DE102022122197A1 - Diagnostic method for prostate cancer - Google Patents

Abstract

The present invention relates to a method for early diagnosis of the pathology of a possible disease in patients suspected of having prostate cancer, comprising the following steps:i. Determine isotope ratios or quantitative ratios of calcium isotopes in a sample of blood from the patient with suspected prostate cancer.ii. Comparison of the value determined in step i with the interval defined by a threshold value −0.91‰<δ44/42CaSerum value<−0.79‰iii. Assignment based on the comparison from step ii to one of three defined situations M0, M1 and M2 whereM0: -0.91‰ < δ44/42caserum value < -0.79‰M1: δ44/42caserum value < -0.91‰ M2: δ44/42Caserum value > -0.79‰ represents.

Description

The invention relates to a method for early diagnosis of the pathology of a disease in patients suspected of having prostate cancer.

According to the German Cancer Society, prostate cancer is the most common cancer among men in Germany at 22.7%. In 2017, 62,230 new cases were diagnosed in Germany.

The prostate-specific antigen (PSA) value is now used for the early detection of prostate cancer, although the significance of the PSA value is often questioned due to its low specificity. That's how it beats US 2014/0322740 A1 a more specific protein-based marker test (HSSK-Hic-Q) as an improved diagnostic method for prostate cancer compared to the PSA test. A distinction can be made here between malignant and benign tumors. However, according to the oncology guideline program (German Cancer Society, German Cancer Aid, AWMF): S3 guidelines for prostate cancer, long version 6.0, 2021, AWMF registration number: 043/022OL, the (PSA) value is still recommended for the early detection of prostate cancer.

The problem of the low specificity of the PSA test is addressed by a phased wait-and-see diagnosis that is aimed at the occurrence of bone metastases. Bone metastases are detected using imaging methods such as dual X-ray absorptiometry (DXA) or magnetic resonance imaging (MRI). The problem with these imaging procedures is that no generally valid objective value is determined; the correct interpretation of the images requires a great deal of experience. In addition, DXA often only reveals abnormalities when a significant portion of the bone mass has already disappeared. Therefore, earlier diagnosis of the pathology of a possible disease in patients with suspected prostate cancer is desirable.

The DE 10 2018 214 660.8 contains a method for diagnosing a disease that is associated with reduced bone density and/or calcium loss, which is based on the determination of isotope ratios.

It is the object of the invention to provide a new method for the early diagnosis of the pathology of a possible disease in patients. This should preferably apply to patients in whom prostate cancer is suspected based on a previous medical history and/or a previous test with a protein-based marker.

1. i. Bestimmen von Isotopenverhältnissen oder Mengenverhältnissen von Calciumisotopen in einer Probe von Blut des Patienten mit Verdacht auf Prostatakrebs.
2. ii. Vergleich des in Schritt i ermittelten Wertes mit dem durch einen Schwellenwert definiertem Intervall $$-\mathrm{0,91}‰<{\mathrm{\delta }}^{44/42}{\text{Ca}}_{\text{Serum}}-\text{Wert}<-\mathrm{0,79}‰$$
   
3. iii. Zuordnung anhand des Vergleichs aus Schritt ii zu einem der drei definierten Situationen M0, M1 und M2 wobei
   • M0: -0,91‰ < δ^44/42 Ca_serum-Wert < -0,79 ‰
   • M1: δ^44/42 Ca_serum-Wert < -0,91‰
   • M2: δ^44/42 Ca_serum-Wert > -0,79‰ darstellt.

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:

1. i. Determining isotope ratios or quantitative ratios of calcium isotopes in a sample of blood from the patient with suspected prostate cancer.
2. ii. Comparison of the value determined in step i with the interval defined by a threshold value $$-0.91‰<{\mathrm{\delta }}^{44/42}{\text{Approx}}_{\text{serum}}-\text{Value}<-0.79‰$$
   
3. iii. Assignment based on the comparison from step ii to one of the three defined situations M0, M1 and M2 where
   • M0: -0.91‰ < δ ^44/42 Ca _serum value < -0.79‰
   • M1: δ ^44/42 Ca _serum value < -0.91‰
   • M2: δ ^44/42 Ca _serum value > -0.79‰.

In the sense of the present invention, the assignment M0 means that there is no change in bone density and therefore no metastasis and the assignment M1 means that an osteolytic stage (osteoporotic or bone-dissolving event) has been reached, there is calcium loss and metastases in the skeleton have already been formed and The assignment M2 means that there is an osteoblastic metastasis finding with proliferating and heavily mineralizing metastases.

For the purposes of the present invention, the term pathology of a possible disease in patients with suspected prostate cancer is understood in particular to mean the absence or presence of an osteolytic or osteoblastic metastasis stage.

In a preferred embodiment of the invention, the isotope ratios or quantitative ratios of calcium isotopes are determined using mass spectrometry.

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

The procedure for determining the isotope ratios or quantitative ratios of calcium isotopes using laser-induced fluorescence can be done by a person skilled in the art, for example US10302565B2 remove.

It has been shown that the method according to the invention has significant advantages over the methods currently used for detecting prostate cancer, consisting of anamnesis, positive PSA test and monitoring of possible bone metastases.

Surprisingly, with the help of the method according to the invention, the pathology in relation to the formation of osteolytic, i.e. bone-dissolving, and osteoblastic, i.e. bone-forming, metastases can be diagnosed much earlier and more correctly.

Compared to early diagnosis methods that are based solely on the use of protein-based markers, the method according to the invention makes it possible to assign not only benign and malignant changes in the prostate, but in the latter case also to the pathology in relation to the formation of osteolytic and osteoblastic Metastases.

In a further embodiment of the method according to the invention, the following steps can additionally be carried out before step i: an anamnesis and/or a test with a protein-based marker.

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

mit m3 > m2 > m1.The δ ^m3/m2 Ca values can also be transferred to other Ca isotope ratios (for example involving ⁴⁰ Ca, ⁴¹ Ca, ⁴⁶ Ca, ⁴⁸ Ca, ⁴³ Ca) using the following formula: $${\ddot{\text{a}}}^{m3/m2}Ca={\ddot{\text{a}}}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

with m3 > m2 > m1.

In this way, within the meaning of the invention, other calcium isotope ratios can also be determined, which are then converted to the claimed δ ^44/42 Ca _serum value of step ii).

The following is intended to explain the method according to the invention and its advantages without restricting the generality of the teaching.

The shows the δ ^44/42 Ca _serum values of 20 randomly selected patients who tested positive for prostate cancer as a function of patient age.

The assignment to the situations M0, M1 and M2 was carried out before the measurement of the Ca isotope ratios using the current state of the art diagnostic procedures (anamnesis and positive PSA test) and additionally using imaging procedures.

The thick dashed line indicates the equilibrium value δ ^44/42 Ca _equilibrium = -0.85%o.

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

There are two cases, designated A and B, in which an assignment based on the traditional procedures (anamnesis, PSA value, imaging procedures) to the M0 situation is not correct.

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

In case B, there is unrecognized and age-appropriate mineralization of the bones, so it must be assumed that there is a proliferating osteoblastic metastasis finding (M2), which was, however, incorrectly classified as “no findings, M0” by traditional methods.

material and methods

Blood sampling:

The doctor will take a blood sample from the patient with a normal amount of blood required for blood tests (approx. 8 ml). The blood sample is left to stand for half an hour and then centrifuged. The resulting blood serum is separated from the blood cake. Only blood serum is used for further chemical preparation.

For further chemical preparation, an amount of serum is removed that corresponds to an absolute amount of 50 µg calcium.

Extraction of calcium from the samples:

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

The blood can be prepared for chemical extraction of calcium using the following procedure:

• Tag 1
  • • Die für den Mikrowellen(MW)aufschluss vorgeschriebenen Gefäße werden mit HNO₃ und H₂O₂ befüllt.
  • • Die vorbereiteten Proben und Standards werden zu dem jeweiligen Gefäß pipettiert.
  • • Gefäße werden verschlossen, das Mikrowellengerät wird gestartet und für 1.5 Stunden in die MW gestellt.
  • • Die aufgeschlossenen Proben werden aus der MW geholt. Im Abzug werden die Lösungen nacheinander in Becher umgefüllt und auf die Heizplatte zum Trocknen (über Nacht) gestellt.
• Tag 2
  • • Die aufgeschlossenen und über Nacht getrockneten Proben werden mit 1ml HNO₃ +0.5ml H₂O₂ aufgenommen und wieder verschlossen für 3 Stunden gekocht.
  • • Becher öffnen und Lösung eintrocknen.
  • • Getrocknete Proben werden in 1ml 2M HNO₃ aufgenommen.
• Tag 3
  • • Die Konzentration des Calciums (Ca) wird an der Q-ICP-MS mittels Standardverfahren gemessen.
• Tag 4
  • • Um für die Säulenchromatographie immer die gleiche absoluten Ca Mengen (50 µg_Ca) zu haben, wird die Menge an Säure, die für die automatisierte Messung an einer ESI PrepFast benötigt wird, berechnet und die Proben entsprechend verdünnt.
  • • Die Proben werden aus den Bechern in die PrepFAST® Röhrchen (Fa. Elemental Scientific) überführt.
  • • Die Abtrennung der Erdalkalielemente erfolgt automatisch mittels des prepFAST® Gerätes (Fa. Elemental Scientific).
• Tag 5
  • • Die nach Elementen getrennten Proben werden aus den Röhrchen wieder in die Becher überführt und zum Eintrocknen auf die Heizplatte gestellt.
  • • Nach Eintrocknung werden diese mit 1ml HNO₃ und 0.5ml H₂O₂ erneut aufgenommen und für weitere 3 Stunden geschlossen gekocht.
• Tag 6
  • • Die Becher mit den Proben werden geöffnet und eingetrocknet.
  • • Die Proben werden in10 ml HNO₃ aufgenommen und 4 Stunden zur Equilibrierung stehen gelassen.
  • • Die Proben werden in die Röhrchen für die massenspektrometrische Messung an der Neptune® (Plasmamassenspektrometer, Fa. ThermoFisher) überführt und dort gemessen.

Chemical sample preparation for calcium isotope determination in blood

• day 1
  • • The vessels prescribed for microwave (MW) digestion are filled with HNO ₃ and H ₂ O ₂ .
  • • The prepared samples and standards are pipetted into the respective vessel.
  • • Vessels are closed, the microwave oven is started and placed in the MW for 1.5 hours.
  • • The digested samples are taken from the MW. In the hood, the solutions are transferred one after the other into beakers and placed on the hot plate to dry (overnight).
• day 2
  • • The digested and dried overnight samples are taken up with 1ml HNO ₃ +0.5ml H ₂ O ₂ and closed again and boiled for 3 hours.
  • • Open the beaker and let the solution dry.
  • • Dried samples are taken up in 1ml 2M HNO ₃ .
• Day 3
  • • The concentration of calcium (Ca) is measured on the Q-ICP-MS using standard methods.
• Day 4
  • • In order to always have the same absolute Ca amounts (50 µg _Ca ) for column chromatography, the amount of acid required for the automated measurement on an ESI PrepFast is calculated and the samples are diluted accordingly.
  • • The samples are transferred from the beakers into the PrepFAST® tubes (Elemental Scientific).
  • • The alkaline earth elements are separated automatically using the prepFAST® device (Elemental Scientific).
• Day 5
  • • The samples, separated according to elements, are transferred from the tubes back into the beakers and placed on the heating plate to dry.
  • • After drying, they are taken up again with 1ml HNO ₃ and 0.5ml H ₂ O ₂ and boiled closed for another 3 hours.
• Day 6
  • • The beakers with the samples are opened and dried.
  • • The samples are taken up in 10 ml HNO ₃ and left to equilibrate for 4 hours.
  • • The samples are transferred to the tubes for mass spectrometric measurement on the Neptune® (plasma mass spectrometer, ThermoFisher) and measured there.

In addition to mass spectrometric methods, spectroscopic methods can also be used to determine the isotope ratios or quantitative ratios of calcium isotopes, which use the mass dependence of the hyperfine structure of the spectral lines by means of light emission.

The procedure for determining the isotope ratios or quantitative ratios of calcium isotopes using laser-induced fluorescence can be done by a person skilled in the art, for example US10302565B2 remove.

In particular, all measured values are given relative to the international standard SRM915a.

Without limiting the generality of the method, the mass spectroscopic determination of the values is described below.

Mass spectrometric measurement:

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 also be used as an alternative measured in a thermionic mass spectrometer. The aim is to determine the calcium isotope composition in blood serum. In the TIMS, ⁴⁴ Ca/ ⁴⁰ Ca ratios are measured and in the MC-ICP-MS, ⁴⁴ Ca/ ⁴² Ca or alternative ratios are measured. Both isotope ratios are equivalent in their statement. The values differ by only one factor: ⁴⁴ Ca/ ⁴⁰ Ca ≅ 2.05 · ⁴⁴ Ca/ ⁴² Ca.

Determination of the calcium isotope composition ⁴⁴ Ca/ ⁴² Ca using a plasma mass spectrometer:

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 so that the atomic masses (u) 42, 43, 43.5 and 44 can be measured simultaneously. To suppress interfering Ca and Ar hydrides (e.g. ⁴⁰ Ar ₁ H ₂ to ⁴² Ca), an APEX IR sample introduction system (Elemental Scientific®) is used. All measurements are carried out in medium resolution (m/Δm - 4000) on the interference-free plateau of the low-mass side of the central measurement peak. This is achieved by selecting an appropriate mean cup mass of 4.687±0.001u and verifying it daily. The instrumental fractionation (“mass bias”) is corrected using the “Standard Sample Bracketing (SSB)” procedure. The measurement of a sample is corrected by measurements of a 5 µg/ml Ca solution prepared from a 10000 µg/ml Ca-ICP standard solution. Each sample is measured at least four times during a session and their average value is used for further procedures. The Ca isotope composition is given as δ ^44/42 Ca in parts per thousand (%o): $${\mathrm{\delta }}^{44/42}\text{Approx}(‰)=\left[{\left({}^{44}{\text{Approx.}}^{42}\text{Approx}\right)}_{\text{sample}}/{\left({}^{44}{\text{Approx.}}^{42}\text{Approx}\right)}_{\text{reference}}\right]-1$$

d.h. alle Messwerte werden relativ zum internationalen Standard SRM915a angegeben.The measured Ca-ICP standard solution serves as the primary reference material. The δ ^44/42 Ca _ICP values are then converted from NIST SRM 1486 to NIST SRM 915a using the measured δ ^44/42 Ca _ICP value: $${\mathrm{\delta }}^{44/42}{\text{Approx}}_{\text{SRM}915\text{a}}\left(\text{sample},‰\right)={\mathrm{\delta }}^{44/42}{\text{Approx}}_{\text{ICP}}\left(\text{sample}\right)-{\mathrm{\delta }}^{44/42}{\text{Approx}}_{\text{ICP}}\left(\text{SRM}915\text{a}\right),$$

i.e. all measured values are given relative to the international standard SRM915a.

During each session, chemically unprocessed NIST SRM 915a material is measured at the beginning and end of each session and these results are compared to those of chemically processed NIST SRM 915a. The average difference between processed and unprocessed SRM 915a should generally be less than 0.01‰, so that Ca isotope fractionation during chemical cleaning can be considered negligible.

The “on-peak” approach is used for background correction. The measured intensities of a 1% HNO ₃ solution are subtracted from the intensities of the subsequent sample measurements.

Additionally, the measurements are checked for residual doubly charged strontium ( ⁸⁴ Sr, ⁸⁶ Sr and ⁸⁸ Sr) to ensure correct measurement of the intensities of the ⁴² Ca, ⁴³ Ca and ⁴⁴ Ca masses. For this purpose, the intensity ratios of the masses 42/43.5, 43/43.5 and 44/43.5 of a 2 µg/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.

For quality assurance, the δ ^44/42 Ca values of NIST SRM 1486 and IAPSO seawater standard measured during the measurement phase are compared with published values. The long-term reproducibility (2 SD = standard deviation) over a period of approximately two months is usually better than ±0.06 ‰ for all reference materials analyzed.

• • |δ^44/42Ca - 2.δ^43/42Ca|>0.2 %o gilt.
• • Eine Probenmessung wird verworfen, wenn die durchschnittliche Intensität außerhalb eines Intensitätsfensters von 70 bis 130 % liegt, verglichen mit der durchschnittlichen Intensität der 5µg/ml Ca ICP -Lösung oder NIST SRM 915a-Lösung derselben Charge.
• • Eine vollständige Sequenz wird abgelehnt, wenn mehr als eines der gemessenen internationalen Referenzmaterialien mehr als 0.2 ‰ vom Literaturwert entfernt ist oder die Daten nicht entlang der massenabhängigen Fraktionierungslinie fielen.

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

• • |δ ^44/42 Ca - 2.δ ^43/42 Ca|>0.2 %o applies.
• • A sample measurement is rejected if the average intensity is outside of an intensity window of 70 to 130% compared to the average intensity of the 5µg/mL Ca ICP solution or NIST SRM 915a solution of the same lot.
• • A complete sequence will be rejected if more than one of the measured international reference materials is more than 0.2‰ away from the literature value or the data did not fall along the mass-dependent fractionation line.

Evaluation of the calcium isotope data:

wobei (⁴⁴Ca/⁴²Ca)_Probe das im Massenspektrometer gemessene Verhältnis der Blutprobe ist und (⁴⁴Ca/⁴²Ca)_Standard das Calcium Isotopenverhältnis eines international bekannten und zertifizierten Verhältnisses ist. Diese Vorgehensweise erlaubt eine internationale Vergleichbarkeit der gemessenen Isotopenverhältnisse.The measured calcium isotope values are reported in the usual δ notation: $${\delta }^{44/42}Ca=\left[\frac{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{PrObe}}{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Standard}}-1\right]\cdot 1000$$

where ( ⁴⁴ Ca/ ⁴² Ca) _sample is the ratio of the blood sample measured in the mass spectrometer and ( ⁴⁴ Ca/ ⁴² Ca) _standard is the calcium isotope ratio of an internationally known and certified ratio. This procedure allows international comparability of the measured isotope ratios.

List of illustrations:

: 844/42 Ca _serum values from patients who tested positive for prostate cancer

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 20140322740 A1 [0003]
• DE 1020182146608 [0005]
• US 10302565 B2 [0012, 0034]

Claims (5)

A method for early diagnosis of the pathology of a possible disease in patients suspected of having prostate cancer, comprising the following steps: i. Determining isotope ratios or quantitative ratios of calcium isotopes in a sample of blood from the patient with suspected prostate cancer. ii. Comparison of the value determined in step i with the interval defined by a threshold value $$-0.91‰<{\mathrm{\delta }}^{44/42}{\text{Approx}}_{\text{serum}}-\text{Value}<-0.79‰$$

iii. Assignment based on the comparison from step ii to one of three defined situations M0, M1 and M2 where M0: -0.91‰ < δ ^44/42 Ca _serum value < -0.79 ‰ M1: δ ^44/42 Ca _serum - Value < -0.91‰ M2: δ ^44/42 Ca _serum value > -0.79‰ represents. Procedure according to Claim 1 , characterized in that the isotope ratios or quantitative ratios of calcium isotopes are determined using mass spectrometry. Procedure according to Claim 1 , characterized in that the isotope ratios or quantitative ratios of calcium isotopes are determined using laser-induced fluorescence. Procedure according to Claim 1 , characterized in that the determination of the isotope ratios or quantitative ratios of calcium isotopes is carried out with the aid of spectroscopic methods which use the mass dependence of the hyperfine structure of the spectral lines by means of light emission. Procedure according to Claim 1 , characterized in that the following additional steps are carried out before step i: an anamnesis and/or a test with a protein-based marker.