)

The detected value of selected parameter is made comparisons with corresponding normal healthy controls person's parameter, and this is relatively preferably finished by the counter in the NMR instrument.In a preferred embodiment,, be 360MHz(8.45T with proton frequency with the numerical value homogenizing of methyl and methene proton), best 400MHz(9.40T) time 33Hz or littler mean value as the indication of malignant tumour.

If obtain positive reading from the water inhibition proton N MR spectrum of patient's blood plasma, the test of then carrying out second level is to make a definite diagnosis.At first, carry out a conventional test, be referred to as the triglyceride analysis usually to determine patient's content of triglyceride.If content of triglyceride is normal, then the positive reading that obtains from water inhibition proton N MR spectroscopic methodology is the true positives result, and showing has cancer to exist in the patient body, in this case, also tackles patient and carries out whole body MRI scanning to determine the position of malignant tumour.

Yet, if content of triglyceride surpasses normal value, then preferably draw the C-13NMR spectrum of patient's plasma sample, because existing proton N MR spectrum by counter indication NMR spectrometer, so C-13NMR spectrum is available, so just can distinguish true and false positive findings.Because the false positive results that hypertriglyceridemia causes can be different from the true positives result reliably by the marked difference of C-13 spectral signature.Therefore, the plasma sample that obtains from suspicious patient to be diagnosed is exposed to magnetic field and radio-frequency (RF) energy to produce the C-13 NMR (Nuclear Magnetic Resonance) spectrum.

At first, the alkene zone (120-140ppm) of check spectrum.Two peaks will occur, and one at about 128-129ppm, and another is at about 130-131ppm, the 2ppm of being separated by approximately.Can make a definite diagnosis cancer at the resonance of 128ppm general area and ratio in the resonance at 130ppm place.In deriving from normal control and non-cancer patient's blood plasma reading, the ratio of the height of above-mentioned two kinds of resonance (128/130ppm) is 0.9 or bigger, that is, the resonance peak at 128ppm place approximates or be higher than the resonance peak at 130ppm place.Measure peak height with counter from center to the summit of baseline noise.In deriving from the blood plasma reading of suffering from the untreated patient of cancer, the ratio of two peak heights is less than 0.86, or the resonance peak at 130ppm place is at least than the resonance peak height 5% at 128ppm place.It should be noted that in the patient of hypertriglyceridemia the ratio of the height of resonance peak (128/130) is identical with the normal control value.Therefore, the counter of NMR spectrometer can calculate the ratio of the peak height that has recorded, is healthy if this ratio, is then diagnosed out patient greater than storage values, otherwise, then diagnose out cancer.In a preferred embodiment, storage values is 0.9.The making a definite diagnosis of cancer means that next step will do aforesaid MRI scanning to patient.

Fig. 5 A and 5B have shown when 125.76MHz the alkene zone to the spectrum that obtains behind normal control and the untreated cancer patient broad band proton-decoupled connection.In Fig. 5 A, normal control's peak height ratios is 1.14, and in Fig. 5 B, untreated cancer patient's peak height ratios is 0.7.In the patient of the hypertriglyceridemia that is studied, peak height ratios is in 1.05～1.68 scopes.

The increase of polyunsaturated fatty acids chain or reduce and explain that oleic acid and linoleic level are best indications in the variation in the alkene zone of untreated cancer patient's spectrum can be by lipoid.

Oleic acid is the monounsaturated fatty acids that is produced by human body.Linoleic acid is two unsaturated fatty acids, and it is not produced by human body, but obtain by consumption.Dietary fat acid comprises polyunsaturated acid, for example linoleic acid.Resonance peak in the general area of 128-129ppm shows to have only linoleic acid in the patient body.Resonance peak in the general area of 130-131ppm shows that existing oleic acid has linoleic acid again in the patient body.

The height of now finding those relevant mutually resonance peaks is subjected to some condition influence of patient.The people who for example has a high triglyceride level has the ratio of high linoleic acid and oleic acid usually because cancer can cause the polyunsaturated fatty acid oxidation of (comprising linoleic acid), therefore estimates can have low-level linoleic acid in untreated cancer patient's body.This is consistent with this hypothesis, and promptly because polyunsaturated fatty acid is the most responsive to oxidation, therefore, lipoid can be by the hydroxyl free radical oxidation among the cancer patient.

Therefore, if the patient has high triglyceride levels and untreated cancer, then the resonance peak in the 130ppm place can be higher, the linoleic acid of reduction be reflected in bimodal in.But as the peak at the 128ppm place weak point unlike the 130ppm place, and short degree is no more than 7%, then do not have linoleic reduction or significantly reduce, and the positive findings that is obtained by proton N MR spectrum is considered to false positive.In this case, the NMR spectrometer is made the diagnosis that no cancer exists.

In addition, more complicated more than in the blood plasma of normal control or hypertriglyceridaemia of 48ppm in untreated cancer blood plasma and the spectral region between 80ppm." more complicated " is meant more resonance peak in this zone.If the height of resonance peak big by 50% than ground unrest at least in normal duration of test, then resonance peak can be calculated by the program of establishment.Just as is known to the person skilled in the art, the data of collection are long more, and resolution characteristic is good more.Fig. 6 A and 6B have represented the district of normal control with the cancer blood plasma that is untreated respectively.These spectrum are at 125-76MHz, use the 5mm sample hose, and accumulation in 14 hours, obtain.The C-13 spectrum that has suitable information also can be in 90.5MHZ, 10mm or longer sample hose, obtain.Certainly, some parameters of test procedure being changed is conspicuous to those skilled in the art.

These parameters comprise the sample hose size, pulse width, pulse fidelity factor and the index multiple of freely inducing decline by different factor affecting.For example, it will be apparent for a person skilled in the art that: the sample of test is big more, and the spectrum of the suitable quality of gained is fast more.Here other change of the condition of giving says it is tangible to those skilled in the art.

Can be at the very start do the method that C-13 spectrum or MRI scanning exist as cancer diagnosis, and needn't obtain proton N MR spectrum as mentioned above earlier to the patient.Obtain proton N MR spectrum and needed for 30 seconds, and the C-13 spectrum needs 1 to 15 hour, therefore, the C-13 spectrum is consuming time many and relatively more expensive.Therefore, in preferred embodiments, C-13 spectrum is used to verify the positive findings that obtained by proton N MR spectrum and from statistics with the notable difference of illustrating plasma C-13 spectrum clinically, thereby distinguishes from proton water and suppress the true and false positive findings that the NMR spectrum is tested.Whole body MRI scanning is also very expensive.Can avoid using another kind of instrument and needn't make with the C-13 spectrum earlier before MRI scanning is with false-positive patient to do other medical inspection step.

In preferred embodiments, use the NMR spectrometer with stationary magnetic field intensity, the NMR signal is to pass through the conversion of Fuli's leaf by useful NMR parameter (proton resonance of methyl and methylene and linoleic C-13 resonance are at the whole line width of half height).

Such as among the U.S. Patent application No833.840 notes, correct specimen preparation and method for making are necessary for the measurement of successfully carrying out plasma sample.Containing 70ml 15%Na ₂Collect blood sample in the test tube of EDTA solution.Blood sample 4 ℃ of preservations until centrifugal.Tell blood plasma and analyze up to being used for NMR 4 ℃ of preservations.Because the freezing integrality that can destroy the fat structure of lipoprotein, so plasma sample is never freezing.Get rid of the sample that any haemolysis sign is arranged.

All spectrums obtain at 20～22C, and magnetic field intensity is 360MHz or bigger.The partly whole line width of height of freely inducing blighted area with computing machine sample to be regulated up to water resonance at proton is 4Hz or littler.Certainly, the careful adjustment is a pith of NMR experimental technique well.

From those experiment conditions as seen, temperature and adjustment are composed C-13NMR and are not crucial, because do not need to measure line width.Certainly, used magnetic field intensity has determined to obtain the required time of spectrum.In addition, diagnosis accurately need be scrutinized patient's case history.

Fig. 9 represents is the process flow diagram of this method operation, and wherein, sample is from body fluid and be sent in the NMR spectrometer to obtain the H-1 spectrum that water suppresses.Resonance line is selected and measured to this instrument then, and the normal value that the line width of gained and 33Hz(are scheduled to) relatively find out the averaging spectrum line width.If the averaging spectrum line width is greater than 33Hz, what then instrument showed is negative diagnosis; Otherwise, measure patient's triglyceride levels.If triglyceride levels is less than 190mg/dl, the diagnosis that then shows cancer is positive, and the patient also will carry out whole body MRI scanning.If but triglyceride levels then will obtain the C-13 spectrum of sample greater than 190mg/dl.Analyze the C-13 spectrum by the ratio of measuring peak, 128ppm place and peak, 130ppm place and the peak number order that calculates between 48ppm to 80ppm then.If this ratio is 0.9 or greater than 0.9, show that then diagnostic result is negative; But less than 0.9 or unusual high peak number is arranged, show then that the patient suffers from cancer as this ratio, aforesaid then the patient is carried out MRI scanning with the location malignant tumour at 44ppm to 80ppm place.The above-mentioned diagnosis algorithm that comprises triglyceride levels and C-13NMR spectrum sees that title is the common pending application of the Eric T Fossel of " detecting method for cancer with nuclear magnetic resonance (C-13) ", U. S. application number, 325,773,1989,3,20 propose, and insert as a reference here.

Fig. 7 has described nuclear magnetic resonance spectrometer 2, and it can carry out proton and C-13NMR spectrum and be preferred in preferred version, but dispensable, the type nuclear magnetic resonance analyser can suppress the NMR signal of water.For producing the H-1 or the C-13 spectrum of reappearing, adjustment is necessary.The step of adjusting is seen process flow diagram among Figure 10 A, wherein, sample is placed in the machine, makes temperature stabilization, adjust parameter, adjust and receive amplification coefficient, operation is adjusted, and the water transport dynamic frequency is that H-1 water suppresses spectrum, receive amplification coefficient and induce the blighted area adjusted in the inhibition proton frequency that water suppresses spectrum, obtain data, charge to reservoir, deal with data.What Figure 10 B represented is the program (charging to Bruker DESNER software program) that is used for carrying out automatically Figure 10 A step.What Figure 10 C represented is the program that is used to regulate the C-13 spectrum.Figure 10 D represents is the step 8(RJ FQSET of the adjustment program represented among Figure 10 C) subroutine.That Figure 10 E represents is the step 6(RJ FQSET of program shown in Figure 10 B) subroutine.Spectrometer 2 is applicable to the detection of sample 4, and sample in this example is the human plasma in test tube 6.Above-mentioned steps is seen the common pending application of EMZ.T.Fossel, and title is " with the instrument and the method for magnetic resonance detection cancer ", and U. S. application number 418,182,1989,10,6 propositions are here as list of references.

According to the present invention, spectrometer 2 contains device 8, and it is used for selecting at least one NMR resonance line and preferred a plurality of NMR resonance lines of the NMR spectrum of sample 4, and it also is used to measure the line width of selected single spectral line or a plurality of spectral lines.Line width is preferably highly measured at half of spectral line, but this is optional, and line width can be measured at any predetermined height of spectral line place.Preferably at half high measurement of spectral line, because this is the canonical measure of carrying out in the NMR spectrum field.Several commercially available computer programs are used in the half whole line width of highly automated measurement.

The device 8 of spectrometer 2 of the present invention is the selection peak of energy measurement C-13NMR spectrum detection also.Spectrometer 2 also is a conventional configurations, and other device, it comprises the device 10 that is used for storage data except that all.According to the present invention, spectrometer 2 also comprises device 12, and it is used for and will directly measures or compare from numerous line width that directly measure and normal patient (the being non-cancer patient) value of obtaining.On the basis of stored information, device 12 also is used for line width measured or that derive, and peak height, peak number order are divided into normally (being non-cancer) or unusual (being cancer stricken).This can be by relatively, deducts or any other suitable mathematical method is carried out.

In preferred embodiments, the device 8 with selection and measurement function is regulated in advance to measure methyl and the line width of methylene, peak height and the peak number of the lipoprotein lipid in the C-13NMR spectrum.This comprises the signal of inhibition water from the NMR spectrum of sample 4, or otherwise directly does, and is enough responsive when wherein spectrometer 2 is done like this.

In preferred embodiments, on average to produce the line width of combination, it is the arithmetic mean of two values to the line width of methyl and methylene by measurement mechanism 8.This combination line width and 33Hz(should be worth preferably be stored in the device 10) relatively, distinguish by installing 12.When this line width that comparison shows that combination during, show that then sample is unusual (promptly suffering from cancer) less than 33Hz.

Embodiment

In this example, method of the present invention is got 135 patients that suffer from tangibly or non-stereognosis thoracic injury and is carried out the chest biopsy.For the chest research of this expection, blood sample collection also remains on 4 ℃ up to centrifugal.With blood specimen collection to containing 70ml 14%Na ₂In the empty test tube of the non-silica gelization of EDTA, tell blood plasma and analyze up to NMR 4 ℃ of storages.Plasma sample is never freezing, because the freezing integrality that can destroy the fat structure of lipoprotein.Any showing has the sample of haemolysis sign all should be removed.

On whole fresh plasma sample, measure triglyceride concentration in the blood plasma (Damon Clinical Laboratories, Wertweed, Massachusstte).NMR spectrometer with type of the present invention obtains proton (H-1) and C-13 spectrum at 360MHz and 90MHz in 21 ℃.In 11.8T Bruker AM spectrometer, obtain C-13 spectrum at 125.7MHz in addition.All researchs all are that (Wilmad, Vineland, New Jersay#507pp or #5288pp) carries out in the 5mmOD sample tube.It is 4Hz or littler that each sample that contains 0.6ml blood plasma is freely induced the halfwidth degree of regulating separately on the blighted area (FID) up to water resonance (FWHH) at proton.The line width of EDTA resonance contrasts as internal soundness.If specimen preparation and regulate and all to carry out, the line width of EDTA resonance (no index expansion) should be 2Hz or littler and generally should be between 1.0-1.5.For finishing this task, numerical control should be lacked of proper care to avoid radiation damping mostly.Making the probe lack of proper care up to 90 ° of radio-frequency pulses is 20msec.In the 8.45T spectrometer, will cause probe about 2MHz that lacks of proper care.Sample rotates at the conditioning period of Z adjustable pipe with during obtaining data.The H-1 spectrum here obtains with presaturation, and this presaturation can suppress water and suppress reverse-recovery sequence to exist to no any methyl lactate proton.The presaturation pulse is 4.0 seconds, and the delay of 180 ° of-90 ° of pulses is about 0.8 second.8 FIDS are carried out Fourier transform then by signal averaging, cause the spectral line of 2Hz to enlarge by the index multiplication subsequently.0.5-1.6ppm spectra part changed so that the baseline values of curved edge is identical the changing not exclusively mutually of this other (non-curveization) that can cause composing part mutually.

At 8.45 and 11.57 signal places, the broad band proton with the 2000-28.000FIDS homogenizing is uncoupled and joined according to signal-noise level and required resolution, obtains the C-13 spectrum.Except adding 100ml D ₂O is used for outside the sealing of magnetic field, and this sample is identical with the sample that is used for the H-1 spectrum.Find that now the minimum 2.000FIDS of need could produce the reliable strength of resonance.The index multiplication that is equivalent to enlarge the 2.5Hz spectral line is used in the spectrum that 8.45T obtains.

In this research, we wish to gather blood plasma from be the bioptic many women of chest.Sample prepares before biopsy, analyzes with NMR, and the result is relevant with the pathology report.Comprise two groups of patients in this research, one group is 63 patients that the tangibly infringement is arranged, and another group is that the breast x-ray photograph is found, and the infringement of non-tangibly needs with 72 fixing patients of silk thread.What the H-1NMR spectrum was assessed the results are shown in Figure 8.In two groups, benign lesions can clearly be separated based on the proton value with malignant lesions (P＜0.0001).For those values, the trigonum shows result and the proton outcome conflict of sample in C-13.Therefore, for the symbol that launches, sample will forward pernicious hurdle to or reform conversely from optimum hurdle.

Patient in this research is one group of non-healthy women (outpatient) that is used to assess, because their normal breast examination or evaluation mammogram abnormality.In this group, susceptibility and specificity are respectively 93% and 95%.The predicted value of positive test is 84%, and the predicted value of negative test is 98%.The patient's who reclassifies on C-13 data basis susceptibility and specificity rise to 97% and 98% respectively, and the predicted value of positive test is 93%.These data show that the H-1NMR line width of being confirmed by the C-13 ratio can be used for assisting to diagnose the patient that the chest infringement is arranged.

5 false positives and two false negative result are arranged here.In the triglyceride levels (265mg/dl and 206mg/dl) that raises and 5 false positives two are relevant.The C-13 ratio is negative in three false positives, these two triglyceride with rising wherein; In all 5 positives, it is pernicious that a chest biopsy is arranged.The false negative that C-13 shows, its H-1 also is negative.Also need the C-13 assessment though have the patient of the triglyceride levels of rising, not all patient with triglyceride of rising has narrow line width.

The present invention can implement in other concrete mode on spirit of the present invention or essential characteristic basis.Therefore, embodiment of the present invention should regard described all aspects as and without limits, scope of the present invention is shown in appended claims, rather than the description of front, and therefore, all changes in these claims scope all comprise in the present invention.

Claims (22)

1, a kind of diagnose cancer among the live body patient to exist and the position and to the harmless method of patient, this method comprises:

A) from the patient, get the liquid sample;

B) preparation is used for the liquid sample of NMR test;

C) make proton N MR spectrum, wherein be suppressed from the undesired signal in the humoral sample;

I) predetermined height of the lipoprotein resonance line in described spectrum is measured preset width;

Ii) by with preassigned relatively, preset width is divided into normal width class and unusual width class, unusual width shows and has cancer;

D) when humoral sample is had unusual width by evaluation, measure the triglyceride levels of used humoral sample;

I) measured triglyceride levels is divided into normal level class and hypernormal level class, wherein normal triglyceride levels and unusual spectrum show and have cancer;

E) the report cancer diagnosis is a positive or negative;

F) patient with positive cancer diagnosis carries out the whole body magnetic resonance radiography:

Whether g) analyze radiography scanning exists with the decision lump; With

H) whether the report cancerous tumors exists.

2, the process of claim 1 wherein that body fluid is blood.

3, the method for claim 2, wherein said blood sample are that the erythrocyte of removing wherein prepares.

4, the process of claim 1 wherein that the undesired signal that is suppressed is a water.

5, the process of claim 1 wherein that the preset width in predetermined height is the whole width of highly locating half.

6, the process of claim 1 wherein that proton resonance is more than 60MHz.

7, the method for claim 6, wherein proton resonance is equivalent to 360MHz or more than it.

8, the process of claim 1 wherein and the measurement of preset width is finished by computing machine in predetermined height.

9, the process of claim 1 wherein preset width classification is finished by computing machine.

10, a kind of existence of diagnosing cancer among the live body patient and position and to the harmless method of patient, this method comprises:

A) get the liquid sample from the patient;

B) preparation is used for the fluid sample of NMR test;

C) system proton N MR spectrum, wherein the undesired signal from humoral sample is suppressed;

ⅰ) predetermined height in the lipoprotein resonance line is surveyed preset width in described spectrum;

ⅱ) with preassigned relatively, institute survey preset width is divided into normal width class and unusual width class, wherein unusual width class shows and has cancer;

D) whether the report preset width is normal;

E) when humoral sample is decided to be the full duration with abnormality, measure the triglyceride levels of used humoral sample;

I) measured triglyceride levels is divided into normal level class and hypernormal level class;

F) whether the report triglyceride is normal;

G) be decided to be when having super normal triglyceride levels when described body fluid, used humoral sample is done C-13 nuclear-magnetism spectrum;

I) C-13 nuclear-magnetism spectrum is divided into normal spectrum or improper spectrum, the bright cancer that exists of wherein improper stave;

H) report C-13 nuclear-magnetism spectrum, the index whether it exists as cancer;

I) patient with the cancer diagnosis positive carries out the whole body magnetic resonance radiography;

J) scanning analyzes with decision whether have lump to radiography: and

K) whether report exists cancerous tumors.

11, the method for claim 10, wherein body fluid is blood.

12, the method for claim 10, wherein said blood sample are to prepare by the erythrocyte of removing wherein.

13, the method for claim 10, the undesired signal that is suppressed is a water.

14, the method for claim 10, wherein the preset width in predetermined height is to be positioned at the half whole width of highly locating.

15, the method for claim 10, wherein proton resonance is more than 60MHz.

16, the method for claim 15, wherein proton resonance is equivalent to 360MHz or more than it.

17, the method for claim 10, wherein the peak ratio at the 128ppm-130ppm intermittent gauging of C-13 nuclear-magnetism spectrum is used for cancer diagnosis.

18, the method for claim 17, wherein at the peak ratio of the 48ppm-80ppm intermittent gauging of C-13 nuclear-magnetism spectrum with deciding cancer.

19, the method for claim 10 is wherein measured preset width in predetermined height and is finished by computing machine.

20, the method for claim 10 is wherein finished by computing machine the classification of preset width.

21, the method for claim 10 is wherein finished by computing machine the classification of triglyceride levels.

22, the method for claim 10 is wherein finished by computing machine the classification of C-13 nuclear-magnetism spectrum.

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