WO2016162037A1

WO2016162037A1 - Lhcgr for use in diagnosing and monitoring of andrological diseases

Info

Publication number: WO2016162037A1
Application number: PCT/DK2016/050097
Authority: WO
Inventors: Martin Blomberg JENSEN
Original assignee: Rigshospitalet Copenhagen Universtiy Hospital
Priority date: 2015-04-08
Filing date: 2016-04-07
Publication date: 2016-10-13
Also published as: EP3281015A1

Abstract

The present invention relates to a method for predicting for a male subject atrisk of having, developing or relapse oftestis cancer; and/or prostate cancer; and/or a need of Androgen replacement therapy (ART); and/or gonadal dysfunction. The method is based on the measurement of the level of LHCGR (LH/hCG receptor) in a biological sample from the male subject, such as a (blood) serum sample.

Description

LHCGR FOR USE IN DIAGNOSING AND MONITORING OF ANDROLOGICAL DISEASES

Technical field of the invention

The present invention relates to the use LHCGR e.g. in body fluids as a diagnostic marker. In particular, the present invention relates to the use of LHCGR or derivatives thereof in body fluids to diagnose or monitor patients at risk of having, developing or relapse of testis cancer; and/or prostate cancer; and/or a need of Androgen replacement therapy (ART); and/or gonadal dysfunction

Background of the invention

The gonadotropic hormones luteinizing hormone (LH) and human chorionic gonadotropin (hCG) play an essential role for male reproduction during

development and in adult life. The action of both hormones is mediated by the LH/hCG receptor (LHCGR), a G protein-coupled receptor, expressed in Leydig, granulosa-lutein, and theca cells. The LHCGR can also bind human

choriongonadotropin (hCG), which is produced in fetal life and in some forms of testicular cancer. Low or absence of LH receptor (LHCGR) lowers testosterone and semen quality because of impaired stimulation of the Leydig cells. Therefore, several groups have investigated mutations in the LHR because they could be relevant for reproductive disorders. The gene for LHR comprises 11 exons on chromosome 2. Ten of the 11 exons encode the extracellular domain, while exon 11 encodes the seven transmembrane and the intracellular domain (Ascoli, Endocr Rev. 23: 141-74, 2002). Several inactivating LHR mutations were described in patients with 46,XY disorder of sex development (46, XY DSD) due to Leydig cell hypoplasia (LCH), an autosomal recessive disease characterized by a female phenotype in subjects with 46, XY karyotype (Themmen, Endocr. Rev. 21 : 551- 583, 2000). However, in a substantial number of patients (50%) with the classical symptoms of LCH no mutations of the LHR are found (Zenteno, J. Clin. Endocrinol. Metab. 84: 3803-3806, 1999; Richter-Unruh, Clin. Endocrinol. (Oxf). 56: 103-12, 2002). Summary of the invention

It is suggested that one of the LHreceptor isoforms may be released from the cell, but it has never been measured in body fluids from any man. It is suggested that LH receptor release to body fluids takes place and is much more abundant when a subject has diseases where the cells that express the LHreceptor are under stress, for instance in highly proliferating cells. Similarly, the LHreceptor may be released from a special subset of cells, for instance Leydig cells. Leydig cells are very heterogeneous and it is believed that a subset of the immature Leydig cell pool may be releasing LHreceptor (LHCGR) and therefore account for the vast majority of the serum level of LHCGR. The reason why exactly these cells release the receptor to serum could be (without being bound by theory) due to alternative splicing of the LHCGR since a transcript without the transmembrane part, encoded by exon 11, may be internalized or secreted because it cannot reside in the cell membrane (ref mauel tempa sempere JCEM 1994). Alternative, enzymes in the membrane may be responsible for cleavage and release of the receptor to body fluids. The mechanism responsible for the release of LHR is important because the size of the soluble LHR in serum will depend on this. Specific detection of LHR in serum or body fluids can therefore be used to discriminate between different diseases if the method can distinguish the different LHR forms.

One possibility is (without being bound by theory) that some forms of testicular cancer actually produce and express LHCGR. Testicular cancer originates from carcinoma in situ cells that eventually will develop into malignant testicular germ cell tumors seminoma or non-seminoma that are invasive. Today several markers of non-seminoma exist, which can be measured in serum and can be used to monitor these patients and identify relapse. There is no reliable serum marker of seminomas and it is therefore suggested that circulating serum LHCGR or maybe seminal plasma levels of LHCGR can be used to monitor not only the invasive seminoma, but also non-seminoma patients, and patients with carcinoma in situ. This suggestion is supported by the marked expression of LHCGR in CIS and invasive tumors, which indicates a novel role of the truncated LHCGR in testis cancer. We have found elevated serum LHR in men with testicular cancer who later have a relapse. This shows that LHR may be used as a prognostic marker for testis cancer patients. Similarly, we have found expression of LHCGR in prostate cancer cells unlike normal prostate cells. This indicates that serum and seminal fluid LHCGR also can assist in diagnosis or at least assist in clinical surveillance of prostate cancer patients. This suggestion is supported by the finding of high serum LHCGR in a man without testis but with an invasive prostate cancer and the fact that the truncated LHR isoform, which is secreted is the only isoform found in the prostate.

Noteworthy, another possibility is that high serum LHCGR levels reduce the availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. This would therefore strongly indicate that this patient should be treated with testosterone because the stimulation of the gonadal testosterone production was impaired by partly blocking the effect of LH. It is suggested that measured serum levels of LHCGR can be used as a marker of impaired Leydig cell function either alone or with improved sensitivity in combination with serum levels of testosterone, LH, INSL3 and/or testis size. This is supported by the finding that men with high LHR in serum also have disturbed inhibin/FSH ratio, which confirms that it is a marker for testicular function. Together with one or more of these factors, LHCGR will provide unique information about the Leydig cell population and to some extent also spermatogenesis, which therefore can be used to diagnose selected patients and to monitor Leydig cell function in men prior to or under infertility evaluation or testosterone supplementation treatment.

Thus, it is an object of the invention to use the detection of the LHCGR protein, by using antibodies or other compounds that specifically binds and detects the LHCGR in a provided sample of body fluids like serum, plasma, seminal fluid, to diagnose and/or monitor specific andrological diseases such as carcinoma in situ testis, and/or invasive testis cancer, and/or prostate cancer, and/or Klinefelter Syndrome and/or a need of Androgen replacement therapy (ART) and/or gonadal dysfunction. Thus, an aspect of the invention relates to a method for predicting for a male subject a risk of having, developing or relapse of

- carcinoma in situ testis; and/or

invasive testis cancer; and/or

prostate cancer; and/or

Klinefelter syndrome; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction such as abnormal puberty or male infertility; the method comprising

• providing a biological sample from a male subject, said sample being a fluidic sample, such as serum, plasma, and/or seminal

• determining the level of LHCGR (LH/hCG receptor) in the sample;

• comparing said determined level to a reference level; and

• determining that

o said subject is at risk of having, developing or relapse of carcinoma in situ testis, and/or, invasive testis cancer, and/or prostate cancer, and/or Klinefelter Syndrome, and/or is in need of androgen supplementation, and/or having gonadal dysfunction if said level is higher than said reference level, or

o determining that said subject is not at risk of having, developing or relapse of carcinoma in situ testis, and/or, invasive testis cancer, and/or prostate cancer, and/or Klinefelter Syndrome, and/or is in need of androgen supplementation, and/or having gonadal dysfunction if said level is equal to or lower than said reference level.

In another aspect the invention relates to a method for predicting for a male subject a risk of having, developing or relapse of

- testis cancer; and/or

prostate cancer; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction; the method comprising

• providing a biological sample from a male subject, said sample being a fluidic sample, such as (blood) serum, plasma, and/or seminal;

• determining the level of LHCGR (LH/hCG receptor) in the sample;

· comparing said determined level to a reference level; and

• determining that

o said subject is at risk of having, developing or relapse of testis

cancer, and/or prostate cancer, and/or a need of androgen supplementation, and/or having gonadal dysfunction, if said level is higher than said reference level, or o determining that said subject is not at risk of having, developing or relapse of testis cancer, and/or prostate cancer, and/or a need of androgen supplementation, and/or having gonadal dysfunction if said level is equal to or lower than said reference level.

A further aspect relates to a method for monitoring a condition in a male subject previously determined of having (or at risk of developing) a condition selected from

- carcinoma in situ testis; and/or

- invasive testis cancer; and/or

prostate cancer; and/or

Klinefelter syndrome; and/or

- a need of Androgen replacement therapy (ART); and/or

gonadal dysfunction including abnormal puberty or male infertility; the method comprising

• providing a first biological sample from a male subject, said sample being a fluidic sample, such as serum, plasma, and/or seminal fluid;

• providing a second biological sample from a male subject, said sample being a fluidic sample, such as serum, plasma, and/or seminal fluid;

wherein said second sample has been obtained from said subject at a later time point than the first sample.

• determining the level of LHCGR (LH/hCG receptor) in the first and the

second sample;

· comparing said determined levels of LHCGR in the samples;

• determining that o the condition has developed negatively for said subject, if said

determined level in the second sample is higher than in the first sample; or

o the condition has not developed for said subject, if said determined level in the second sample is equal to the level in the first sample: or o the condition has developed positively for said subject, if said determined level in the second sample is lower than in the first sample. Brief description of the figures

Figure 1

Figure 1 shows serum total LHR (LHCGR), LHR bound to LH (HCG-LHCGR) and the ratio between soluble and total LHR in men with normal health, infertile, and Klinefelter syndrome, mean + sem.

Figure 2

Figure 2 shows serum total LHR (LHCGR), LHR bound to LH (HCG-LHCGR) and the ratio between soluble and total LHR in men with normal health, infertile,

Klinefelter syndrome, and orichectomized due to previous testis cancer (hCG-test is a stimulation test with injection of human choriongonadotropin that stimulates LHR). mean + sem.

Figure 3

Figure 3 shows association between serum total LHR and A) serum LH, B) testosterone, C) sperm concentration, D) FSH, and E) estradiol.

Figure 4

Figure 4 shows serum levels of LHCGR in normal men, infertile men, HCG-test, and men with Klinefelter syndrome. Circle indicates serum LHCGR in men with tumors in the testicles.

Figure 5

Figure 5 shows serum LHR in men with normal health, infertile, Klinefelter syndrome, orichectomized due to previous testis cancer, current testis cancer and a man without testis who developed prostate cancer, mean + sem.

Figure 6

Figure 6 shows expression of LHR using two different antibodies targeting the extracellular domain of LHR in Leydig cells and testicular carcinoma in situ cells (CIS) residing in the abnormal tubules (Top). Below negative controls without primary antibodies.

Figure 7

Figure 7 shows expression of LHR using two different antibodies targeting the extracellular domain of LHR in invasive embryonal carcinoma cells, which have invaded the stromal compartment (Top). Below negative controls without primary antibodies.

Figure 8

Differential expression of LHR in prostate tissue.

A, IHC with two different antibodies against LHR. In the lower part the prostate glands are normal with well-differentiated glands with tall columnar epithelial lining cells and virtually no LHR expression. In the upper part, both LHR antibodies react with the more irregular glandular structures forming prostatic dysplasia with enlargement of nuclei, prominence of nucleoli, overlapping cells, and not classical tall and columnar epithelial cells.

B, Western analysis performed on prostate biopsies from 5 patients. Patients 1-3 have prostate cancer while patients 4-5 are healthy controls. A tissue sample from testis CIS was included as a positive control. Antibodies against full length LHR (exon 1-11, 75 KDa) and truncated LHR (exon 1 -6, 32 KDa) were used, β-actin was used as loading control.

Figures 9-10

Serum LHR and reproductive hormones in normal men. When stratifying the normal men in quartiles of serum LHR or in high versus low LHR there was a significant difference in serum Inhibin B/FSH (Fig. 9) ratio and estradiol levels (Fig. 10) (*p<0.05). data presented as means + sem.

Figure 11

Serum LHR in men with testis cancer. Men with relapse of cancer had significantly higher LHR at baseline prior to the initial surgery (A). Moreover serum LHR was associated with testicular histology (B), hCG levels and LHCGR (C), LDH levels above upper reference (+) and LHCGR levels in men with testis cancer and stratified in in non-seminomas and seminomas (D), alphafetoprotein in testis cancer (E) and Data presented as mean + sem. * p<0.05.

Figure 12

mRNA of LHR in various organs.

A) picture show all isoforms of LHR in all the investigated organs. Several isoforms exist on the RNA level in the normal testis, testicular cancer, prostate and cancer cell lines. B) An investigation of the truncated LHR RNA without the transmembrane domain. Figure 13

A) Western blot of LHR in human testis, cancer specimens and prostate and B) in testicular cancer cell lines. Lanes: 1) Normal testis, 2) CIS, 3) Seminom, 4) EC, 5) Normal prostate, and 6) Kidney.

Figure 14

Tumor volume in an NTera2 xenograft model following treatment with vehicle, hCG, LH and cisplatin. Data presented as Median tumor volume and sem.

The present invention will now be described in more detail in the following.

Detailed description of the invention

Method for predicting for a male subject a risk of having, developing or relapse of a condition

As described above, in an aspect the present invention relates to a method for predicting for a male subject a risk of having, developing or relapse of

- carcinoma in situ testis; and/or

invasive testis cancer; and/or

prostate cancer; and/or

Klinefelter syndrome; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction including abnormal puberty or male infertility; the method comprising

· determining the level of LHCGR (LH/hCG receptor) in the sample;

• comparing said determined level to a reference level; and

• determining that

As also described above, in a more preferred aspect the invention relates to a method for predicting for a male subject a risk of having, developing or relapse of

- testis cancer; and/or

prostate cancer; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction; the method comprising

• determining the level of LHCGR (LH/hCG receptor) in the sample;

• comparing said determined level to a reference level; and

• determining that

o said subject is at risk of having, developing or relapse of testis

cancer, and/or prostate cancer, and/or a need of androgen supplementation, and/or having gonadal dysfunction, if said level is higher than said reference level, or

o determining that said subject is not at risk of having, developing or relapse of testis cancer, and/or prostate cancer, and/or a need of androgen supplementation, and/or having gonadal dysfunction if said level is equal to or lower than said reference level.

In the present context the term "a risk of having, developing or relapse of

- testis cancer; and/or

- prostate cancer; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction"; is also to be understood as "a risk of having any of the conditions listed under a) - d), OR developing any of the conditions listed under a) - d) OR having a relapse of any of the conditions listed under a) - d) : wherein

a) testis cancer;

b) prostate cancer;

c) a need of Androgen replacement therapy (ART);

d) gonadal dysfunction;"

A similar understanding is to be understood through-out the document for the different disease indications.

LHCGR

The LH/hCG receptor (LHCGR), a G protein-coupled receptor has several synonyms. Thus, in the present context the terms "LHCGR", LHR, "LHreceptor" and "LH/hCG receptor" are used interchangeable and describe the same protein. Mammal

Reference to a "mammal", "subject" or a "individual" includes a human or non- human species including primates, livestock animals (e.g. sheep, cows, pigs, horses, donkey, goats), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs, hamsters) and companion animals (e.g. dogs, cats). The present invention has applicability, therefore, in human medicine as well as having livestock and veterinary and wild life applications. In a preferred embodiment, the mammal is a human. In a particular preferred embodiment, the mammal is a man/male.

Carcinoma in situ ( CIS) testis

In an embodiment, said method is for predicting a risk of having, developing, or relapse of carcinoma in situ testis. Carcinoma in situ (CIS) testis is the preinvasive form of testicular cancer with a gonocytes like appearance that develops into invasive testicular cancer (Skakkebaek 1972 Lancet). As shown in example 3 and figure 4, subjects have been identified having testis cancer and an increased level of LHCGR in serum.

Prostate cancer

In another embodiment, said method is for predicting a risk of having, developing, or relapse of prostate cancer. Prostate cancer, also known as carcinoma of the prostate, is the development of cancer in the prostate, a gland in the male reproductive system. As shown in example 4 and figure 5 subjects have been identified having prostate cancer and an increased level of LHCGR in serum.

In a further embodiment, said method is for predicting a risk of having Klinefelter Syndrome. Klinefelter syndrome or Klinefelter's syndrome, also known as 47,XXY or XXY, is the set of symptoms resulting from a genetic disorder in which there is at least one extra X chromosome to a standard human male karyotype, for a total of 47 chromosomes rather than the 46 found in genetically typical humans.

Genetic testing is a reliable test method but is also time consuming and

expensive. As shown in example 1 and e.g. figure 1 subjects have been identified having Klinefelter Syndrome and an increased level of LHCGR in serum.

Invasive testis cancer

In a further embodiment, said method is for predicting a risk of having,

developing, or relapse of testis cancer (such as invasive testis cancer), such as seminomas or non-seminomas. When tumour cells have invaded the extratubular compartment, it is considered an invasive testis cancer. Testicular germ cell tumours (TGCT) are the most frequent solid tumours in young men, and their embryonic stem cell-like phenotype separates them from somatic cancers. TGCTs originate from a pre-cursor cell, carcinoma in situ (CIS), which has been identified as transformed fetal gonocytes or primordial germ cells (see above). The non- invasive CIS cells undergo malignant transformation after puberty and form either an invasive seminoma, which retains germ cell characteristics, or the more aggressive non-seminoma that contains a de-differentiated component

(embryonal carcinoma EC) resembling embryonic stem cells (ESC) and more differentiated somatic (teratomas) or extra-embryonic (chorioncar-cinoma or yolk sac) components. As shown in example 3 and figure 4 subjects have been identified having testis cancer and an increased level of LHCGR in serum.

In an embodiment, said testis cancer is invasive testis cancer. In another embodiment, said invasive testis cancer is seminomas and/or non- seminomas.

In yet an embodiment (preferred) said method is for predicting a risk of having, developing or relapse of testis cancer. Developing

In the present context, it is to be understood that the term "developing" and indication according to the present invention may relate both to the development for the first time of the disease/state and/or the development of a relapse of the disease/state.

Androgen replacement therapy (ART).

In yet a further embodiment, said method is for predicting a risk of having a need of androgen replacement therapy (ART). Androgen replacement therapy (ART), often referred to as testosterone replacement therapy (TRT), is a class of hormone replacement therapy in which androgens, often testosterone, are replaced. ART is often prescribed to counter the effects of male hypogonadism. It typically involves the administration of testosterone, either by injection or by use of testosterone skin creams or gels.

As described above, high serum LHCGR levels may reduce the availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. This would therefore strongly indicate that this patient should be treated with testosterone because the stimulation of the gonadal testosterone production will be impaired by partly blocking the effect of LH. Thus, measured serum levels of LHCGR can be used as a marker of impaired Leydig cell function either alone or with improved sensitivity in combination with serum levels of testosterone, LH, INSL3 or testis size. Together with one or more of these factors, LHCGR will provide unique information about the Leydig cell population, which therefore can be used to diagnose selected patients and to monitor Leydig cell function in men prior to or under testosterone supplementation treatment.

Different types of androgen replacement therapy (ART) exists. Thus, in an embodiment said androgen replacement therapy is selected from the group consisting of testosterone replacement therapy, such as by supplementation of testosterone, testosterone derivatives having testosterone activity, such as Testosteronundecanoat. Commercial products are testoviron, Nebido, gel Tostra testogel and oral tablets Andriol. Thus, in an embodiment, said method is for predicting a risk of having, developing or relapse of a need of androgen replacement therapy (ART).

Gonadal dysfunction or gonadal dysgenesis

In an embodiment, said method is for predicting a risk of having, developing or relapse of gonadal dysfunction. Gonadal dysfunction or gonadal dysgenesis is any congenital developmental disorder of the reproductive system characterized by a progressive loss of germ cells on the developing gonads of an embryo. In male subjects, this condition may occur if there is an absence of both Mullerian inhibiting factor and testosterone. The absence of testosterone will result in regression of the Wolffian ducts; normal male internal reproductive tracts will not develop. The absence of Mullerian inhibiting factor will allow the Mullerian ducts to differentiate into the oviducts and uterus. In sum, this individual will possess female-like internal and external reproductive characteristics, lacking secondary sex characteristics. The genotype may be either 45, XO, 46, XX or 46, XY. As explained in example 2, high levels of LHCGR may be indicate of gonadal dysfunction and a relevant therapy may be androgen replacement therapy (ART). In yet an embodiment, said gonadal dysfunction is selected from the group consisting of impaired sperm out, low sperm motility, Sertoli cell only, Leydig cell hyperplasia, testis cancer, carcinoma in situ, and/or hyalinised testis.

Further biomarkers

In an embodiment, said method further includes determining the level of one or more of Testosterone (T), LH, T/LH ratio, Estradiol, FSH, AMH, Inhibin B and/or INSL3 in a sample from said subject. As explained in example 2, several advantages may exist by combining the method of the invention with analysis of further biomarkers. Preferably, the different levels are determined in the same sample to save cost and time. Thus, in an embodiment, said further determination is done on the same sample material.

Determination of biomarkers

A protein marker may be determined both at the RNA level and at the protein level. Thus, in an embodiment, said level is determined by measuring the level of mRNA and/or protein, preferably the protein level. Different techniques for making such determination also exists. Thus, in an embodiment said determination of the at least one protein level is performed using a method selected from the group consisting of immunohistochemistry, immunocytochemistry, FACS, ImageStream, Western Blotting, qPCR, RT-PCR, qRT-PCR, ELISA, Luminex, Multiplex, Immunoblotting, TRF-assays,

immunochromatographic lateral flow assays, Enzyme Multiplied Immunoassay Techniques, RAST test, Radioimmunoassays, immunofluorescence and

immunological dry stick assays. In one preferred embodiment, said determination is performed by immunocytochemistry. In another preferred embodiment, said determination is performed by ELISA.

Immunohistochemistry

Immunohistochemistry refers to the process of localizing antigens e.g. proteins in cells of a sample exploiting the principle of antibodies binding specifically to antigens in biological samples. There are two strategies used for the

immunohistochemical detection of antigens, the direct method and the indirect method. In both cases, many antigens also need an additional step for

unmasking, which often makes the difference between staining and no staining. Unlike immunocytochemistry, the tissue does not need to be permeabilized because this has already been accomplished by the microtome blade during sample preparation. Detergents like Triton X-100 are generally used in

immunohistochemistry to reduce surface tension, allowing less reagent to be used to achieve better and more even coverage of the sample. In another presently preferred embodiment, the invention relates to a method where the determination is performed by immunocytochemistry.

Immunocytochemistry

Immunocytochemistry refers to the process of localizing antigens e.g. proteins in cells collected in a suspension/fluid exploiting the principle of antibodies binding specifically to antigens. The procedure depends on the type and amount of cells in question, but usually the cells in the suspension/fluid is fixed to a microscope glass and subsequently treated as immunohistochemical slides. Special systems may be used to transfer the cells but not the fluid to a hard support material that can be analysed. Special reagents, which facilitate permeabilization may be applied.

ELISA

Immunoassays, in their most simple and direct sense, are binding assays.

Antibody binding to for instance LHCGR can be detected by any immunoassay means known in the art. Preferably, antibody binding is detected by an assay selected from the group consisting of protein microarray assay,

radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluoroimmunoassay, immunofluorometric assay, and immunoradiometric assay. Most preferably, antibody binding is detected by ELISA. Thus a preferred embodiment relates to a method wherein the determination of the expression of the at least one protein is performed by ELISA. Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step, a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.

Orchiectomiza tion

In an embodiment, said subject has one or both testicles removed, e.g. due to testicular cancer. Since invasive testis cancer may show up after an

orchiectomization, it may be relevant to predicting a risk for a subject having one or both testicles removed for invasive testis cancer.

Truncated versions of LHCGR

As previously mentioned it may be relevant to determine the levels of truncated versions of LHCGR lacking the transmembrane domain (see also example section). Thus, in an embodiment, the LHCGR (LH/hCG receptor) is a truncated version lacking the transmembrane domain. In a more specific embodiment,

said transmembrane domain is encoded by exon 11 of the LHCGR gene. Without being bound by theory, the lack of the transmembrane domain may be due to alternative splicing events or post-translational modifications.

Preferred method

In a preferred aspect of the invention, the invention relates to a method for predicting for a male subject a risk of having, developing or relapse of

- carcinoma in situ testis; and/or

invasive testis cancer; and/or

prostate cancer; and/or

Klinefelter syndrome; and/or

- a need of Androgen replacement therapy (ART); and/or

gonadal dysfunction including abnormal puberty or male infertility; said method comprising the steps of;

a) providing a biological sample from a male mammal said sample being a fluidic sample, such as serum, plasma, seminal fluid;

b) adding some phosphate buffered saline to the sample;

c) spinning down the sample on a slide;

d) optionally incubating the slide at 4 °C for 1-1000 days;

e) determining the level of LHCGR (LH/hCG receptor) in the sample;

f) comparing said determined level to a reference level; and

g) determining that said subject is at risk of having, developing or relapse of carcinoma in situ testis, and/or, invasive testis cancer, and/or prostate cancer, and/or Klinefelter Syndrome, and/or is in need of androgen supplementation, and/or having gonadal dysfunction if said level is higher than said reference level, or determining that said subject is not at risk of having, developing or relapse of carcinoma in situ testis, and/or, invasive testis cancer, and/or prostate cancer, and/or Klinefelter Syndrome, and/or is in need of androgen supplementation, and/or having gonadal dysfunction if said level is equal to or lower than said reference level. It is to be understood that embodiments of each aspects may also find use in other aspects of the present invention.

Treatment protocols

When/if an increased risk for a condition is determined, further analysis may be required. Thus, in an embodiment, an indication of an increased risk, further comprises referring said subject to further analysis for determining the presence of one or more of the indicated conditions. Conditions may be carcinoma in situ testis, and/or invasive testis cancer, and/or prostate cancer, and/or Klinefelter Syndrome, and/or need of androgen supplementation, and/or having gonadal dysfunction. The further analysis may lead to the initiation of a treatment protocol. Thus, in an embodiment, said further analysis may lead to initiation of a treatment protocol in the form of surgery, such as testicle removal,

chemotherapy, radiation therapy, androgen replacement therapy (ART), with the proviso that the indication is carcinoma in situ testis; and/or invasive testis cancer; and/or prostate cancer; and/or wherein said further analysis may lead to initiation of a treatment protocol in the form of hormone treatment, such as Androgen replacement therapy (ART); and or chromosomal analysis, with the proviso that the indication is Klinefelter

syndrome; and/or a need of androgen replacement therapy (ART) and/or gonadal dysfunction.

Monitoring a condition

The method of the invention may also be used to monitor or follow the

development of an identified condition. Thus, yet an aspect of the invention relates to a method for monitoring a condition in a male subject previously determined of having, a risk of developing or relapse a condition selected from

- carcinoma in situ testis; and/or

- invasive testis cancer; and/or

prostate cancer; and/or

Klinefelter syndrome; and/or

- a need of Androgen replacement therapy (ART); and/or

• providing a second biological sample from a male subject, said sample

being a fluidic sample, such as serum, plasma, and/or seminal fluid;

• determining the level of LHCGR (LH/hCG receptor) in the first and the

second sample;

· comparing said determined levels of LHCGR in the samples;

determined level in the second sample is higher than in the first sample; or

o the condition has not developed for said subject, if said determined level in the second sample is equal to the level in the first sample: or

o the condition has developed positively for said subject, if said determined level in the second sample is lower than in the first sample.

By having samples obtained at different time points it is thus possible to follow the development of a condition. Such development may e.g. development due to an initiated treatment protocol. Thus, in an embodiment a treatment regime has been performed between the sampling of the two samples. In yet an embodiment, the treatment protocol is surgery, such as testicle removal, chemotherapy, radiation therapy, androgen replacement therapy (ART), with the proviso that the condition is carcinoma in situ testis; and/or invasive testis cancer; and/or prostate cancer; and/or wherein the treatment protocol is hormone treatment, such as Androgen replacement therapy (ART); with the proviso that the condition is Klinefelter syndrome; and/or a need of Androgen replacement therapy (ART) and/or gonadal dysfunction.

Since comparisons between identical sample types are the most reliable, preferably the samples are of the same type. Thus, in yet a further embodiment, the first sample and the second sample are from the same sample types, such as blood serum.

Further Definitions

Relapse

In the present context the term "relapse" relate to malignant disease, which was initially cured, but after a certain time comes back and requires treatment.

Expression level

In one embodiment of the present invention, the term "expression level" or "level" is to be understood as the relative amount of in a sample. In another embodiment of the present invention, the level is to be understood as absolute number in a sample.

The presence or level of the biomarker (such as LHCGR) may be determined at the level of the molecule itself or to the extent to which a gene is expressed. The level is measured by conventional analytical methods, such as immunological methods known to the art. Measurements can be performed at RNA or protein level in accordance with the teachings herein.

Reference level

In the context of the present invention, the term "reference level" relates to a standard in relation to quantity which other values or characteristics can be compared, such as e.g. a standard curve.

As stated above, detection may be made at the protein (or nucleic acid) levels. Consequently, reference to presence or level includes direct and indirect data.

To determine a reference level for an ELISA test the expression level of the protein in samples may be determined from patients who already have been investigated for the expression of e.g. LHCGR. Based on these results a standard curve may be obtained that shows the relationship between the protein level detected by ELISA and persons at risk. The standard curve can thereby be used to determine the amount of protein, which corresponds to for instance an increased risk of seminomas. The same approach for determining a reference level applies to all the proteins, which may be combined according to the present invention. The same approach may be applied for other techniques as known in the art.

In one preferred embodiment of the present invention, the reference means is an internal reference means and/or an external reference means.

In the present context the term "internal reference means" relates to a reference which is not handled by the user directly for each determination, but which is incorporated into a device for the determination of the concentration of the marker in question, whereby only the ^" final result^" or the ^" final measurement^" is presented. The terms the "final result" or the "final measurement" relate to the result presented to the user when the reference value has been taken into account. In a further embodiment of the present invention, the internal reference means is provided in connection to a device used for the determination of the concentration of the at least one marker in question.

In yet an embodiment of the present invention, the device is selected from the group consisting of an assay, a stick, a dry-stick, an electrical device, an electrode, a reader (spectrophotometric readers, IR-readers, isotopic readers and similar readers), histochemistry, and similar means incorporating a reference.

In the present context, the term "external reference means" relates to a reference which is handled directly by the user in order to determine the concentration of the at least one marker in question, before obtaining the ^" final result^" or the ^" final measurement^" .

In yet a further embodiment of the present invention, external reference means are selected from the group consisting of a table, a diagram and similar reference means where the user can compare the measured signal to the selected reference means. The external reference means relates to a reference used as a calibration, value reference, information object, etc. for the marker in question and which has been excluded from the device used.

One embodiment of the present invention relates to a method, wherein said reference level/predetermined value is indicative of a normal individual (not at risk). Although any of the known analytical methods for measuring the level of the marker in question will function in the present invention, as obvious to one skilled in the art, the analytical method used for said protein must be the same method used to generate the reference data for said protein. If a new analytical method is used for the marker in question, a new set of reference data, based on data developed with the method, must be generated. Thus, the technique utilized to analyse the sample should preferably be the same for the reference data and the samples to be screened.

Risk Assessment

The present inventors have successfully developed a new method to predict the risk for different conditions in a male mammal. The results presented in the examples shows that LHCGR appears to be an efficient marker for monitoring and diagnostic usage in a male mammal. The discrimination is considered better than with conventional analysis. To determine whether the subject has an increased risk according to the invention, a cut-off must be established. This cut-off may be established by the laboratory, the physician or on a case-by-case basis by each subject. The cut-off level could be established using a number of methods, including : percentiles, mean plus or minus standard deviation(s); multiples of median value; patient specific risk or other methods known to those who are skilled in the art.

The multivariate discriminant analysis and other risk assessments can be performed on the commercially available computer program statistical package Statistical Analysis system (manufactured and sold by SAS Institute Inc.) or by other methods of multivariate statistical analysis or other statistical software packages or screening software known to those skilled in the art.

As obvious to one skilled in the art, in any of the embodiments discussed above, changing the risk cut-off level of a positive or using different a priori risks, which may apply to different subgroups in the population, could change the results of the discriminant analysis for each patient.

When expression levels of a specific protein in a sample are compared to a reference level, they can either be different (above or below the reference value) or equal. However, using today's detection techniques is an exact definition of different or equal result can be difficult because of noise and variations in obtained expression levels from different samples. Hence, the usual method for evaluating whether two or more expression levels are different or equal involves statistics.

Statistics enables evaluation of significantly different expression levels and significantly equal expressions levels. Statistical methods involve applying a function/statistical algorithm to a set of data. Statistical theory defines a statistic as a function of a sample where the function itself is independent of the sample's distribution: the term is used both for the function and for the value of the function on a given sample. Commonly used statistical tests or methods applied to a data set include t-test, f-test or even more advanced test and methods of comparing data. Using such a test or methods enables a conclusion of whether two or more samples are significantly different or significantly equal.

The significance may be determined by the standard statistical methodology known by the person skilled in the art. The chosen reference level may be changed depending on the mammal for which the test is applied. The chosen reference level may also be changed if desiring a different specificity or sensitivity.

Sensitivity

As used herein, the sensitivity refers to the measures of the proportion of actual positives, which are correctly identified as such - in analogy with a diagnostic test, i.e. the percentage of mammals or people having a fertility potential below normal who are identified as having a fertility potential below normal.

Usually the sensitivity of a test can be described as the proportion of true positives of the total number with the target disorder i.e. a fertility potential below normal. All patients with the target disorder are the sum of (detected) true positives (TP) and (undetected) false negatives (FN).

Specificity

As used herein the specificity refers to measures of the proportion of negatives, which are correctly identified - The ideal diagnostic test is a test that has 100 % specificity, i.e. only indicates mammal with a condition according to the present invention and therefore no false positive results.

For any test, there is usually a trade-off between each measure. For example in a manufacturing setting in which one is testing for faults, one may be willing to risk discarding functioning components (low specificity), in order to increase the chance of identifying nearly all faulty components (high sensitivity). This trade-off can be represented graphically using a ROC curve.

Selecting a sensitivity and specificity, make it is possible to obtain the optimal outcome in a detection method. In determining the discriminating value distinguishing mammals having a fertility potential below normal, the person skilled in the art has to predetermine the level of specificity. The ideal diagnostic test is a test that has 100% specificity, i.e. only detects mammals with a condition according to the present invention and therefore no false positive results, and 100% sensitivity, and i.e. detects all mammals with a condition according to the present invention and therefore no false negative results. However, due to biological diversity no method can be expected to have 100% sensitive without including a substantial number of false negative results. The chosen specificity determines the percentage of false positive cases that can be accepted in a given study/population and by a given institution. By decreasing specificity, an increase in sensitivity is achieved. One example is a specificity of 95% that will result in a 5% rate of false positive cases. As will be generally understood by those skilled in the art, methods for screening are processes of decision-making and therefore the chosen specificity and sensitivity depends on what is considered the optimal outcome by a given institution/clinical personnel.

In a preferred embodiment, the invention relates to a method with a high specificity, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% such as at least 95% such as 100%. In another preferred embodiment, the invention relates to a method with a high sensitivity, such as at least 60% such as at least 70% such as at least 80% such as at least 90% such as 100%.

The cut-off level could be established using a number of methods, including :

percentiles; mean plus or minus standard deviation(s); multiples of median value; patient specific risk or other methods known to those who are skilled in the art.

Another aspect of the invention relates to a method wherein the ratio between at least two markers is used to predict whether a male mammal has a condition according to the present invention.

In a most preferred embodiment, the levels are determined at the protein level.

Ligands to the proteins are particularly useful in detecting and/or quantifying these proteins. Antibodies to the proteins are particularly useful. Techniques for the methods contemplated herein, are known in the art and include sandwich assays, xMAP multiplexing, Luminex, ELISA and ELISpot. Reference to antibodies includes parts of antibodies, mammalinized (e.g. humanized) antibodies, recombinant or synthetic antibodies and hybrid and single chain antibodies.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the immune signalling molecules or antigenic fragments thereof and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well-known in the art.

Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the immune signalling molecule, or antigenic part thereof, collecting serum or plasma from the animal and isolating specific sera by any of the known immuno-adsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques, which are well known to those who are skilled in the art.

Several techniques are known to the skilled addressee for determination of biological markers such as LHCGR.

In a presently preferred embodiment, the invention relates to a method where the determination is performed by immunohistochemistry.

Examples

Material and methods

Cross-sectional association study of young healthy men

From an ongoing monitoring study of semen quality of young men from the general Danish population (J0rgensen et al., 2010A; J0rgensen et al., 2010B), 10 men participating were retrospectively investigated for an association between serum LHCGR and semen quality. The men delivered one semen sample, one blood sample (stored at - 20°C until analysis), had a physical examination performed and answered a comprehensive questionnaire. The questionnaire included information on age, previous or current diseases, any known history of fertility, medication, etc.

Men with Klinefelters syndrome (KS)

Five patients with KS confirmed with karyotype was used in this study. They were aged 5-55 years and were followed in our endocrine outpatient clinic.

Chromosome analysis was performed on peripheral blood lymphocytes in each case, Karyotypes were established on 30 metaphases from each patient. One was XX-males which also was SRY-positive (detected by polymerase chain reaction). All Klinefelter subjects and their parents (when the patient was < 18 years of age) gave informed consent for clinical and biochemical follow-up. Data from routine clinical visits were obtained from patient files and used for this study. Registration of clinical data was approved by the Danish Data Protection Agency (2005-41- 5479).

Men referred for androloqical work due to male infertility

15 subfertile men from the andrology clinic were included. All included men underwent a physical examination including ultrasound of the testicles, had a blood sample drawn and produced two semen samples by masturbation. Two men had a tumor suspect lesion following ultrasound of the testicles and were later orchiectomized.

After analyses of LHCGR the pathology report concerning the testis specimens and the patient had a bilateral seminoma and a Sertoli cell tumor were retrieved, respectively. Men referred for androloqical work after orchiectomy due to testis cancer

Five men who had one testis removed due to testis cancer were included. All included men underwent a physical examination including ultrasound of the testicles and had a blood sample drawn. All these men underwent human choriongonadotropin stimulation test to evaluate their residual capacity in their Leydig cells. We analyzed serum LHCGR prior to and 72 hours after hCG stimulation. Interestingly, one of these men with total anorchia (no testis) developed an invasive prostate cancer.

Men referred for semen banking due to testis cancer prior to orchiectomy

272 men who had one testis removed due to testis cancer were included. All included men underwent a physical examination including ultrasound of the testicles and had a blood sample drawn. The men were followed for 5 year in the oncology department and the histological tumor subtype, size, levels of biomarkers and relapse within 5 years were determined. It should be noted that testis cancer patients today are monitored differently depending on tumor histology. Non-seminomas release markers such as alfa-fetoprotein and hCG, which are important for determining relapse. At baseline are serum levels of these markers normally not prognostic for relapse. Seminomas produces no markers and monitoring relapse is thus difficult.

LHCGR ELISA

A method for determining LHCGR by ELISA is described in "Quantitative ELISAs for serum soluble LHCGR and hCG-LHCGR complex: potential diagnostics in first trimester pregnancy screening for stillbirth, Down's syndrome, preterm delivery and preeclampsia, Reprod Biol Endocrinol. 2012 Dec 17;10:113".

Assessment of LHCGR, reproductive hormones and other biochemical variables Serum LHCGR was assessed using ELISA. The intra- variation for measurements of LHCGR was 1.9%. Serum FSH levels were determined using a time-resolved immunofluorometric assay (Delfia; Wallac, Turku, Finland) and Inhibin-B by a specific two-sided enzyme linked immunoassay (Bio-Innovation Ltd, Oxford, UK). The intra- and inter-assay variation for measurements of FSH and Inhibin B were 3% and 4.5%, 15% and 18% respectively. Semen analysis

The participants produced a semen sample by masturbation. Self-reported information of duration of ejaculation abstinence was obtained. Trained

technicians conducted semen analysis and a detailed description of assessment of semen samples has previously been published. Semen volume was estimated by weighing. For sperm motility assessment, duplicates of 10 μΙ of well-mixed semen were placed on a glass slide, examined on a heating stage kept at 37 °C, under a microscope at x 400 magnification, and spermatozoa were classified as

progressive motile (WHO class A+B), non-progressive motile (class C) or immotile (class D). The average of the two motility assessments was used. For the assessment of the sperm concentration, the samples were diluted in a solution of 0.6 mol/l NaHC03 and 0.4% (v/v) formaldehyde in distilled water, and

subsequently assessed using Burker-Turk haemocytometer. Only spermatozoa with tails were counted. Finally, smears were prepared, Papanicolaou stained and spermatozoa morphology assessed according to strict criteria (Menkveld et al., 1990).

Statistics

For a basic description mean and sem were used. Between-group differences were tested by t-test. Correlations between LHCGR and both semen variables and reproductive hormone levels were examined using the non-parametric Spearman's rank test. To test for the effect of LHCGR, regression analyses were also performed. In all regression models, semen volume, sperm concentration, total sperm count, LH, testosterone, FSH and Inhibin-B were natural logarithm transformed and motility variables logit transformed before analysis to obtain normal distribution of the residuals. The following co-variates were tested :

duration of ejaculation abstinence, hour-of-day of blood sampling, smoking, body- mass-index (BMI), age, medication, fever, time from ejaculation to motility assessment, morphology observer, history of cryptorchidism, season, albumin- corrected calcium, For all analyses, p<0.05 was considered statistically significant. Analyses were performed using PASW GradPack 18.0 (SPSS Inc., Chicago, IL, USA). Disease model

Tumors were established via subcutaneous inoculation of NTERA2 testis cancer cells into the right flank and the left flank of the mice. Cells were cultured in vitro as follows:

NTERA2 cells:

DMEM with 100 u/ml penicillin, 0.1 mg/ml streptomycin, foetal bovine serum (10%) and 5 ml L-glutamin (Gibco # 25030-024).

Procedure used for preparation of cell for inoculations

1. Wash the cell layer twice with 10 ml PBS.

2. Wash the cell layer once with 5 ml of TrypleSelect solution.

3. Add 3 ml of TrypleSelect solution and incubate the cells for 2-5 min at

37°C, checking regularly. As cells begin to detach, dislodge the remaining cells by gently hitting the flask (or let it detach almost completely, since the hitting can facilitate aggregating of cells).

4. Once cells have detached from the flask, resuspend trypsin/cell

suspension with 5 ml pipette to break up any cell aggregates formed. 5. Add 9.0 ml DMEM, 10% FCS and pipette to obtain a single-cell suspension.

6. Optional : If necessary run the cell suspension through a Falcon filter with a 70 μηι pore size (according to pilot-experiments, this will not be necessary).

7. Centrifuge the cell suspension at 900 rpm for 4 min.

8. Carefully resuspend the cells in 5 ml PBS with a 5 ml pipette and then add another 15 ml PBS.

9. Centrifuge the cell suspension at 900 rpm for 4 min.

10. In the following points the cells should be re-suspended and pooled in

PBS to be sure to have at least the concentration required in point 13.

11. Count the cells by making 10-fold or 20-fold dilutions; 50 μΙ trypan blue and 50 μΙ cell suspension.

12. Aliqoutes, each enough for injection of 5 mice, are prepared from the pool/batch of cells. The aliquots will be placed on ice in the time before injection.

13. Dilute the solution in PBS in order to reach a concentration of 4xl0⁷ cells/ml, mix 1 : 1 with matrigel(*) to have 2xl0⁷ cells/ml for SC injection of 2xl0⁶ in 0.1 ml.

14. Take care that the cells are resuspended before they are drawn into the syringe and the mixture is kept cold throughout the procedure. 0.1 ml cell/matrigel suspension will be injected per mouse. Draw suspension into the syringe for 5 animals. The solution will be swirled carefully before each volume is drawn into the syringe. The 5 mice will be dosed in one continues routine.

*) Matrigehcell suspension (1 : 1). Matrigel will be purchased from BD biosciences.

Procedure used for inoculation of tumor cells at the flanks of the mouse:

The needle is inserted in the cranial direction in the entire length of the needle 16 mm (25G) and cell suspension is injected and after some seconds the needle will be retracted. Care is taking that the needle is not twisted, pointed upward to skin and the skin should not be stretched during the procedure. This is to ensure a narrow injection channel, to avoid intradermal injection and damage of skin from the inside. The inoculum is placed at the flank of the mouse (in this protocol, both in the right and left flank), which is at the middle of the right side of the mouse free from the abdominal and thoracic mammary fat pads and just as far in cranial direction that the rib bones will serve as back-support for the tumor during the measurements of tumor dimensions.

Example 1

Expression of different LHCGR isoforms in various organs from normal men and men with various diseases including cancer. We investigated LHR expression on mRNA and protein level in multiple organs. On the RNA level, at least one LHR isoform was expressed in many tissues. Several isoforms exist on the RNA level in the normal testis, testicular cancer, prostate and cancer cell lines. However, an investigation of the truncated LHR RNA without the transmembrane domain showed that this isoform is exclusively expressed in testis specimens, seminoma cells, ovary, epididymis and cell lines from seminoma. The RNA data indicate that It may be a novel marker of seminoma tumors (figure 12). On the protein level, IHC (figure 8) and WB (Figure 13) confirmed the presence of multiple LHR isoforms in testis, while the testicular tumors CIS seminoma and embryonal carcinoma (EC) only have the truncated isoform on protein level. Normal prostate and kidney do not express normal LHR. Right panel shows expression of both isoform in testicular germ cell cancer cell lines (NT@, 2102EP) as in NT supporting the presence of LHR in testis cancer.

Conclusion

We are the first to show that the truncated LHreceptor isoform is found not only in normal testis, but also in testicular and prostate cancer cells. This finding implies that LHreceptor may be released from these cells as the transmembrane domain is not existing and presence of this isoform in fluids may be used as a diagnostic or prognostic marker of these diseases. Moreover, the truncated LHreceptor has another signaling ability compared with the full-length receptor and presence of the truncated LHreceptor isoform in these cancers indicates a novel unidentified role of hCG and LH in these cancer cells. Especially since the truncated LHR receptor signals differently than the full-length receptor.

Example 2

Serum levels of LHCGR in normal men and men with Klinefelters syndrome (KS) Serum levels of LHCGR in normal men, infertile men and men with Klinefelters syndrome (KS) were measured. From figure 1, it is evident that men with Klinefelters syndrome have a significantly higher mean serum level compared with both normal and infertile men. Interestingly, all Klinefelter patients independent of age (one of them is a child under 10 years) have a high serum level of LHCGR, which indicates that a high serum level of LHCGR may be associated with an increased risk of having Klinefelter.

The high serum LHCGR in Klinefelter patients could be due to high serum LH levels in these patients. To determine this, we compared serum levels of LHCGR with a group of men who had one testis removed due to testicular cancer. These patients have a very high serum LH and as shown in the last group in Figure 2 (hCG-test), serum LHCGR levels are low and comparable with the normal and infertile men.

Conclusion

The above results indicate that serum LHCGR can be used as a predictive marker in diagnosing and potentially monitoring Klinefelter patients by a simple test.

Example 3

Combination of markers

It is believed that combining the level of serum LHCGR with other markers the predictive value may be increased.

Setup

Levels of testosterone, LH, FSH, Inhibin B were measured together with LHCGR in normal and Klinefelter patients.

Results

From figure 3A-E it can be seen that LHCGR is not directly influenced by serum levels of gonadotropins and sex hormones. This is important, because it indicates that serum LHCGR provides novel information, which combined with one or more of the traditional marker of gonadal function may improve monitoring of men in need of testosterone supplementation. The presumed effect combined with INSL3 may be higher, because INSL3 is exclusively produced in mature Leydig Cells and is thus inversely proportional to serum LHCGR. Conclusion

From this example, it can be seen the difference between Klinefelter patients and normal men can be increased by including e.g. ratios between LHCGR and testicular hormones. Thus, the diagnostic value becomes stronger compared with LHCGR alone. On the other hand it obvious that none of the hormones can predict the serum level of LHCGR. This indicates that this marker alone or in combination with traditional markers for gonadal dysfunction may assist in diagnose and monitoring of men in need of testosterone supplementation. Moreover, it is indicated to use INSL3 in combination with LHCGR, because INSL3 is exclusively produced in mature Leydig Cells and is thus inversely proportional to serum LHCGR and may therefore provide better data.

Example 4

LHCGR and testicular cancer

When evaluating some of the above individual values, it became clear that two men in the infertile group had a serum value of LHCGR, which were comparable to the serum levels found in men with Klinefelters syndrome (see figure 4, circle).

These two patients were referred to closer examination. Both of them were referred for andrological work up due to male infertility. During the clinical evaluation, both surprisingly presented with a tumor suspect lesion in the testis as visualized by ultrasound. They were therefore referred for surgical evaluation and were subsequently orchiectomized. One of them had an invasive seminoma while the other had a Sertoli cell tumor. These results indicate that serum levels of LHCGR, besides diagnosing impaired Leydig cell function in Klinefelter, also is elevated when you have invasive testicular cancer such as seminoma besides Sertoli cell tumors, which mainly are benign. All these diseases is part of what is referred as testicular dysgenesis syndrome (TDS). We suggest that serum LHCGR can be used as marker of the severe forms of TDS. This was indeed supported when we investigated expression of LHCGR in Carcinoma in situ and invasive testicular cancers. We found a marked expression of these proteins indicating that the cells express LHCGR, which enables us to measure elevated levels in serum of LHCGR. Conclusion High serum levels in two testicular tumors were detected indicating that LHCGR may be a marker for specific types of testicular cancer, such as seminoma.

Example 5

LHCGR and testicular cancer

When evaluating the 272 men with testis cancer who had serum LHR measured prior to orchiectomy, it became clear that serum LHR has important prognostic value. In men with TGCTs we have found that LHR is associated with an increased risk of relapse (figure 11A, presence of non-seminoma (Figure 11B) and higher LDH levels in blood. It will therefore be a useful prognostic marker and an important marker for biochemical relapse, which can be used to determine who should be extra-ordinary CT-scanned or at best to determine whether

chemotherapy should be initiated to cure advanced disease. This finding is important and can be used clinically, because serum levels of LHRR in normal men are remarkable stable over time. Therefore we will be able to detect minor changes in serum level of LHCGR in men treated testis cancer and compare the new levels with their values before and after surgery to identify potential relapse of the malignant disease. Moreover, only LHR was able to predict relapse. We also found associations between LHR and alpha fetoprotein in non-seminomas indicating that the histological phenotype is important for LHR production. While in seminomas it appears that size and LDH production is most important. When we can use serum levels of LHR to identify men with relapse, non-seminoma etc. then we are also confident that we can use the concentration of LHR as a marker of CIS, TGCTs, and prostate cancer in seminal plasma because here we have no disturbance from other organs and the concentration is most likely higher.

Western blot analysis performed on testicular patients with CIS, seminoma and non-seminoma including cancer cell lines confirmed the presence of LHR in testis cancer cells. Using different LHR antibodies, we were able to demonstrate that more than one isoform of LHR protein was detectable and the quantity and isoforms varied according to the histological subtype. Therefore, the assay for detection LHR can be specified for the different histological subtypes.

Conclusion

We have found that different subtypes of LHR are expressed in invasive testis cancer cells. In men with invasive testis tumors we have found that LHR is associated with an increased risk of relapse, presence of non-seminoma and specific phenotypes of these and indicative for higher LDH levels and seminoma with higher tumor burden. It will therefore be a useful prognostic marker and an important marker for biochemical relapse, which can be used to determine who should be extra-ordinary CT-scanned or at best to determine whether

chemotherapy should be initiated to cure advanced disease. Noteworthy, in non- seminomas LHR was associated with histological subtype and not tumor burden. This indicates that it to some extend follows alfa-fetoprotein. However, in seminomas LHR levels were dependent of tumor burden and LDH levels, which indicates that most seminoma cells produce and release LHR. This finding is intriguing as an increase in LHR in serum may be an indicator of seminoma relapse. Especially since LHR was not dependent on hCG levels and thereby provides information which cannot be obtained through other sources as LDH can increase from multiple reasons. So it may not be a prognostic marker in seminomas at baseline prior to surgery as it is for non-seminomas but instead be used to monitor relapse in seminomas and in non-seminomas.

Example 6

LHCGR and prostate cancer

When evaluating some of the above individual values, it became clear that one man without any testicles had highly elevated serum LHCGR compared with all other groups including men with Klinefelters syndrome (see figure 5). We took a closer look on this patient because it was not anticipated that he should have any measurable LHCGR with no testis. During the clinical and biochemical evaluation, it was evident that he had elevated PSA levels (a diagnostic marker for prostate cancer). His PSA levels remained elevated and a biopsy revealed an invasive prostate cancer, which was removed.

The above data was further supported, when we investigated expression of LHCGR in normal and malignant prostate cancer tissue. We found a marked expression of LHCGR in dysplatic and malignant prostate but not normal cells (Figure 8A) indicating that the cancer cells express LHCGR, which enables us to measure elevated levels in serum of LHCGR in prostate cancer patients. Western blot analysis were performed on prostate biopsies from 5 patients (Figure 8B). Patients 1-3 have prostate cancer while patients 4-5 are healthy controls. Using different LHR antibodies, we were able to demonstrate that only the truncated LHR protein is detectable in prostate cancer, which may explain the high serum levels in men with prostate cancer

Conclusion

These descriptive studies clearly show that high blood (serum) levels of LHCGR may be used for diagnosis and bio-monitoring of prostate cancer patients while following these patients in the andrology or oncology department. Thus, serum LHCGR can be used as predictive marker for prostate cancer. Especially the truncated version of LHCGR lacking the transmembrane domain may be a potential marker.

Conclusion

In men with testis cancer we have found that LHR is associated with an increased risk of relapse, presence of non-seminoma and higher LDH levels in blood. It will therefore be a useful prognostic marker and an important marker for biochemical relapse, which can be used to determine who should be extra-ordinary scanned or at best to determine whether chemotherapy should be initiated to cure advanced disease. Example 7

LHCGR and testicular function in normal men and men with testicular problems

We also measured serum LH in a large group of normal young men because another important aspect of measuring serum LHR is to investigate the

physiological role of serum LHR. It is possible that high serum LHR levels reduce the availability of LH for the hormone's cognate and membrane bound LHR, leading to lack of hormone utilization. This would strongly indicate that such patients should be treated with testosterone because the normal LH stimulation of the gonadal testosterone production will be impaired by reduced level of free LH. In normal men, we could see that men with high circulating LHR have an impaired testicular function as illustrated by lower inhibin B and Inhibin B/FSH ratio (Figure 9). Lower Inhibin B/FSH ratio is a fundamental characteristic of male infertility and LHR may thus assist in determining the severity of male infertility. During puberty Inhibin B/FSH ratio changes dramatically and LHR may be a novel marker for puberty. Interestingly, LHR also is associated with serum estradiol (figure 10). The same link may also be consistent in men with testis cancer. Moreover, We have found that men treated for testis cancer undergoing a hCG stimulation test, which is used to evaluate testosterone producing capacity, have significant decrease in both total and albumin corrected calcium within 72 hours following injection of 5000 IU hCG (Figure 5). Interestingly, 10% of hCG-treated men had a marked drop in serum calcium, which indicates that either serum LHR or expression level of LHR in organs relevant for calcium absorption (intestine), regulation (Parathyroid), storage (Bone) and excretion (Kidney) will be important. Presence of LHR in one of these organs will indicate that it is a novel target organ and circulating levels of LHR may be a marker of this.

Example 8

Stimulation of LHCGR and growth of testicular tumors in vivo

In the present study Embryonic cancer cells (NTERA2 cells) were cultured and cells were inoculated in two locations, the left and the right flank of

immunodeficient NMRI nude male mice. 48 mice were inoculated with 2xl0⁶ cells (with matrigel) to have 4 groups with tumors of 10 mice that was included for each treatment. When the mice have established tumors at study day 16 and the average tumor volume was approximately 150 mm³, they were randomized by cages with the aim to have a homogeneous distribution of the animals according to tumor size. Treatment of the animals was started at study day 17. Groups were as follows: LH (3 IE/kg, 5 time/week, i.p.), hCG (1000 IE/kg, 3 times/week, i.p.), Cisplatin (6 mg/kg, one dose, i.p), and one group with vehicle (3 times/week). The mice were treated for 11 days with the different bone drugs. The animals were terminated when the tumors have reached the size of the human end point, i.e. 864 mm³, or at termination of the study. Tumor volume was measured 3 times a week and body weight was measured once a week from the time of inoculation until the termination. At the termination, serum was prepared and the tumors, kidney, epididymis, testis and long bones (femur) was weighed. Growth curve presenting the development of the group mean of the tumor volumes are presented to indicate the efficacy of inhibition/stimulation of tumor growth. In the group treated with cisplatin, the tumor growth were clearly inhibited (Figure 14). For LH the tumor growth was similar to the vehicle group, however hCG treated mice showed a tendency towards growth stimulation. Median tumor burden was significantly different between Lh and hCG treated mice indicating a differential action of these agonist on testiscancer cells. This finding is of great interest since hCG and LH apparently do not have similar actions on these testicular cancer cells. Conclusion

LH and hCG stimulation in mice with testis cancer do not mediate the same effect on tumor growth. hCG stimulates tumor growth, while LH acts like vehicle. This indicates that the presence of a truncated LHR in cancer cells may have clinical value because hCG and Lh do not mediate the same effect and it is known that hCG is produced in many testis cancers besides some prostate cancers. This study shows that the presence of LHR in cancer cells has clinical relevance due to the growth regulatory response indicated by increased tumor volume in hCG treated mice compared with vehicle or LH treated mice. Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is set out by the accompanying claim set. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

Claims

1. A method for predicting for a male subject a risk of having, developing or relapse of

- testis cancer; and/or

- prostate cancer; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction; the method comprising

· providing a biological sample from a male subject, said sample being a fluidic sample, such as serum, plasma, and/or seminal;

• determining the level of LHCGR (LH/hCG receptor) in the sample;

• comparing said determined level to a reference level; and

• determining that

o said subject is at risk of having, developing or relapse of testis cancer, and/or prostate cancer, and/or a need of androgen supplementation, and/or having gonadal dysfunction, if said level is higher than said reference level, or

2. The method according to claim 1, wherein said testis cancer is invasive testis cancer.

3. The method according to claim 2, wherein said invasive testis cancer is seminomas and/or non-seminomas.

4. The method according to claim 1, wherein said method is for predicting a risk of having, developing or relapse of testis cancer.

5. The method according to claim 1, wherein said method is for predicting predicting a risk of having, developing or relapse of prostate cancer.

6. The method according to claim 1, wherein said method is for predicting a risk of having, developing or relapse of a need of androgen replacement therapy (ART).

7. The method according to claim 6, wherein said androgen replacement therapy is selected from the group consisting of testosterone replacement therapy, such as by supplementation of testosterone, testosterone derivatives having

testosterone activity, such as Testosteronundecanoat and hCG.

8. The method according to claim 1, wherein said method is for predicting a risk of having, developing or relapse of gonadal dysfunction.

9. The method according to claim 8, wherein said gonadal dysfunction is selected from the group consisting of impaired sperm out, low sperm motility, Sertoli cell only, Leydig cell hyperplasia, testis cancer, carcinoma in situ, and hyalinised testis.

10. The method according to any of the preceding claims, wherein said method further includes determining the level of one or more of Testosterone (T), LH, T/LH ratio, Estradiol, FSH, AMH, Inhibin B and/or INSL3 in a sample from said subject.

11. The method according to claim 10, wherein said further determination is done on the same sample material.

12. The method according to any of the preceding claims, wherein said level is determined by measuring the level of mRNA and/or protein, preferably the protein level.

13. The method according to claim 12, wherein said determination of the at least one protein level is performed using a method selected from the group consisting of immunohistochemistry, immunocytochemistry, FACS, ImageStream, Western Blotting, qPCR, RT-PCR, qRT-PCR, ELISA, Luminex, Multiplex, Immunoblotting, TRF-assays, immunochromatographic lateral flow assays, Enzyme Multiplied Immunoassay Techniques, RAST test, Radioimmunoassays, immunofluorescence and immunological dry stick assays.

14. The method according to claim 13, wherein said determination is performed by immunocytochemistry.

15. The method according to claim 13, wherein said determination is performed by ELISA.

16. The method according to any of the preceding claims, wherein said patient has one or both testicles removed, e.g. due to testicular cancer.

17. The method according to claim 16, wherein said method is for predicting a risk of having, developing or relapse invasive testis cancer.

18. The method according to claim 17, wherein said method is for predicting a risk of relapse of invasive testis cancer.

19. The method according to any of the proceeding claims, wherein an indication of an increased risk, further comprising referring said subject to further analysis for determining the presence of one or more of the indicated conditions.

20. The method according to claim 19, wherein said further analysis may lead to initiation of a treatment protocol in the form of surgery, such as testicle removal, chemotherapy, radiation therapy, androgen replacement therapy (ART), with the proviso that the indication is carcinoma in situ testis; and/or invasive testis cancer; and/or prostate cancer; and/or wherein said further analysis may lead to initiation of a treatment protocol in the form of hormone treatment, such as Androgen replacement therapy (ART); and or chromosomal analysis, with the proviso that the indication is a need of Androgen replacement therapy (ART) and/or gonadal dysfunction.

21. A method for monitoring a condition in a male subject previously determined of having, a risk of developing or relapse of a condition selected from

- testis cancer; and/or

prostate cancer; and/or

- a need of Androgen replacement therapy (ART); and/or

- gonadal dysfunction; the method comprising

wherein said second sample has been obtained from said subject at a later time point than the first sample;

· determining the level of LHCGR (LH/hCG receptor) in the first and the

second sample;

• comparing said determined levels of LHCGR in the samples;

determined level in the second sample is higher than in the first sample; or

o the condition has developed positively for said subject, if said

determined level in the second sample is lower than in the first sample.

22. The method according to claim 21, wherein a treatment regime has been performed between the sampling of the two samples.

23. The method according to claim 22, wherein the treatment protocol is surgery, such as testicle removal, chemotherapy, radiation therapy, Androgen replacement therapy (ART), with the proviso that the condition is carcinoma in situ testis; and/or invasive testis cancer; and/or prostate cancer; and/or wherein the treatment protocol is hormone treatment, such as Androgen replacement therapy (ART); with the proviso that the condition is a need of Androgen replacement therapy (ART) and/or gonadal dysfunction.

24. The method according to any of claims 21-23, wherein the first sample and the second sample are from the same sample types, such as blood serum.

25. The method according to any of the preceding claims, wherein the LHCGR (LH/hCG receptor) is a truncated version lacking the transmembrane domain.

26. The method according to claim 25, wherein said transmembrane domain is encoded by exon 11 of the LHCGR gene.