WO2023153998A1 - Method for determining risks associated with hormone-dependent cancers - Google Patents

Method for determining risks associated with hormone-dependent cancers Download PDF

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WO2023153998A1
WO2023153998A1 PCT/SE2023/050122 SE2023050122W WO2023153998A1 WO 2023153998 A1 WO2023153998 A1 WO 2023153998A1 SE 2023050122 W SE2023050122 W SE 2023050122W WO 2023153998 A1 WO2023153998 A1 WO 2023153998A1
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hormone
subject
expression
cancer
akr1
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Jonas Nilsson
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Rjan Holding Ab
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the present disclosure relates the prediction of the risks in connection with hormone-dependent cancers using a hormone-dependent cancer biomarker.
  • Hormone dependent cancers like cancers dependent on androgens and estrogens, often responds well to therapies reducing the hormone levels.
  • Both androgen and estrogen are built from the same starting material, cholesterol, that is enzymatically modified in a series of steps to end up with active hormones (e.g. androgens respectively estrogens). Only one enzymatic step differs between an active androgen respectively an active estrogen molecule, so therapies affecting the synthesis of one hormone often can affect the synthesis of the opposite hormone.
  • Androgen deprivation therapy (ADT) in men or Estrogen deprivation therapy (HDT) in female are an anti-hormonal treatment aimed at lowering the circulating levels of sex- hormones in prostate or breast cancer patients.
  • GnRH gonadotropin-releasing hormone
  • LHRH hormone-releasing hormone
  • the patients with a primary diagnosed prostate cancer are assessed into risk groups receiving different treatments based on the classification of the tumour according to the national care program (www.cancercentrum.se) using the Tumour Node Metastasis (TNM) classification system together with graduation with the Gleason score grades and serum PSA levels.
  • TPM Tumour Node Metastasis
  • Today, the low-risk patients are subjected to curative treatment (surgery or RT) or through watchful waiting monitoring the disease. More advanced diseases are depending on the grading of the disease where combinations of ADT and RT will be used to treat the patients. This approach is now slowly starting to change with new targeted therapies being used earlier in the progression, but still ADT will be used for all high-risk patients with advanced disease at some point during their treatment.
  • More personalized and targeted therapies for prostate cancer will be implemented in a near future where we will use genetic information from the tumour to stratify treatment that have a higher chance of effective response.
  • Still therapies targeting the androgen receptor signalling will continue to be a major therapeutic approach due to its effective response and curative ability in combination with RT.
  • the main resistance mechanisms to treatments of primary tumours are the upregulation of androgen receptor signalling, through mutations and amplifications of the androgen receptor, so that the tumour cells survive under low hormone levels.
  • Other ways include neuroendocrine differentiation of the tumour cells, losing their dependence of androgens. Therefore, the development of tests that can help differentiate patients into androgen targeted therapy responders respectively nonresponders are of outermost importance.
  • prostate cancer has been to reduce the androgen signalling, androgen deprivation therapy (ADT), unfortunately the disease still reappears with a spread of tumour cells mainly to the bones forming distal metastases.
  • ADT androgen deprivation therapy
  • tumour cells are still responsive to androgens, but they have circumvented the sensitivity to low levels of circulating androgens, either through enhanced activity of the receptors or intratumorally synthesis of androgens.
  • chemotherapies like docetaxel, etc.
  • the largest prostate cancer subtype is driven by androgen receptor signalling, with higher serum PSA levels compared to the two other subtypes.
  • the androgen signal driven subtype responds better to androgen signalling therapies, like abiraterone acetate, or enzalutamide and thereby also more susceptible for secondary resistance mechanisms like androgen receptor amplification, expression of constitutive active receptors or through extragonadal synthesis of androgens to compensate to the suppressed receptor activity and low hormone levels.
  • Abiraterone acetate is often used as first or second line treatment of CRPC targeting the androgen synthesis pathway via suppression of CYP17A1 to further reduce the extragonadal synthesis of androgens within the tumour cells themselves.
  • Abiraterone acetate treatment are being used in combination with standard of care.
  • the abiraterone acetate treatment its effective in a subpopulation of patients, but resistance can occur through upregulation of downstream enzymes in the androgen synthesis process, like AKR1 C3, increasing the output of androgens. Therefore, tests that can help differentiate patients into responders respectively non-responders are of outermost importance.
  • Platelets and androgens Pho-art documents have indicated that platelets can be a source of extragonadal androgen synthesis with levels of synthesized androgens that can in vitro support tumour cells under castrated hormone levels.
  • Another interesting aspect of the potential for platelet to synthesis androgens is their interact with circulating tumour cells (CTCs) in the blood circulation where they may provide platelet-derived androgens to the cancer cells.
  • CTCs circulating tumour cells
  • This cross-talk have been described for interactions between platelet-derived molecules and circulating tumour cells (CTC) where platelet-derived transforming growth factor B1 (TGFbl ) promote extravasation and formation of metastases.
  • TGFbl platelet-derived transforming growth factor B1
  • AKR1 C3 is an enzyme in the steroid synthesis pathway that has been shown to be overexpressed in prostate cancer and also associated with resistance to treatment in CRPC. Prostate tissue levels of AKR1C3 increase with Gleason grading and targeted therapies against AKR1 C3 are under development.
  • the risk for recurrence of a hormone dependent cancer is quite high in some patients and being able to identify these already before the treatment starts would increase the chances for a successful treatment. Also, being able to identify subjects having an initial higher risk of developing a hormone-dependent cancer would mean that it would be possible to monitor these patients closer to be able to quickly react and start treatment if a cancer develops, e.g. by cancer-screening or more frequent monitoring during treatment. In addition, being able to identify those subjects that will or will not respond, or respond to a lesser degree, to hormone-deprivation therapy would also provide a personal treatment plan increasing the chances for recovery and survival of subjects with hormone-dependent cancer.
  • the object of the present invention is to alleviate or overcome at least some of the above identified problems associated with hormone dependent cancers.
  • the document discloses a method for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the risk for non-responsiveness to hormone- deprivation therapy in a subject with a hormone-dependent cancer, said method comprising: a) isolating platelets and/or extracellular vesicles derived from platelets from a blood or plasma sample from said subject; b) determining in said platelets and/or extracellular vesicles derived from platelets i) the expression of AKR1 C3 and ii) determining the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins to obtain a relative expression of AKR1 C3; c) comparing the relative expression of AKR1 C3 of step b) to a reference expression level derived from a relevant patient-control cohort information; d) based on the comparison in step c) predicting the risk for a subject of
  • said hormone-dependent cancer is prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and/or breast cancer.
  • said prostate cancer is castration resistant.
  • said expression of AKR1 C3 is determined by digital or quantitative PCR (such as qPCR and/or ddPCR), proximity ligation assay and/or enzyme-linked sorbent assay (ELISA).
  • digital or quantitative PCR such as qPCR and/or ddPCR
  • proximity ligation assay and/or enzyme-linked sorbent assay (ELISA).
  • ELISA enzyme-linked sorbent assay
  • the expression of AKR1 C3 and expression of one or more reference genes and/or total RNA is determined by analysing mRNA expression. Any suitable method for analysing mRNA known by the skilled person may be used.
  • the expression of AKR1C3 and expression of one or more reference genes and/or total RNA is determined by PCR, such as qPCR and/or ddPCR.
  • said expression of AKR1 C3 and said expression of one or more reference proteins and/or total proteins is determined by analysing protein levels, such as ELISA.
  • said reference genes is GADPH, bAKT and/or ITGA2B.
  • said method further comprises a step of determining the level of prostate specific antigen (PSA) in said subject.
  • PSA prostate specific antigen
  • said method further comprises a step of deciding on a treatment regimen for said subject having a hormone-dependent cancer.
  • said method further comprises a step of determining the expression of prostate specific antigen (PSA) from the blood or plasma sample of said subject.
  • PSA prostate specific antigen
  • i) for subjects with low expression of PSA and low expression of AKR1 C3 or with low expression of PSA and high expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen not comprising hormone-deprivation therapy, such as with Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check Point Inhibitors (ICI), EZH2-directed inhibitors, chemotherapy or any combination thereof; ii) for subjects with high expression of PSA and low expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen comprising hormone-deprivation therapy, such as abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof; and/or iii) for subjects with high expression of PSA and high expression of AKR1C3 it is decided that said subject should be treated with a treatment regimen not comprising androgensynthesis inhibitor, such as a combination of AR-directed therapy with one
  • High expression of PSA may be defined as an above median value compared to a relevant patient population.
  • said method further comprising the step of treating said subject with the treatment regimen decided upon.
  • the method for treating said subject may comprise administering an effective amount of a drug typically used in a hormone-deprivation therapy or may comprise administering an effective amount of a drug not typically used in a hormone-deprivation therapy. Examples of such drugs are disclosed elsewhere herein.
  • said method further comprises communicating to the subject and/or to a healthcare provider the risk for said subject of developing a hormone dependent cancer, the risk for the subject for relapse in a hormone dependent cancer, and/or the risk for non-responsiveness to hormone- deprivation therapy, such as by means of electronic communication and/or paperbased communication.
  • said subject belongs to a sub-group of subjects having a high expression of PSA.
  • Another aspect of the disclosure relates to a use of platelet-derived AKR1 C3 as a biomarker for hormone-dependent cancer, such as prostate cancer, castration- resistant prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer.
  • said biomarker may predict the risk for a subject of developing a hormone dependent cancer, predict the risk for a subject for relapse in a hormone dependent cancer, and/or predict the risk for non- responsiveness to hormone-deprivation therapy in a subject with a hormone- dependent cancer,.
  • AKR1 C3 may be combined with the use of PSA as a biomarker for hormone-dependent cancer.
  • the combination of the use of AKR1 C3 and PSA as biomarkers may provide a more precise information to assist cancer treatment decisions, particularly for prostate cancer.
  • Subjects classified as high risk or low risk may receive different treatments, such as a different dosage regimen and/or a different drug treatment.
  • the present document discloses methods for determining different risks associated with hormone dependent cancers.
  • AKR1 C3 is an enzyme in the steroid synthesis pathway. It was found by the present inventor that subjects with a hormone dependent cancer have a higher mean expression value of AKR1 C3 in platelets than control subjects without a hormone dependent cancer. Without wishing to be bound by theory, since hormone dependent cancers share a common steroid synthesis pathway and AKR1C3 is part of said pathway, the inventors have surprisingly found that the expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets would be an excellent biomarker for the prognosis of any hormone dependent cancer, including but not limited to prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer.
  • Platelet derived AKR1 C3 can thereby provide an increased amount of downstream steroid products, like androgens in hormone dependent cancers.
  • the platelet produced steroids thereby help supplying the hormone dependent cancer with low levels of hormones e.g. promoting cancer cell proliferation and survival during androgen deprivation therapy or when CTCs circulates the blood.
  • the present method can be used to determine risks as defined herein associated with any hormone dependent cancer.
  • hormone dependent cancers include but are not limited to hormone-dependent cancer is prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer.
  • the prostate cancer may e.g. be castration resistant prostate cancer.
  • a number of different diagnosis and/or treatment decisions can be taken. For example, subjects identified to have a high risk of developing a hormone dependent cancer, can be screened (e.g., regular early cancer screening programs) more regularly/often in order to diagnose the cancer as soon as possible, should it develop. Likewise, a subject identified as having a high risk for relapse in a hormone dependent cancer can be more regularly screened for relapse. Moreover, the subject may be more predisposed to having an increased number of metastases. Further, a subject identified as having a high risk for non-responsiveness to hormone-deprivation therapy can be treated with alternative therapies not based on hormone deprivation to improve the chances of recovery/survival.
  • AKR1 C3 For subjects with a high expression of AKR1 C3 relative to the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or extracellular vesicles derived from platelets, it may be decided that these subjects should be treated with a treatment regimen not comprising hormone-depletion therapy. For subjects with a low expression of AKR1 C3 relative to the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or extracellular vesicles derived from platelets, it may be decided that these should be treated with a treatment regimen comprising hormonedeprivation therapy.
  • CRPC patients having low (below median) levels of serum PSA have low responsiveness to androgen synthesis inhibitory therapy (ASI), regardless of AKR1C3 levels in the platelets.
  • ASI androgen synthesis inhibitory therapy
  • Said patients may be treated by for instance Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check Point Inhibitors (ICI), chemotherapy, EZH2-directed inhibitors, or any combination thereof.
  • BETinh Bromodomain and Extra-Terminal Domain Protein Inhibitors
  • ICI Immune Check Point Inhibitors
  • chemotherapy EZH2-directed inhibitors, or any combination thereof.
  • AKR1 C3 and reference gene(s)/total RNA can be studied by analysing mRNA expression. Amounts of mRNA expression may e.g. be determined by PCR, such as ddPCR and/or qPCR, RNA expression arrays, or RNA sequencing.
  • the kit for predicting the risk of developing a hormone dependent cancer advantageously allows comparing the AKR1 C3 expression with the expression of housekeeping genes and reference materials for both a positive and negative control evaluation. Said comparison advantageously provides a reliable and effective kit for predicting the risk of developing a hormone dependent cancer in the subject having or not said cancer.
  • the present document is also directed to a method for treating and/or preventing a hormone dependent cancer in a subject, said method comprising: a) administering to said subject a pharmaceutically effective amount of a drug for treating and/or preventing said hormone dependent cancer; or b) performing surgical removal of said hormone dependent cancer; wherein the predicted risk for said subject of developing a hormone dependent cancer, predicted the risk for said subject for relapse in a hormone dependent cancer, and/or predicted the risk for non-responsiveness to hormonedeprivation therapy for said subject is high or low, as defined by the method as defined elsewhere herein.
  • the present document is also directed to a drug for use in the treatment of a hormone-dependent cancer in a subject, said drug being selected from an androgen synthesis inhibitor and/or an androgen receptor-directed inhibitor, wherein said subject belongs to a sub-group of subjects having i) a low expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets relative to an expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or said extracellular vesicles derived from platelets from said subject; and ii) a high expression of PSA in a blood sample from said subject.
  • said subgroup of subjects has an improved treatment outcome when treated with a treatment regimen comprising hormone-deprivation therapy, such as abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof.
  • hormone-deprivation therapy such as abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof.
  • subjects having a high PSA expression and low AKR1 C3 expression belong to a specific sub-group of subjects that responds significantly better to said treatment, thereby increasing the overall survival rate and reducing risks and costs associated with choosing a less effective treatment for this sub-group of subjects.
  • the platelets can be obtained e.g. by pelleting them to the bottom of the tube by centrifugation, e.g. for 10 min 800xG in room temperature. The plasma is then removed, the platelets are preferably washed, e.g. with PBS. The platelets can then be snap frozen and stored in -80°C or directly analysed.
  • Plasma derived extracellular vesicles can be isolated from the plasma fraction after pelleting the platelets. Extracellular vesicles can be extracted using size exclusion chromatography (SEC). In brief, the platelet depleted plasma is loaded on a size exclusion column (Sepharose CL-2b) and the plasma components are separated by their size. The ECV fraction is collected and ECVs are isolated for downstream processing (e.g. according to Bdnig et al. 2014)
  • RNA preparation see “RNA preparation” in the below
  • protein preparation see “Protein preparation” in the below
  • ECV RNA isolating ECV RNA
  • Another way of isolating ECV RNA can be achieved by pellet the extracellular vesicles fraction using ultracentrifugation or through ECV precipitation methods where the isolated ECVs RNA is extracted with a standard RNA isolation kit (like RNeasy Mini Kit (Qiagen), miRVANA (Invitrogen)) per manufacturer's instructions. RNA concentrations are measured e.g. with Qubit method (Invitrogen) and correlated to volume of plasma used for isolation.
  • RNA isolation kit like RNeasy Mini Kit (Qiagen), miRVANA (Invitrogen)
  • RNA concentrations are measured e.g. with Qubit method (Invitrogen) and correlated to volume of plasma used for isolation.
  • RNA from platelet rich plasma can be processed using a standard total RNA isolation kit to extract RNA from the platelet rich plasma suspension (like TRIZOL LS (Invitrogen) per manufacturer's instructions.
  • the method may include a silica-based column binding and purification step. For example, 1 part of platelet-rich plasma suspension (250ul) is lysed in 3 parts Trizol LS (750ul) is used in the protocol. The eluted RNA concentration is measured with Qubit method (Invitrogen) and correlated to volume of platelet rich plasma used for isolation.
  • the ECV are mainly derived from the platelets, they will contain the same information as the original platelets and therefore the platelet-derived ECV population can be used as a surrogate marker for AKR1 C3 levels in the platelets.
  • Platelet levels of AKR1C3 correlates with survival and therapy response.
  • the patients were divided into quartiles (Q1 -4) based on their expression of AKR1C3.
  • PSA and AKR1C3 levels predict a favourable response to abiraterone therapy.
  • AKR1C3 holds no information in the PSA low group (e.g. a subgroup of cancer patients with low AR activity), neither regarding PFS nor OS (figure 5E-F).
  • AKR1C3 expression as a prognostic and/or predictive biomarker when used alone and together with high PSA levels.
  • Patients from the PSA high group with AKR1 C3-low expression had a three-time longer PFS and five-times longer OS then AKR1 C3-high (9.9 vs 3.0-month respectively 34.0 vs 6.1 - month).

Abstract

The present document is directed to methods predicting risks associated with hormone dependent cancers. The method is based on determining the expression of a biomarker, AKR1C3, in platelets and/or extracellular vesicles derived from platelets and relating the expression to the expression of other RNA and/or proteins in platelets and/or extracellular vesicles.

Description

METHOD FOR DETERMINING RISKS ASSOCIATED WITH HORMONEDEPENDENT CANCERS
TECHNICAL FIELD
The present disclosure relates the prediction of the risks in connection with hormone- dependent cancers using a hormone-dependent cancer biomarker.
BACKGROUND ART
Hormone dependent cancers, like cancers dependent on androgens and estrogens, often responds well to therapies reducing the hormone levels. Both androgen and estrogen are built from the same starting material, cholesterol, that is enzymatically modified in a series of steps to end up with active hormones (e.g. androgens respectively estrogens). Only one enzymatic step differs between an active androgen respectively an active estrogen molecule, so therapies affecting the synthesis of one hormone often can affect the synthesis of the opposite hormone. Androgen deprivation therapy (ADT) in men or Estrogen deprivation therapy (HDT) in female are an anti-hormonal treatment aimed at lowering the circulating levels of sex- hormones in prostate or breast cancer patients. This can be achieved by surgery (orchiectomy) in prostate cancer or by chemical castration using GnRH (gonadotropin-releasing hormone) I Luteinising hormone-releasing hormone (LHRH) targeting treatments (like leuprolide, etc.) or through direct hormone receptor inhibitors (like flutamide, tamoxifen, etc.). These therapies are often first line therapies in patients with prostate cancer or breast cancer.
The patients with a primary diagnosed prostate cancer are assessed into risk groups receiving different treatments based on the classification of the tumour according to the national care program (www.cancercentrum.se) using the Tumour Node Metastasis (TNM) classification system together with graduation with the Gleason score grades and serum PSA levels. Today, the low-risk patients are subjected to curative treatment (surgery or RT) or through watchful waiting monitoring the disease. More advanced diseases are depending on the grading of the disease where combinations of ADT and RT will be used to treat the patients. This approach is now slowly starting to change with new targeted therapies being used earlier in the progression, but still ADT will be used for all high-risk patients with advanced disease at some point during their treatment. More personalized and targeted therapies for prostate cancer will be implemented in a near future where we will use genetic information from the tumour to stratify treatment that have a higher chance of effective response. Still therapies targeting the androgen receptor signalling will continue to be a major therapeutic approach due to its effective response and curative ability in combination with RT.
The main resistance mechanisms to treatments of primary tumours are the upregulation of androgen receptor signalling, through mutations and amplifications of the androgen receptor, so that the tumour cells survive under low hormone levels. Other ways include neuroendocrine differentiation of the tumour cells, losing their dependence of androgens. Therefore, the development of tests that can help differentiate patients into androgen targeted therapy responders respectively nonresponders are of outermost importance.
The main therapeutic option for prostate cancer has been to reduce the androgen signalling, androgen deprivation therapy (ADT), unfortunately the disease still reappears with a spread of tumour cells mainly to the bones forming distal metastases.
The main therapeutic option of metastases has previously been treatment with ADT in combination with chemotherapies (like docetaxel, etc.) suppressing the circulating androgens forcing the tumour cells to find alternative ways of survival where they later reappear as castration resistant prostate cancer (CRPC). In many cases of CRPC the tumour cells are still responsive to androgens, but they have circumvented the sensitivity to low levels of circulating androgens, either through enhanced activity of the receptors or intratumorally synthesis of androgens.
With new understanding of the CRPC tumours and their different subtypes provide a new route or an alternative approach to alter the treatment based the tumour transcription pattern and cellular phenotypes, similar to what is known for breast cancer and their subtypes. The largest prostate cancer subtype is driven by androgen receptor signalling, with higher serum PSA levels compared to the two other subtypes. The androgen signal driven subtype responds better to androgen signalling therapies, like abiraterone acetate, or enzalutamide and thereby also more susceptible for secondary resistance mechanisms like androgen receptor amplification, expression of constitutive active receptors or through extragonadal synthesis of androgens to compensate to the suppressed receptor activity and low hormone levels.
Abiraterone acetate is often used as first or second line treatment of CRPC targeting the androgen synthesis pathway via suppression of CYP17A1 to further reduce the extragonadal synthesis of androgens within the tumour cells themselves. Also, earlier in the disease process, prior CRPC, Abiraterone acetate treatment are being used in combination with standard of care. The abiraterone acetate treatment its effective in a subpopulation of patients, but resistance can occur through upregulation of downstream enzymes in the androgen synthesis process, like AKR1 C3, increasing the output of androgens. Therefore, tests that can help differentiate patients into responders respectively non-responders are of outermost importance.
Platelets and androgens. Pho-art documents have indicated that platelets can be a source of extragonadal androgen synthesis with levels of synthesized androgens that can in vitro support tumour cells under castrated hormone levels. Another interesting aspect of the potential for platelet to synthesis androgens is their interact with circulating tumour cells (CTCs) in the blood circulation where they may provide platelet-derived androgens to the cancer cells. This cross-talk have been described for interactions between platelet-derived molecules and circulating tumour cells (CTC) where platelet-derived transforming growth factor B1 (TGFbl ) promote extravasation and formation of metastases. The shear pressure in the blood stream makes CTCs dependent of shielding during circulation and platelets are known to provide a protective cover and together with transfer of growth factors they may increase CTC survival in circulation. Platelets also facilitate the extravasation of CTCs from the circulation and the formation of new tumour niches and daughter tumours. Androgen synthesis in platelets can thus have dual effects, both as a source for androgen synthesis to support tumour survival and a direct platelet-tumour cell communication in circulation promoting survival and the ability to form new daughter tumours. AKR1 C3 is an enzyme in the steroid synthesis pathway that has been shown to be overexpressed in prostate cancer and also associated with resistance to treatment in CRPC. Prostate tissue levels of AKR1C3 increase with Gleason grading and targeted therapies against AKR1 C3 are under development.
The risk for recurrence of a hormone dependent cancer is quite high in some patients and being able to identify these already before the treatment starts would increase the chances for a successful treatment. Also, being able to identify subjects having an initial higher risk of developing a hormone-dependent cancer would mean that it would be possible to monitor these patients closer to be able to quickly react and start treatment if a cancer develops, e.g. by cancer-screening or more frequent monitoring during treatment. In addition, being able to identify those subjects that will or will not respond, or respond to a lesser degree, to hormone-deprivation therapy would also provide a personal treatment plan increasing the chances for recovery and survival of subjects with hormone-dependent cancer.
It is therefore of uttermost importance to be able to identify subjects having higher risks associated with hormone dependent cancers or later when the patients receive androgen directed therapies when the tumours become castration resistant, but still respond to androgens.
Summary of the invention
The object of the present invention is to alleviate or overcome at least some of the above identified problems associated with hormone dependent cancers.
The document discloses a method for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the risk for non-responsiveness to hormone- deprivation therapy in a subject with a hormone-dependent cancer, said method comprising: a) isolating platelets and/or extracellular vesicles derived from platelets from a blood or plasma sample from said subject; b) determining in said platelets and/or extracellular vesicles derived from platelets i) the expression of AKR1 C3 and ii) determining the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins to obtain a relative expression of AKR1 C3; c) comparing the relative expression of AKR1 C3 of step b) to a reference expression level derived from a relevant patient-control cohort information; d) based on the comparison in step c) predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the response to hormone-deprivation therapy in a subject with a hormone-dependent cancer, i) wherein a high expression of AKR1 C3 relative to said reference expression in said platelets and/or said extracellular vesicles derived from platelets from said subject indicates a high risk, ii) wherein a low expression of AKR1 C3 relative to said reference expression in said platelets and/or said extracellular vesicles derived from platelets from said subject indicates a low risk.
According to one exemplary embodiment, said hormone-dependent cancer is prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and/or breast cancer. According to another exemplary embodiment, said prostate cancer is castration resistant.
According to another exemplary embodiment, said expression of AKR1 C3 is determined by digital or quantitative PCR (such as qPCR and/or ddPCR), proximity ligation assay and/or enzyme-linked sorbent assay (ELISA). However, any suitable method for measuring the expression of AKR1 C3 known by the skilled person may be used.
According to one exemplary embodiment, the expression of AKR1 C3 and expression of one or more reference genes and/or total RNA is determined by analysing mRNA expression. Any suitable method for analysing mRNA known by the skilled person may be used.
According to another exemplary embodiment, the expression of AKR1C3 and expression of one or more reference genes and/or total RNA is determined by PCR, such as qPCR and/or ddPCR. According to one exemplary embodiment, said expression of AKR1 C3 and said expression of one or more reference proteins and/or total proteins is determined by analysing protein levels, such as ELISA.
According to another exemplary embodiment, said reference genes is GADPH, bAKT and/or ITGA2B.
According to one exemplary embodiment, said method further comprises a step of determining the level of prostate specific antigen (PSA) in said subject. The skilled person is aware of such methods for measuring PSA level in said subject.
According to another exemplary embodiment, said method further comprises a step of deciding on a treatment regimen for said subject having a hormone-dependent cancer.
According to one exemplary embodiment, i) for subjects with a high expression of AKR1 C3 relative to said expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in said platelets and/or said extracellular vesicles from said subject it is decided that these should be treated with a treatment regimen not comprising hormone-depletion therapy; and/or ii) for subjects with a low expression of AKR1 C3 relative to said of one or more reference genes and or reference proteins and/or total RNA or total proteins in said platelets and/or said extracellular vesicles from said subject it is decided that these should be treated with a treatment regimen comprising hormone-deprivation therapy.
According to another exemplary embodiment, said method further comprises a step of determining the expression of prostate specific antigen (PSA) from the blood or plasma sample of said subject.
According to another exemplary embodiment i) for subjects with low expression of PSA and low expression of AKR1 C3 or with low expression of PSA and high expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen not comprising hormone-deprivation therapy, such as with Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check Point Inhibitors (ICI), EZH2-directed inhibitors, chemotherapy or any combination thereof; ii) for subjects with high expression of PSA and low expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen comprising hormone-deprivation therapy, such as abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof; and/or iii) for subjects with high expression of PSA and high expression of AKR1C3 it is decided that said subject should be treated with a treatment regimen not comprising androgensynthesis inhibitor, such as a combination of AR-directed therapy with one or more of Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check-point Inhibitors (ICI), bone/cancer targeting radionuclidesEZH2-directed inhibitors and/or AR-degraders.
High expression of PSA may be defined as an above median value compared to a relevant patient population.
According to one exemplary embodiment, said method further comprising the step of treating said subject with the treatment regimen decided upon. The method for treating said subject may comprise administering an effective amount of a drug typically used in a hormone-deprivation therapy or may comprise administering an effective amount of a drug not typically used in a hormone-deprivation therapy. Examples of such drugs are disclosed elsewhere herein.
According to one exemplary embodiment, said method further comprises communicating to the subject and/or to a healthcare provider the risk for said subject of developing a hormone dependent cancer, the risk for the subject for relapse in a hormone dependent cancer, and/or the risk for non-responsiveness to hormone- deprivation therapy, such as by means of electronic communication and/or paperbased communication.
According to one exemplary embodiment, said subject belongs to a sub-group of subjects having a high expression of PSA.
Another aspect of the disclosure relates to a use of platelet-derived AKR1 C3 as a biomarker for hormone-dependent cancer, such as prostate cancer, castration- resistant prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer. According to one exemplary embodiment, said biomarker may predict the risk for a subject of developing a hormone dependent cancer, predict the risk for a subject for relapse in a hormone dependent cancer, and/or predict the risk for non- responsiveness to hormone-deprivation therapy in a subject with a hormone- dependent cancer,.
According to another exemplary embodiment, the use of AKR1 C3 may be combined with the use of PSA as a biomarker for hormone-dependent cancer. The combination of the use of AKR1 C3 and PSA as biomarkers may provide a more precise information to assist cancer treatment decisions, particularly for prostate cancer.
Another aspect of the disclosure relates to a kit for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the response to hormone- deprivation therapy in a subject with a hormone-dependent cancer, said kit comprising: a) reagents for performing a quantitative RNA analysis of AKR1C3 and housekeeping gene levels in sample using a digital or quantitative PCR system and reference material for positive control and negative control evaluation; and/or b) reagents for performing a semi-quantitative ELISA analysis of AKR1 C3 protein levels and reference material to make AKR1 C3 protein standard curve and a negative control; and optionally instructions regarding the method for how to calculate the relative expression of AKR1 C3 to reference expression and the risk associated with the score.
Another aspect of the disclosure relates to a method for treating and/or preventing a hormone dependent cancer in a subject, said method comprising: a) administering to said subject a pharmaceutically effective amount of a drug for treating and/or preventing said hormone dependent cancer; or b) performing surgical removal of said hormone dependent cancer; wherein the predicted risk for said subject of developing a hormone dependent cancer, predicted the risk for said subject for relapse in a hormone dependent cancer, and/or predicted the risk for non-responsiveness to hormone- deprivation therapy for said subject is high or low, as defined by the method as defined elsewhere herein.
Said drug may be a drug used in a hormone-deprivation therapy or a drug not typically used in a hormone-deprivation therapy. Said drug may be abiraterone. Said drug may be selected from Enzalutamide, Apalutamide, Darolutamide, or any combination thereof.
Said hormone dependent cancer may be a prostate cancer, castration-resistant prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and/or breast cancer.
Subjects classified as high risk or low risk may receive different treatments, such as a different dosage regimen and/or a different drug treatment.
Another aspect of the disclosure relates to a drug for use in the treatment of a hormone-dependent cancer in a subject, said drug being selected from an androgen synthesis inhibitor and/or an androgen receptor-directed inhibitor, wherein said subject belongs to a sub-group of subjects having i) a low expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets relative to an expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or said extracellular vesicles derived from platelets from said subject; and ii) a high expression of PSA in a blood sample from said subject.
Said sub-group of subjects may have been classified by the method for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the response to hormone-deprivation therapy in a subject with a hormone-dependent cancer, as defined elsewhere herein. Said sub-group of subjects may have been classified by said method(s) as a high risk or a low risk.
The androgen synthesis inhibitor may be abiraterone.
The androgen receptor-directed inhibitor may be selected from Enzalutamide, Apalutamide, Darolutamide, or any combination thereof. Said hormone dependent cancer may be prostate cancer, castration-resistant prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and/or breast cancer.
Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims.
Definitions
By “hormone dependent cancer” is meant a cancer that it dependent on a hormone for growth. A hormone dependent cancer is hormone sensitive (dependent) in that the binding of certain hormones to the receptor, the cancer can grow and spread. Examples of hormone dependent cancers are e.g. breast cancer, which may be dependent on estrogens, such as estradiol for growth, and prostate cancer, which may be dependent on androgens, such as testosterone, for its growth. One type of hormone dependent cancers are prostate cancers that are castration resistant, but still responds to androgens and are sensitive to antiandrogen therapies. Hormone dependent cancers may in some cases lose their hormone dependency after treatment. However, such cancers are still included in the term “hormone dependent cancer” in the present document.
“AKR1C3 refers to the enzyme ”aldo-keto reductase family 1 member C3”, also denoted “17[3-hydroxysteroid dehydrogenase type 5”. AKR1 C3 is an enzyme in the steroid synthesis pathway.
The term “high” used in the context of AKR1 C3 refers to the relative expression of said AKR1 C2 in platelets and/or extracellular vesicles derived from platelets which is above median/defined cut off levels of AKR1 C3 expression when compared to a reference expression level derived from a relevant patient-control cohort information. Said expression level may refer to RNA and/or protein expression.
The term “low” used in the context of AKR1 C3 refers to the relative expression of said AKR1 C3 in platelets and/or extracellular vesicles derived from platelets which is below median/defined cut off levels of AKR1 C3 expression when compared to a reference expression level derived from a relevant patient-control cohort information. Said expression level may refer to RNA and/or protein expression. The terms “subject” and “patient” may be used interchangeably herein.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 describes the expression of AKR1 C3 of different stages of primary prostate cancer patients at diagnosis (G1-5) and healthy controls, as well as CRPC patients, according to one exemplary embodiment.
Figures 2a and 2b describe a Kaplan-Meier analysis of patients with metastasized disease at diagnosis, according to one exemplary embodiment.
Figures 3a and 3b describe a Kaplan-Meier analysis of overall survival (OS) of the patients, according to one exemplary embodiment.
Figures 4a and 4b describe a correlation between AKR1 C3 levels (quartiles) (A) respectively ROC-curve analysis (below or above cut-off) (B) for defining the cut-off of AKR1 C3 expression in the risk groups, according to one exemplary embodiment.
Figures 5a to 5f describe that a combination of high serum PSA levels and low AKR1 C3 levels identify a responsive subgroup of patients, according to one exemplary embodiment.
Figure 6 describes an analysis of therapy response after abiraterone therapy in patients divided to good responders (PSA-High/AR1C3 - low) as compared to the rest of the patients, according to one exemplary embodiment.
DETAILED DESCRIPTION
The present document discloses methods for determining different risks associated with hormone dependent cancers.
The present inventor has surprisingly identified a biomarker, AKR1C3, that can be used for personalized medicine in hormone dependent cancers, such as prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer. AKR1C3 was found to be expressed in platelets and extracellular vesicles derived from platelets and by relating the expression of this biomarker to the expression of other RNA/protein in platelets and/or extracellular vesicles (ECVs), it is surprisingly possible to predict the risk for a subject of developing a hormone dependent cancer, the risk for a subject for early relapse in a hormone dependent cancer, and/or the risk for non-responsiveness to hormone-deprivation therapy in a subject with a hormone- dependent cancer.
AKR1 C3 is an enzyme in the steroid synthesis pathway. It was found by the present inventor that subjects with a hormone dependent cancer have a higher mean expression value of AKR1 C3 in platelets than control subjects without a hormone dependent cancer. Without wishing to be bound by theory, since hormone dependent cancers share a common steroid synthesis pathway and AKR1C3 is part of said pathway, the inventors have surprisingly found that the expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets would be an excellent biomarker for the prognosis of any hormone dependent cancer, including but not limited to prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer. Platelet derived AKR1 C3 can thereby provide an increased amount of downstream steroid products, like androgens in hormone dependent cancers. The platelet produced steroids thereby help supplying the hormone dependent cancer with low levels of hormones e.g. promoting cancer cell proliferation and survival during androgen deprivation therapy or when CTCs circulates the blood.
The present document discloses a method for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the risk for non-responsiveness to hormone-deprivation therapy in a subject with a hormone-dependent cancer, said method comprising: a) isolating platelets and/or extracellular vesicles derived from platelets from a blood or plasma sample from said subject; b) determining in said platelets and/or extracellular vesicles derived from platelets i) the expression of AKR1 C3 and ii) determining the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins to obtain a relative expression of AKR1 C3; c) comparing the relative expression of AKR1 C3 of step b) to a reference expression level derived from a relevant patient-control cohort information; d) based on the comparison in step c) predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the response to hormone-deprivation therapy in a subject with a hormone-dependent cancer, i) wherein a high expression of AKR1C3 relative to said reference expression in said platelets and/or said extracellular vesicles derived from platelets from said subject indicates a high risk, ii) wherein a low expression of AKR1 C3 relative to said reference expression in said platelets and/or said extracellular vesicles derived from platelets from said subject indicates a low risk.
The present method can be used to determine risks as defined herein associated with any hormone dependent cancer. Examples of hormone dependent cancers include but are not limited to hormone-dependent cancer is prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer. The prostate cancer may e.g. be castration resistant prostate cancer.
Based on the personal expression level of AKR1 C3, a number of different diagnosis and/or treatment decisions can be taken. For example, subjects identified to have a high risk of developing a hormone dependent cancer, can be screened (e.g., regular early cancer screening programs) more regularly/often in order to diagnose the cancer as soon as possible, should it develop. Likewise, a subject identified as having a high risk for relapse in a hormone dependent cancer can be more regularly screened for relapse. Moreover, the subject may be more predisposed to having an increased number of metastases. Further, a subject identified as having a high risk for non-responsiveness to hormone-deprivation therapy can be treated with alternative therapies not based on hormone deprivation to improve the chances of recovery/survival.
The comparison of the relative expression of AKR1C3 to a reference expression level may be derived from a relevant patient-control cohort information. The relevant patient-control cohort may be a group of patients having cancer, such as prostate cancer (e.g., castration resistant prostate cancer), uterine, endometrial, ovarian cancer and/or breast cancer. The method of the present document may further comprise a step of deciding on a suitable treatment regimen for a subject having a hormone-dependent cancer based on the results of the AKR1 C3 levels and the risk determination. For subjects with a high expression of AKR1 C3 relative to the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or extracellular vesicles derived from platelets, it may be decided that these subjects should be treated with a treatment regimen not comprising hormone-depletion therapy. For subjects with a low expression of AKR1 C3 relative to the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or extracellular vesicles derived from platelets, it may be decided that these should be treated with a treatment regimen comprising hormonedeprivation therapy.
The method of the present document may therefore comprise a further step of treating said subject with the treatment regimen decided upon. Hormone deprivation therapy may e.g. include the use of compounds inhibiting the synthesis of the hormone in question and/or the use of compounds blocking the hormone in question’s receptor(s).
When it comes to prostate cancer, almost all prostate cancer patients with advanced disease get recurrence. The time to recurrence can take several years though (up to 10 years) while as much as 10% of diagnosed patients already have a primary metastasized disease when diagnosed with prostate cancer. Androgen deprivation therapy works very well in prostate cancer and provide curative treatment for many patients, still prostate cancer often recurs.
Subjects of high risk of developing prostate cancer have a high expression level of the AKR1 C3 in their platelets and/or ECVs. These subjects may therefore benefit from early cancer screening in order for a quick diagnosis of prostate cancer and curative therapy.
The present method can also be used to identify prostate cancer patients with good response to androgen deprivation therapy (ADT) as first line therapy, especially in patients diagnosed with a primary metastatic cancer. The good response is associated with a lower AKR1 C3 expression level in the platelets. Prostate cancer patients identified to have a high level of AKR1 C3 expression may need a therapy alternative to ADT to prevent early recurrence.
Combining measurements of AKR1 C3 expression levels with PSA level measurements allows even more precision when it comes to prostate cancer treatment decisions.
The method of the present document may therefore also comprise a step of determining the level of prostate specific antigen (PSA) in the subject.
CRPC patients having low (below median) levels of serum PSA have low responsiveness to androgen synthesis inhibitory therapy (ASI), regardless of AKR1C3 levels in the platelets. Said patients may be treated by for instance Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check Point Inhibitors (ICI), chemotherapy, EZH2-directed inhibitors, or any combination thereof.
In CRPC patients having high (above median) levels of PSA and low levels of AKR1 C3 are expected to show a good response to ASI. These patients may e.g. be treated with androgen synthesis inhibitors and/or androgen receptor inhibitors such as e.g. abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof.
In CRPC patients having high levels of PSA and high levels of AKR1 C3 are expected to show low response to ASI. These patients may need alternative treatments (s), such as e.g. treatment with BET inhibitors, novel AKR1 C3 targeting inhibitors, androgen receptor (AR) directed therapies, EZH2 inhibitors and/or epigenetic regulator inhibitors. Particularly, said patients may be treated by a combination of AR-directed therapy with at least one of the following: Bromodomain and ExtraTerminal Domain Protein Inhibitors (BETinh), Immune Check-point Inhibitors (ICI), bone/cancer targeting radionuclides, EZH2-directed inhibitors, AR-degraders (such as PROTAC).
By determining the expression of AKR1 C3, optionally in combination with determination of PSA levels, in accordance with the present document, a treatment decision may be taken that increases the chances of recovery/survival of a prostate cancer patient.
The test can also be used to identify potential responders in other steroid synthesis driven diseases, like BrCa, OvCa, etc.
According to the present document, the expression level of AKR1 C3 and references genes in platelets and/or extracellular vesicles (ECV) is determined. From the work of (Arraud et al. 2014) the ECV fraction of blood plasma was quantified and the largest fraction of ECVs were derived from platelets. Therefore, the same analysis as can be performed on platelets can be performed on ECVs.
Platelets can be isolated from a blood or a plasma sample as exemplified elsewhere herein. Typically, different centrifugation steps are used to stepwise separate the platelets from blood. Likewise, typically, different centrifugation steps are used to stepwise separate ECVs from blood. Blood may contain small amounts of AKR1 C3 produced by other cells (like the tumour) and some of this may remain in the sample analysed. However, this amount of tumour derived AKR1C3 is negligible compared to the amount expressed in platelets or platelet derived ECVs and does therefore not have to be taken into consideration in the method of the present document.
The expression of AKR1 C3 and reference gene(s)/total RNA can be studied by analysing mRNA expression. Amounts of mRNA expression may e.g. be determined by PCR, such as ddPCR and/or qPCR, RNA expression arrays, or RNA sequencing.
Alternatively, or in addition, expression levels may be determined by measuring the expression of protein levels of AKR1 C3 and reference protein/total protein. Protein expression levels may e.g. be determined by techniques such as western blot, proximity ligation assay and/or ELISA.
Techniques for analysing expression of mRNA and/or protein are well known to the person skilled in the art and other methods but the ones disclosed herein may also be employed.
Examples of suitable reference genes to measure expression of include, but is not limited to, housekeeping genes, such as GADPH, bAKT and/or ITGA2B. The present document is also directed to the use of platelet-derived AKR1 C3 as a biomarker for hormone dependent cancer(s). AKR1 C3 may as disclosed herein thus be used as a biomarker to predict the risk of a subject of developing a hormone- dependent cancer, predict the risk of a subject for relapse in a hormone-dependent cancer and/or predict the response to hormone-deprivation therapy in a subject with a hormone-dependent cancer.
The present document also discloses a kit for predicting the risk associated with a hormone dependent cancer as disclosed herein. Such a kit may e.g. comprise reagents for performing a quantitative RNA analysis of AKR1C3 and housekeeping gene levels in sample using a digital or quantitative PCR. Reference material for positive control and negative control evaluation is also included.
Such a kit may alternatively or in addition comprise reagents for performing a semi- quantitative ELISA analysis of AKR1 C3 protein levels. In this case, reference material to make AKR1 C3 protein standard curve and a negative control is also included.
The kit for predicting the risk of developing a hormone dependent cancer advantageously allows comparing the AKR1 C3 expression with the expression of housekeeping genes and reference materials for both a positive and negative control evaluation. Said comparison advantageously provides a reliable and effective kit for predicting the risk of developing a hormone dependent cancer in the subject having or not said cancer.
A kit according to the present document may also optionally comprise instructions regarding the method for how to calculate the relative expression of AKR1C3 to reference expression and the risk associated with the score.
The present document is also directed to a method for treating and/or preventing a hormone dependent cancer in a subject, said method comprising: a) administering to said subject a pharmaceutically effective amount of a drug for treating and/or preventing said hormone dependent cancer; or b) performing surgical removal of said hormone dependent cancer; wherein the predicted risk for said subject of developing a hormone dependent cancer, predicted the risk for said subject for relapse in a hormone dependent cancer, and/or predicted the risk for non-responsiveness to hormonedeprivation therapy for said subject is high or low, as defined by the method as defined elsewhere herein.
The drug to be administered to the subject may defined according to the predicted risk for said subject (i.e. , high or low risk), wherein said drug may be a drug used in a hormone-deprivation therapy or a drug not typically used in a hormone-deprivation therapy, as defined elsewhere herein. The skilled person knowns which drugs may be used and in which dosage regimen.
The present document is also directed to a drug for use in the treatment of a hormone-dependent cancer in a subject, said drug being selected from an androgen synthesis inhibitor and/or an androgen receptor-directed inhibitor, wherein said subject belongs to a sub-group of subjects having i) a low expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets relative to an expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or said extracellular vesicles derived from platelets from said subject; and ii) a high expression of PSA in a blood sample from said subject.
As shown in the experimental section, it has been surprisingly found that said subgroup of subjects has an improved treatment outcome when treated with a treatment regimen comprising hormone-deprivation therapy, such as abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof. In other words, subjects having a high PSA expression and low AKR1 C3 expression belong to a specific sub-group of subjects that responds significantly better to said treatment, thereby increasing the overall survival rate and reducing risks and costs associated with choosing a less effective treatment for this sub-group of subjects. Said subgroup of subjects is physiologically distinguishable from the general group of subjects having prostate cancer due to the high expression of PSA in the blood and the low expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets. The skilled person knows which drugs belong to the group of androgen synthesis inhibitor and/or an androgen receptor-directed inhibitor, as well as the dosage regimen for each one of said drugs. The drug for use in the treatment of a hormone-dependent cancer is preferably used for treating prostate cancer, more preferably castration-resistant prostate cancer.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXPERIMENTAL SECTION
Exemplary methods for analysis
Alternative A) Analysis of platelet derived AKR1 C3 or extracellular vesicles (ECVs) derived AKR1 C3:
Isolation of platelet or extracellular vesicle samples
Blood (typically around 10 ml) is collected e.g. in EDTA Vacutainer tubes (or similar tubes for platelet isolation). Ideally, the blood is processed and platelets isolated between 30 min to two hours after blood draw.
A suspension of platelet rich plasma is then obtained. For example, this can be achieved by centrifuging the blood, such as about 20 min at 120xG in room temperature, to separate the nucleated cells and erythrocytes from the platelet rich plasma (including platelets, extracellular vesicles and plasma). After centrifugation the upper part of the platelet rich plasma (typically the upper 2/3) is transferred to a new tube and centrifuged again, such as for another 10 min for 120xG, to pellet aggregates. The above protocol is one example of many variations to achieve a suspension of platelet rich plasma that can be used for downstream processing.
The platelet rich plasma can be used to isolate platelets and/or extracellular vesicles.
From the platelet rich plasma, the platelets can be obtained e.g. by pelleting them to the bottom of the tube by centrifugation, e.g. for 10 min 800xG in room temperature. The plasma is then removed, the platelets are preferably washed, e.g. with PBS. The platelets can then be snap frozen and stored in -80°C or directly analysed.
Plasma derived extracellular vesicles can be isolated from the plasma fraction after pelleting the platelets. Extracellular vesicles can be extracted using size exclusion chromatography (SEC). In brief, the platelet depleted plasma is loaded on a size exclusion column (Sepharose CL-2b) and the plasma components are separated by their size. The ECV fraction is collected and ECVs are isolated for downstream processing (e.g. according to Bdnig et al. 2014)
Using ddPCR to analyse biomarker levels within the sample is one way of doing this, but other RNA analyses approaches can be used, like hybrid capture arrays, nanostring technology, RNA-sequencing approaches that can be used to measure levels of AKR1 C3 and references genes.
From this point either an RNA (see “RNA preparation” in the below) based and/or a protein (see “Protein preparation” in the below) based isolation protocol is applied.
RNA preparation
Isolation of platelet RNA. The platelet pellet prepared, e.g. as described above, is processed using a standard total RNA isolation kit to extracted RNA from the platelet fraction (like RNeasy Mini Kit (Qiagen) or miRVANA (Invitrogen)) per manufacturer's instructions. RNA concentrations are measured e.g. with Qubit method (Invitrogen) and correlated to volume of platelet rich plasma used for isolation.
Isolation extracellular vesicle RNA. Eluted ECVs from the isolated plasma be processed using a standard total RNA isolation kit to extract RNA from the eluate (like TRIZOL LS (Invitrogen)) per manufacturer's instructions. The method may include a silica-based column binding and purification step. For example, 1 part of platelet-rich plasma suspension (250ul) is lysed in 3 parts Trizol LS (750ul) and used in the protocol. Another way of isolating ECV RNA can be achieved by pellet the extracellular vesicles fraction using ultracentrifugation or through ECV precipitation methods where the isolated ECVs RNA is extracted with a standard RNA isolation kit (like RNeasy Mini Kit (Qiagen), miRVANA (Invitrogen)) per manufacturer's instructions. RNA concentrations are measured e.g. with Qubit method (Invitrogen) and correlated to volume of plasma used for isolation.
Digital PCR Analysis of RNA. 1 -50 ng (preferably about 10 ng) depending on the input source of RNA can be used in a one-step-RT-PCR analysis to detect the absolute transcript number using digital PCR (like the Droplet Digital PCR QX-200 system, Bio-Rad) using assays for a FAM-probe AKR1 C3 assay and a reference gene HEX-probe (like GAPDH, IGT2B, etc.). Biomarker status in the sample can e.g. be defined as AKR1 C3 transcripts per reference gene transcripts, AKR1 C3 transcripts per total RNA levels, or AKR1 C3 transcripts per ml of plasma.
Read-out. The expression value of the AKR1 C3 in comparison to reference gene or correlated to platelet total RNA per ml plasma is calculated. In the analysis the most precise AKR1 C3-transcript levels compared to the reference is preferably used to differentiate between a positive (low risk) respectively negative (high risk) result. For example, the use of Receiver Operating Characteristic (ROC)-curve analysis to determine the most accurate cut-off value provides higher sensitivity and specificity in the read-out to identify high risk patients from low risk patients, compared to use the median expression value.
Protein preparation
Isolation of platelet proteins. Platelet protein can be extracted with a combined RNA/protein extraction kit (like mirVana PARIS (Invitrogen)) according to manufacturer’s instructions. E.g. 600 ml of ice-cold disruption buffer from the kit is used for each platelet sample. One third of the samples is then used for protein extraction. Protein concentration can be determined using standard techniques like the BCA protein assay, Bradford Protein Assay or similar.
Analysis of AKR1C3 protein levels. Protein from the platelet or ECV sample can be analysed with SDS-page western blotting. For example., between 2-50 ug of platelet proteins (preferably 10 ug) is used and the expression levels analysed with antibodies against AKR1C3 and a protein reference (like B-Aktin or CD41), where the protein expression of AKR1 C3 is correlated to an internal standard of AKR1 C3 protein levels. Protein levels can also be analysed with alternative protein detection methods (like OLINK PEA-technology, ELISA etc.).
Read-out. The expression value of the AKR1 C3 in comparison to reference protein or correlated to platelet total protein per ml plasma is determined. In the analysis the most precise AKR1 C3-protein levels compared to the reference is preferably used to differentiate between a positive (low risk) respectively negative (high risk) result. The use of Receiver Operating Characteristic (ROC)-curve analysis to determine the most accurate cut-off value provides high sensitivity and specificity in the read-out to identify high risk patients from low risk patients.
Alternative B) Analysis the platelet rich plasma directly
In this approach the platelets and extracellular vesicles are not separated, instead the combined platelet rich plasma fraction is used. Since plasma contains low levels of free-circulating RNA the much higher RNA levels from platelets and to a lesser extent RNA from the extracellular vesicles will provide the majority of the signal analysed in platelet-rich plasma, with no/low impact from the free-circulating plasma RNA. The amount of circulating AKR1 C3 derived from the tumour will thus be negligible.
RNA preparation
Isolation of RNA from platelet rich plasma. The platelet rich plasma can be processed using a standard total RNA isolation kit to extract RNA from the platelet rich plasma suspension (like TRIZOL LS (Invitrogen) per manufacturer's instructions. The method may include a silica-based column binding and purification step. For example, 1 part of platelet-rich plasma suspension (250ul) is lysed in 3 parts Trizol LS (750ul) is used in the protocol. The eluted RNA concentration is measured with Qubit method (Invitrogen) and correlated to volume of platelet rich plasma used for isolation.
Digital PCR Analysis of RNA. 1-50 ng (preferably 10 ng) depending on the input source of RNA are used in the one-step-RT-PCR analysis to detect the absolute transcript number using digital PCR (like the Droplet Digital PCR QX-200 system, Bio-Rad) using assays for a FAM-probe AKR1 C3 assays and a reference HEX-probe (like GAPDH, IGT2B, etc.). Biomarker status in the sample can be defined as AKR1 C3 transcripts per reference gene transcripts, AKR1 C3 transcripts per total RNA levels, or AKR1 C3 transcripts per ml of plasma.
Read-out. The read-out will be compared to the median expression value of the AKR1 C3 in comparison to reference gene, to total RNA levels in sample or correlated to total RNA per ml plasma. In the analysis the most precise AKR1 C3-transcript levels will be used to differentiate between a positive (low risk) respectively negative (high risk) result. The use of Receiver Operating Characteristic (ROC)-curve analysis to determine the most accurate cut-off value provides high sensitivity and specificity in the read-out to identify high risk patients from low risk patients.
Example 1 : Early predictive marker for therapy response in patients with metastasized prostate cancer at diagnosis.
Sample cohort, collection and processing
Blood was collected prospectively from patients with prostate cancer and healthy controls after which platelets were isolated within 12h. RNA was isolated and processed for RNA sequencing as previously described (Best et al. 2015). The disease risk stages were defined according as following; Stage 1 (G1 ): T1 -2, Gleason score 6; PSA<10, Stage 2 (G2): T1 -2, Gleason score 7; and/or 10<PSA<20, Stage 3 (G3): T1 - 3, Gleason score 8-10; and/or 10<PSA<20, Stage 4 (G4): T4 and/or N1 ; 50<PSA<100, Stage 5 (G5): M1 , and/or PSA>100, and CRPC, castration resistant prostate cancer. The patients were followed for recurrence monitoring and Progression free-survival and Overall survival was determined. The quantitative evaluation of the metastases at diagnosis was determined, 0, lymph node metastases; 1 , low bone metastatic burden; and 2, high metastatic burden.
Data analysis
Low quality bases and adapters were trimmed off using Trim Galore v.0.4.1 and the sequence data was thereafter aligned to the human genome (hg19) with STAR v.2.5.3a. Following alignment, gene body coverage analysis was performed with Qualimap v.2.2.2. Spliced reads were extracted using Bamlltil v.1.0.14 findCigars with the cskip flag. Gene counts were further generated by HTSeq-count v.0.9.1 using union intersection. All subsequent statistical analyses were performed in R-studio v.3.4.0. The count matrix was further TMM (Trimmed Mean of M-values) normalized. The normalized expression of AKR1 C3 was used in the analyses.
Results
Fig. 1 shows the expression of AKR1 C3 (NGS sequencing data) of different stages of primary prostate cancer patients at diagnosis (G1 -5) and healthy controls as well as CRPC patients. Normal controls are significantly lower than in prostate patients (P=0,05). Horizontal line in figure 1 is set as the median value of the healthy control group AKR1 C3 expression. The y-axis is the normalized expression of AKR1 C3 in the cohort.
Analysis of the expression of AKR1 C3 in normal individuals and prostate cancer patients, shows that there is a slightly increased expression in cancer patients over controls (Fig. 1 ). Still all groups have a broad gradient expression indicating that there is an individual difference in expression and that the AKR1 C3 levels seen in the platelets are mainly derived from the platelets, and not through transfer of tumour- derived RNAs.
Progression-free survival (PFS) was determined according to clinical observation of progression of the disease (PSA relapse). Overall survival was determined by the time of death from any cause. The time-to-event (either PFS (Figure 2) or OS (Figure 3)) was evaluated using the Kaplan-Meier method and the log rank test was used to determine significance results. The results presented is in the stage 5 group (G5), the primary metastatic group, that had a metastatic disease at diagnosis (figure 2). In figure 2a, and 2b, the median expression of AKR1 C3 respectively the ROC-assisted levels in the platelets was used as the cut-off between high risk and low risk patients. Moreover, Fig. 2a shows that AKR1 C3 above median expression can distinguish patients that have a short time to recurrence (p=0.010), highlighting the patients that are at high risk of recurrence (within 15,6 months vs. 44.6 months). Further, in Fig. 2b the ROC-curve analysis was preformed to identify the most accurate cut-off for the AKR1 C3 expression increasing the sensitivity of the Kaplan-Meier analysis (p<0.001 ). The legend of those figures are as follows: Full line, below cut-off, and dashed line, above cut-off of AKR1 C3 expression. PFS, progression-free survival, ROC-curve analysis, Receiver Operating Characteristic- curve analysis.
The high risk was also seen when comparing stage 5 group to overall survival, where AKR1 C3 above median was associated with a significant shorter overall survival (figure 3a), and in the AKR1 C3 low (full line) group only 1 Z15 (7%) patient did die during the follow-up in stark contrast to the AKR1 C3 high group (dashed line) where 11/15 (73%) died during the follow-up period. A similar distinction could be seen when using ROC-curve analysis to define cut-off between high versus low risk groups (figure 3B). In other words, dividing patients into risk groups either through using a median cut-off of expression (Fig. 3a) or using ROC-curve analysis to determine cut-off of AKR1 C3 (Fig.3b) can predict patients that have a short time to live (P<0.001 respectively P<0.001 ). The legend of those figures are as follows: OS, overall survival, LogR, Log rank test, Above cut-off, dashed line, below cut-off, full line.
These experiments demonstrate that one can use the RNA expression levels of AKR1 C3 in platelets to determine a high risk of early recurrence and a high risk of early death following a cancer diagnosis in patients with a metastatic disease at diagnosis.
As disclosed elsewhere herein, since the ECV are mainly derived from the platelets, they will contain the same information as the original platelets and therefore the platelet-derived ECV population can be used as a surrogate marker for AKR1 C3 levels in the platelets.
Example 2: Androgen directed therapy response prediction using AKR1 C3 levels in blood platelets prior therapy start.
Sample cohort, collection and processing
Blood samples from 25 CRPC patients prior abiraterone therapy were used in this study. The RNA and cDNA were previously prepared for that study (Tjon-Kon-Fat et al. 2018; doi: 10.1002/pros.23443), AKR1 C3 levels was later analysed according to the following outline. Serum PSA levels was collected routinely in the clinic to follow progression of the cancer.
RNA and cDNA preparation. Total RNA was extracted from the platelet fraction with RNeasy Mini Kit (Qiagen) or miRVANA (Invitrogen). The eguivalent of RNA extracted from 1 ml of whole blood was used for reverse transcription and amplification using whole transcriptomic amplification 2 - kit (WTA-2, Sigma-Aldrich).
Digital PCR Analysis of cDNA. Amplified cDNA (50 ng) was used for PCR analysis. The absolute transcript number was determined using ddPCR (QX-200, Bio-Rad). TagMan (Applied Biosystems) assays for AKR1C3 (Hs00366267_m1 ) and GAPDH (Hs99999905_m1 ) mRNA analysis was used. Platelet biomarker status was expressed as no. transcripts per 5000 GAPDH transcripts. Clinical Outcomes. Positive therapy response (PSA response) was defined as a > 50% decline in PSA level from baseline during therapy. PFS was defined as PSA > 2ng/ml and a 25% increase from nadir. OS was determined by the time of death from any cause. (Tjon-Kon-Fat et al. 2018; doi: 10.1002/pros.23443)
Statistical Evaluations. Analyses were performed with SPSS v.22. Time-to-event was evaluated using the Kaplan-Meier method and the log rank test was used to determine significance. Student’s t-tests or one-way ANOVA were used to test for difference in means between two groups respectively more than two groups. Significance was defined by two-sided tests with a p-value of 0.05 or less.
Results:
Platelet levels of AKR1C3 correlates with survival and therapy response.
The patients were divided into quartiles (Q1 -4) based on their expression of AKR1C3. Low (Q1 ) levels of platelet AKR1C3 levels have a longer time-to-progression in a Kaplan-Meyer analysis, the Q1 group had a significantly longer progression-free- survival (PFS; 9.9month; log-rank, p=0.013), than the other groups, Q2 - 4 (PFS; Q2 - 3.3 month, Q3 - 3.97 respectively Q4 - 3.1 month; Figure 4A). In Figure 4B, the ROC- curve analysis was used to identify the most accurate cut-off of AKR1 C3 expression and used in the Kaplan-Mayer analysis to identify responding patients. As previously, below cut-off was associated with a significant longer time to PFS and a good therapy response (P=0.043).
PSA and AKR1C3 levels predict a favourable response to abiraterone therapy.
Plasma levels of PSA can be viewed as a surrogate marker for AR (androgen receptor) signalling activity and/or tumour burden. In this study serum PSA levels (above respectively below median) did not support any information regarding PFS or OS on how the patients responded to abiraterone therapy (figure 5A-B). It was also investigated if AKR1C3 expression (below versus above median) was associated with therapy response in patients with high PSA levels (e.g. a subgroup of cancer patients with high AR activity) and there was a significantly shorter PFS in patients with high AKR1C3 expression (figure 5C; PFS = 3.0 month vs. 9.9 month; log-rank, p=0.030) and a significantly shorter OS (figure 5D; OS = 6.1 respectively 34.0 month; log-rank, p=0.015). However, AKR1C3 holds no information in the PSA low group (e.g. a subgroup of cancer patients with low AR activity), neither regarding PFS nor OS (figure 5E-F).
When looking at therapy response the combined high PSA and low AKR1C3 was a positive predictive biomarker (t-test, p=0.05) for therapy response (Figure 6), identifying 28% (7/25) of the patients responding well to abiraterone therapy. This is close to the 29% therapy response seen in a similar patient cohort with in a large clinical study (De Bono et al. 2011 ; doi: 10.1056/NEJMoa1014618).
Conclusions:
In conclusion, the above data identified AKR1C3 expression as a prognostic and/or predictive biomarker when used alone and together with high PSA levels. Patients from the PSA high group with AKR1 C3-low expression had a three-time longer PFS and five-times longer OS then AKR1 C3-high (9.9 vs 3.0-month respectively 34.0 vs 6.1 - month).
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.
REFERENCES
Arraud N., Linares R., Tan S., Gounou C., Pasquet J.M., Mornet S., Brisson A.R. 2014. Extracellular vesicles from blood plasma: Determination of their morphology, size, phenotype and concentration. J. Thromb. Haemost. 12, 614-627.
Best, M. G., et al. (2015). "RNA-Seq of Tumor-Educated Platelets Enables Blood- Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics." Cancer Cell. Tjon-Kon-Fat, L. A. et al. Platelets harbor prostate cancer biomarkers and the ability to predict therapeutic response to abiraterone in castration resistant patients. Prostate 78, 48-53, doi:10.1002/pros.23443 (2018). de Bono , J. S. et al. Abiraterone and Increased Survival in Metastatic Prostate Cancer. New England Journal of Medicine 364, 1995-2005, doi: 10.1056/NEJMoa1014618 (2011 ).
Tjon-Kon-Fat, L. A., Sol, N., Wurdinger, T. & Nilsson, R. J. A. Platelet RNA in Cancer Diagnostics. Semin Thromb Hemost 44, 135-141 , doi:10.1055/s-0037-1606182
(2018).

Claims

CLAIMS A method for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the risk for non-responsiveness to hormone-deprivation therapy in a subject with a hormone-dependent cancer, said method comprising: a) isolating platelets and/or extracellular vesicles derived from platelets from a blood or plasma sample from said subject; b) determining in said platelets and/or extracellular vesicles derived from platelets i) the expression of AKR1 C3 and ii) determining the expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins to obtain a relative expression of AKR1 C3; c) comparing the relative expression of AKR1 C3 of step b) to a reference expression level derived from a relevant patient-control cohort information; d) based on the comparison in step c) predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the response to hormone-deprivation therapy in a subject with a hormone-dependent cancer, i) wherein a high expression of AKR1C3 relative to said reference expression in said platelets and/or said extracellular vesicles derived from platelets from said subject indicates a high risk, ii) wherein a low expression of AKR1 C3 relative to said reference expression in said platelets and/or said extracellular vesicles derived from platelets from said subject indicates a low risk. The method according to claim 1 , wherein said hormone-dependent cancer is prostate cancer, castration-resistant prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and/or breast cancer. The method according to claim 1 or 2, wherein said expression of AKR1 C3 is determined by digital or quantitative PCR, proximity ligation assay and/or enzyme-linked sorbent assay (ELISA).
4. The method according to any one of the preceding claims, wherein the expression of AKR1 C3 and expression of one or more reference genes and/or total RNA is determined by analysing mRNA expression.
5. The method according to any one of the preceding claims, wherein the expression of AKR1 C3 and expression of one or more reference genes and/or total RNA is determined by PCR, such as qPCR and/or ddPCR.
6. The method according to any one of the preceding claims, wherein said expression of AKR1 C3 and said expression of one or more reference proteins and/or total proteins is determined by analysing protein levels, such as ELISA.
7. The method according to any one of the preceding claims, wherein said reference genes is GADPH, bAKT and/or ITGA2B.
8. The method according to any one of the preceding claims, wherein said method further comprises a step of deciding on a treatment regimen for said subject having a hormone-dependent cancer.
9. The method according to claim 8, wherein i) for subjects with a high expression of AKR1 C3 relative to said expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in said platelets and/or said extracellular vesicles derived from platelets from said subject it is decided that these should be treated with a treatment regimen not comprising hormone-depletion therapy; ii) for subjects with a low expression of AKR1 C3 relative to said of one or more reference genes and or reference proteins and/or total RNA or total proteins in said platelets and/or said extracellular vesicles derived from platelets from said subject it is decided that these should be treated with a treatment regimen comprising hormone-deprivation therapy.
10. The method according to any one of the preceding claims, wherein said method further comprises a step of determining the expression of prostate specific antigen (PSA) from the blood or plasma sample of said subject. The method according to claim 10, wherein i) for subjects with low expression of PSA and low expression of AKR1 C3 or with low expression of PSA and high expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen not comprising hormone-deprivation therapy, such as with Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check Point Inhibitors (ICI), chemotherapy, EZH2-directed inhibitors, or any combination thereof; ii) for subjects with high expression of PSA and low expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen comprising hormone-deprivation therapy, such as abiraterone, enzalutamide, apalutamide, darolutamide, or any combination thereof; and/or iii) for subjects with high expression of PSA and high expression of AKR1 C3 it is decided that said subject should be treated with a treatment regimen not comprising androgen-synthesis inhibitor, such as a combination of AR- directed therapy with one or more of Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETinh), Immune Check-point Inhibitors (ICI), bone/cancer targeting radionuclides, EZH2-directed inhibitors and/or AR- degraders. The method according to any one of claims 8-11 , said method further comprising the step of treating said subject with the treatment regimen decided upon. The method according to any one of the preceding claims, further comprising communicating to the subject and/or to a healthcare provider the risk for said subject of developing a hormone dependent cancer, the risk for the subject for relapse in a hormone dependent cancer, and/or the risk for nonresponsiveness to hormone-deprivation therapy, such as by means of electronic communication and/or paper-based communication. The method according to any one of the preceding claims, wherein said subject belongs to a sub-group of subjects having a high expression of PSA. Use of platelet-derived AKR1 C3 as a biomarker for hormone-dependent cancer, such as prostate cancer, castration-resistant prostate cancer, uterine, endometrial, ovarian cancer and/or breast cancer. The use according to claim 15, wherein said biomarker predicts the risk of a subject of developing a hormone-dependent cancer, predicts the risk of a subject for relapse in a hormone-dependent cancer and/or predicts the response to hormone-deprivation therapy in a subject with a hormone- dependent cancer. A kit for predicting the risk for a subject of developing a hormone dependent cancer, predicting the risk for a subject for relapse in a hormone dependent cancer, and/or predicting the response to hormone-deprivation therapy in a subject with a hormone-dependent cancer, said kit comprising: a) reagents for performing a quantitative RNA analysis of AKR1C3 and housekeeping gene levels in sample using a digital or quantitative PCR system and reference material for positive control and negative control evaluation; and/or b) reagents for performing a semi-quantitative ELISA analysis of AKR1 C3 protein levels and reference material to make AKR1 C3 protein standard curve and a negative control; and c) optionally instructions regarding the method for how to calculate the relative expression of AKR1 C3 to reference expression and the risk associated with the score. A method for treating and/or preventing a hormone dependent cancer in a subject, said method comprising: a) administering to said subject a pharmaceutically effective amount of a drug for treating and/or preventing said hormone dependent cancer; or b) performing surgical removal of said hormone dependent cancer; wherein the predicted risk for said subject of developing a hormone dependent cancer, predicted the risk for said subject for relapse in a hormone dependent cancer, and/or predicted the risk for non-responsiveness to hormonedeprivation therapy for said subject is high or low, as defined by the method as defined in any one of claims 1 -14.
19. The method according to claim 18, wherein said drug is a drug used in a hormone-deprivation therapy or a drug not typically used in a hormonedeprivation therapy.
20. A drug for use in the treatment of a hormone-dependent cancer in a subject, said drug being selected from an androgen synthesis inhibitor and/or an androgen receptor-directed inhibitor, wherein said subject belongs to a sub-group of subjects having i) a low expression of AKR1 C3 in platelets and/or extracellular vesicles derived from platelets relative to an expression of one or more reference genes and/or reference proteins and/or total RNA or total proteins in platelets and/or said extracellular vesicles derived from platelets from said subject; and ii) a high expression of PSA in a blood sample from said subject.
21 .The drug for use in the treatment of a hormone-dependent cancer in a subject according to claim 20, wherein the androgen synthesis inhibitor is abiraterone.
22. The drug for use in the treatment of a hormone-dependent cancer in a subject according to claim 20 or 21 , wherein the androgen receptor-directed inhibitor is selected from Enzalutamide, Apalutamide, Darolutamide, or any combination thereof.
23. The drug for use in the treatment of a hormone-dependent cancer in a subject according to any one of claims 20-22, wherein said hormone dependent cancer is prostate cancer, castration-resistant prostate cancer, uterine cancer, endometrial cancer, ovarian cancer and/or breast cancer.
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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ARRAUD N.LINARES R.TAN S.GOUNOU C.PASQUET J.M.MORNET S.BRISSON A.R.: "Extracellular vesicles from blood plasma: Determination of their morphology, size, phenotype and concentration", J. THROMB. HAEMOST., vol. 12, 2014, pages 614 - 627
BEST, M. G. ET AL.: "RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics", CANCER CELL, 2015
DE BONO , J. S. ET AL.: "Abiraterone and Increased Survival in Metastatic Prostate Cancer", NEW ENGLAND JOURNAL OF MEDICINE, vol. 364, 2011, pages 1995 - 2005, XP002725563, DOI: 10.1056/NEJMoa1014618
MARKN STEIN ET AL: "Androgen synthesis inhibitors in the treatment of castration-resistant prostate cancer", ASIAN JOURNAL OF ANDROLOGY, vol. 16, no. 3, 1 January 2014 (2014-01-01), US, pages 387 - 400, XP055316057, ISSN: 1008-682X, DOI: 10.4103/1008-682X.129133 *
MICHAEL STANBROUGH ET AL: "Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 66, no. 5, 1 March 2006 (2006-03-01), pages 2815 - 2825, XP002639099, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-05-4000 *
TJON-KON-FAT, L. A. ET AL.: "Platelets harbor prostate cancer biomarkers and the ability to predict therapeutic response to abiraterone in castration resistant patients", PROSTATE, vol. 78, 2018, pages 48 - 53
TJON-KON-FAT, L. A.SOL, N.WURDINGER, TNILSSON, R. J. A.: "Platelet RNA in Cancer Diagnostics", SEMIN THROMB HEMOST, vol. 44, 2018, pages 135 - 141
YUANTONG TIAN ET AL: "AKR1C3 overexpression may serve as a promising biomarker for prostate cancer progression", DIAGNOSTIC PATHOLOGY, BIOMED CENTRAL LTD, LO, vol. 9, no. 1, 26 February 2014 (2014-02-26), pages 42, XP021178900, ISSN: 1746-1596, DOI: 10.1186/1746-1596-9-42 *

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