Diagnostic method for prostate cancer

Abstract

The present invention relates to a method for the early diagnosis of the pathology of a possible illness in patients suspected of having prostate cancer, comprising the following steps: i. determining isotope ratios or quantity ratios of calcium isotopes in a sample of the blood of the patient suspected of having prostate cancer. ii. comparing the value determined in step i with the interval -0.91‰ < δ44 / 42 CaSerum value < -0.79 ‰ defined by a threshold value, iii. assigning, based on the comparison from step ii, to one of three defined situations M0, M1 and M2 where M0: -0.91‰ < δ44 / 42 CaSerum value < -0.79 ‰ M1: δ44 / 42 CaSerum value < -0.91‰ M2: δ44 / 42 CaSerum value > -0.79‰.

Description

[0001] Diagnostic method for prostate cancer

[0002] The invention relates to a method for the early diagnosis of the pathology of a disease in patients with suspected prostate cancer.

[0003] According to the German Cancer Society, prostate cancer is the most common cancer among men in Germany, accounting for 22.7% of cases. In 2017, 62,230 new cases were diagnosed in Germany.

[0004] The prostate-specific antigen (PSA) level is currently used for the early detection of prostate cancer, although its validity is often questioned due to its low specificity. US 2014 / 0322740 A1 proposes a more specific protein-based marker test (HSSK-Hic-Q) as an improved diagnostic method for prostate cancer compared to the PSA test. This test can distinguish between malignant and benign tumors. However, according to the Oncology Guidelines Program (German Cancer Society, German Cancer Aid, AWMF): S3 Guideline Prostate Cancer, Long Version 6.0, 2021, AWMF Registry Number: 043 / 0220L, the PSA level is still recommended for the early detection of prostate cancer.

[0005] The problem of the low specificity of the PSA test is addressed by a wait-and-see approach aimed at detecting the presence of bone metastases. Bone metastases are detected using imaging techniques such as dual X-ray absorptiometry (DXA) or magnetic resonance imaging (MRI). The problem with these imaging techniques is that no universally valid objective value is determined; instead, correct interpretation of the images requires considerable experience. Furthermore, DXA often only reveals abnormalities after a significant portion of the bone mass has already disappeared. An earlier diagnosis of the pathology of a possible disease in patients with suspected prostate cancer is therefore desirable.

[0006] DE 10 2018 214 660.8 describes a method for diagnosing a disease associated with reduced bone density and / or calcium loss, based on the determination of isotope ratios. Prostate cancer is not necessarily considered such a disease, as there are both osteolytic and osteoblastic stages of prostate cancer.

[0007] US 2014 / 0273248 A1 primarily describes the observational monitoring of drug therapy for bone cancer through analytical measurement of calcium isotopes in urine, blood, or other tissue. Reference is also made to the possible detection of incipient metastasis in osteoblastic prostate cancer through a potential increase in bone growth and the resulting positive shift in the bone mineral balance of calcium isotopes. US 2014 / 0273248 A1 further states that, in practice, an evaluation of the bone mineral balance of calcium isotopes is performed through serial measurements, which are compared with a patient-specific baseline of isotopes. However, with regard to cancer types other than bone cancer, which is the focus of the document, only an assumption is revealed that this procedure could be applied to these types of cancer.

[0008] The object of the invention is to provide a novel method for the early diagnosis of the pathology of a potential disease in patients. This should preferably apply to patients in whom prostate cancer is suspected based on a prior medical history and / or a prior test with a protein-based marker. The method should be simpler and faster to perform than previously known methods.

[0009] The object of the invention is achieved by a method for the early diagnosis of the pathology of a possible disease in patients with suspected prostate cancer, comprising the following steps: i. Determining isotope ratios or quantitative ratios of calcium isotopes in a blood sample of the patient with suspected prostate cancer. ii. Comparing the value determined in step i with the interval defined by a threshold value.

[0010] -0.91%o < δ 44 / 42 CaSerum -value < -0.79 %o iii. Assignment based on the comparison from step ii to one of the three defined situations MO, M1 and M2 where

[0011] MO: -0.91%o < δ 44 / 42 Ca Serum -value < -0.79 %o

[0012] M1 : δ 44 / 42 Ca Serum -value < -0.91%o

[0013] M2: δ 44 / 42 Ca Serum -value > -0.79%o.

[0014] For the purposes of the present invention, the MO classification means that there is no change in bone density and thus no metastasis. The M1 classification means that an osteolytic stage (osteoporotic or bone-dissolving process) has been reached, calcium loss is present, and metastases have already formed in the skeleton. The M2 classification means that osteoblastic metastases with proliferating and highly mineralizing metastases are present. For the purposes of the present invention, the term "pathology" of a possible disease in patients with suspected prostate cancer refers, in particular, to the absence or presence of an osteolytic or osteoblastic metastasis stage.

[0015] In a preferred embodiment of the invention, the isotope ratios or quantity ratios of calcium isotopes are determined using mass spectrometry.

[0016] In an alternative embodiment of the invention, the isotope ratios or quantity ratios of calcium isotopes are determined using laser-induced fluorescence.

[0017] The procedure for determining the isotope ratios or quantity ratios of calcium isotopes using laser-induced fluorescence can be found, for example, in US10302565B2.

[0018] It was shown that the method according to the invention offers significant advantages over the methods currently used for the detection of prostate cancer, which consist of anamnesis, a positive PSA test and the monitoring of possible bone metastases.

[0019] Surprisingly, with the aid of the method according to the invention, the pathology relating to the formation of osteolytic, ie bone-dissolving, and osteoblastic, ie bone-forming, metastases can be diagnosed much earlier and more accurately.

[0020] Compared to early diagnosis methods that rely solely on the use of protein-based markers, the method according to the invention makes it possible to classify not only benign and malignant changes in the prostate, but in the latter case also to the pathology related to the formation of osteolytic and osteoblastic metastases.

[0021] From the measured calcium isotope data in comparison to the threshold values ​​in urine and blood serum, a statement can be made about a person's calcium balance, i.e. whether the calcium loss via urine is higher than the amount of calcium required for bone mineralization (osteoporotic process). The difference between the method according to the invention and the methods of the prior art can be seen in particular in the fact that the determination of whether an osteoporotic process is present or not is a sufficient but not necessary condition for the presence of metastatic prostate cancer. In a preferred embodiment of the present method, a pre-selection of critical cases is carried out using a second indicator, e.g.a positive PSA test (usually >4 - 10 ml / l depending on age), even if the sensitivity of the PSA test is only 21% and the person affected still has a chance of 1 in 5 of not having the cancer (false positive assumption).

[0022] Nevertheless, based on a risk assessment, all patients who test positive in a PSA test will then have their calcium isotope ratio measured weekly, for example, to determine if the value changes. Statistically, it can be expected that 70% of suspected prostate cancer cases will not develop metastases at all (MO cases), and only approximately 30% of suspected prostate cancer cases will develop metastases in the bone (M1 cases).

[0023] Initially, "micro-metastases" form, which must be detected as early as possible to guarantee a cure. However, these are inadequately or not at all detected by imaging techniques in the early stages. As a result, the bone metastases develop very rapidly, transitioning from the initial osteolytic state of pure bone dissolution, as in osteoporosis, but at higher rates, to the osteoblastic state, with then high rates of uncontrolled mineralization (M2 cases). This is precisely where the method according to the invention comes in. The method according to the invention can already detect changes caused by early cancer-related osteolysis of micro-metastases in the range of 0.1 g and less, which corresponds to one ten-thousandth of the bone mass, or 0.1% of the bone mass.

[0024] The inventive method for detecting micrometastases in prostate cancer thus exhibits a very high sensitivity of less than 0.1% and is therefore suitable for the first time for distinguishing between the MO, M1, and M2 stages using a measurement that is simpler and faster than imaging techniques. This very early detection of the transition from MO to M1 then allows for targeted treatment / therapy of the patient with a very high expectation of cure.

[0025] In other words, the core of the invention can be seen in the applicability of fixed threshold values ​​for the diagnostic classification of prostate cancer into three different stages, which was previously neither known nor expected. US 2014 / 0273428 A1 proposes the measurement of the ratio of Ca isotopes for the diagnosis and monitoring of bone cancer. Among other things, the detection of metastases resulting from prostate and breast cancer is envisaged. According to the findings of US 2014 / 0273428 A1, in the case of prostate cancer, only osteoblastic metastases occur, whereas in breast cancer, osteolytic metastases are observed. An early diagnosis in patients in whom there is an uncertain suspicion of prostate cancer, for example, based on a positive PSA test, is neither intended nor possible.

[0026] The calcium isotope values ​​in US 2014 / 0273428 A1 do not refer to a threshold value independent of the dataset, so no clear statement can be made as to whether mineralization or demineralization is actually present. Although Figure 2 in US 2014 / 0273428 A1 mentions a δ44Caine as an equilibrium value, it is not quantified, so a clear assignment to "mineralization" or "demineralization" due to osteolytic or osteoblastic conditions cannot be made in everyday clinical practice. The classification of "healthy" or "diseased" made in US 2014 / 0273428 A1 always refers to the statistical mean of the dataset under consideration, which, however, is subject to a high statistical error due to the small dataset, which generally complicates interpretation in one direction or the other, not to mention that the data are always from patients with bone cancer.

[0027] Furthermore, it is not addressed how a patient suffering from osteoporosis can be distinguished from metastatic prostate cancer in an osteolytic state. Therefore, pre-selection must be carried out using a criterion independent of calcium isotopes, the PSA test, as provided by the invention. The problem here is detecting the transition from the MO to the M1 stage using calcium isotopes; this is only possible with clearly defined threshold values ​​that enable delimitation and differentiation between the MO, M1, and M2 stages. In contrast to US 2014 / 0273428 A1, this is achieved with the method of the present invention.

[0028] Based on US 2014 / 0273428 A1, which teaches that metastasis following prostate cancer leads to osteoblastic metastases, the skilled person has no reason to assume that an early diagnosis in patients in whom prostate cancer is only suspected could yield meaningful results or even the classification of distinguishable conditions. In particular, a generally valid threshold value for distinguishing between the MO, M1, and M2 states and the detection of the state of absence of metastases and the associated possibility of preserving the prostate within the framework of a watchful waiting diagnosis is not suggested by US 2014 / 0273428 A1. In a further embodiment of the method according to the invention, the following additional steps can be carried out before step i: a medical history and / or a test with a protein-based marker.

[0029] The diagnostic method according to the invention is therefore preferably used to further diagnose and / or supplement a previously established suspicion based on an anamnesis and / or a positively evaluated protein-based marker test.

[0030] The particularly advantageous aspect of the invention, in a preferred embodiment, is therefore the combination of a test with an insensitive marker such as PSA, typically with a sensitivity in the range of only 21%, with a very sensitive threshold value determination.

[0031] The invention further relates to a device for the early diagnosis of the pathology of a possible prostate cancer, comprising a) means for determining isotope ratios or quantity ratios of calcium isotopes in a blood sample of a patient, b) a storage medium for storing the determined value of the isotope ratios or quantity ratios, c) means for comparing the stored value with an interval defined as a threshold value -0.91%o < δ 44 / 42 Ca Serum -value < -0.79 %o, which is stored in the storage medium, d) evaluation unit for assigning the comparison to one of three previously defined situations MO, M1 and M2 stored in the storage medium, where

[0032] MO: -0.91%o < δ 44 / 42 Ca Serum -value < -0.79 %o M1: δ 44 / 42 Ca Serum -value < -0.91%o

[0033] M2: δ 44 / 42 Ca Serum-value > -0.79%o, and e) output unit for outputting the situation MO, M1 or M2 determined during the evaluation. The 8 m3 / m2 Ca values ​​can also be converted to other Ca isotope ratios (for example, involving 40 Approx. 41 Approx. 46 Approx. 48 Approx. 43 Ca) transferred: ln(m3 / m2) ä m3 / m2 Ca = ä m3 / ml Ca x with m3 > m2 > m1.

[0034] In this way, other calcium isotope ratios can also be determined in accordance with the invention, which can then be applied to the claimed δ 44 / 42 Ca Serum -value of step ii).

[0035] In the following, the generality of the teaching will be explained in a non-limiting manner, the method according to the invention and its advantages.

[0036] Figure 1 shows the δ 44 / 42 Ca Serum-Values ​​of 20 randomly selected prostate cancer positive patients as a function of patient age.

[0037] The assignment to the situations MO, M1 and M2 was made before the measurement of the Ca isotope ratios using the diagnostic procedures corresponding to the current state of the art (anamnesis and positive PSA test) and additionally by imaging procedures.

[0038] The thick dashed line shows the equilibrium value δ 44 / 42 CaEquilibrium= -0.85 %o.

[0039] The two dotted lines show the tolerance range (±0.06 %o) of the threshold value (-0.85 %o).

[0040] There are two cases, designated A and B, in which an assignment to the MO situation based on traditional procedures (anamnesis, PSA value, imaging procedures) is not appropriate.

[0041] In case A, there is a reduction in bone density, which may be due to undetected osteolytic metastases (M1).

[0042] Case B shows undetected and age-inappropriate bone mineralization, suggesting a proliferating osteoblastic metastasis (M2), which was still incorrectly classified as "no findings, MO" by traditional methods. Materials and Methods

[0043] Blood sample collection:

[0044] The doctor takes a blood sample from the patient, a volume equivalent to the amount required for blood tests (approximately 8 ml). The blood sample is allowed to stand for half an hour and then centrifuged. The resulting blood serum is separated from the blood clot. Only blood serum is used for further chemical processing.

[0045] For further chemical processing, a serum quantity corresponding to an absolute amount of 50 pg calcium is taken.

[0046] Extraction of calcium from the samples:

[0047] In a chemical process, the calcium is extracted from the blood until a solution with a concentration of approximately 5 ppm is available for mass spectrometric measurement.

[0048] The preparation of blood for chemical extraction of calcium can be carried out using the following procedure:

[0049] Chemical sample preparation for calcium isotope determination in blood

[0050] Day 1

[0051] • The vessels required for microwave (MW) digestion are filled with HNO3 and H2O2.

[0052] • The prepared samples and standards are pipetted into the respective vessel.

[0053] • Containers are closed, the microwave oven is started and placed in the MW for 1.5 hours.

[0054] • The digested samples are removed from the microwave oven. In the fume hood, the solutions are transferred one after the other into beakers and placed on the hotplate to dry overnight.

[0055] Day 2

[0056] • The digested and overnight dried samples are taken up with 1ml HNO3+0.5ml H2O2 and boiled again for 3 hours.

[0057] • Open the beaker and allow the solution to dry. • Dissolve the dried samples in 1 ml of 2M HNO3.

[0058] Day 3

[0059] • The concentration of calcium (Ca) is measured on the Q-ICP-MS using standard methods.

[0060] Day 4

[0061] • In order to always use the same absolute Ca amounts (50 g Ca), the amount of acid required for automated measurement on an ESI PrepFast is calculated and the samples are diluted accordingly.

[0062] • The samples are transferred from the beakers into the PrepFAST® tubes (Elemental Scientific).

[0063] • The separation of the alkaline earth elements takes place automatically using the prepFAST® device (Elemental Scientific).

[0064] Day 5

[0065] • The samples separated by elements are transferred from the tubes back into the beakers and placed on the hot plate to dry.

[0066] • After drying, these are taken up again with 1ml HNO3 and 0.5ml H2O2 and boiled in a closed container for a further 3 hours.

[0067] Day 6

[0068] • The cups containing the samples are opened and dried.

[0069] • The samples are taken up in 10 ml HNO3 and left to stand for 4 hours for equilibration.

[0070] • The samples are transferred into the tubes for mass spectrometric measurement on the Neptune® (plasma mass spectrometer, ThermoFisher) and measured there.

[0071] In addition to mass spectrometric methods, spectroscopic methods can also be used to determine the isotope ratios or quantity ratios of calcium isotopes, which use light emission to exploit the mass dependence of the hyperfine structure of the spectral lines.

[0072] The procedure for determining the isotope ratios or quantity ratios of calcium isotopes using laser-induced fluorescence can be found, for example, in US10302565B2.

[0073] All measured values ​​are given relative to the international standard SRM915a. The mass spectroscopic determination of the values ​​is described below, without limiting the generality of the method.

[0074] Mass spectrometric measurement:

[0075] This solution is usually measured in a plasma mass spectrometer (MC-ICP-MS: Multi-Collector Ionization Coupled Mass Spectrometer) (e.g., ThermoFisher, Neptune) (This procedure is briefly described below), but can alternatively be measured in a thermionic mass spectrometer. The goal is to determine the calcium isotopic composition in blood serum. In the TIMS, 44 Ca / 40 Ca ratios and in the MC-ICP-MS 44 Ca / 42 Ca or alternative ratios. Both isotope ratios are equivalent in their significance. The values ​​differ only by one factor: 44 Ca / 40Ca = 2.05 · 44 Ca / 42 Approx.

[0076] Determination of calcium isotope composition 44 Ca / 42 Ca using a plasma mass spectrometer:

[0077] Calcium isotope measurements are performed on an MC-ICPMS (Neptune®, Thermo Fisher Scientific). The mass spectrometer is equipped with nine Faraday cups, eight of which are movable. These are set up to measure the atomic masses (u) 42, 43, 43.5, and 44 simultaneously. To eliminate interfering Ca and Ar hydrides (e.g., 40 AriH2 on 42 To suppress the presence of Ca), an APEX IR sample introduction system (Elemental Scientific®) is used. All measurements are performed at medium resolution (m / Am - 4000) on the interference-free plateau of the low-mass side of the central measurement peak. This is achieved by selecting a suitable mean cup mass of 4.687±0.001 u and verifying it daily.

[0078] Instrumental fractionation (“mass bias”) is corrected by applying the “standard sample bracketing” (SSB) procedure. The measurement of a sample is corrected by measurements of a 5 pg / ml Ca solution prepared from a 10,000 pg / ml Ca ICP standard solution. Each sample is measured at least four times during a session, and the average value is used for further processing. The Ca isotopic composition is expressed as δ 44 / 42 Ca expressed in parts per thousand (%o): δ 44 / 42 Ca (%o) = [( 44 Ca / 42 Ca) Probe / ( 44 Ca / 42 Ca) Referenz ] - 1

[0079] The measured Ca-ICP standard solution serves as the primary reference material. The δ 44 / 42 Ca ICP - Values ​​are then calculated using the measured δ 44 / 42 Ca ICP -value converted from NIST SRM 1486 to NIST SRM 915a: δ 44 / 42 Ca SR M9i5a (sample, %o) = δ 44 / 42 Ca ICP (sample) - δ44 / 42 Ca ICP (SRM 915a), ie all measured values ​​are given relative to the international standard SRM915a.

[0080] During each session, chemically unprocessed NIST SRM 915a material is measured at the beginning and end of each session, and these results are compared with those of chemically processed NIST SRM 915a. The average difference between processed and unprocessed SRM 915a is typically less than 0.01%, so Ca isotope fractionation during chemical purification can be considered negligible.

[0081] The "on-peak" approach is used for background correction. The measured intensities of a 1% HNO3 solution are subtracted from the intensities of the subsequent sample measurements.

[0082] In addition, the measurements are carried out for residues of doubly charged strontium ( 84 Sir, 86 Sr and 88Sr) to ensure the correct measurement of the intensities of the masses of 42 Approx. 43 Ca and 44 Ca. To do this, the intensity ratios of the masses 42 / 43.5, 43 / 43.5, and 44 / 43.5 of a 2 pg / ml Sr solution are measured at the beginning of each session. These ratios are then used to calculate the intensities of doubly charged Sr from the measured intensities of mass 43.5 of a given sample.

[0083] For quality assurance, the δ measured during the measurement phase 44 / 42 Ca values ​​from NIST SRM 1486 and IAPSO seawater standards were compared with published values. Long-term reproducibility (2 SD = standard deviation) over a period of approximately two months is generally better than ±0.06% for all analyzed reference materials.

[0084] For further quality assurance, several criteria are applied to reject data from a single measurement, a single sample, and entire sequences. A single measurement or sequence is discarded if:

[0085] • |δ 44 / 42 Ca - 2 δ 43 / 42 Ca|>0.2 %o applies.

[0086] • A sample measurement is discarded if the average intensity is outside a 70 to 130% intensity window compared to the average intensity of the 5pg / ml Ca ICP solution or NIST SRM 915a solution of the same lot.

[0087] • A complete sequence is rejected if more than one of the measured international reference materials is more than 0.2 %o from the literature value or the data did not fall along the mass-dependent fractionation line.

[0088] Evaluation of calcium isotope data:

[0089] The measured calcium isotope values ​​are reported in the usual ö-notation: where ( 44 Ca / 42 Ca) Probe is the ratio of the blood sample measured in the mass spectrometer and ( 44 Ca / 42 Ca) Standard the calcium isotope ratio is an internationally known and certified ratio. This procedure allows for international comparability of the measured isotope ratios.

[0090] List of figures:

[0091] Figure 1 : δ44 / 42 Ca Serum -Values ​​of patients tested positive for prostate cancer

Claims

AMENDED CLAIMS received by the International Bureau on 29 January 2024 (29.01.2024) 1. A method for the early diagnosis of the pathology of a possible disease in patients with suspected prostate cancer, comprising the following steps: Determination of isotope ratios and quantity ratios of Calcium isotopes in a blood sample from a patient suspected of having prostate cancer. ii. Comparison of the value determined in step i with the interval defined by a threshold value -0.91 %o < δ 44 / 42 Ca Serum -value < -0.79 %o iii. Allocation based on the comparison from step ii to one of three defined Situations MO, M1 and M2, where MO: -0.91%o < δ 44 / 42 Ca Serum -value < -0.79 %o M1 : δ 44 / 42 Ca Serum -value < -0.91%o M2 : δ 44 / 42 Ca Serum-value > -0.79%o, and the assignment MO means that there is no change in bone density and therefore no metastasis, the assignment M1 means that an osteolytic stage has been reached, calcium loss is present and metastases have already formed in the skeleton and the assignment M2 means that there is an osteoblastic metastasis finding.

2. Method according to claim 1, characterized in that the determination of the isotopic ratios or quantitative ratios of calcium isotopes is carried out by means of mass spectrometry.

3. Method according to claim 1, characterized in that the determination of the isotope ratios or quantity ratios of calcium isotopes is carried out by means of laser-induced fluorescence.

4. Method according to claim 1, characterized in that the determination of the isotopic ratios or quantity ratios of calcium isotopes is carried out by means of spectroscopic methods which use the mass dependence of the hyperfine structure of the spectral lines by means of light emission. AMENDED SHEET (ARTICLE 19) 5. The method according to claim 1, characterized in that the following additional steps are carried out before step i: a medical history and / or a test with a protein-based marker.

6. Device for the early diagnosis of the pathology of a possible prostate cancer, comprising a) means for determining isotope ratios or quantity ratios of calcium isotopes in a blood sample of a patient, b) a storage medium for storing the determined value of the isotope ratios or quantity ratios, c) means for comparing the stored value with an interval defined as a threshold value -0.91%o < δ44 / 42 Ca Serum -value < -0.79 %o, which is stored in the storage medium, d) evaluation unit for assigning the comparison to one of three previously defined situations MO, M1 and M2 stored in the storage medium, where MO: -0.91 %o < δ 44 / 42 Ca Serum -value < -0.79 %o M1 : δ 44 / 42 Ca Serum -value < -0.91%o M2: δ 44 / 42 Ca Serum -value > -0.79%o, and e) output unit for outputting the situation MO, M1 or M2 determined during the evaluation, whereby the assignment MO means that there is no change in bone density and therefore no metastasis, the assignment M1 means that an osteolytic stage has been reached, calcium loss is present and metastases have already formed in the skeleton and the assignment M2 means that an osteoblastic metastasis finding is present. AMENDED SHEET (ARTICLE 19)

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