How prostate cancer is diagnosed

Abstract

The present invention relates to a method for early diagnosis of a possible disease state in a patient suspected of having prostate cancer. The present invention provides a method for determining the isotope ratio or isotope quantity ratio of calcium isotopes in a blood sample of a patient suspected of having prostate cancer, the method comprising the steps of: i. determining the isotope ratio or isotope quantity ratio of calcium isotopes in a blood sample of a patient suspected of having prostate cancer; ii. determining the isotope ratio or isotope quantity ratio of calcium isotopes in a range defined by the value determined in step i and a threshold value; 44 / 42 Ca 血清 iii. based on the comparison of step ii, assigning the value to one of three states M0, M1, and M2 defined below, where M0: -0.91‰<δ 44 / 42 Ca 血清 Value<-0.79‰ M1:δ 44 / 42 Ca 血清 Value<-0.91‰ M2:δ 44 / 42 Ca 血清 Including values ​​> -0.79‰.

Description

[Technical Field]

[0001] The present invention relates to a method for early diagnosis of disease pathology in patients suspected of having prostate cancer. [Background technology]

[0002] According to the German Cancer Society (Deutsche Krebsgesellschaft), prostate cancer is the most common cancer among German men, with an incidence rate of 22.7%. In 2017, 62,230 new cases were diagnosed in Germany.

[0003] Currently, prostate-specific antigen (PSA) levels are used for the early detection of prostate carcinoma, but their significance is often questioned due to their low specificity. Therefore, Patent Document 1 proposes a specific protein marker test (HSSK-Hic-Q) as an improved method for diagnosing prostate cancer compared to PSA testing. This test can distinguish between malignant and benign tumors. However, the S3 Prostate Carcinoma Guideline, Extended Version 6.0, 2021, AWMF Registration Number: 043 / 022OL, of the German Oncology Testing Guidelines Program (Leitlinienprogramm Onkologie) (German Cancer Society, German Cancer Support Association (Deutsche Krebshilfe), German Association for Scientific and Medical Research (AWMF)) still recommends PSA levels for the early detection of prostate carcinoma.

[0004] The low specificity of PSA testing has been addressed by using a stepwise, cautious diagnostic approach that emphasizes the presence of bone metastases. Bone metastases are detected using imaging modalities such as dual-energy x-ray absorptiometry (DXA) or magnetic resonance imaging (MRT). These modalities generally lack valid objective values ​​and require considerable skill to interpret. Furthermore, DXA abnormalities often become apparent only after significant bone loss has occurred. Therefore, early diagnosis of potential disease pathology in patients with suspected prostate cancer is crucial.

[0005] Patent Document 2 discloses a method for diagnosing diseases accompanied by decreased bone density and / or calcium loss based on the determination of isotope ratios. Prostate cancer is not necessarily counted as one of such diseases, as it has both osteolytic and osteoblastic stages.

[0006] Patent Document 3 is based on the continuous monitoring of drug therapy for bone cancer by analytical measurement of calcium isotopes in urine, blood, or other tissues. It also mentions the possibility that in the case of osteoblastic prostate cancer, the onset of metastasis can be detected by the assumed increase in bone growth and the resulting positive shift in the calcium isotope mineral balance in the bone. Furthermore, Patent Document 3 describes that the assessment of calcium isotope mineral balance in the bone in clinical practice is performed by comparing the baseline calcium isotope values ​​with measurements over time for each patient. However, this document focuses on bone cancer and merely speculates that this method may be applicable to cancer types other than bone cancer. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Patent Application Publication No. 2014 / 0322740 A1 [Patent Document 2] German Patent Application Publication No. 10 2018 214 660.8 [Patent Document 3] US Patent Application Publication No. 2014 / 0273248 A1 Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to provide a novel method for early diagnosis of a probable disease state in a patient. This method is preferably applied to a patient suspected of having prostate cancer based on medical history and / or prior testing using protein markers. This method is intended to be simpler and faster than conventionally known methods. [Means for solving the problem]

[0009] The object of the present invention is to provide a method for early diagnosis of a possible disease pathology in a patient suspected of having prostate cancer, comprising: i. determining the isotope ratio or isotope quantity ratio of calcium isotopes in a blood sample from a patient suspected of having prostate cancer; ii. The value determined in step i.) and the interval defined by the threshold value -0.91‰<δ 44 / 42 Ca 血清 a step of comparing the value with a value <-0.79‰; iii. The comparison of step ii.) results in classification into one of three defined states M0, M1, and M2, wherein: M0:-0.91‰<δ 44 / 42 Ca 血清 Value < -0.79‰, M1:δ 44 / 42 Ca 血清 Value < -0.91‰, M2:δ 44 / 42 Ca 血清 Value > -0.79‰, This is achieved by a method comprising:

[0010] In the sense of the present invention, category M0 means that there is no change in bone density and therefore no metastasis; category M1 means that the osteolytic stage (osteoporotic or bone degrading event) has been reached with calcium loss and metastasis already occurring within the skeleton; category M2 means that osteoblastic metastasis is present with highly calcified proliferating metastatic foci.

[0011] In the present invention, the term "pathological condition" should be understood to mean the presence or absence of the disease, particularly osteolytic or osteoblastic stage metastasis, that may be expected in a patient suspected of having prostate cancer.

[0012] In a preferred embodiment of the present invention, the determination of the isotopic ratio or isotopic abundance ratio of calcium isotopes is carried out using mass spectrometry.

[0013] In another embodiment of the present invention, the determination of the isotopic ratio or isotopic abundance ratio of calcium isotopes is carried out using laser-induced fluorescence.

[0014] Those skilled in the art can obtain, for example, from US Pat. No. 10,302,565 B2, how to determine the isotopic ratio or isotopic mass ratio of calcium isotopes using laser-induced fluorescence.

[0015] The method of the present invention has been shown to have significant advantages over previously used methods of detecting prostate cancer, which consist of monitoring medical history, positive PSA tests, and the appearance of possible bone metastases.

[0016] Surprisingly, the method of the present invention allows for earlier and more accurate diagnosis of the pathology of osteolytic, i.e., bone-degrading, and osteoblastic, i.e., bone-forming, metastases.

[0017] Unlike early diagnostic methods based solely on the use of protein markers, the method of the present invention makes it possible not only to classify benign and malignant changes in the prostate, but also, in the latter case, to classify the pathological conditions relating to the occurrence of osteolytic and osteoblastic metastases.

[0018] By comparing the measurement data of calcium isotopes in urine and serum with the threshold values, it is possible to predict the subject's calcium balance, i.e., whether or not there is a high level of calcium loss through urine compared to the amount of calcium required for bone mineralization (osteoporosis event).

[0019] In particular, the difference between the method of the present invention and the prior art methods is that determining whether or not an osteoporotic event has occurred is a sufficient condition for confirming the presence of metastatic prostate carcinoma, but is not a necessary condition. In a preferred embodiment of the method of the present invention, even if the sensitivity of the PSA test is only 21%, meaning that there is a 1 in 5 chance that the patient does not have cancer (considered a false positive), serious cases are first pre-selected based on another indicator, such as a positive PSA test (typically >4-10 mL / L, depending on age).

[0020] At the same time, as a risk assessment, calcium isotope ratios should be determined, for example, weekly, in all patients with a positive PSA test to see if the values ​​change. Statistically, it can be assumed that 70% of suspected prostate cancer cases will not actually metastasize (M0 cases), and only about 30% of suspected prostate cancer cases will progress to bone metastasis (M1 cases).

[0021] Micrometastases develop first, and their early detection is essential for a cure. However, imaging techniques often fail to detect them early enough, or even at a sufficient level. As a result, bone metastases progress extremely rapidly, transitioning from an early osteolytic state, where bone breakdown occurs like osteoporosis, to a more osteoblastic state, followed by a high rate of uncontrolled calcification (M2). This is precisely where the method of the present invention is applicable. The method of the present invention can detect early cancer-related osteolytic changes in micrometastases at a level of 0.1 g or less, equivalent to one ten-thousandth of bone mass, or 0.1‰ of bone mass. Therefore, the method for detecting prostate cancer micrometastases of the present invention has extremely high sensitivity in the sub-0.1‰ range, making it the first method suitable for distinguishing between M0, M1, and M2 states through simpler and more rapid measurements than image analysis. Thus, detecting the transition from M0 to M1 at a very early stage allows for targeted treatment / therapy for patients, with a very high chance of recovery.

[0022] Thus, the focus of the present invention can be seen as the applicability of the defined thresholds for diagnostic classification of prostate cancer into three different stages, which was previously unknown and unanticipated.

[0023] US Patent Application Publication No. 2014 / 0273428 A1 proposes measuring Ca isotope ratios for the diagnosis and monitoring of bone cancer. Detection of metastases from prostate and breast cancer is specifically envisioned. According to the teachings of US Patent Application Publication No. 2014 / 0273428 A1, only osteoblastic metastases occur in prostate cancer, while osteolytic metastases occur in breast cancer. Early diagnosis of patients with uncertain prostate cancer suspicion due to, for example, a positive PSA test is neither anticipated nor possible.

[0024] The calcium isotope values ​​in US Patent Application Publication No. 2014 / 0273428 A1 are not related to a threshold independent of the dataset, and therefore no clear prediction can be made regarding whether mineralization or demineralization is actually occurring. In Figure 2 of US Patent Application Publication No. 2014 / 0273428 A1, δ44Ca is actually listed as a balance value but not quantified, so a clear classification into "mineralization" or "demineralization" based on osteolytic or osteoblastic states cannot be made in daily clinical practice. The distinction between "healthy" and "diseased" in US Patent Application Publication No. 2014 / 0273428 A1 always involves the statistical mean of the dataset under consideration, but because the dataset is small, there is a problem with large statistical error in the mean, which makes it difficult to interpret the results in one direction or the other. Furthermore, all of these data are from bone cancer patients.

[0025] Furthermore, it has not been addressed how to distinguish between patients with osteoporosis and patients with osteolytic metastatic prostate cancer. Therefore, preselection by calcium isotope-independent criteria, i.e., PSA testing as envisioned in the present invention, is necessary. The challenge here is to detect the transition from M0 to M1 stage using calcium isotopes, which cannot be achieved without using clearly defined thresholds that allow for the division and differentiation of M0, M1, and M2 stages. Unlike U.S. Patent Application Publication No. 2014 / 0273428 A1, this challenge is achieved by the method of the present invention.

[0026] Starting from US Patent Application Publication No. 2014 / 0273428 A1, which teaches that metastasis of prostate cancer leads to osteoblastic metastasis, it is hard to imagine that a person skilled in the art would be able to conceive that early diagnosis of patients who are only suspected of having prostate cancer can provide reasonable results or further classify them into different states. In particular, it is not clear from US Patent Application Publication No. 2014 / 0273428 A1 that there are generally reasonable thresholds for dividing patients into M0, M1, and M2 states in careful diagnosis, and that there is a possibility of detecting a state without metastasis and associated prostate cancer.

[0027] In yet another embodiment of the method of the present invention, prior to step i.), an anamnesis and / or testing with protein markers may be carried out as an additional step.

[0028] Therefore, the diagnostic methods of the present invention are preferably used to provide an additional diagnosis and / or to complement a suspicion previously established based on medical history and / or a positive test result using protein markers.

[0029] It is therefore particularly advantageous that in one preferred implementation the present invention provides a combination of highly sensitive threshold determination with testing using a low sensitivity marker such as PSA, which typically has a sensitivity of only 21%.

[0030] Furthermore, the present invention relates to an apparatus for early diagnosis of a prostate cancer disease condition, a) means for determining the isotopic ratio or isotopic mass ratio of calcium isotopes in a patient's blood sample; b) a storage medium for storing the determined isotope ratio or isotope quantity ratio values; c) The interval defined by the stored value and the threshold value stored in the storage medium: -0.91‰<δ 44 / 42 Ca 血清 Means compared with values ​​<-0.79‰, d) an evaluation unit for classifying the result of the comparison into one of three predefined states M0, M1, and M2 stored on a storage medium, wherein M0:-0.91‰<δ 44 / 42 Ca 血清 Value < -0.79‰, M1:δ 44 / 42 Ca 血清 Value < -0.91‰, M2:δ 44 / 42 Ca 血清 value >-0.79‰, and e) an output unit for outputting the state M0, M1, or M2 determined by the evaluation; Includes.

[0031] δ m3 / m2 Ca values ​​can be calculated using other Ca isotope ratios (e.g., 40 Ca, 41 Ca, 46 Ca, 48 Ca, 43 Ca) can be replaced with

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[0032] Thus, in the present invention, it is also possible to determine other calcium isotope ratios, and these ratios can be calculated by the δ 44 / 42 Ca 血清 can be converted to a value. [Brief explanation of the drawings]

[0033] [Figure 1] δ44 / 42 Ca serum levels in prostate cancer-positive patients. DETAILED DESCRIPTION OF THE INVENTION

[0034] Without limiting the generality of the disclosure, the method of the present invention and its advantages will now be described.

[0035] Figure 1 shows the delta-positive prostate cancer test results for 20 randomly selected patients. 44 / 42 Ca 血清 Values ​​are expressed relative to the age of the patient.

[0036] Prior to measuring Ca isotope ratios, classification into status M0, M1, and M2 was performed using state-of-the-art diagnostic methods (medical history and positive PSA test) and additional imaging modalities.

[0037] The thick dashed line indicates the balance value δ 44 / 42 Ca バランス =-0.85‰.

[0038] The two dotted lines indicate the tolerance range (±0.06‰) of the threshold value (−0.85‰).

[0039] The two cases shown as A and B are distinguishable, and classification into status M0 based on traditional methods (medical history, PSA level, imaging) is inaccurate.

[0040] Case A shows decreased bone mineral density, possibly due to undetected osteolytic metastasis (M1).

[0041] In case B, the starting point was the finding of proliferative osteoblastic metastasis (M2) due to the suggestion of undetected but age-inappropriate bone mineralization, which would have been erroneously classified as "negative, M0" using conventional methods.

[0042] material and method Blood collection: For blood tests, a doctor draws a standard volume (approximately 8 mL) of blood sample from the patient. The blood sample is allowed to stand for 30 minutes and then centrifuged. The resulting supernatant serum is separated from the clot. Only the serum is used for further chemical preparations.

[0043] To carry out this further chemical preparation, an amount of serum corresponding to 50 μg absolute calcium is isolated.

[0044] Extraction of calcium from samples: Calcium is extracted from the blood by chemical means until a solution of approximately 5 ppm is obtained, a concentration that can be used for mass spectrometry measurements.

[0045] Blood for chemical extraction of calcium can be prepared in the following manner.

[0046] Chemical preparation of samples for calcium isotope determination in blood Day 1 ·Fill the specified vessel for microwave (MW) digestion with HNO3 and H2O2. Pipette the prepared samples and standards into their respective containers. Seal the container, turn on the microwave generator, and microwave for 1.5 hours. Remove the digested sample from the MW. Transfer the solution into cups in a fume hood and dry on a hot plate overnight.

[0047] Day 2 After drying the digested sample overnight, add 1 mL of HNO3 + 0.5 mL of H2O2, seal again, and heat for 3 hours. Open the cup and allow the solution to dry. Add 1 mL of 2M HNO3 to the dried sample.

[0048] Day 3 Calcium (Ca) concentrations are measured by Q-ICP-MS using standard methods.

[0049] Day 4 Column chromatography ensures that the absolute amount of Ca is always the same (50 μg Ca Calculate the amount of acid required for automated ESI PrepFast measurement and dilute the sample appropriately so that the Transfer the sample from the cup into a PrepFAST® tube (Elemental Scientific). The separation of alkaline earth elements is performed automatically using a PrepFAST® instrument (Elemental Scientific).

[0050] Day 5 The elementally separated samples are returned from the tubes to the cups and placed on a hot plate to dry. After the sample has dried, add 1 mL of HNO3 and 0.5 mL of H2O2 again, seal, and heat for another 3 hours.

[0051] Day 6 Open the sample cup and allow it to dry. Add 10 mL of HNO3 to the sample and let it stand for 4 hours to equilibrate. The sample is transferred to a mass spectrometry measurement tube for Neptune (registered trademark) (plasma mass spectrometer, ThermoFisher) and measurement is performed.

[0052] In addition to mass spectrometry, spectroscopic analysis utilizing the mass dependence of the hyperfine structure of emission spectral lines can also be used to determine the isotope ratio or isotope quantity ratio of calcium isotopes.

[0053] Those skilled in the art can obtain, for example, from US Pat. No. 10,302,565 B2, how to determine the isotopic ratio or isotopic mass ratio of calcium isotopes using laser-induced fluorescence.

[0054] Specifically, all measurements are expressed relative to the international standard material SRM915a.

[0055] Below we will explain how to determine the values ​​by mass spectrometry, without limiting the general validity of this method.

[0056] Mass spectrometry measurements: This solution is typically measured using a plasma mass spectrometer (MC-ICP-MS: multi-collector inductively coupled plasma mass spectrometer) (e.g., ThermoFisher, Neptune) (this method will be briefly described later), but alternatively, measurement using a surface ionization mass spectrometer is also possible. The purpose of the measurement is to determine the calcium isotopic composition of the serum. In TIMS, 44 Ca / 40 The Ca ratio was measured, and MC-ICP-MS 44 Ca / 42 Measure the Ca ratio or other ratios. Both isotope ratios have equal predicted values. The values ​​differ by only one factor.

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[0057] Plasma mass spectrometer 44 Ca / 42 Determination of calcium isotopic composition Measurement of calcium isotopes is performed using an MC-ICPMS (Neptune®, Thermo Fisher Scientific). The mass spectrometer is equipped with nine Faraday cups, eight of which are movable. The Faraday cups are configured to simultaneously measure atomic masses (u) 42, 43, 43.5, and 44. Ca and Ar hydrides (e.g., 42 Ca 40To suppress ArH interference, an APEX IR sample guide system (Elemental Scientific®) was used. All measurements were performed at moderate resolution (m / Δm 4,000 or so) on an interference-free plateau below the central measurement peak. This was achieved by selecting an appropriate mean cup mass of 4.687 ± 0.001 u and performing daily validation.

[0058] Correction for instrument-induced fractionation ("mass bias") is performed using the "standard sample bracketing (SSB)" method. Sample measurements are corrected using measurements of a 5 μg / mL Ca solution prepared from a 10,000 μg / mL Ca-ICP standard. Each sample is measured at least four times during one session, and the average value is used in the next step. The Ca isotopic composition is determined by δ 44 / 42 Expressed as Ca in parts per thousand (‰): δ 44 / 42 Ca(‰)=[( 44 Ca / 42 Ca) サンプル / ( 44 Ca / 42 Ca) 標準液 ]-1

[0059] The Ca-ICP standard solution used in the measurement serves as the primary standard. 44 / 42 Ca ICP δ based on NIST SRM 1486 using the value 44 / 42 Ca ICP Convert values ​​to values ​​based on NIST SRM 915a: δ 44 / 42 Ca SRM915a (sample, ‰) = δ 44 / 42 Ca ICP (Sample)-δ 44 / 42 Ca ICP (SRM 915a) In other words, all measured values ​​are expressed as relative values ​​based on the international standard material SRM915a.

[0060] During each session, untreated NIST SRM 915a material is measured at the beginning and end of each session, and the results are compared with those of treated NIST SRM 915a material. The average difference between treated and untreated SRM 915a is typically expected to be less than 0.01‰, so Ca isotope fractionation during chemical purification can be considered negligible.

[0061] For background correction, an "on-peak" approach is used: the measured intensity of the 1% HNO3 solution is subtracted from the measured intensity of the subsequent sample.

[0062] In addition, the residue of strontium with double charge ( 84 Sr, 86 Sr, and 88 Check the Sr) measurement value, 42 Ca, 43 Ca, and 44 To verify the accuracy of the Ca mass intensity measurements, the 42 / 43.5, 43 / 43.5, and 44 / 43.5 mass intensity ratios of a 2 μg / mL Sr solution are measured at the beginning of each session. These ratios are then used to calculate the intensity of Sr, which has twice the charge, from the measured intensity of the 43.5 mass for a given sample.

[0063] For quality control purposes, the δ values ​​of NIST SRM 1486 and IAPSO standard seawater were measured during the measurement stage. 44 / 42 The Ca values ​​are compared with published values. Generally, the long-term reproducibility (2 SD = standard deviation) of all analytical standards over a period of approximately 2 months is better than ±0.06‰.

[0064] Additionally, for quality control purposes, several criteria are used to reject data from single measurements, single samples, and entire sequences. A single measurement or sequence is discarded if: ·|δ 44 / 42 Ca-2·δ 43 / 42 Ca|>0.2‰ Discard the sample if its mean intensity is outside the 70%-130% intensity range compared to the mean intensity of the same batch of 5 μg / mL Ca-ICP solution or NIST SRM 915a solution. If the deviation from the literature value for more than one of the international reference materials used in the measurement is greater than 0.2‰, or if the data does not lie on a mass-dependent fractionation line, the entire sequence is rejected.

[0065] Analysis of calcium isotope data Calcium isotope measurements are reported in conventional δ notation.

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Claims

1. A method for early diagnosis of a probable disease state in a patient suspected of having prostate cancer, comprising: i. determining the isotope ratio or isotope quantity ratio of calcium isotopes in a blood sample of a patient suspected of having prostate cancer; ii. The value determined in step i.) and the interval defined by the threshold value -0.91‰<δ ４４／４２ Ca 血清 a step of comparing with a value <-0.79‰; iii. The comparison of step ii.) results in a classification into one of three states M0, M1, and M2 defined below, wherein: M0: -0.91‰<δ ４４／４２ Ca 血清 Value < -0.79‰, M1:δ ４４／４２ Ca 血清 Value < -0.91‰, M2:δ ４４／４２ Ca 血清 value > -0.79‰; A method comprising:

2. 2. The method of claim 1, wherein the determination of the isotopic ratio or isotopic abundance ratio of the calcium isotopes is performed using mass spectrometry.

3. 2. The method of claim 1, wherein the determination of the isotope ratio or isotope quantity ratio of the calcium isotopes is performed using laser-induced fluorescence.

4. 2. The method according to claim 1, wherein the determination of the isotope ratio or isotope quantity ratio of the calcium isotopes is carried out using a spectroscopic analysis method utilizing the mass dependence of the hyperfine structure of the emission spectral lines.

5. 2. The method according to claim 1, characterized in that before step i.), as an additional step, a medical history and / or a test using protein markers is carried out.

6. A device for early diagnosis of a prostate cancer disease condition, comprising: a) means for determining the isotopic ratio or isotopic mass ratio of calcium isotopes in a patient's blood sample; b) a storage medium for storing the determined isotope ratio or isotope quantity ratio value; c) An interval defined by the stored value and a threshold value stored in the storage medium: −0.91‰<δ ４４／４２ Ca 血清 means for comparing with a value < -0.79‰; d) an evaluation unit for classifying the result of the comparison into one of three predefined states M0, M1 and M2 stored on said storage medium, M0: -0.91‰<δ ４４／４２ Ca 血清 Value < -0.79‰, M1:δ ４４／４２ Ca 血清 Value < -0.91‰, M2:δ ４４／４２ Ca 血清 value > -0.79 ‰, and e) an output unit for outputting the state M0, M1, or M2 determined by the evaluation; An apparatus comprising:

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