FIELD OF THE INVENTION

The present invention is in the field of medicine, in particular in tumor therapy, more particular in targeted tumor therapy.

BACKGROUND

Successful tumor therapy is dependent of the time point of the detection of the tumor and the selection of the right therapy. Identification of specific biomarkers in tissue samples of a tumor, help to determine both, the stage of malignancy and the presence of cell structures, which can serve as targets for therapy. Also, the detection of tumor-associated biomarkers in the blood is used to detect malignancies. However, expectations on biomarkers have not been met in many cases, because these are not specific enough or do not have the necessary sensitivity to be able to detect malignancies at an early stage in a reliable way. Furthermore, known biomarkers can often just be applied on specific tumor types. Therefore, biomarkers are increasingly being sought, which allow the general detection of tumors. Such a general tumor test would considerably simplify the diagnosis of a wide variety of tumors and thus significantly improve both, therapy and monitoring.

So far, small benign tumors and small malignant tumors cannot be detected with tumor markers, which are determined in blood samples. Small tumors release relatively small amounts of biomarker, which are then diluted in the blood volume, consequently no significant increase in the concentration of biomarkers in blood is measurable.

A novel method for detecting biomarkers in specific cells in the blood, which carry tumor material has solved this problem, since the tumor material in these specific cells is not diluted in the whole blood and remains in high concentration inside said cells.

This novel method is based on the principle of the immune system to detect, phagocyte and digest undesirable cell structures. Undesirable cell structures comprise intruders from outside like bacteria and parasites or even degenerated body ^' s own cells like tumor cells. During the process of eliminating undesirable cell structures, immune cells, such as monocytes, phagocyte these structures and present them. Thus, tumor cells are recognized by said immune cells, which then phagocyte and attack them. Therefore, the immune cells migrate from the blood vessels into the tumors to phagocyte tumor cells. Consequently, the immune cells enclose fragments of one or more tumor cells, which are then presented over a period of several days. The aim is to inform other immune cells what the target is. After a while, the immune cell leaves the tumor and gets back into the bloodstream containing still tumor material. A sort of immune cells, which carry tumor material in the cytoplasm are characterized by the fact that they carry the surface markers CD14 and CD16, which can be easily detected in blood samples by flow cytometry. With this method it is possible to detect these immune cells, count them and even further characterize them.

Merely recognition and counting of immune cells, such as monocytes in blood samples is not sufficient to detect tumors at an early stage, since the number of these cells is not significantly different between healthy and tumor patients. Only through the detection of tumor material in these immune cells, a population of immune cells can be identified which has a significant numerical difference between healthy and tumor patients. The detection of these immune cells is done by an elaborate method in which specific biomarkers expressed in the inside of the specific immune cells, are identified. This new technology is called "EDIM", epitope detection in monocytes. Several studies have shown that the detection of biomarkers with the EDIM technology is much more sensitive and specific than the detection of biomarkers in blood. With the EDIM technology, biomarkers can be used to detect tumors earlier and more specifically.

In search of a general biomarker for tumors, it was noticed that there is a fundamental process that is alike in all tumor cells. All forms of tumor cells have a disorder in the process of planned cell death. Only if this process is disturbed, a tumor cell can arise. Therefore, biomarkers for apoptosis disorders are of big interest. One of the known biomarkers is based on the DNaseX-enzyme, which normally performs the last step of the apoptosis and has been shown to be overexpressed in tumors. Nevertheless, the enzymatic activity of the DNase-enzyme is inhibited by the increased release of inhibitors by the tumor cells. The ApolO epitope of the DNaseX protein sequence is particularly well detectable, which therefore, can be used for diagnosis in patients with tumors. Further studies have identified a gene which is associated with the invasive growth behavior of tumors and additionally leads to an increased cell proliferation and an inactivation of the apoptosis. Therefore, TKTL1 is a further important biomarker for tumors. The detection of biomarkers ApolO and TKTL1 in immune cells by means of blood test allows early detection of all types of carcinoma examined so far, but also of sarcomas and hematological tumors.

For some types of cancer, most patients will have an appropriate target for a particular targeted therapy. Those targets are specific molecules that are involved in the growth, progression and spread of the cancer. In a targeted therapy, drugs or other substances are administered to the patient, which block the growth or the spread of the cancer by interfering with the targets.

The use of a targeted therapy is only appropriate for patients whose tumor has a specific gene mutation that codes for the target; patients who do not have the mutation would not be candidates because the therapy would have no target.

A limitation of the targeted tumor therapy is, that the amount of the cells comprising the target can change during the therapy. Cells might become resistant to the therapy, maybe through mutation of the target, so that the targeted drugs or substances no longer interact with it or the tumor finds a new pathway for tumor growth independent on the target. For this reason, it is important to proof, if a special target in the tumor cells is existing before and during the therapy. This is highly important for deciding, which therapy to use and if the therapy already used has to be changed, respectively. For the evaluation if a tumor is susceptible to a special targeted therapy, also the proportion of the tumor cells comprising the target to the whole tumor is important. One approach is the detecting of targets in tumor cells, which have separated from the tumor and are circulating in the blood of the patient.

This approach does not provide realistic and reliable information about the tumor, as these circulating tumor cells differ from the tumor tissue and do not reflect the exact structure of the tumor. A realistic estimation about the proportion of the tumor cells comprising the target to the whole tumor is not possible.

Therefore, a reliable method for detecting the susceptibility of a tumor to a targeted tumor therapy is needed. SUMMARY OF THE INVENTION

Based on the background above, it is the objective of the present invention to provide a method of efficiently and reliably detecting the susceptibility of a tumor to a targeted tumor therapy.

This objective is attained by a method for detecting the susceptibility of a tumor to targeted therapy in a subject, the method comprising a. detecting at least one type of monocytes in a sample obtained from said subject and determining a first quantity of monocytes from that monocytes which are TKTL1, ApolO, Epcam and/or other tumor specific marker positive; b. determining a second quantity of said monocytes of a. which are additionally positive for at least one marker of a target used in tumor targeted therapy; and c. calculating the ratio of the first quantity to the second quantity.

The invention further relates to a kit for detecting the susceptibility of a tumor to targeted therapy in a subject, the kit comprising instructions for performing the method according to the invention, markers for detecting monocytes, comprising CD14 and CD16 marker, TKTL1, ApolO, Epcam and/or other tumor specific marker, at least one marker of a target used in tumor targeted therapy and reaction agents selected from the group comprising reaction buffer, wash buffer, secondary markers if applicable.

The invention also relates to marker for TKTL1, ApolO, Epcam and/or other tumor specific marker and at least one tumor marker for targeted therapy for use in a method for diagnosis, prediction or risk stratification for mortality or disease outcome of a subject that has or is suspected to have a tumor.

Furthermore, the invention relates to marker for TKTL1, ApolO, Epcam and/or other tumor specific marker and at least one tumor marker for targeted therapy for use in a method for monitoring a tumor therapy as well as to a method for monitoring, prediction or risk stratification of a tumor targeted treatment of a subject that has or is suspected to have a tumor. DETAILED DESCRIPTION

The present invention relates to a method for detecting the susceptibility of a tumor to targeted therapy in a subject, the method for detecting the susceptibility of a tumor to targeted therapy in a subject, the method comprising: a. detecting at least one type of monocytes in a sample obtained from said subject b. determining a first quantity of those at least one type of monocytes of step a) which are additionally TKTL1, ApolO, Epcam and/or other tumor specific marker positive; c. determining a second quantity of said monocytes of step b) which are additionally positive for at least one marker of a target used in tumor targeted therapy; and d. calculating the ratio of the first quantity to the second quantity.

The inventors surprisingly found that detecting the quantity of monocytes migrating into the tumor tissue and phagocyting tumor tissue and calculating the ratio of the quantity of that monocytes to the quantity of said monocytes, which are additionally positive to a marker of a target for targeted tumor therapy, gives reliable and correct information about the structure of that tumor.

By using this method, it is possible to determine the proportion of target positive cells of the tumor at any given time, even without having directly access to tumor material, and to make it dependent on whether a targeted therapy promises success.

The term "tumor" is used in the present application and is understood to encompass malignant and non-malignant tumors, including solid tumors and hematological tumors. Solid tumors are exemplified by tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g. thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, lover, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva. In addition, it includes primary tumors in said organs and corresponding secondary tumors in distant organs (tumor metastases). Hematological tumors are exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia, acute lymphoblastic leukemia, Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes and tumors of unknown primary site.

Herein "targeted tumor therapy" refers to each therapy which targets one or more specific molecules that are involved in the growth, progression and/ or spread of the cancer. In a targeted therapy, drugs or other substances are administered to the patient, which block the growth or the spread of the cancer by interfering with the targets. Many different targeted therapies have been approved for use in cancer treatment. These therapies include, but are not limited to hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, and toxin delivery molecules

Herein the term "cut off value" defines the dividing point on measuring scales where the test results are divided into different categories; typically positive (indicating someone has the condition of interest), or negative (indicating someone does not have the condition of interest).

Monocytes are the largest type of white blood cells. They are part of the innate immune system of vertebrates including all mammals (humans included), birds, reptiles, and fish. They are amoeboid in shape, having a granulated cytoplasm. Monocytes constitute 2% to 10% of all leukocytes in the human body. They play multiple roles in immune function. Monocytes are produced by the bone marrow from precursors called monoblasts, which are bipotent cells that differentiated from hematopoietic stem cells. Monocytes typically circulate in the bloodstream for about one to three days and then move into tissues throughout the body. Monocytes in tissues mature to different types of macrophages depending on the anatomical location, i.e. osteoclasts, microglia cells, histiocytes, and Kupfer cells. Monocytes can move quickly to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response.

There are at least three types of monocytes in human blood:

The classical monocyte, characterized by high quantity expression of the CD14 cell surface receptor (CD14++ CD16- monocyte) The non-classical monocyte shows low quantity expression CD14 and additional co-expression of the CD16 receptor (CD14+ CD16++ monocyte).

The intermediate monocyte with high quantity expression of CD14 and low quantity expression of CD16 (CD14++ CD16+ monocytes).

Monocyte markers comprise CD2, CDllb, CD14, CD16, CD31, CD33, CD56, CD62L, CD115, CD163, CD192, CX3CR1, CXCR3, CXC 4, CCR2, CDllb, CD16/CD32, CD31, CD43, CD44, CD45, CD62L, CD115,

CX3CR1, F4/80, Grl, Ly-6C, LFA-1, VEGF, and further.

Since hardly any surface markers are known which specifically detect only monocytes, therefore the combination of surface markers is used to identify monocytes alone in the flow cytometer, for example a combination of the markers CD14 and CD16. Macrophages engulf and digest cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have types of proteins specific to healthy body cells on its surface in a process called phagocytosis. Human macrophages can be identified by using flow cytometry or immunohistochemical staining by their specific expression of proteins such as, but not limited to CDllb, CD14, CD16, CD40, CDllb, CD56, CD64, CD68, and CD163. Macrophages are attracted to a damaged site through chemotaxis, triggered by a range of stimuli (immune response) including pathogens and cytokines released by macrophages already at site. Macrophages phagocyting tumor cells, move away from the tumor and can be found circulating in blood. These macrophages contain debris of tumor cells. Herein the term "subject" is used to refer to an animal (e.g. a mammal, a fish, an amphibian, a reptile, a bird and an insect). In a specific embodiment, a subject is a mammal (e.g., a non-human mammal and a human). In another embodiment, a subject is a primate (e.g., a chimpanzee and a human). In another embodiment, a subject is a human. In another embodiment the subject is a human with one or more tumors. As used herein, the term "diagnosis" refers to the identification of the disease (herein a tumor) at any stage of its development, and also includes the determination of predisposition of a subject to develop the disease. In a preferred embodiment of the invention, diagnosis of a tumor occurs prior to the manifestation of symptoms. Subjects with higher risk of developing a tumor are of particular concern. The diagnostic method of the invention also allows confirmation of the presence of a tumor in a subject suspected to have a tumor.

The term "biomarker" (biological marker) and "marker" are used synonymously. The term herein relates to measurable and quantifiable biological parameters (e.g., specific enzyme concentration, specific hormone concentration, specific gene phenotype, presence of biological substances) which serve as indices for health- and physiology-related assessments, such as disease risk, disease diagnosis, etc. Furthermore, a biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or responses to a therapeutic intervention. A biomarker may be measured on a sample. Biomarkers can be classified as antecedent biomarkers (identifying the risk of developing an illness), screening biomarkers (screening for disease), diagnostic biomarkers (recognizing diseases), staging biomarkers (categorizing disease severity), or prognostic biomarkers (predicting future disease course, including recurrence and response to therapy, and monitoring efficacy of therapy). A biomarker may be a protein, peptide or nucleic acid molecule. In context of the present invention a biomarker is a biomolecule, more specifically a protein or even more specifically a protein which is expressed on the cell surface or intracellularly of immune cells. Furthermore, a biomarker is a biomolecule used to detect a further biomarker in and/or on immune cells, these comprise primary and/or secondary antibodies. Also, a marker according to this invention is a biomolecule to detect a target for tumor targeted therapy in cells.

In the present invention, the term "monitoring" denotes the observation of the state or progression of a subject's medical condition by measuring the level of a certain diagnostic marker or markers for said medical condition at various points of time.

Flerein, the term "beneficial outcome" refers to the outcome a subject may experience after tumor therapy, which leads to an improvement in at least one sign of a tumor, which comprise tumor shrinkage, decrease in growth rate or suppression of tumor growth. Further, a reduction of the number of tumor cells, reduction of tumor size, inhibition of tumor cell infiltration into peripheral organs, retarded, slowed or stopped cell infiltration, inhibition of tumor metastasis or slowed tumor metastasis, prevention or delayed recurrence of tumor. Also, it may mean that the disease is no longer progressive. Further, it refers to the relieve of one, more, or even all symptoms associated with tumors.

As used herein, a "kit" is a packaged combination optionally including instructions for use of the combination and/or other reactions and components for such use.

All other terms are unless explicitly defined otherwise, used as understood by those skilled in the art.

In one embodiment of the invention, the sample of the subject is obtained before initiation of the therapy. Thereby it can be tested if a tumor is susceptive to targeted tumor therapy.

The sample of said subject can also be obtained at one or more further time points during the therapy. Thereby it can be tested if the tumor is still susceptive to the applied targeted tumor therapy.

In a preferred embodiment of the invention said monocytes are selected from the group comprising monocytes positive for CD14 and CD16, CD2, CDllb, CD14, CD16, CD16/CD32, CD31, CD33, CD40, CD43, CD44, CD45, CD56, CD62L, CD64, CD68, CD115, CD163, CD192, CX3CR1, CXCR3, CXC 4, CCR2, CDllb, CD16/CD32, CD31, CD43, CD44, CD45, CD62L, CD115, CX3CR1, F4/80, Grl, Ly-6C, LFA-1, or VEGF. In a preferred embodiment the monocytes are macrophages. Especially preferred the monocytes are CD 14 and CD 16 positive macrophages.

According to the invention, all markers appropriate for detecting a target of tumor targeted therapy can be used. In a preferred embodiment the at least one marker of a target used in tumor targeted therapy is selected from the group of HER2, AFP, Beta-HCG, CEA, PSA, CA 125, CA 15-3, CA 19-9, CA 72-4, Calcitonin, CgA, CYFRA 21-1, NSE-Tumormarker, Protein S100, PD-L1, PSMA-Ligand and SCC- Tumormarker.

In one embodiment according to the invention, the first quantity of the monocytes is set to 100%. Correlating the second quantity to the first quantity in percent, the percentage of monocytes in the tumor that are susceptible to targeted tumor therapy can be obtained.

In an alternative embodiment of the invention a beneficial outcome is associated to the targeted therapy using the detected target, if the ratio of the first quantity to the second quantity is beyond a determined cut off value.

There are several methods existing for detecting a quantity of cells, which can be used to determine a first and a second quantity of the specific monocytes.

In a preferred embodiment, methods for direct and indirect cell counting are used. Especially preferred is the method of flowcytometry.

In a preferred embodiment of the invention, the first quantity of monocytes and the second quantity of monocytes are determined by flowcytometry. In one embodiment according to the invention, the method according to the invention is for use in medical decision making for individual targeted tumor therapy in a subject.

Another embodiment of the invention relates to a kit for detecting the susceptibility of a tumor to targeted therapy in a subject, the kit comprising: a) instructions for performing the method of any of the claims 1 to 8 b) markers for detecting monocytes, comprising CD14 and CD16 marker c) marker for TKTL1, ApolO, Epcam and/or other tumor specific marker, d) at least one marker of a target used in tumor targeted therapy e) reaction agents selected from the group comprising reaction buffer, wash buffer, secondary markers if applicable

The invention also relates to TKTL1, ApolO, Epcam and/or other tumor specific marker and at least one tumor marker for targeted therapy for use in a method for diagnosis, prediction or risk stratification for mortality or disease outcome of a subject that has or is suspected to have a tumor.

The invention further relates to TKTL1, ApolO, Epcam and/or other tumor specific marker and at least one tumor marker for targeted therapy for use in a method for monitoring a tumor therapy.

In a preferred embodiment the invention also relates to a method for monitoring, prediction or risk stratification of a tumor targeted treatment of a subject that has or is suspected to have a tumor, the method comprising: a) detecting at least one type of monocytes in a sample obtained from said subject b) determining a first quantity of those at least one type of monocytes of step a) which are additionally TKTL1, ApolO, Epcam and/or other tumor specific marker positive; c) determining a second quantity of said monocytes of step b) which are additionally positive for at least one marker of a target used in tumor targeted therapy; and d) calculating the ratio of the first quantity to the second quantity.

In another embodiment the invention relates to a method of treating a tumor patient in need thereof, wherein the tumor therapy is initiated or altered depending on the outcome of the method of monitoring according to the invention.