Determining an individual's HLA pattern, its use as a prognostic factor, target genes, and therapeutic agents

Abstract

The present disclosure relates to in vitro methods for determining individual HLA patterns (adult and / or embryonic) in body samples (tissue or blood samples) from cancer patients and / or patients suffering from disorders related to autoimmune diseases, and methods for stratifying such patients for tailored treatment. The present disclosure further relates to corresponding kits and uses thereof, as well as nucleic acid molecules as prognostic biomarkers for neoplastic diseases such as cancer, autoimmune diseases, infectious diseases, and pregnancy-related conditions. The present disclosure also relates to therapeutic agents and methods for manufacturing the therapeutic agents.

Description

[Technical Field]

[0001] The present invention relates to an in vitro method for determining the individual HLA pattern (adult and / or embryonic) in a body sample, in particular a tissue or blood sample, of a patient suffering from cancer and / or a disorder related to an autoimmune disease, and to a method for stratifying said patients for a tailored treatment. [Background technology]

[0002] Chemical or biological tumor treatments do not focus on individual tumor cell types in a single patient, but generally detect all rapidly dividing cells, regardless of whether they are malignant or not, and regardless of the individual patient.

[0003] The end result is that this type of treatment is often ineffective and / or has severe side effects. More targeted, highly personalized treatments would be extremely useful.

[0004] The basis for the well-regulated existence of a whole organism is communication between cells, or cellular dialogue. This dialogue and its regulation allows the existence of the whole organism to be maintained even though cells are constantly dying and / or regenerating. As a result of this dialogue, cell differentiation is also regulated, as is known from stem cell research. This dialogue allows the well-regulated cooperation of two different cell clones, even if one of them shows very rapid growth, as is the case during pregnancy.

[0005] The basis of cellular communication in humans is the MHC (major histocompatibility complex) with its HLA groups. Cellular discrimination by HLA groups is the basis of all cellular communication. Cellular communication develops in coordination with specific receptors such as killer immunoglobulin-like receptors (KIR) or leukocyte immunoglobulin-like receptors (LILR) on natural killer cells (NK cells), and then further factors such as cytokines and growth factors become involved.

[0006] Various HLA groups are known which can be described as follows: HLA classes A, B, and C (MHC I): These identify virtually all adult and somatic cells. HLA D group (DR, DP, DQ, etc.; MHC II): These play an important role in immunocompetent cells and / or antigen presentation. HLA groups E, F, and G: These identify germ cells, especially at the so-called invasion front.

[0007] In addition, the MHC complex also contains further substances such as complement factors belonging to class III.

[0008] Tumor cells essentially have the same genetic code as other cells in the entire organism. Therefore, tumor cells have no information regarding cell division and differentiation other than that of the entire organism. As a result, every malignant tumor disease is unique and individual, i.e., specific to each organism.

[0009] In some tumor diseases, additional genetic information, such as so-called oncogenes, is introduced into cells from outside, for example, by viral vectors. However, oncogenes can also form an integral part of the genetic material from reproduction. Oncogenes and / or their activation, as well as other external factors, can permanently affect the biology of tumor cells. Nevertheless, tumor cells continue to participate in the cellular dialogue and effective regulation of the entire organism.

[0010] Organisms with a high degree of cellular differentiation, such as humans, "compensate" for their high differentiation by a loss of pluripotency or totipotency. In the case of organ loss, restoration to the original state is no longer possible, and only repair by connective tissue is possible. If the organism is less strongly differentiated, such as a starfish, the loss of totipotency or pluripotency is less pronounced, and therefore, for example, if an arm is lost, a new arm can be regrow, albeit small.

[0011] Totipotency is essentially encoded in the genetic material of higher organisms as well. This is evidenced by the simple fact that this genetic material had previously to control the development of a fertilized egg cell into a differentiated organism. Cloning experiments also show that the genetic material of even highly differentiated cells, such as the mammary cells of the cloned sheep "Dolly," can be "reset" to "zero" ("reprogrammed") in the nucleus. In the final analysis, this also applies to the reproduction and / or fertilization of an egg cell, where the genetic material of two relatively older individuals (father and mother) is reset to "zero" and re-coded for the development of a new organism.

[0012] Correspondingly, it is clear that tumor cells are also provided with genetic material that essentially codes for all possible growth and differentiation processes in the entire organism, i.e., the mechanisms of early embryonic-maternal cell interaction and subsequent early embryonic implantation in embryo-fetal development.

[0013] All tumor cells lose their differentiation (hence "dedifferentiate") to varying degrees and make their "way back" to varying degrees. The essential features of this "way back" are the loss of cell differentiation and loss of specific cellular capabilities, and the (re)gain of uncontrolled cell proliferation.

[0014] It is known that tumor cells can express typical embryonic HLA groups on their surface. Although the respective investigations are still fragmentary, this expression of embryonic HLA groups contributes to the situation in which tumor cells avoid the attack of the non-specific immune defense of the own organism. The expression of these typical HLA groups on the surface allows the cells to not only activate the corresponding receptors on, for example, NK cells, but also lymphocytes and even immunocompetent cells, so that not only are there no attacks by non-specific immune defenses, i.e., NK cells and lymphocytes, but in individual cases, tumor cells (and embryonic cells) can "put the immune defense to work for them", i.e., by synthesizing growth factors and cytokines that are beneficial for their own development.

[0015] Here, mention should be made, for example, of the phenomenon of TAMs (tumor-associated macrophages) or MDSCs (tolerogenic "myeloid-derived suppressor cells"), which support (i.e., are "redirected") tumor growth in the malignant tumor microenvironment. The same applies equally to cytokines such as MIF (macrophage migration inhibitory factor), which are likely produced in tumors (probably by NK cells) and have a pro-angiogenic effect, thus supporting tumor cell proliferation and migration.

[0016] Tumor cells do not necessarily have to be very resistant. As is known, they are more sensitive to chemotherapy and more sensitive to radiation than "healthy" differentiated standard cells. Nor do they have a particularly high rate of cell division. The danger posed by malignant tumor cells is, above all, that they can "force" progressive, uncontrolled growth based on cell communication.

[0017] This situation is also illustrated by the fact that, according to current knowledge, metastases are primarily formed due to the spread and colonization of malignant cells, which can be termed "malignant stem cells." If this is correct, such "malignant stem cells" should also be able to locally evade growth control and differentiation pressures through cellular communication with neighboring tissues. Even if formed from dedifferentiation, stem cells generally behave like stem cells, and in this case we focus specifically on the manner in which embryo-maternal communication (embryo-maternal dialogue) functions.

[0018] Malignant cell degeneration is an inherent process specific to every individual. This is not altered by the fact that there are pathologically well-classified (always recurring) tumor types across individuals. This situation is rather evidence of the fact that malignant tumors are not formed by all dedifferentiation and all "backtracking." Rather, only a specific subset is likely to "survive" the "backtracking" and thus result in a typical tumor entity across individuals.

[0019] Cell dedifferentiation or "degeneration" is probably a relatively ubiquitous process throughout all living organisms. However, it rarely leads to the formation of tumor diseases, since only a small proportion of these cells possess the cell biological and (cell-to-cell) communication prerequisites necessary for survivability. Survivable cells probably use a combination of these two mechanisms: on the one hand, they exploit the restoration of embryo-maternal communication to evade immune attack and even obtain support from this communication during cell growth; on the other hand, they are protected by fully or partially expressed (originally adult) HLA patterns (and those corresponding to their maternal patterns (see below)), allowing them to evade the attack of specific immune defenses and thus proliferate.

[0020] "Acquired" immunity develops during pregnancy and means that the body is not only tolerant to its own HLA groups, but is also always tolerant to the foreign adult HLA groups of one's own mother.

[0021] According to current knowledge, tumor cells express essentially the same adult HLA group as all other somatic cells in the entire organism, as transmembrane proteins that present peptides as antigens in a cleft formed by the extracellular alpha 1 and alpha 2 domains, while binding beta-microglobulin as a cofactor in a complex. As a result, tumor cells are protected from the attack of specific immune defenses. This is also true, in principle, if part of the original HLA pattern is lost "on the way back," is expressed at a lower density, or is available in an altered, i.e., corrupted, form (which is not anomalous for tumor cells).

[0022] A useful comparison here is made with the germ cells of an embryo that are shed and remain in the mother's organism (a phenomenon known as "microchimerism"). Germ surface structures on the germ cells (mainly placental or trophoblast cells), particularly HLA-G, E, and F, prevent the mother's immune system from attacking the cells.

[0023] Once specific differentiation of embryonic cells (usually embryonic cells) has already occurred, they integrate specifically into the corresponding organs, a behavior that very strongly mirrors that of malignant tumors, which often favor specific patterns of metastasis formation.

[0024] If the germ cell's embryonic surface structure is maintained, the germ cell, like tumor cells, will likely not be attacked throughout the mother's life.

[0025] If a large number of embryonic cells flow into the maternal organism during pregnancy, this resistance can also result in an attempt to "take over" the mother's body, i.e., a "graft-versus-host" reaction occurs, as in the case of, for example, the very dangerous HELLP syndrome (hemolysis, elevated liver enzymes, decreased platelet count).

[0026] However, there may also be adverse reactions to microchimerism, possibly due to the fact that embryonic cells at least partially lose HLA-G, E, F protection and optionally undergo further differentiation.

[0027] Then, as the embryonic cells differentiate, they display an "adult" HLA structure that differs from that of the maternal organism, resulting in an inflammatory counter-reaction by the maternal organism. The typical result is the formation of connective tissue around the site of inflammation, which can lead to certain immune disorders (see Hashimoto's thyroiditis).

[0028] This adverse inflammatory reaction of the organism does not occur in the case of tumor cells, since during further differentiation they form an HLA profile on their surface which does not differ from that of the host organism.

[0029] As is known and described for tumor cells, this complete or incomplete expression of the adult HLA pattern prevents the attack of specific immune defenses, even if the tumor cells resist by expressing (or overexpressing) additional antigens. The protection resulting from the complete or incomplete expression of the original adult HLA pattern is apparently so effective that tumor cells are able to express their specific antigen pattern without being effectively attacked by (specific) immune responses, i.e., the existing immune responses of B and T lymphocytes (thus becoming "resistant"). It is noteworthy that tumor antigen expression patterns are relatively specific to individual tumor types. It is also possible that altered and / or mutated MHC / HLA groups render the antigen presentation cascade (APM (antigen processing machinery)) increasingly defective, so that typically human-associated or self-specific antigens are rarely or (no longer) presented. However, the molecular mechanisms underlying such a putative process are unknown and have yet to be identified.

[0030] When the whole organism is confronted with somatic cells whose HLA expression pattern is strongly or completely different from that of the organism or its mother, one must assume that there will be an attack of the immune defense accompanied by the formation of memory cells. "Memory" is naturally directed primarily against diverse HLA groups. However, it is known that such an attack by the immune system and the subsequent destruction of foreign cells also involves the formation of antibodies against other surface structures. In individual cases, antibodies are even formed against ubiquitous cellular components, such as phospholipids ("antiphospholipid syndrome").

[0031] Against this background, it is necessary to consider both mechanisms as a whole, i.e., how "tumor cells on their way back" communicate with other cells and the immune system, and / or how the communication features lost during fetal development are restored, and from this understanding, to develop therapeutic concepts and corresponding medicines that take these fundamental principles into account.

[0032] In addition to specific immune defenses (i.e., T lymphocytes and B lymphocytes), monocytes and the macrophages derived from them also play a role in tumor growth. However, macrophages can only be activated in the presence of nonspecific immune defense cells (such as NK killer cells) or specific immune defense cells (such as T cells and B cells). However, this requires "priming," in which antigen-presenting cells (such as dendritic cells) present mutant or "foreign" proteins, thus leading to the formation of cytotoxic T cells. Here, numerous cytokines, such as interferon (IFN-γ) and tumor necrosis factor (TNF-α), also play a role.

[0033] A comparison between embryonic and tumor cells also occurs in the case of macrophages. Macrophages can be found in the endometrial basement during pregnancy. They normally have an inhibitory effect on the invasive behavior of the embryo, forming a sort of "protective barrier" between the implanted embryo and the myometrium. On the other hand, the embryo secretes macrophage migration inhibitory factors, i.e., factors that limit and inhibit macrophage attack. This is equally true for malignant tumors (see above).

[0034] In conclusion, the current understanding is based on direct cell-to-cell communication between tumor cells and immune cells based on membrane-bound MHC I-mediated peptide presentation and subsequent binding to specific receptors (such as KIR and LILR) on immune cells.

[0035] The molecular basis of communication between tumor cells and the immune system is still poorly understood. With the advent of antibody-based therapies attacking so-called "checkpoints," determining the respective target structure (e.g., quantifying PD-L1 expression on an mRNA or protein basis) and / or determining tumor mutational burden and / or MSI status has shown some impact in predicting response to treatment or long-term survival (DFS, MFS, DSS, or OS).

[0036] As used herein, the term "predicting the outcome" of a disease is meant to include both the prediction of the outcome of a patient receiving a given treatment and the prognosis of an untreated patient. The term "predicting the outcome" may particularly relate to the risk of a patient experiencing an event such as metastasis or death, preferably within a given time frame.

[0037] In conclusion, there is a great medical need to better stratify cancer patients for their individual risk of recurrence and response to chemotherapy or alternative treatment options for multiple tumor types.

[0038] This includes, on the one hand, mutations (KRAS, NRAS, EGFR, cMET, HER2, ESR1, FGRF3, etc.), molecular subtyping transcripts (ESR1 PGR, ERBB2, etc.; proliferation genes such as MKI67, RACGAP1, BIRC5, MYBL1, FOXM1; keratins such as KRT4, KRT5, KRT17, KRT18, KRT19, KRT20; EMT markers such as SNAI1, SNAI2, FOXA1), immune genes (CD3, CD8, CD19, CD68, CD168, CSF1R, IGKC, IGHM, IFNG, etc.), checkpoint genes (PD-L1, PD-L2, CD86, CD80, L-ICOS, B7-H3, B7-H4; PD1, CTLA4, CD28, ICOS, etc.), as well as determining HLA expression patterns ("HLA typing") as described in the present disclosure.

[0039] For example, in a highly heterogeneous group of both bladder cancer stages, the optimal treatment procedure is unclear because current clinicopathological features cannot reliably assess prognosis.

[0040] The standard methodology currently applied worldwide to detect receptor status in cancers, such as breast cancer, is immunohistochemistry (IHC) of formalin-fixed and paraffin-embedded (FFPE) biopsies or resected tissues. In breast cancer, endocrine therapy or targeted systemic therapy (e.g., with trastuzumab) is mostly based on IHC. However, IHC-based approaches that determine individual risk by testing multiple markers do not provide reliable prognostic subclassification for bladder cancer, which could help resolve the diagnostic / therapeutic challenges mentioned above. Furthermore, IHC testing generally suffers from a lack of sensitivity.

[0041] Therefore, there is a clear need for a reliable, objective, quantitative, and reproducible test system for molecular subtyping of bladder cancer that allows reliable individual risk assessment, facilitates selection of appropriate tumor treatment regimens (i.e., patient stratification), and allows prediction of prognosis and treatment success. Furthermore, such a test system should allow for decentralized testing that is suitable for a significant proportion of cancer patients. [Prior art documents] [Patent documents]

[0042] [Patent Document 1] WO 02 / 42759 [Patent Document 2] WO 02 / 41992 [Patent Document 3] International Publication No. 02 / 097413 [Non-patent literature]

[0043] [Non-Patent Document 1] J.Sambrook et al. eds.,2000, Molecular Cloning:A Laboratory Manual,3rd Edition,Cold Spring Harbor Laboratory Press,Cold Spring Harbor

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0044] These and other objects are achieved by the invention as described below.

[0045] The present invention relates to an in vitro method for determining individual HLA patterns (adult and / or embryonic) in body samples (tissue or blood samples) of cancer patients and / or patients suffering from disorders related to autoimmune diseases, and to a method for stratifying said patients for tailored treatment.

[0046] The invention further relates to corresponding kits and methods of use thereof, as well as nucleic acid molecules as prognostic biomarkers for neoplastic diseases such as cancer, autoimmune diseases, infectious diseases, and pregnancy-related conditions. The invention also relates to therapeutic agents and methods for producing therapeutic agents.

[0047] As used herein, the terms "sample," "biological sample," or "clinical sample" refer to a sample obtained from a patient. A sample can be any biological tissue or biological fluid. Such samples include, but are not limited to, saliva, blood, serum, plasma, blood cells (e.g., white blood cells), tissue, core or fine needle biopsy samples, cell-containing body fluids, suspended nucleic acids, urine, ascites, and pleural fluid, cerebrospinal fluid, tears, or cells derived therefrom. Biological samples can also include sections of tissue, such as frozen or fixed sections taken for histological purposes, or microdissected cells or their extracellular portions. The biological sample analyzed is tissue material from a neoplastic lesion obtained by aspiration or puncture, resection, or other surgical method resulting in biopsy or excised cellular material. Such biological samples can include cells obtained from a patient. Cells can be found in cellular "smears" of solid tumor material, lavage fluids, or body fluids. A sample can also be a processed sample, such as a frozen, fixed, embedded sample, etc. A preferred type of sample is a formalin-fixed, paraffin-embedded (FFPE) sample. Preparation of FFPE samples is standard medical practice, and these samples can be stored for long periods of time.

[0048] The term "patient" as used herein refers to any organism, such as a vertebrate, particularly any mammal, including both humans and other mammals, such as rodents, rabbits, or monkeys. Rodents can be mice, rats, hamsters, guinea pigs, or chinchillas. Preferably, the patient is a human.

[0049] In one aspect, the present invention relates to a method for determining an individual's HLA pattern from a tumor, the method comprising: determining a first expression level of an RNA transcript encoding a first region of a first HLA gene; determining a second expression level of an RNA transcript of a second region of a second HLA gene; and comparing the determined first and second expression levels to obtain the individual's HLA pattern, wherein the first HLA gene and the second HLA gene are selected from the group consisting of genes encoding HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H to HLA-J.

[0050] In some embodiments, the first HLA gene and the second HLA gene can encode different HLA groups. For example, the first HLA gene can encode one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J. Similarly, the second HLA gene can encode another gene selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J.

[0051] In such cases, the comparison of the expression levels of the first and second genes may be referred to as "intergenic."

[0052] In particular, the first HLA gene may encode a classical HLA gene, i.e., selected from the group consisting of HLA-A, HLA-B, and HLA-C, while the second HLA gene may encode a non-classical HLA gene or pseudogene, i.e., selected from the group consisting of HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J.

[0053] In other embodiments, the first and second HLA genes may be identical or may encode the same HLA group, for example, the first and second HLA genes may both encode one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J.

[0054] In such cases, the comparison of the first and second expression levels may be referred to as "intragenic."

[0055] In particular, the present disclosure relates to an in vitro method for determining the individual HLA pattern of a tumor in a patient, e.g., a cancer patient, comprising determining the expression level of an RNA transcript of at least one gene selected from the group consisting of adult HLA groups (e.g., HLA-A, HLA-B, HLA-C; MHC I), HLA D groups (DR, DP, DQ, etc.; MHC II), "embryonic" HLA (e.g., HLA-E, HLA-F, HLA-G), and HLA pseudogenes (e.g., HLA-H, HLA-J) in a sample of tumor tissue or blood, e.g., from the cancer patient.

[0056] According to the present invention, the term "RNA transcript" relates to transcripts in the sense and / or antisense orientation.

[0057] Preferably, the term includes and relates to "mRNA," which means "messenger RNA," and relates to a "transcript" that encodes a peptide or protein. mRNA typically comprises a 5'-untranslated region (5'-UTR), a protein or peptide coding region, and a 3'-untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro.

[0058] In other cases, such as antisense RNA (aRNA) transcripts, the RNA may not encode a peptide or protein. However, the RNA may be complementary to an mRNA and thereby regulate the translation of the corresponding sense mRNA into a peptide or protein. For the purposes of this disclosure, antisense RNA transcripts may be considered to be directed to an additional "region" (antisense region) of the respective "HLA gene" that is not directly translated into a protein or peptide of the HLA group.

[0059] In some cases, both the first expression level and the second expression level may be related to sense RNA transcripts. In other cases, the first expression level may be related to sense RNA transcripts, while the second expression level may be related to antisense RNA transcripts (or vice versa). Preferably, but not necessarily, the sense and antisense transcripts are related to the same HLA group. For example, the antisense transcript may be at least partially complementary to the sense transcript of the same HLA group.

[0060] The term "expression level" refers to, for example, the determined level of gene expression. The term "expression level pattern" refers to the determined level of gene expression compared to a reference gene, such as a housekeeper or counter-regulatory gene, or to the average expression value calculated, for example, in DNA chip analysis. The pattern is not limited to the comparison of two genes, but also relates to multiple comparisons of genes to a reference gene or sample. A particular "expression level pattern" can also be obtained and determined by comparing and measuring several genes as disclosed herein, and can indicate the relative abundance of these transcripts relative to each other. The expression level can also be evaluated by comparing the expression of the gene in different tissues, for example, cancer tissues versus non-cancerous tissues.

[0061] The term "expression level" as used herein refers to the expression of a particular gene (e.g., HLA-E, HLA-F, HLA-G) to produce a transcript and / or protein. According to the present invention, the expression level is determined at the RNA transcription level, particularly the mRNA level (transcription level), for example, by measuring the transcribed mRNA (e.g., by Northern blot), by reverse transcription (RT) quantitative PCR (RT-qPCR), or by directly staining the mRNA (e.g., by in situ hybridization).

[0062] In some embodiments, the expression level is normalized to the (average) expression level of one or more reference genes in tumor samples. As used herein, the term "reference gene" refers to a gene that has a relatively stable expression level at the RNA transcript / mRNA level in the system being tested, i.e., cancer. Such genes may be referred to as housekeeping genes. In some embodiments, the one or more reference genes are selected from the group including CALM2, B2M, RPL37A, GUSB, HPRT1, and GAPDH, preferably CALM2 and / or B2M. Other suitable reference genes are known to those skilled in the art.

[0063] Each of the first and second regions may include an exon-exon boundary, or may include a portion of one or less exons (ie, not including an exon-exon boundary).

[0064] For example, one of the first region and the second region (e.g., the first region) spans parts of two exons (i.e., includes an exon-exon boundary), and the other of the first region and the second region (e.g., the second region) includes parts of no more than one exon (i.e., does not include an exon-exon boundary).

[0065] Alternatively, the first region may include an exon-exon boundary (i.e., span portions of two exons), and the second region may include an exon-exon boundary. The first and second regions may or may not include portions of a common exon. For example, the first region may include the boundary between exon 2 and exon 3 (i.e., include the exon 2 / exon 3 boundary). In such cases, the second region may include a portion of any exon (e.g., exon 1, exon 2, exon 3, exon 4), or may include any exon-exon boundary (e.g., exon 3 / exon 4 boundary, exon 4 / exon 5 boundary, etc.). The group of exon-exon boundaries also includes boundaries formed by exon skipping, such as the exon 2 / exon 4 boundary.

[0066] In a further alternative, the first region includes a portion of no more than one exon and the second region includes a portion of no more than one exon.

[0067] In some embodiments, the first region may encode a signal peptide region of an HLA group, and the second region may encode a transmembrane region of an HLA group.

[0068] Generally, the method for determining an individual's HLA pattern may also include determining whether the individual's HLA pattern is primarily soluble or membrane-bound based on a comparison of the first and second expression levels. In particular, the individual's HLA pattern may be determined to be primarily soluble if the expression level of the region encoding the signal peptide region of the HLA group exceeds the expression level of the region encoding the transmembrane region of the HLA group. The individual's HLA pattern may be determined to be primarily membrane-bound if the expression level of the region encoding the transmembrane region of the HLA group is essentially equal to or exceeds the expression level of the region encoding the signal peptide region of the HLA group.

[0069] Generally, the method for determining an individual's HLA pattern may also include determining an HLA isoform based on a comparison of the first and second expression levels. In particular, if the expression level of the region encoding a portion of the first exon exceeds the expression level of the region encoding a portion of the second exon, the individual's HLA pattern may be determined to include one or more isoforms that include the first exon and do not include the second exon.

[0070] Generally, the method for determining the HLA pattern of an individual may also include determining one or more further expression levels (e.g., a third expression level) of one or more further regions (e.g., a third region) of the HLA group, and the comparison is further based on the determined further expression levels to obtain the HLA pattern of the individual.

[0071] The term "RNA expression level" refers to the determined level of transcribed RNA, the nascent unspliced ​​RNA transcript, or the DNA gene sequence information converted into mature mRNA. RNA expression can be monitored by measuring the level of either the total RNA of a gene or a subsequence.

[0072] In some embodiments, determining the expression level of an RNA transcript includes determining whether the expression level of the RNA transcript is lower or higher (= dichotomous) than a defined expression threshold for the RNA transcript. If the expression level is equal to the defined expression threshold, the expression level can be considered to belong to a group of expression levels higher than the defined expression threshold. Thus, as used herein, the phrase "higher than the defined expression threshold" includes expression levels that are equal to or greater than the defined expression threshold. An expression level "higher than the defined expression threshold" can also be referred to as "expression positive," and an expression level "lower than the defined expression threshold" can also be referred to as "expression negative."

[0073] In some embodiments, the expression levels of RNA transcripts encoding homologous regions of signal peptides of one or more HLAs are determined and set relative to the expression levels encoding homologous transmembrane regions of one or more HLAs and / or the diverse cytoplasmic tails of one or more HLAs are set relative to each other, so that the ratio of secreted alpha domains to transmembrane-localized HLAs can be determined for individual HLAs and HLA isoforms.

[0074] The step of "determining the expression levels of RNA transcripts" includes (i) measuring the expression levels of the RNA transcripts and (ii) analyzing the measured expression levels of the RNA transcripts (e.g., by comparison with a reference expression level, such as a defined expression threshold), where the order in which the expression levels of the RNA transcripts are measured may or may not be related to the order in which the measured RNA transcript expression levels are analyzed.

[0075] In some embodiments, the expression levels of RNA transcripts of at least one, two, three, or four genes selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, and HLA pseudogenes (HLA-H and / or HLA-J) are determined.

[0076] In some embodiments, the resulting individual's HLA pattern may indicate the presence and / or absence and / or expression level of one or more isoforms of an HLA group. For example, known isoforms of HLA-G include HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7. The same applies to the additional embryonic HLA groups HLA-E and HLA-F, and the HLA pseudogenes HLA-H and HLA-J. In such embodiments, it may be determined that additional, currently unknown, isoforms of an HLA group exist.

[0077] The resulting individual's HLA pattern, indicating the presence and / or expression levels of one or more isoforms of the HLA group, can further be used to identify the molecular subtype of a tumor and / or in methods to generate therapeutics.

[0078] Additionally or alternatively, indicating the presence and / or absence and / or expression levels of isoforms, particularly soluble isoforms, can be used (a) to stabilize embryo implantation in assisted reproductive technologies, e.g., in vitro fertilization (IVF), (b) to reduce the risk of transplant rejection, e.g., in host-versus-graft reactions, and / or (c) to reduce the risk of or minimize the effects of autoimmune outbreaks. Such uses can include, inter alia, producing media or therapeutic agents containing soluble, biologically active isoforms of HLA-E, HLA-F, and / or HLA-G. Examples of such soluble, biologically active isoforms include HLA-G5.

[0079] In some embodiments, the method further comprises determining the expression level of an RNA transcript of at least one gene selected from a group of immune genes (CD3, CD8, CD19, CD68, CD168, CSF1R, IGKC, IGHM, IFNG, etc.) in the tumor sample.

[0080] In one embodiment of the present disclosure, the combination of HLA typing with checkpoint signatures, exemplified by protein- and / or mRNA-based assessment of PD-L1, PD-L2, CD86, CD80, L-ICOS, B7-H3, B7-H4, PD1, CTLA4, CD28, and / or ICOS, is of particular interest when targeting checkpoint genes with specific inhibitors, such as humanized antibodies, in the clinical setting of advanced cancer. HLA typing provides HLA expression pattern information that adds value to exclusively quantifying checkpoint target genes for the prediction of response to chemotherapeutic agents and / or checkpoint inhibitors (such as anti-PD1 or anti-PD-L1 or anti-CTLA4 agents).

[0081] As one embodiment of the present disclosure, the combination of HLA typing with quantification of immune cell infiltration, exemplified by protein- and / or mRNA-based assessment of immune genes (CD3, CD8, CD19, CD68, CD168, CSF1R, IGKC, IGHM, IFNG, etc.), adds value to exclusively quantifying checkpoint target genes for response prediction to chemotherapeutic agents and / or anti-checkpoint drugs, especially when predicting response to neoadjuvant treatment strategies.

[0082] According to one or more embodiments of the invention, the immune checkpoint inhibitor comprises at least one selected from the group consisting of an antibody, an engineered antibody format, an antibody derivative or fragment that retains target binding properties, an antibody-based binding protein, an oligopeptide binder, and an antibody mimetic.

[0083] An "antibody," also referred to interchangeably as an "immunoglobulin" (Ig), generally refers to a multimeric protein comprising four polypeptide chains: two heavy (H) chains and two light (L) chains; and thus includes full-length functional mutants, variants, or derivatives thereof (including murine, chimeric, humanized, and fully human antibodies that retain the essential epitope-binding function of an Ig molecule, including bispecific, dual-specific, multispecific, and dual variable domain immunoglobulins), or their equivalent Ig homologs (e.g., camelid nanobodies, which comprise only heavy chains; single-domain antibodies (dAbs), which can be derived from either heavy or light chains). Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and allotype.

[0084] As used herein, an "antibody-based binding protein" refers to a protein that contains at least one antibody-derived V in the context of other non-immunoglobulin or non-antibody-derived components. H , V L , or C HIt can refer to any protein that contains an immunoglobulin domain. Such antibody-based proteins include: (i) immunoglobulin C H F binding proteins, including receptors or receptor components having all or part of the domain c fusion protein, (ii) V H and / or V L a binding protein, or (iii) immunoglobulin V, in which the domain is attached to an alternative molecular scaffold. H and / or V L , and / or C H These include, but are not limited to, molecules in which domains are joined and / or assembled in a manner not normally found in naturally occurring antibodies or antibody fragments.

[0085] As used herein, "antibody derivative or fragment" refers to a molecule that comprises at least one polypeptide chain derived from an antibody that is not full-length, including, but not limited to, (i) a variable light chain (V L ), variable heavy chain (V H ), constant light chain (C L ), and constant heavy chain 1 (C H (1) a Fab fragment, which is a monovalent fragment consisting of a domain; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and C H (iv) the heavy chain portion of the Fab (Fd) fragment, consisting of one domain, and (v) the V of a single arm of the antibody. L and V H (v) a domain antibody (dAb) fragment comprising a single variable domain; (vi) an isolated complementarity-determining region (CDR); (vii) a single-chain Fv fragment (scFv); (viii) a V H and V L The domains are expressed on a single polypeptide chain, but are too short to allow pairing between the two domains on the same chain 1644457389430_0 by pairing a domain with a complementary domain on another strand to form two 1644457389430_1 (ix) a pair of tandem Fv segments (V) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions; H -C H 1-V H -C H (i) a molecule comprising at least one polypeptide chain derived from an antibody that is not full length, including, but not limited to, (i) a linear antibody, including, but not limited to, (ii) other non-full-length portions of immunoglobulin heavy and / or light chains, or mutants, variants, or derivatives thereof, in any case, such derivatives or fragments retain target binding properties.

[0086] As used herein, the term "modified antibody format" encompasses antibody-drug conjugates, polyalkylene oxide modified scFvs, monobodies, diabodies, camelid antibodies, domain antibodies, bi- or triabodies, IgA or two IgG structures linked by a J chain and secretory component, shark antibodies, New World primate framework + non-New World primate CDRs, IgG4 antibodies with the hinge region removed, IgGs with two additional binding sites engineered into the CH3 domain, antibodies with Fc regions altered to enhance affinity for Fcγ receptors, and dimerization constructs comprising CH3 + VL + VH, etc.

[0087] The term "antibody mimic" as used herein refers to proteins that do not belong to the immunoglobulin family, as well as non-protein or synthetic polymers such as aptamers. Some species have an antibody-like beta-sheet structure. Potential advantages of "antibody mimics" or "alternative scaffolds" over antibodies include good solubility, high tissue penetration, high thermal and enzymatic stability, and relatively low production costs.

[0088] Several antibody mimetics can be provided in large libraries that provide specific binding candidates for all possible targets. As with antibodies, target-specific antibody mimetics can be developed using high-throughput screening (HTS) techniques and established display technologies such as phage display, bacterial display, yeast display, or mammalian display. Antibody mimetics currently in development include, for example, ankyrin repeat proteins (called DARPins), C-type lectins, A-domain proteins of S. aureus, transferrin, lipocalin, the tenth type III domain of fibronectin, Kunitz domain protease inhibitors, ubiquitin-derived binders (called affilins), gamma-crystallin-derived binders, cysteine ​​knots or knottins, thioredoxin A scaffold-based binders, SH-3 domains, stradobodies, the "A domains" of disulfide-bonded and Ca2+-stabilized membrane receptors, CTLA4-based compounds, Fyn SH3, and aptamers (peptide molecules that bind to specific target molecules).

[0089] According to one or more embodiments of the present invention, the immune checkpoint inhibitor comprises at least one selected from the group set forth in Table 1. In Table 1, DART stands for "Dual-Affinity Re-Targeting," mAb stands for "Monoclonal Antibody," and NA stands for "Not Applicable."

[0090] [Table 1] TIFF0007762135000002.tif61170

[0091] In another aspect, the present invention relates to the use of the above method in the treatment of cancer, comprising as a first step stratifying a cancer patient for tumor treatment and as a second step providing the cancer patient with a selected anti-HLA tumor treatment regimen.

[0092] "Stratifying cancer patients for tumor treatment" according to the present invention includes allocating cancer patients to patient groups with specific molecular tumor subtypes, which allows medical professionals to select the most suitable tumor treatment regimen.

[0093] In some embodiments of the present disclosure, the treatment involves the use of humanized antibodies or RNA or protein-based immunizations raised against specific HLA isoforms thereof for patients suffering from neoplastic diseases.

[0094] In some embodiments, the use may include generating soluble HLA domains ex vivo. For example, the generation may include combining any of the naturally occurring alpha domains as synthetic monomers (α1, α2, α3), or multimers in naturally occurring order (e.g., α1α2α3, α1α2, α1α3) or de novo ordered multimers (α2α3), or de novo concatemers (e.g., α1α1α1, α2α2α2, α3α3α3, α1α1α2, α1α1α3, α1α1α1α1α2α2α2α3α3α3, etc.).

[0095] The therapeutic agents may be applied to patients suffering from disorders associated with autoimmune diseases or to pregnant women at potential risk of early miscarriage to alleviate autoimmune symptoms and enhance immune tolerance to enable the pregnancy to continue to term.

[0096] Generally, a method for producing a therapeutic may involve determining an individual's HLA pattern using the methods described above and producing the therapeutic.

[0097] In some embodiments, a therapeutic agent may comprise a protein, protein domain, and / or polypeptide such that the therapeutic agent specifically binds to the determined individual's HLA pattern. Such binding of the therapeutic agent may result in the disruption of binding or interaction between the determined individual's HLA pattern and a ligand or receptor, for example, on an immunocompetent cell.

[0098] For example, the therapeutic agent may comprise a soluble HLA domain or an antibody based on the determined HLA pattern of the individual.

[0099] In some embodiments, the therapeutic agent may comprise a nucleic acid, particularly RNA. Such RNA may encode an antigen that can be synthesized by the immune system after injection of the therapeutic agent in accordance with known RNA vaccination techniques. As a result of such translation of the therapeutic agent into an antigen, an immunocompetent cell response can be triggered, regardless of the individual's determined HLA pattern.

[0100] For example, the therapeutic agent may comprise a soluble HLA domain or an antibody based on the determined HLA pattern of the individual.

[0101] In some embodiments, all of the above combinations of synthetic alpha domains can occur in cis (i.e., by combining the alpha domains of only one single HLA gene, such as HLA-G or HLA-F or HLA-E or HLA-A) or in trans (i.e., by combining the alpha domains of two or more HLA genes, such as HLA-G and HLA-E or HLA-F or HLA-A; HLA-A and HLA-E or HLA-F or HLA-G).

[0102] In some embodiments, the above synthetic alpha domains of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F are bioengineered to contain additional cysteines in positions similar to the substitutions in HLA-G to obtain soluble HLA alpha domains capable of dimerization and / or multimerization to reduce diffusion and increase local retention of the applied HLA alpha domain.

[0103] In yet another aspect, the present invention relates to a kit for identifying the molecular subtype of a tumor, for example in a bladder cancer patient, by reverse transcription (RT) quantitative PCR (RT-qPCR), comprising at least one pair of primers and at least one probe specific for a gene selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J.

[0104] The present invention also relates to the use of the above kit for identifying the molecular subtype of a tumor.

[0105] The term "bladder cancer" refers to a type of cancer that originates in bladder or urethral tissue. In some embodiments, the bladder cancer is non-muscle-invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC). Occasionally, bladder cancer is metastatic. Common sites of metastasis include bone, liver, lung, and brain. Bladder cancer occurs in humans and other mammals. The majority of human cases occur in men, but bladder cancer can also occur in women. In general, treatment for bladder cancer can include surgery, drug therapy (such as immunotherapy and / or chemotherapy and / or immunotherapy with BCG), radiation therapy, and / or targeted therapy.

[0106] Nearly all bladder cancers begin in the urothelium. The cancer progresses as it grows into or through other layers within the bladder, and stages are determined according to the tumor's progression through deeper tissue layers. Stages are defined as follows: Ta (pTa): papillary carcinoma in situ; Tis (pTis): flat carcinoma in situ (flat carcinoma in situ, or CIS); T1 (pT1): the tumor has grown from the inner cell layer of the bladder into the underlying connective tissue but is still considered NMIBC; T2 (pT2): the tumor has grown into the muscle layer (MIBC); T3 (pT3): the tumor has grown through the muscle layer of the bladder into the surrounding fatty tissue layer; and T4 (pT4): the tumor has spread beyond the fatty tissue to nearby organs or structures. Tumors can grow into any of the following: the stroma (main tissue) of the prostate, seminal vesicles, uterus, vagina, pelvic wall, or abdominal wall.

[0107] "Primer pair" and "probe" within the meaning of the present invention shall have the ordinary meaning of these terms, which are well known to those skilled in the art of molecular biology. In preferred embodiments of the present invention, "primer pair" and "probe" shall be understood to be polynucleotide molecules having a sequence identical to, complementary to, or homologous to the complement of a region of a target polynucleotide to be detected or quantified. In some embodiments, nucleotide analogs are also included for use as primers and / or probes. Probe technologies used in kinetic or real-time PCR applications may be, for example, the TaqMan® system available from Roche Molecular Diagnostics, extension probes such as Scorpion® Primers, Dual Hybridization Probes, Amplifluor® available from Chemicon International, Inc., or Minor Groove Binders.

[0108] In some embodiments, the kit comprises specific primer pairs and specific probes for at least two, three, or four genes selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J.

[0109] In some embodiments, the kit comprises: - at least one pair of HLA-G specific primers and at least one HLA-E specific probe; at least one pair of HLA-G-specific primers and at least one HLA-F-specific probe; and / or - at least one pair of HLA-F specific primers and at least one HLA-G specific probe.

[0110] In some embodiments, the kit comprises at least one pair of HLA group-specific primers and at least one HLA group-specific probe, wherein both the primer pair and the probe are specific for a gene selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J.

[0111] Preferably, primers for use in accordance with the present invention have a length of 15-30 nucleotides, particularly deoxyribonucleotides. In some embodiments, primers (1) are specific for a target mRNA sequence (e.g., HLA-E, HLA-F, or HLA-G), (2) provide an amplicon size of less than 120 bp (preferably less than 100 bp), (3) detect all known protein-coding splicing variants, (4) are free of known polymorphisms (e.g., single nucleotide polymorphisms, SNPs), (5) are mRNA-specific (taking into account exons / introns; preferably without DNA amplification), (6) are not prone to dimerization, and / or (7) have a melting temperature T in the range of 58°C to 62°C. m (preferably T m is approximately 60°C).

[0112] As used herein, the term "nucleotide" includes natural (naturally occurring) nucleotides, which include a nucleobase selected from the group consisting of adenine (A), thymidine (T), cytosine (C), guanine (G), and uracil (U), a sugar selected from the group consisting of ribose, arabinose, xylose, pyranose, and deoxyribose (the base-sugar combination commonly referred to as a "nucleoside"), and one to three phosphate groups capable of forming a phosphodiester internucleoside linkage. Additionally, as used herein, "nucleotide" refers to a nucleotide analog. As used herein, "nucleotide analog" refers to an analog of A, G, C, T, or U (i.e., an analog of a nucleotide containing the base A, G, C, T, or U) that is recognized by a DNA or RNA polymerase (whichever is appropriate) and incorporated into a DNA or RNA strand (whichever is appropriate). Examples of such nucleotide analogs include, but are not limited to, 5-propynylpyrimidines (i.e., 5-propynyl-dTTP and 5-propynyl-dCTP), 7-deazapurines (i.e., 7-deaza-dATP and 7-deaza-dGTP), aminoallyl-dNTPs, biotin-AA-dNTPs, 2-amino-dATP, 5-methyl-dCTP, 5-iodo-dUTP, 5-bromo-dUTP, 5-fluoro-dUTP, N4-methyl-dCTP, 2-thio-dTTP, 4-thio-dTTP, and alpha-thio-dNTPs. Also included are labeled analogs, e.g., fluorescent analogs such as DEAC-propylenediamine (PDA)-ATP, analogs based on morpholino nucleoside analogs, and locked nucleic acid (LNA) analogs.

[0113] The phrase "specific for a target mRNA sequence" when used in connection with primers for use in accordance with the present invention refers to the ability of the primer to hybridize (i.e., anneal) to the cDNA of the target mRNA sequence under appropriate conditions of temperature and solution ionic strength, particularly under PCR conditions. These conditions determine the stringency of hybridization. Hybridization requires that the two nucleic acids (i.e., the primer and the DNA) contain complementary sequences, although mismatches between bases may occur depending on the stringency of hybridization. In some embodiments, "appropriate conditions of temperature and solution ionic strength" refer to a temperature in the range of 58°C to 62°C (preferably a temperature of about 60°C) and the ionic strength of the solution typically used in PCR reaction mixtures. In some embodiments, the sequence of the primer is 80%, preferably 85%, more preferably 90%, and even more preferably 95%, 96%, 97%, 98%, 99% or 100% complementary to the corresponding sequence of the cDNA of the target mRNA sequence as determined by sequence comparison algorithms known in the art.

[0114] For example, a primer may hybridize to a cDNA of a target mRNA sequence under stringent or moderately stringent hybridization conditions. "Stringent hybridization conditions" as described herein may involve hybridization in 5xSSC / 5xDenhardt's solution / 1.0% SDS at 68°C, followed by washing in 0.2xSSC / 0.1% SDS at room temperature, or an art-recognized equivalent thereof (e.g., hybridization in 2.5xSSC buffer at 60°C, followed by several wash steps at 37°C with low buffer concentrations to maintain stability). "Moderately stringent hybridization conditions" as defined herein involve washing in 3xSSC at 42°C, or an art-recognized equivalent thereof. Salt concentration and temperature parameters may be varied to achieve an optimal level of identity between the primer and the target nucleic acid. Guidance regarding such conditions can be found, for example, in J. Sambrook et al. eds., 2000, Molecular Cloning: A Laboratory Manual, 3 rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor; (Non-Patent Document 1) and Ausubel et al. eds., 1995, Current Protocols in Molecular Biology, John Wiley and Sons, NY (Non-Patent Document 2).

[0115] In some embodiments, the probe hybridizes to the (amplified) cDNA of the target mRNA sequence under stringent or moderately stringent hybridization conditions as defined above.

[0116] Preferably, probes for use in accordance with the present invention have a length of 20 to 35 nucleotides, particularly deoxyribonucleotides. In some embodiments, the probes (1) are specific for a target mRNA sequence (e.g., HLA-E, HLA-F, or HLA-G), (2) contain no known polymorphisms (e.g., single nucleotide polymorphisms, SNPs), and / or (3) have a melting temperature, T, of the corresponding primer. m The melting temperature T is approximately 5 to 8 degrees higher than m The device is designed to have:

[0117] The phrase "specific for a target mRNA sequence" when used in connection with a probe for use in accordance with the present invention refers to the ability of the probe to hybridize (i.e., anneal) to the (amplified) cDNA of the target mRNA sequence under suitable conditions of temperature and solution ionic strength, particularly under PCR conditions. The conditions of temperature and solution ionic strength determine the stringency of hybridization. Hybridization requires that the two nucleic acids (i.e., the probe and the cDNA) contain complementary sequences, although mismatches between bases may occur depending on the stringency of hybridization. In some embodiments, "suitable conditions of temperature and solution ionic strength" refer to temperatures ranging from 63°C to 70°C and the ionic strength of solutions typically used in PCR reaction mixtures. In some embodiments, the sequence of the probe is 80%, preferably 85%, more preferably 90%, and even more preferably 95%, 96%, 97%, 98%, 99% or 100% complementary to the corresponding sequence of the (amplified) cDNA of the target mRNA sequence as determined by sequence comparison algorithms known in the art.

[0118] In some embodiments, the method comprises the use of HLA-A specific primers comprising or having the sequences of SEQ ID NOs: 1, 2, 3, and / or HLA-B / C specific and / or HLA-G specific primers comprising or having the sequences of SEQ ID NOs: 4, 5, 6, and / or HLA-H specific primers comprising or having the sequences of SEQ ID NOs: 7-27, and / or HLA-H specific primers comprising or having the sequences of SEQ ID NOs: 28-33.

[0119] In some embodiments, the quantitative PCR is fluorescence-based quantitative real-time PCR.

[0120] In some embodiments, detection of the probe is based on amplification-mediated probe displacement.

[0121] In some embodiments, the probe is a dual-labeled probe comprising a fluorescent reporter moiety and a fluorescent quencher moiety.

[0122] In some embodiments, the kit further comprises a reverse transcriptase and a DNA polymerase.

[0123] In some embodiments, the reverse transcriptase and DNA polymerase are provided in the form of an enzyme mixture that allows for one-step reverse transcription (RT) quantitative PCR (RT-qPCR).

[0124] In some embodiments, the kit further comprises at least one pair of reference gene-specific primers and at least one reference gene-specific probe.

[0125] In some embodiments, the reference gene is one or more selected from the group consisting of CALM2, B2M, RPL37A, GUSB, HPRT1, and GAPDH. As used herein, CALM2 refers to calmodulin-2, phosphorylase kinase, delta (Reference Sequence (mRNA): NM_001743), B2M refers to beta-2 microglobulin (Reference Sequence (mRNA): NM_004048), RPL37A refers to 60S ribosomal protein L37a (Reference Sequence (mRNA): NM_000998), GUSB refers to beta-glucuronidase (Reference Sequence (mRNA): NM_000181), HPRT1 refers to hypoxanthine-phosphoribosyltransferase 1 (Reference Sequence (mRNA): NM_000194), and GAPDH refers to glyceraldehyde-3-phosphate-dehydrogenase (Reference Sequence (mRNA): NM_002046).

[0126] In some embodiments, the kit further comprises at least one control RNA sample.

[0127] In some embodiments, the primers provide an amplicon size of less than 120 bp.

[0128] In some embodiments, the HLA-A-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 1, 2, or 3, and / or the HLA-B / C-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 4, 5, or 6, and / or the HLA-G-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of one of the sequences of SEQ ID NO: 7-27, and / or the HLA-H-specific primer has a length of 15 to 30 nucleotides and comprises at least 10 consecutive nucleotides of one of the sequences of SEQ ID NO: 28-33.

[0129] In some embodiments, the cancer indication is breast cancer, ovarian cancer, lung cancer, bladder cancer, gastric cancer, or colon cancer.

[0130] In a further aspect, the present invention relates to the use of the expression levels of HLA-E RNA transcripts, and / or the expression levels of HLA-F RNA transcripts, and / or the expression levels of HLA-G RNA transcripts as prognostic or predictive biomarkers for cancer, in particular as predictive biomarkers indicative of resistance to chemotherapy or indicative of resistance to immunotherapy.

[0131] The term "marker" or "biomarker" refers to a biological molecule, e.g., a nucleic acid, peptide, protein, hormone, etc., whose presence or concentration can be detected and correlated, e.g., by a mathematical algorithm, with a known condition, such as a disease state, or a combination thereof.

[0132] The term "prognostic marker" as used herein refers to a marker that provides information about the likely course of a respective disease (e.g., bladder cancer) in treated or untreated patients. In some embodiments, the prognosis includes one or more of disease-specific survival (DSS), recurrence-free survival (RFS), progression-free survival (PFS), and distant recurrence-free survival. In some embodiments, the prognosis includes DSS. The term "single prognostic biomarker" as used herein means that no additional prognostic markers are used / analyzed for prognosis.

[0133] In some embodiments, the expression level of an HLA-E RNA transcript or the expression level of an HLA-F RNA transcript or the expression level of an HLA-G RNA transcript is used as a single prognostic biomarker.

[0134] In some embodiments, - an expression level of the RNA transcript of HLA-E lower than a defined expression threshold of the RNA transcript of HLA-G indicates a positive prognosis, and / or - an expression level of the HLA-F RNA transcript lower than a defined expression threshold of the HLA-F RNA transcript indicates a negative prognosis, and / or - An expression level of HLA-F RNA transcripts lower than a defined expression threshold of HLA-G RNA transcripts indicates a positive prognosis.

[0135] In some embodiments, a positive prognosis includes an increased probability of one or more of prolonged disease-specific survival (DSS), recurrence-free survival (RFS), progression-free survival (PFS), and distant recurrence-free survival, preferably DSS.

[0136] In some embodiments, - an expression level of HLA-E RNA transcripts higher than a defined expression threshold of HLA-E RNA transcripts indicates a negative prognosis, and / or - an expression level of HLA-F RNA transcripts higher than a defined expression threshold of HLA-F RNA transcripts indicates a positive prognosis, and / or - An expression level of HLA-G RNA transcripts higher than a defined expression threshold of HLA-F RNA transcripts indicates a negative prognosis.

[0137] In some embodiments, a negative prognosis comprises a reduced probability of one or more of prolonged disease-specific survival (DSS), recurrence-free survival (RFS), progression-free survival (PFS), and distant recurrence-free survival, preferably DSS.

[0138] In another aspect, the present invention relates to the use of a pair of primers as defined herein and / or a probe as defined herein for identifying the molecular subtype of a tumor in a bladder cancer patient, e.g. in a method as defined herein, wherein the pair of primers and / or the probe are specific for a gene selected from the group consisting of HLA-E, HLA-F, and HLA-G.

[0139] In some embodiments, the probe is a dual-labeled probe comprising a fluorescent reporter moiety and a fluorescent quencher moiety.

[0140] In yet another aspect, the present invention relates to the use of a pair of primers as defined herein and / or a probe as defined herein for the manufacture of a kit for identifying the molecular subtype of a tumor in a bladder cancer patient by reverse transcription (RT) quantitative PCR (RT-qPCR), wherein the pair of primers and / or the probe are specific for a gene selected from the group consisting of HLA-E, HLA-F, HLA-G, HLA-H, or HLA-J.

[0141] In some embodiments, the probe is a dual-labeled probe comprising a fluorescent reporter moiety and a fluorescent quencher moiety.

[0142] In one aspect, the present invention relates to an in vitro method for identifying the molecular subtype of a tumor in a patient with cancer, comprising determining the expression level of an RNA transcript and then determining HLA expression as described above.

[0143] As used herein, the term "molecular subtype of tumor" (or "molecular subtype of cancer") refers to a subtype of tumor / cancer characterized by a distinct molecular profile, e.g., gene expression profile.

[0144] In some embodiments, the method comprises determining the expression levels, particularly the expression levels of RNA transcripts, of one or more additional non-reference genes.

[0145] The term "non-reference gene" as used herein is meant to refer to a gene that has variable levels of expression at the RNA transcript / mRNA level in the system being tested, i.e., cancer, and can therefore be used, for example, for tumor / cancer subtyping and / or assessing cancer progression. In some embodiments, the non-reference gene is selected from tumor markers, e.g., those known from the prior art. Non-reference genes that can be used in accordance with the present invention can be bladder-specific or non-bladder-specific genes.

[0146] In some embodiments, the method does not include determining the expression levels, particularly the expression levels of RNA transcripts, of four or more, three or more, or two or more additional non-reference genes.

[0147] In some embodiments, the method does not include determining the expression levels of any additional non-reference genes, in particular the expression levels of their RNA transcripts, i.e., the expression levels of genes other than HLA-E, HLA-F, HLA-G, HLA-H, or HLA-J, and optionally at least one gene selected from HLA-A, HLA-B, HLA-C, and HLA-D, and optionally one or more reference genes, in particular the expression levels of their RNA transcripts, are not determined.

[0148] In some embodiments, the expression levels of RNA transcripts of up to seven, preferably six, more preferably five, and even more preferably four different non-reference genes are determined.

[0149] In some embodiments, the method does not include any other diagnostic steps, such as histological grading or determination of lymph node status, hi some embodiments, the method does not include any steps involving immunohistochemistry (IHC).

[0150] In some embodiments of the invention, the tumor is a solid tumor. In some embodiments, the tumor is a bladder or urethral tumor, or originates from a bladder or urethral tumor (e.g., by metastasis). As used herein, the term "bladder" refers to the urinary bladder.

[0151] In some embodiments, the tumor sample can be a tumor tissue sample isolated from a cancer patient (e.g., a tumor biopsy or resected tissue). In preferred embodiments, the tumor tissue sample is a frozen section of a tumor tissue sample or a chemically fixed tumor tissue sample. In more preferred embodiments, the tumor tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tumor tissue sample. In some embodiments, the tumor sample is (total) RNA extracted from a tumor tissue sample. In particularly preferred embodiments, the tumor sample is (total) RNA extracted from an FFPE tumor tissue sample. Those skilled in the art can perform the RNA extraction procedure. For example, total RNA from 5-10 μm FFPE tumor tissue rolls can be extracted using the High Pure RNA Paraffin Kit (Roche, Basel, Switzerland), the XTRAKT RNA Extraction Kit XL (Stratifyer Molecular Pathology, Cologne, Germany), or the RNXtract® Extraction Kit (BioNTech Diagnostics GmbH, Mainz, Germany). It is also possible to store the sample materials to be used / tested in a freezer and thaw each sample material before carrying out the method of the present invention at an appropriate time. Samples may be obtained from cancer patients before the start of therapeutic treatment, during therapeutic treatment, and / or after therapeutic treatment, i.e., before, during, or after administration of a cancer therapeutic agent.

[0152] In a further aspect, the present invention relates to a method for stratifying patients, e.g., bladder cancer patients, for tumor treatment, comprising, as a first step, identifying a molecular subtype of the tumor in the cancer patient using the in vitro method defined above, and, as a second step, selecting a tumor treatment regimen based on the molecular subtype identified by the in vitro method.

[0153] In some embodiments, the method for stratifying bladder cancer patients for tumor treatment does not include any other diagnostic steps, such as histological grading or determination of lymph node status, other than identifying the molecular subtype of the tumor in the cancer patient using the in vitro method defined above. In some embodiments, the method does not include any step involving immunohistochemistry (IHC).

[0154] In some embodiments, the molecular subtype is selected from the group consisting of HER2-positive, triple-negative (also referred to as "basal-like"), luminal A, and luminal B. The term "basal-like" refers to the fact that such tumors share some similarity in gene expression to that of basal epithelial cells. The term "luminal" is derived from the similarity in gene expression between the tumor and luminal epithelium.

[0155] In some embodiments, the expression levels of HER2, ESR1, and Ki67 RNA transcripts are determined, and molecular subtypes are selected from the group comprising, preferably consisting of, HER2+, HER2- / ESR1+, HER2- / ESR1- / Ki67+, and HER2- / ESR1- / Ki67-. In some embodiments, the molecular subtypes are associated with MIBC. Molecular subtypes can vary significantly in clinical outcome and response to treatment.

[0156] In some embodiments, - the molecular subtype is HER2-positive and the tumor treatment regimen includes transurethral resection and / or BCG instillation and / or chemotherapy and / or anti-HER2 therapy and / or administration of antibodies targeting immune checkpoints and / or cystectomy, followed by administration of anti-HER2 therapeutic agents and / or chemotherapy agents; - the molecular subtype is triple-negative and the tumor treatment regimen includes transurethral resection and / or BCG instillation and / or chemotherapy, in particular neoadjuvant chemotherapy, and / or administration of antibodies targeting immune checkpoints and / or cystectomy; the molecular subtype is luminal A and the tumor treatment regimen comprises transurethral resection and / or BCG instillation and / or cystectomy and / or chemotherapy, in particular adjuvant chemotherapy, and / or (adjuvant) endocrine therapy; and / or - the molecular subtype is luminal B and the tumor treatment regimen comprises transurethral resection and / or BCG instillation and / or endocrine therapy and / or chemotherapy, in particular adjuvant or perioperative chemotherapy, and / or cystectomy.

[0157] In some embodiments, - the molecular subtype is luminal A, the bladder cancer is NMIBC, and the tumor treatment regimen comprises transurethral resection (TUR) and / or Bacillus Calmette-Guerin (BCG) instillation, preferably TUR and BCG instillation; - the molecular subtype is luminal B, the bladder cancer is NMIBC, and the oncotherapy regimen includes adjuvant chemotherapy with or without adjuvant endocrine therapy; - The molecular subtype is HER2-positive, the bladder cancer is NMIBC, and the tumor treatment regimen includes (neo)adjuvant chemotherapy with or without (neo)adjuvant anti-HER2 therapy; - The molecular subtype is triple-negative, the bladder cancer is NMIBC, and the tumor treatment regimen includes neoadjuvant chemotherapy; - the molecular subtype is luminal A, the bladder cancer is MIBC, and the tumor treatment regimen comprises (i) cystectomy and (ii) adjuvant chemotherapy and / or adjuvant endocrine therapy, preferably adjuvant chemotherapy or adjuvant endocrine therapy; - the molecular subtype is luminal B, the bladder cancer is MIBC, and the tumor treatment regimen comprises (i) cystectomy and (ii) adjuvant chemotherapy and / or adjuvant endocrine therapy, preferably adjuvant chemotherapy and adjuvant endocrine therapy; - the molecular subtype is HER2 positive, the bladder cancer is MIBC, and the tumor treatment regimen comprises (i) cystectomy and (ii) adjuvant chemotherapy and / or adjuvant anti-HER2 therapy, preferably adjuvant chemotherapy and adjuvant anti-HER2 therapy; and / or -The molecular subtype is triple negative, the bladder cancer is MIBC, and the oncotherapy regimen includes neoadjuvant chemotherapy with or without subsequent cystectomy.

[0158] In some embodiments, the molecular subtype is HER2-positive, the cancer is preferably NMIBC, and the tumor treatment regimen comprises anti-HER2 therapy in combination with endocrine therapy, hormonal therapy, and / or chemotherapy, all of which may be in the form of adjuvant or neoadjuvant therapy.

[0159] In another embodiment, the molecular subtype is HER2-positive, the cancer is preferably NMIBC, and the tumor treatment regimen comprises intravenous instillation therapy, for example, BCG instillation.

[0160] In some embodiments, the molecular subtype is luminal B, the bladder cancer is preferably NMIBC, and the tumor treatment regimen comprises intravenous therapy (e.g., BCG instillation) and / or neoadjuvant / adjuvant chemotherapy, preferably in combination with endocrine therapy, e.g., in combination with tamoxifen (Nolvadex®), fulvestrant (Faslodex®), or an aromatase inhibitor.

[0161] The meaning of the term "anti-HER2 therapy" is known to those skilled in the art. In some embodiments, anti-HER2 therapy involves the administration of an anti-HER2 antibody, particularly a monoclonal anti-HER2 antibody. Monoclonal anti-HER2 antibodies include trastuzumab (Herceptin®) and pertuzumab (Perjeta®), which can be administered alone or in combination. Trastuzumab is effective only in cancers in which HER2 is overexpressed. Other monoclonal antibodies, such as ertumaxomab (Rexomun®), are currently undergoing clinical trials. Anti-HER2 antibodies can be further modified to include a therapeutic moiety / therapeutic agent, such as a cytotoxic agent, a drug (e.g., an immunosuppressant), a chemotherapeutic agent, or a radionuclide, or a radioisotope. Thus, when a tumor treatment regimen includes anti-HER2 therapy and chemotherapy (in combination), an anti-HER2 antibody conjugated to a chemotherapeutic agent can be used. A cytotoxin or cytotoxic agent includes any agent that is harmful to, and in particular kills, cells. Examples include mertansine or emtansine (DM1), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, dione, mitoxantrone, mithramycin, actinomycin D, amanitin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. In some embodiments, the antibody conjugate is trastuzumab (T)-DM1, e.g., trastuzumab emtansine.Other suitable therapeutic agents for forming antibody conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimetabolites (e.g., vincristine and vinblastine). In preferred embodiments, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In some embodiments, the therapeutic agent is GM-CSF. In another preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A. Additional therapeutic moieties include therapeutic moieties that affect mRNA and / or protein synthesis. Several transcription inhibitors are known. For example, actinomycin D, which is both a transcription inhibitor and a DNA damaging agent, intercalates into DNA, thus inhibiting the initiation step of transcription. Flavopiridol targets the elongation step of transcription. α-amanitin binds directly to RNA polymerase II, thereby inhibiting both the initiation and elongation steps. Anti-HER2 antibodies can also be conjugated to radioisotopes, such as iodine-131, yttrium-90, or indium-111, to generate cytotoxic radiopharmaceuticals. An alternative to administering anti-HER2 antibodies is the administration of small compounds that target HER2, such as lapatinib (Tykerb® or Tyverb®), afatinib, or neratinib. Anti-HER2 therapy can also be supplemented with endocrine therapy (also called anti-hormonal therapy), e.g., hormone therapy with progestins, and / or chemotherapy.

[0162] Chemotherapy involves the administration of chemotherapeutic agents, such as cytostatic or cytotoxic compounds. Traditional chemotherapeutic agents act by killing rapidly dividing cells, one of the primary characteristics of most cancer cells. The term "chemotherapeutic agent" includes taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines and anthracycline analogs, etoposide, bleomycin, vinorelbine, cyclophosphamide, antimetabolites, antimitotic agents, and alkylating agents, including those disclosed above in connection with antibody conjugates, and combinations thereof. In some embodiments, chemotherapy is platinum-based, i.e., involves the administration of platinum-based compounds, such as cisplatin. Reference to a chemotherapeutic agent may include any prodrugs, e.g., derivatives such as esters, salts, or conjugates of the agent. An example is a conjugate of the agent with a carrier substance, e.g., protein-bound paclitaxel, such as albumin-bound paclitaxel. Preferably, the salts of the agent are pharmaceutically acceptable. Chemotherapy drugs are often administered in combination, usually for three to six months. One of the most common treatments is cyclophosphamide and doxorubicin (Adriamycin; a member of the anthracycline and anthracycline analogues group), known as AC. Sometimes, a taxane drug, such as docetaxel, is added, and the regimen is known as CAT. The taxane attacks microtubules in cancer cells. Another common treatment with comparable results is cyclophosphamide, the antimetabolite methotrexate, and the nucleoside analogue (CMF) fluorouracil. Another standard chemotherapy regimen includes fluorouracil, epirubicin, and cyclophosphamide (FEC), which can be supplemented with a taxane, such as docetaxel, or vinorelbine.

[0163] In some embodiments, the molecular subtype is luminal B and the tumor treatment regimen comprises administration of a chemotherapeutic agent. In some embodiments, the molecular subtype is luminal B and the tumor treatment regimen comprises administration of a taxane, preferably docetaxel. In some embodiments, the taxane is administered in combination with platinum-based chemotherapy.

[0164] Endocrine therapy (also called antihormonal therapy) targets cancers that require a constant supply of estrogen by administering drugs that block / downregulate estrogen and / or progesterone receptors, such as tamoxifen (Nolvadex®) or fulvestrant (Faslodex®), or drugs that prevent estrogen production with aromatase inhibitors, such as anastrozole (Arimidex®) or letrozole (Femara®). However, aromatase inhibitors are only suitable for postmenopausal patients. This is because these drugs are not effective at inhibiting the predominant aromatase in premenopausal women, as the active aromatase in postmenopausal women is different from the form typically found in premenopausal women.

[0165] In another aspect, the present invention can be used for the treatment of cancer, the method comprising, as a first step, stratifying a bladder cancer patient for tumor treatment using the method defined above, and, as a second step, providing the bladder cancer patient with a selected tumor treatment regimen, wherein the tumor treatment regimen is selected based on the molecular subtype identified by the in vitro method defined above.

[0166] The first and second steps of the above method may be performed separately from each other in terms of time and / or location. The first step may, for example, provide a treatment guideline that is used to perform the second step at a different time and / or location. The second step may be performed immediately after the first step.

[0167] In some embodiments, the above methods comprise using the quantitative results obtained by the in vitro methods defined above for direct decision making in favor of or against adjuvant / neoadjuvant chemotherapy.

[0168] In another aspect, the present invention may be used to treat cancer, wherein the bladder cancer is characterized by a molecular subtype as defined herein, and the method comprises providing a tumor treatment regimen that is selected based on the molecular subtype.

[0169] In another aspect, the present invention relates to a method for producing a therapeutic agent, the method comprising determining an HLA pattern of an individual using the method described above, and producing soluble HLA domains or antibodies based on the determined HLA pattern of the individual. The present invention also relates to a therapeutic agent produced as described above for use in treating cancer. [Brief explanation of the drawings]

[0170] [Figure 1] FIG. 1 shows a sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at potential translation start sites according to Example 2. [Figure 2a] Figure 2 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at the junction from exon 4 to exon 5. [Figure 2b] Figure 2 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at the junction from exon 4 to exon 5. [Figure 2c]Figure 2 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at the junction from exon 4 to exon 5. [Figure 2d] Figure 2 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at the junction from exon 4 to exon 5. [Figure 3a] Figure 3 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H in exon 8. [Figure 3b] Figure 3 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H in exon 8. [Figure 4a] Figure 4 shows the data distribution of gene expression for luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 as determined by RT-qPCR in FFPE tissues from patients with muscle-invasive bladder cancer. [Figure 4b] Figure 4 shows the data distribution of gene expression for luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 as determined by RT-qPCR in FFPE tissues from patients with muscle-invasive bladder cancer. [Figure 5] Figure 5 shows intergenic Spearman correlations of luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 gene mRNA expression determined by RT-qPCR in XX tissues from muscle-invasive bladder cancer patients. [Figure 6] Figure 6 shows intergenic Spearman correlations of HLA gene mRNA expression determined by RT-qPCR in FFPE tissues from patients with muscle-invasive bladder cancer. [Figure 7]Figure 7 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from muscle-invasive bladder cancer patients based on stratification by combined HLA-A exon 8, HLA-G exon 8, and HLA-G exon 5 mRNA expression. [Figure 8] Figure 8 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from muscle-invasive bladder cancer patients stratified by the gene-by-gene combination of HLA-A exon 8 and HLA-G exon 8 mRNA expression. [Figure 9] Figure 9 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from muscle-invasive bladder cancer patients based on stratification by intragenic combinations of HLA-G exon 8 and exon 5 mRNA expression. [Figure 10] Figure 10 shows a Kaplan-Meier plot showing the disease-specific survival (DSS) probability of FFPE tissue from muscle-invasive bladder cancer patients (n=61) based on stratification by the single gene determination of HLA-G exon 8 and exon 5 mRNA expression only. [Figure 11a] FIG. 11 shows the data distribution of relative mRNA expression of HLA-F isoforms (40-DCT) and antisense HLA-F expression as determined by RT-qPCR. [Figure 11b] FIG. 11 shows the data distribution of relative mRNA expression of HLA-F isoforms (40-DCT) and antisense HLA-F expression as determined by RT-qPCR. [Figure 12] FIG. 12 shows the data distribution for the relative mRNA expression (40-DCT) of ESR1, HLA-F3, and HLA-F AS1 expression as determined by RT-qPCR. [Figure 13] FIG. 13 shows a split-screen study of HLA-F3 mRNA expression in pre-treatment biopsy samples of neoadjuvant-treated ovarian cancer patients as determined by RT-qPCR to predict progression-free survival. [Figure 14]Figure 14 shows a Kaplan-Meier plot showing progression-free survival (PFS) probability based on stratification by the determination of a single gene, HLA-F3 only, as quantified by RT-qPCR assay of fresh tissue from patients with advanced ovarian cancer (n=27). [Figure 15] Figure 15 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratification by the determination of a single gene, HLA-F3 only, as quantified by RT-qPCR assay of fresh tissue from patients with advanced ovarian cancer (n=27). [Figure 16] Figure 16 shows a multivariate analysis for OS using a Cox proportional hazards model including grade, FIGO stage, primary site, and HLA-F3 mRNA expression. [Figure 17] FIG. 17 shows a split-screen study of ESR1 and HLA-F3 mRNA expression in pre-treatment biopsy samples of neoadjuvant-treated ovarian cancer patients as determined by RT-qPCR to predict progression-free survival. [Figure 18] Figure 18 shows a Kaplan-Meier plot showing progression-free survival (PFS) probability based on stratified ESR1 and HLA-F3 mRNA expression, quantified by RT-qPCR assay in fresh tissues from patients with advanced ovarian cancer (n=27). [Figure 19] Figure 19 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratified ESR1 and HLA-F3 mRNA expression, quantified by RT-qPCR assay in fresh tissues from patients with advanced ovarian cancer (n=27). [Figure 20] Figure 20 shows a multivariate analysis of PFS using a Cox proportional hazards model including a combination of grade, FIGO stage, primary site, and ESR1 and HLA-F3 mRNA expression. [Figure 21] Figure 21 shows a multivariate analysis for OS using a Cox proportional hazards model including a combination of grade, FIGO stage, primary site, and ESR1 and HLA-F3 mRNA expression. [Figure 22]Figure 22 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratified HLA-F3 and HLA-F AS1 mRNA expression, quantified by RT-qPCR assay in fresh tissues from patients with advanced ovarian cancer (n=27). [Figure 23] Figure 23 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratified HLA-F3 and HLA-F AS1 mRNA expression, quantified by RT-qPCR assay in fresh tissues from patients with advanced ovarian cancer (n=27). [Figure 24] Figure 24 shows a multivariate analysis for PFS using a Cox proportional hazards model including grade, FIGO stage, primary site, and a combination of HLA-F3 and HLA-F AS1. [Figure 25] Figure 25 shows a multivariate analysis for OS using a Cox proportional hazards model including grade, FIGO stage, primary site, and a combination of HLA-F3 and HLA-F AS1. [Figure 26] Figure 26 shows the consort diagram for the advanced or metastatic urothelial cancer cohort. [Figure 27] Figure 27 shows a Kaplan-Meier plot showing the disease-specific survival (DSS) probability of muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-F1 / F2 expression, as quantified by RT-qPCR assay. [Figure 28] Figure 28 shows a Kaplan-Meier plot showing the disease-specific survival (DSS) probability of muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-F1 / F2 and HLA-G exon 8 mRNA expression, as quantified by RT-qPCR assay. [Figure 29] Figure 29 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-F1 / F2 and HLA-B / C exon 8 mRNA expression, as quantified by RT-qPCR assay. [Figure 30] Figure 30 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-B / C exon 8 mRNA expression, as quantified by RT-qPCR assay. DETAILED DESCRIPTION OF THE INVENTION

[0171] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0172] Specific components of the present invention are described below. While these components may be listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred components. Furthermore, any permutation and combination of all components described in this application should be considered disclosed by the description of this application unless the context dictates otherwise. For example, as will be apparent to one of skill in the art, specific embodiments disclosed herein relating to the expression level of RNA transcripts of specific genes (higher or lower than a defined expression threshold of the RNA transcripts of specific genes) and molecular subtypes based thereon can be combined to enable the identification of molecular subtypes of a given tumor.

[0173] Before proceeding to the description of the embodiments and examples, some general notes on terminology are provided below: Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995) (Non-Patent Document 3).

[0174] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature in the art (e.g., Molecular Cloning: A Laboratory Manual, 3 rd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2000) (Non-Patent Document 4).

[0175] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," are understood to mean the inclusion of a stated member, integer, or step, or group of members, integers, or steps, but not the exclusion of any other members, integers, or steps, although in some embodiments such other members, integers, or steps, or group of members, integers, or steps may be excluded, i.e., the subject matter consists of including a stated member, integer, or step, or group of members, integers, or steps. As used in the context of describing the invention (particularly in the context of the claims), the terms "a," "an," and "the," and similar references, should be interpreted to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each value is incorporated herein as if set forth individually. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention, as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0176] The term "classification of a patient sample" as used herein refers to the association of the sample with at least one of at least two categories. These categories can be, for example, "high risk" and "low risk," high risk, intermediate risk, and low risk, where risk is the probability of a particular event occurring in a particular period, such as the occurrence of metastasis, disease-free survival, etc. It can also refer to categories such as favorable or unfavorable clinical outcomes of a disease, or response or non-response to a given treatment, etc. Classification can be performed using an algorithm, particularly a discrimination function. A simple example of an algorithm is classification according to a first quantitative parameter, such as whether the expression level of a gene of interest is above or below a certain threshold. Classification of a patient sample can be used to predict disease outcomes. Instead of using the expression level of a single gene of interest, a combined score of several genes of interest can be used. Furthermore, additional data can be used in combination with the first quantitative parameter. Such additional data can be the patient's clinical data, such as the patient's gender, age, weight, tumor grade, or stage.

[0177] The term "metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that relies on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then invasion of the target organ after transport by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential.

[0178] A "discriminant function" is a function of a set of variables used to classify an object or event. Thus, a discriminant function allows a patient, sample, or event to be classified into a category or multiple categories according to data or parameters available from the patient, sample, or event. Such classification is a standard means of statistical analysis well known to those skilled in the art. For example, a patient can be classified into "high risk" or "low risk," "high probability of metastasis" or "low probability of metastasis," "treatment required" or "treatment not required" according to the data obtained from the patient, sample, or event. Classification is not limited to "high vs. low," but can be performed into multiple categories, rankings, etc. Examples of discriminant functions that enable classification include support vector machines (SVMs), k-nearest neighbors (kNNs), (naive) Bayesian models, or discriminant functions defined by piecewise defining functions, such as in subgroup discovery, decision trees, and logical analysis of data (LAD), etc.

[0179] The term "prediction" as used herein relates to the likelihood that a patient will respond favorably or unfavorably to a given treatment. In particular, the term "prediction" as used herein relates to an individual assessment of the aggressiveness of a tumor or the expected survival rate of a patient (DFS, disease-free survival) if the tumor is treated with a given therapy. In contrast, the term "prognosis" relates to an individual assessment of the aggressiveness of a tumor or the expected survival rate of a patient (DFS, disease-free survival) if the tumor remains untreated.

[0180] The term "response marker" refers to a marker that can be used to predict a patient's clinical response to a given treatment. Response includes direct observation of tumor shrinkage during neoadjuvant or palliative treatment, as indicated by, for example, CT scans and / or serum biomarkers, as well as impact on disease-free survival (DFS), overall survival (OAS), metastasis-specific survival (MSS), disease-specific survival, and related assessments.

[0181] As used herein, the term "clinical response" of a patient refers to the effectiveness of a particular treatment in a patient, meaning an improvement in any measure of the patient's condition, including measures commonly used in the art such as overall survival, progression-free survival, recurrence-free survival, and distant recurrence-free survival. Recurrence-free survival (RFS) refers to the time (in years) from surgery to the first local, regional, or distant recurrence. Distant recurrence-free survival (DFRS) refers to the time (in years) from surgery and / or initial diagnosis to the first anatomically distant recurrence. The actual calculation of these measures may vary from study to study, depending on the definition of censored or unaccounted events.

[0182] The term "neoplastic disease" refers to cancerous tissue. This includes carcinomas, e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma, and premalignant conditions, neomorphologic changes regardless of histological origin. The term "adenocarcinoma" refers to malignant tumors derived from glandular tissue.

[0183] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell proliferation. The term "cancer" is not limited to any stage, grade, histomorphological features, invasiveness, aggressiveness, or degree of malignancy of the affected tissue or cell aggregate. It specifically includes stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, primary cancer, and all other types of cancer, malignant tumors, and alterations specifically associated with gynecological cancers. The terms "neoplastic disease" or "cancer" are not limited to any tissue or cell type. They also include primary, secondary, or metastatic lesions in cancer patients, including lymph nodes affected by cancer cells or minimal residual disease cells, either locally deposited or free-floating throughout the patient's body.

[0184] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. As used herein, the term cancer also includes cancer metastasis. The terms "tumor" and "cancer" may be used interchangeably herein.

[0185] The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0186] As used herein, the term "lung cancer" refers to cancer or malignant tumors diagnosed in the lung, and is meant to include all cancers, neoplastic growths, and cancerous changes in lung tissue. Examples of lung cancer include, but are not limited to, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly lung squamous cell carcinoma, adenocarcinoma, bronchoalveolar carcinoma, large cell lung carcinoma, and others such as pleuropulmonary blastoma and carcinoid tumor.

[0187] The term "neoplastic cell" refers to an abnormal cell that grows more rapidly than normal due to increased cell proliferation, altered symmetry of cell division, or decreased cell death mechanisms. Thus, the neoplastic cells of the present invention can be cells of a benign neoplasm or cells of a malignant neoplasm.

[0188] Furthermore, the term "characterizing the status" of a neoplastic disease or cancer refers to the measurement and evaluation of one or more of the following statuses, but is not limited to: tumor type, histomorphological appearance, dependence on external signals (e.g., hormones, growth factors), invasiveness, motility, status according to the TNM classification of malignant tumors (TNM), a cancer staging system developed and maintained by the International Union Against Cancer, or similarity, aggressiveness, grade, metastatic potential, and response to a particular treatment.

[0189] The terms "therapeutic method," "therapeutic modality," "regimen," or "chemo-regimen" and "therapeutic regimen" refer to the timed, sequential or simultaneous administration of anti-tumor and / or anti-vascular and / or immunostimulatory and / or blood cell proliferation agents, and / or radiation therapy and / or hyperthermia and / or hypothermia for cancer treatment. These administrations may be carried out in an adjuvant and / or neoadjuvant manner. The composition of such "protocols" may vary in the dose of single agents, the time frame of application, and the frequency of administration within a defined therapeutic window. Currently, various combinations of different drugs and / or physical modalities as well as various schedules are being investigated.

[0190] The term "endocrine therapy" refers to various therapeutic methodologies known as hormone therapy or antihormonal therapy, which produce a desired therapeutic effect by altering hormone / hormone levels. Treatment may involve administering hormones or hormone analogs, synthetic hormones, or other drugs to a patient, or reducing hormone levels in the body through the use of hormone antagonists, hormone receptor antagonists, or hormone ablation therapy, either through surgical removal of the ovaries or chemical suppression of hormone synthesis. Endocrine therapy may include hormone therapies such as selective estrogen reuptake inhibitors, selective estrogen receptor downregulators, aromatase inhibitors, and ovarian ablation. Such endocrine therapy may include administering hormones or hormone analogs, synthetic hormones, or other drugs, such as tamoxifen, raloxifene, and / or goserelin (trade name Zoladex®), to a patient. In a preferred embodiment, such endocrine therapy includes the administration of tamoxifen or tamoxifen and goserelin. Furthermore, the endocrine treatment may include the administration of an anti-estrogen selected from the group including anastrozole, letrozole, exemestane, fulvestrant, toremifene, and megasterol acetate. The endocrine treatment may also include the administration of an estrogen, a progestin, and / or a gestagen.

[0191] The term "determining gene expression levels on a non-protein basis" refers to methods that do not focus on secondary gene translation products, i.e., proteins, but rather on other levels of gene expression based on RNA and DNA analysis. In some embodiments of the invention, the analysis uses mRNA, including precursor forms of mRNA. Exemplary determinable traits are the amount of HLA mRNA, i.e., HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, or portions thereof.

[0192] Alternatively, the differentially expressed genes disclosed herein can be used in methods for identifying reagents and compounds and the use of these reagents and compounds for cancer treatment, as well as in therapeutic methods. Differential regulation of genes is not limited to specific cancer cell types or clones, but indicates interactions between cancer cells, muscle cells, stromal cells, connective tissue cells, other epithelial cells, adipocytes, vascular endothelial cells, and immune system cells, such as lymphocytes, macrophages, and killer cells.

[0193] The term "pattern of RNA expression" refers to the determined level of RNA expression compared to either a reference RNA or a calculated average expression value. The pattern is not limited to the comparison of two RNAs, but also relates to multiple comparisons of RNA with a reference RNA or sample. A particular "pattern of expression levels" can also be obtained and determined by comparing and measuring several RNAs, and can indicate the relative abundance of these transcripts relative to each other.

[0194] A "reference pattern of expression levels" within the meaning of the present invention should be understood to be any pattern of expression levels that can be used for comparison with another pattern of expression levels. In a preferred embodiment of the present invention, the reference pattern of expression levels is e.g. the average pattern of expression levels observed in a group of healthy or diseased individuals serving as a reference group.

[0195] As used herein, the terms "modulated" or "modulation" or "regulation" or "regulation" and "differentially regulated" refer to both upregulation, i.e., activation or stimulation, e.g., by agonism or enhancement, and downregulation, i.e., inhibition or suppression, e.g., by antagonism, reduction, or inhibition.

[0196] The phrases "response," "treatment success," or "response to treatment" refer to the observation of a defined tumor-free, recurrence-free, or progression-free survival period (e.g., 2 years, 4 years, 5 years, 10 years) in the setting of neoadjuvant, adjuvant, and palliative chemotherapy. This disease-free, recurrence-free, or progression-free survival period may vary for different tumor entities, but is significantly longer than the average time for most recurrences to occur. In neoadjuvant and palliative therapy methodologies, response can be further monitored by measuring tumor shrinkage and regression due to apoptosis and necrosis of the tumor mass or reduced blood supply due to altered angiogenic events.

[0197] The term "recurrence" or "recurrent disease" includes distant metastasis, which may appear many years after the initial diagnosis and treatment of the tumor, or local events such as invasion of tumor cells into regional lymph nodes, or the recurrence of tumor cells at the same site and organ of origin within a reasonable time.

[0198] "Predicting recurrence" or "predicting treatment success" refers to the method described in the present invention, in which a tumor specimen is analyzed, for example, for its gene expression, genomic status, and / or histopathological parameters (such as TNM and grade), and / or imaging data, and further classified based on correlation of expression patterns with those known from a reference sample. This classification can be a statement that such a given tumor will undergo recurrence and therefore be considered a tumor "unresponsive" to a given treatment, or can be classified as a tumor with an extended disease-free interval after treatment.

[0199] As used herein, the term "marker gene" refers to a differentially expressed gene whose expression pattern can be utilized as part of a predictive, prognostic, or diagnostic process in the evaluation of malignant neoplasms or cancers, or can be used in methods to identify compounds useful in the treatment or prevention of malignant neoplasms and gynecological cancers, among others. A marker gene can also have the characteristics of a target gene.

[0200] As used herein, "target gene" refers to a differentially expressed gene that is involved in cancer, e.g., lung cancer, in such a way that modulation of target gene expression levels or target gene product activity levels can act to ameliorate symptoms of the malignant neoplasm. A target gene may also have the characteristics of a marker gene.

[0201] The term "receptor" as used herein relates to a protein on a cell membrane or in the cytoplasm or nucleus of a cell that binds to a specific molecule (ligand), such as a neurotransmitter, hormone, or other substance, particularly a hormone such as estrogen, and initiates a cellular response. Ligand-induced changes in the behavior of the receptor protein result in physiological changes that constitute the biological effect of the ligand.

[0202] The term "signal transduction pathway" refers to the intracellular or intercellular process by which cells convert one type of signal or stimulus into another, and most often involves an orderly sequence of intracellular and extracellular biochemical reactions carried out by enzymes and linked via hormones and growth factors (intercellular) and second messengers (intracellular), the latter resulting in what is considered a "second messenger pathway." In many signal transduction pathways, the number of proteins and other molecules involved in these events increases as the process proceeds from an initial stimulus, resulting in a "signal cascade," often resulting in a relatively small stimulus that elicits a large response.

[0203] As used herein, the term "small molecule" is meant to refer to a compound having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic (carbon-containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and / or biological mixtures, often fungal, bacterial, or algal extracts, that can be screened in any of the assays of the present invention to identify compounds that modulate biological activity.

[0204] When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that is capable of hybridizing to the single-stranded nucleic acid sequence (i.e., it is the complement of the single-stranded nucleic acid sequence) under conditions of low stringency as described above.

[0205] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids.

[0206] The term "hybridization-based method" as used herein refers to a method that provides a process for combining complementary single-stranded nucleic acids or nucleotide analogs into a single double-stranded molecule. Because nucleotides or nucleotide analogs bind to their complements under normal conditions, two perfectly complementary strands easily bind to each other. In bioanalysis, single-stranded probes, which are often labeled, are used to find complementary target sequences. If such a sequence is present in a sample, the probe hybridizes to the sequence and can then be detected by the label. Other hybridization-based methods include microarray and / or biochip methods. In these methods, the probe is immobilized on a solid phase and then exposed to the sample. If complementary nucleic acids are present in the sample, they hybridize to the probe and can therefore be detected. These techniques are also known as "array-based methods." Yet another hybridization-based method is PCR, which will be described below. Regarding the determination of expression levels, hybridization-based methods can be used, for example, to determine the amount of mRNA of a given gene.

[0207] "Array" refers to an arrangement of addressable locations or "addresses" on a device. The locations can be arranged in a two-dimensional array, a three-dimensional array, or other matrix format. The number of locations can range from a few to at least hundreds of thousands. Most importantly, each location represents an independent reaction site. Arrays include, but are not limited to, nucleic acid arrays, protein arrays, and antibody arrays. "Nucleic acid array" refers to an array containing nucleic acid probes, such as oligonucleotides, polynucleotides, or larger portions of genes. The nucleic acids on the array are preferably single-stranded. Arrays in which the probes are oligonucleotides are referred to as "oligonucleotide arrays" or "oligonucleotide chips." "Microarrays" herein are also referred to as "biochips" or "biological chips" and have a density of at least about 100 / cm 2 , preferably at least about 1000 / cm 2A microarray is an array of regions having a density of distinct regions of approximately 10 to 250 μm in diameter, and is spaced approximately the same distance from other regions in the array.

[0208] The term "oligonucleotide" refers to a relatively short polynucleotide, including, but not limited to, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids, and double-stranded DNA. The oligonucleotide is preferably a single-stranded DNA probe oligonucleotide. Furthermore, in the context of applicable detection methodologies, the term "oligonucleotide" also refers to nucleotide analogs such as PNAs and morpholinos.

[0209] As used herein, the term "PCR-based method" refers to a method involving polymerase chain reaction (PCR). This is a technique for exponentially amplifying nucleic acids, such as DNA or RNA, through enzymatic replication without the use of living organisms. Because PCR is an in vitro technique, it can be performed on any form of DNA and can be significantly modified to perform diverse genetic manipulations. With regard to determining expression levels, for example, a PCR-based method can be used to detect the presence of a given mRNA by (1) reverse transcribing the entire mRNA pool (the so-called transcriptome) into cDNA using reverse transcriptase and (2) detecting the presence of the given cDNA using respective primers. This technique is commonly known as reverse transcriptase PCR (rtPCR). The term "PCR-based method" includes both the application of end-point PCR and kinetic / real-time PCR techniques that apply special fluorophores or intercalating dyes that emit a fluorescent signal as a function of the amplified target, allowing for target monitoring and quantification. Quantification methods can be either absolute using an external standard curve or relative to a comparative internal standard.

[0210] The term "methods based on electrochemical detection of molecules" refers to methods that utilize electrode systems to which molecules, particularly biomolecules such as proteins, nucleic acids, antigens, and antibodies, bind under the generation of a detectable signal. Such methods are disclosed, for example, in International Publication Nos. WO 02 / 42759, WO 02 / 41992, and WO 02 / 097413, the contents of which are incorporated herein by reference. These detectors include a substrate having a plane formed, for example, by the crystallographic surface of a silicon chip, and an electric detector that can take the form of, for example, an interdigitated electrode or a two-dimensional electrode array. These electrodes carry probe molecules, e.g., nucleic acid probes, that can specifically bind to target molecules, e.g., target nucleic acid molecules. The probe molecules are immobilized, for example, by thiol-gold bonding. For this purpose, the probes are modified at their 5' or 3' end with a thiol group that binds to an electrode containing a gold surface. These target nucleic acid molecules may carry enzyme labels, such as horseradish peroxidase (HRP) or alkaline phosphatase. After the target molecule binds to the probe, a substrate is added (e.g., α-naphthyl phosphate or 3,3'5,5'-tetramethylbenzidine, which is converted by the enzyme in a redox reaction). The product of the reaction or the current generated by electron exchange can then be detected in a site-specific manner using an electrical detector.

[0211] The term "nucleic acid molecule" is intended to denote any single- or double-stranded nucleic acid and / or analog molecule, including DNA, cDNA and / or genomic DNA, RNA, preferably mRNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), and / or morpholino.

[0212] The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize particularly at elevated temperatures. Generally, stringent conditions are those that meet the thermal melting point (T) of a particular sequence at a defined ionic strength and pH. m ) is chosen to be approximately 5°C lower than T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (Target sequences are generally present in excess, so T m (At equilibrium, 50% of the probes are occupied.) Typically, stringent conditions are those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes. Stringent conditions can also be achieved by adding destabilizing agents such as formamide.

[0213] The term "fragment of a nucleic acid molecule" is intended to indicate a nucleic acid comprising a subset of a nucleic acid molecule according to one of the claimed sequences. The same applies to the term "fragment of a nucleic acid molecule."

[0214] The term "variant of a nucleic acid molecule" as used herein refers to a nucleic acid molecule that is substantially similar in structure and biological activity to a nucleic acid molecule according to one of the claimed sequences.

[0215] The term "homologue of a nucleic acid molecule" refers to a nucleic acid molecule whose sequence has one or more nucleotides added, deleted, substituted or otherwise chemically modified compared to a nucleic acid molecule according to one of the claimed sequences, always provided that the homologue retains substantially the same binding properties as the latter.

[0216] The term "derivative" as used herein refers to a nucleic acid molecule that has similar binding properties with a target nucleic acid sequence as a nucleic acid molecule according to one of the claimed sequences.

[0217] As used herein, the term "hybridizing partner" refers to a nucleic acid molecule that can hybridize to a nucleic acid molecule under stringent conditions.

[0218] The term "anamesis" relates to patient data obtained by a physician or other health care professional by asking specific questions of either the patient or others who know the person and can provide relevant information, with the aim of obtaining information useful in formulating a diagnosis and providing medical care to the patient (in this case it is sometimes referred to as heteroanamesis). This type of information is referred to as symptoms, in contrast to clinical signs, which are confirmed by direct examination.

[0219] The term "pathogenesis" relates to the course of the disease, ie its duration, its clinical manifestations, and its outcome.

[0220] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

[0221] The term "clinical outcome" is defined as the clinical result of a disease, particularly after treatment, such as a reduction or improvement in symptoms. In some embodiments, poor clinical outcome includes a relative decrease or worsening of disease-specific survival (DSS), recurrence-free survival (RFS), progression-free survival (PFS), and distant recurrence-free survival. The term "recurrence" in relation to cancer includes local events such as the recurrence of tumor cells in the same site and organ of the original disease, metastasis, which may appear many years after the initial diagnosis and treatment of the cancer, or tumor cell invasion into regional lymph nodes. "Distant recurrence" refers to a scenario in which cancer cells have spread (metastasized) beyond the regional lymph nodes to distant parts of the body (i.e., another organ). Recurrence-free survival is generally defined as the time from randomization to first recurrence, relapse, second cancer, or death. Progression-free survival is the time elapsed from a specific date (generally the first day of treatment or the date the patient enrolled in a clinical trial) to the date the disease "progressed" or the date the patient died from any cause. The terms "DSS" and "CSS" (which stands for "cancer-specific survival time") may be used interchangeably herein.

[0222] The term "(therapeutic) treatment," particularly in relation to cancer treatment as used herein, relates to any treatment that improves the health and / or extends (prolongs) the lifespan of a patient. The treatment may eliminate cancer, reduce the size or number of tumors in a patient, prevent or delay the development of cancer in a patient, inhibit or delay the development of new cancers in a patient, reduce the frequency or severity of symptoms in a patient, and / or reduce recurrence in a patient who currently has or previously had cancer. In some embodiments, the terms "treatment" and "therapeutic treatment" are meant to refer to one or more of surgical removal of a primary tumor, chemotherapy, antihormonal therapy, radiation therapy, and immunotherapy / targeted therapy.

[0223] Adjuvant therapy is treatment given in addition to primary, definitive, or initial treatment. Due to the surgeries and complex treatment regimens used in cancer treatment, the term has come to be used primarily to describe adjuvant cancer treatment. An example of adjuvant therapy is additional treatment (such as chemotherapy) given usually after surgery (post-op) to remove all detectable disease, although there remains a statistical risk of recurrence due to occult disease. Neoadjuvant therapy is treatment given before primary, definitive, or initial treatment (e.g., pre-operative chemotherapy).

[0224] As used herein, the term "defined expression threshold of an RNA transcript" may refer to an average cutoff value (simply, cutoff) calculated from several samples (this number of samples is obtained from several subjects, particularly subjects with cancer). To obtain the threshold, the number of subjects may include subjects with tumors of different molecular subtypes, for example, subjects with HER2-positive tumors and / or subjects with triple-negative tumors and / or subjects with luminal A tumors and / or subjects with luminal B tumors. The threshold may represent the amount or concentration of an RNA transcript. In some embodiments, the threshold is given as a CT (cycle threshold; also called quantification cycle, Cq) value (see below). In some embodiments, the (relative) expression level and expression threshold are expressed as 40-ΔCT or 40-ΔΔCT values ​​(see below).

[0225] The term "subject" as used herein refers to any organism, such as a vertebrate, particularly any mammal, including both humans and other mammals, for example, rodents, rabbits, monkeys, and the like. Rodents can be mice, rats, hamsters, guinea pigs, or chinchillas. Preferably, the subject is a human. In some embodiments, the subject is a subject suspected of having or having a disease, particularly cancer, also referred to herein as a "patient." To determine the average cutoff value, at least two subjects, preferably at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, or at least 2000 subjects are examined.

[0226] Returning to the description of preferred embodiments, in some embodiments, the cutoff / threshold is defined based on one or more previous clinical studies. Furthermore, additional clinical studies may be conducted to establish and validate the cutoff / threshold. The cutoff / threshold may be determined / defined by techniques known in the art. Various clinical studies have already been conducted using the genetic markers used in accordance with the present invention. Concordance studies in training test settings may be sufficient to define and validate clinical cutoff / thresholds for dichotomizing quantitative results into "expression positive" or "expression negative."

[0227] In some embodiments, the cutoff / threshold is determined / defined based on clinicopathological parameters such as IHC-ISH and / or data on overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS) in a training cohort by split testing (such as SAS Software JMP® 9.0.0).

[0228] In some embodiments, the expression level of RNA transcripts is determined by reverse transcription (RT) quantitative PCR (RT-qPCR). Because RNA cannot be directly amplified by PCR, it must be reverse transcribed into cDNA using the enzyme reverse transcriptase. For this purpose, one-step RT-qPCR can be used, which combines reverse transcription and PCR-based DNA amplification in the same reaction. In one-step RT-qPCR, RNA templates are mixed in a reaction mixture containing reverse transcriptase, DNA polymerase, primers and probes, dNTPs, salts, and detergents. In the first PCR step, target RNA is reverse transcribed by reverse transcriptase using a target-specific reverse primer. The cDNA is then amplified using primers / probes and DNA polymerase.

[0229] For example, fluorescence-based quantitative real-time PCR can be used. Fluorescence-based quantitative real-time PCR involves the use of fluorescently labeled probes. Preferably, the fluorescently labeled probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye (=dual-labeled probe). Suitable fluorescent reporter and quencher dyes / moieties are known to those skilled in the art and include, but are not limited to, reporter dyes / moieties 6-FAM™, JOE™, Cy5™, Cy3™, and quencher dyes / moieties Dabcyl, TAMRA™, BHQ™-1, -2, or 3. Amplification of the probe-specific product causes cleavage of the probe (=amplification-mediated probe displacement), thereby resulting in an increase in reporter fluorescence. The increase in fluorescence in the reaction is directly proportional to the increase in target amplification product. The increase in fluorescence can be measured in real time using a LightCycler 480 II system (Roche), a Versant kPCR system (Siemens), an Mx3005P system (Agilent Technologies), or an equivalent real-time instrument to detect fluorescence emitted from the probe. The analytical output is the CT value of each target. The CT (cycle threshold; also referred to as the quantification cycle, Cq) value is determined by the number of PCR amplification cycles after which the fluorescent signal of the probe exceeds a certain background signal, and the CT value is a measure of the amount of target molecules in the sample before PCR amplification. Preferably, the CT value is further analyzed using appropriate software (e.g., Microsoft Excel™) or statistical software packages (e.g., SAS JMP® 9.0.0, GraphPad Prism4, Genedata Expressionist™). The CT value can be converted to absolute target molecular weight (e.g., ng / μl or molecules / μl) based on the CT results of a standard curve using known target concentrations. Alternatively, the amount of target can be reported as x-fold decrease or increase (=ΔCT) based on the reference, where a lower ΔCT value (small difference) indicates a higher amount of target relative to the reference compared to a higher ΔCT (large difference).It is preferable to recalculate the ΔCT by subtracting it from a fixed value (such as the number of PCR cycles, e.g., 40). The result is a value that directly correlates with the amount of target (higher values ​​= higher amounts) and is expressed as 40-ΔCT, where one integer indicates twice the amount of target (e.g., a value of 34 indicates twice the amount of a value of 33). Depending on the desired reproducibility and precision of the system, multiple reference assays can be paneled, or the ΔCT of a calibrator can be used to recalculate / normalize the ΔCT of the sample (one-point calibration; Pfaffl (2001), Nucleic Acid Res., 29(9):e45). It is also possible to multiplex different target assays in the same reaction by using different fluorophores for specific probes. During PCR, each target in the multiplex is amplified in parallel but detected separately using different fluorescence emissions.

[0230] In some embodiments, the 40-ΔCT value is calculated as follows: 40 - [CT of each biomarker (e.g., HLA-E, HLA-F, or HLA-G) in the patient sample - CT of a reference gene (e.g., CALM2) in the patient sample] (=Calculation Method 1). When two or more reference genes are used, the 40-ΔCT value is calculated as follows: 40 - (CT of each biomarker in the patient sample - average CT of selected reference genes in the patient samples) (=Calculation Method 2). Alternatively, the 40-ΔΔCT value can be used, and 40-ΔΔCT can be calculated as follows: ΔΔCT = 40 - [(CT biomarker in the patient sample - CT biomarker in the reference sample) - (CT reference gene in the patient sample - CT reference gene in the reference sample)] (=Calculation Method 3); for example, 40 - ΔΔCT = 40 - [(CT HLA-G patient sample - CT HLA-G reference sample) - (CALM2 in the CT patient sample - CALM2 in the CT reference sample)]. In some embodiments, CALM2 is used as a reference gene.

[0231] For example, the relative expression level of a biomarker is given as 40-ΔΔCT value, which is calculated as follows: 40-[(CT biomarker in patient sample-CT reference gene in patient sample)-(CT biomarker in control sample-CT reference gene in control sample)] (=Calculation Method 4); for example, 40-ΔΔCT=40-[(CT HLA-G patient sample-CT average CombRef patient sample)-(CT HLA-G control sample-CT average CombRef control sample)]. In some embodiments, CT is the median CT. The CT of a reference gene can be the CT of a single reference gene or the average CT of two or more reference genes (referred to as average CombRef). Preferably, the same control sample (also referred to as calibrator) is used in all analyses to produce the same RT-qPCR or qPCR results. In some embodiments, the control sample is cell line RNA, in vitro transcribed artificial RNA, or an equimolar mixture of DNA oligonucleotides representing a fixed ratio of biomarker mRNA or cDNA or biomarker amplicon or portion of a biomarker amplicon. In some embodiments, CALM2 and / or B2M are used as reference genes, and a positive control (e.g., in vitro transcribed artificial RNA) is used as the control sample (calibrator).

[0232] In another exemplary embodiment, the mean cutoff value is given as the 40-ΔΔCT value according to calculation method 4, and the mean cutoff value for HLA-G is a 40-ΔΔCT value of 40.10.

[0233] In some embodiments, the method steps (e.g., steps (a), (b), (c), and (d)) are performed in random order. In preferred embodiments, step (a) is performed first, i.e., followed by steps (b), (c), and (d). In some embodiments, step (d) is performed after steps (a), (b), and (c). In some embodiments, step (a) is performed before step (b), step (b) is performed before step (c), and step (c) is performed before step (d).

[0234] The probes defined above are preferably labeled, for example with a label selected from a fluorescent label, a fluorescence-quenching label, a luminescent label, a radioactive label, an enzymatic label, and combinations thereof. Preferably, the probes defined above are dual-labeled probes comprising a fluorescent reporter moiety and a fluorescent quencher moiety.

[0235] The novelty of the present invention includes not only the mRNA-based determination of HLA-based cancer biomarkers in bladder cancer, but also the algorithmic inclusion of subtypes.

[0236] All these novel aspects of the present invention contribute to the prognostic value of the RT-qPCR-based method and kit of the present invention. Indeed, these novelties provide more accurate and meaningful HLA typing, especially in the molecular subtypes of advanced stage patients, which ultimately provides a prognostic tool for more personalized cancer treatment decisions.

[0237] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0238] Example 1: Determination of mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR) RNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissues. More specifically, total RNA from 5-10 µm FFPE tumor tissue sections was extracted using the RNXtract® Extraction Kit (BioNTech Diagnostics GmbH, Mainz, Germany) and quantified by real-time fluorescent RT-qPCR for a fragment of the reference gene CALM2. Typically, 2.5 µl of RNA from each quantification extract (approximately 50-100 ng) was assayed by RT-qPCR as described below.

[0239] For detailed analysis of gene expression by RT-qPCR, we utilized primers flanking the region of interest and fluorescently labeled probes hybridizing between them. Target-specific primers and probes were selected using the NCBI primer design tool (www.ncbi.nlm.nih.go). RNA-specific primer / probe sequences were used to position primer / probe sequences across exon / exon boundaries, enabling RNA-specific measurements. Furthermore, primers / probes were selected to avoid binding to sequence regions with known single nucleotide polymorphisms (SNPs). In cases where multiple isoforms of the same gene exist, primers were selected to amplify all relevant splice variants. The specificity of all primer pairs was tested by conventional PCR reactions.

[0240] TaqMan® validation experiments demonstrated that the amplification efficiencies of the target and control were approximately equal, a prerequisite for relative quantification of gene expression using the comparative ΔCT method. To perform expression analysis of genes of interest in biological samples, four duplicate assay mixtures were prepared by mixing the respective primers / probes of two specific assays. To separately detect CT values, the assay probes were modified with different fluorescent probes. Each of the four assay mixtures contained 2 μM unmodified forward and reverse primers and 1.2 μM probe. For each reaction, 2.5 μl of total RNA extracted from FFPE sections (see above) was mixed with 2.5 μl of assay mixture, 2.5 μl of enzyme mixture, and 2.5 μl of water in one well of a 96-well optical reaction plate. PCR reactions were measured using a Versant kPCR Cycler (Siemens) or a Light Cycler 480 (Roche) according to the manufacturer's instructions under appropriate conditions (5 min at 50°C for 1 cycle; 20 s at 95°C for 1 cycle; 15 s at 95°C for 1 min at 60°C for 40 cycles). Prior to measuring previously unclassified biological samples, control experiments using, for example, cell lines, healthy control samples, or samples of defined molecular tumor subtypes can be used to standardize the experimental conditions.

[0241] Example 2: Comparison of classical and non-classical HLA genes by DNA sequence Genome analysis and sequence alignment were performed by accessing the UCSC Genome Browser (https: / / genome.ucsc.edu / cgi-bin / hgGateway) and downloading the genome sequences of HLA-A1 (NM_002116.7), HLA-A2 (NM_001242758.1), HLA-G (NM_002127.5), HLA-F1 (NM_001098479.1), HLA-F2 (NM_018950.2), HLA-F3 (NM_001098478.1), and HLA-J (NR_024240.1), as well as the predicted sequence of HLA-H (NR_001434.4). Initial alignment analysis focused on potential translation initiation regions and potential transitions from the extracellular alpha domain to the transmembrane region. HLA-H is thought to be a pseudogene because a single base pair deletion in exon 4 causes a frameshift, resulting in a premature stop codon in exon 4 (Chorney et al., 1990. Transcription analysis, physical mapping, and molecular characterization of a non-classical human leukocyte antigen class I gene. Mol. Cell. Biol. 10:243-253 (Non-Patent Document 6) and Zemmour et al., 1990. HLA-AR, an inactivated antigen-presenting locus related to HLA-AJ Immunol. 144:3619-3629 (Non-Patent Document 7)). Such pseudogene definition is potentially defined by loss of function of the protein-coding capacity due to mutation. However, sequence analysis revealed that the nucleotides surrounding the ATG at the 5' and 3' ends conform to the essential Kozak sequence.

[0242] Figure 1 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at potential translation start sites. Potential start codons are highlighted with black boxes.

[0243] Figure 2 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H at the junction of exon 4 to exon 5. Sequences with premature stop codons are shown with a yellow background.

[0244] As mentioned above, HLA-H has been defined in the literature as a pseudogene due to a premature stop codon in exon 4. They identified the sequence GAC-CAG-ACC-CA-CAC (a single-nucleotide deletion highlighted in red), which causes an in-frame shift. Comparing the sequence from Chorney et al. (shown in Figure 2 with a yellow background), the researchers failed to observe a single base pair deletion (shown in Figure 2 with a red background). This observation led to the assumption that HLA-H is also a full-length protein and therefore not a pseudogene. Furthermore, the researchers identified a sequence in exon 5, which encodes the alpha 3 domain. The single base pair deletion would result in a premature stop codon at the end of exon 5, which would imply that HLA-H lacks the transmembrane and cytoplasmic domains. In the case of HLA-G, a transcript variant with a premature stop codon in intron 5 is known to result in the translation of the soluble isoform HLA-G5. Thus, HLA-H may be a soluble analog of soluble HLA-G5. Soluble HLA-G forms are active proteins that cause immune cell inhibition by interacting with various receptors, such as leukocyte immunoglobulin-like receptors 1 and 2 (LILRB1 and LILRB2), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), and CD8 (Rajagopalan, S. and E. Long, KIR2DL4 (CD158d): An activation receptor for HLA-G. Frontiers in Immunology, 2012. 3(258) (Non-Patent Document 8) and Carosella, et al., Beyond the increasing complexity of the immunomodulatory HLA-G molecule. Blood, 2008. 111(10):4862-70 (Non-Patent Document 9)).

[0245] The literature points to a deletion of a second single nucleotide in exon 7 at the end of the sequence ctc-acg-gcg-tg-. The researchers identified this sequence in exon 8, which encodes an untranslated region and is not involved in protein translation.

[0246] Figure 3 shows the sequence alignment of HLA-A1, HLA-A2, HLA-B, HLA-E, HLA-F1, HLA-F2, HLA-F3, HLA-J, HLA-G, and HLA-H in exon 8. Sequences with premature stop codons are shown in yellow.

[0247] The researchers also identified a second pseudogene, the sequence GAC-CAG-ACC-CA-, in exon 5 of HLA-J, which also contains the predicted single-base pair deletion from Chorney et al. Sequence comparison of HLA-H and HLA-J revealed that the two pseudogenes share 69% sequence identity in RNA but only 20% identity between their amino acid sequences (Table 3).

[0248] [Table 2]

[0249] Table 4 summarizes the homology between HLA-H and HLA class I genes (HLA-A, B, C), non-classical HLA class I genes (HLA-E, F, and G), and additional pseudogenes (HLA-J, L, V, and Y). HLA-H RNA is 77.4% homologous and 22.6% heterologous to HLA class I genes (HLA-A, B, C) and non-classical HLA class I genes (HLA-E, F, and G). Considering the protein sequence, HLA-H is 27.3% heterologous to classical and non-classical HLA class I genes (HLA-A1, A2, B, C, E, F1, F2, F3, and G) and 58.8% heterologous to HLA-J.

[0250] [Table 3]

[0251] Example 3: Determination of HLA mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR) in a cohort of urothelial cancer treated with immunotherapy Seventy-two newly diagnosed patients with histologically confirmed urothelial carcinoma, including bladder cancer and upper tract urothelial carcinoma, were enrolled in the study between 2016 and 2018. After excluding six patients with insufficient biopsy specimens and five patients due to lymph node metastasis, the initial study population of 72 patients was narrowed to 61. Within the urothelial carcinoma (UC) cohort, 49 patients had urothelial bladder cancer (UBC) and 12 patients had upper tract urothelial carcinoma. Nivolumab, pemprolizumab, and atezolizumab were administered as first-, second-, and third-line monotherapy according to approved instructions.

[0252] Survival analyses used disease-specific survival (DSS) for Kaplan-Meier survival estimates and Cox regression analysis. Complete survival data were available for 61 patients. At the end of data collection, the median DSS was 4.32 months.

[0253] Gene-specific TaqMan-based primer / probe sets were used to assess HLA gene expression. For detailed analysis of gene expression by RT-qPCR, primers flanking the region of interest and fluorescently labeled probes hybridizing between them were utilized. Target-specific primers and probes were selected using the NCBI primer design tool (www.ncbi.nlm.nih.go). RNA-specific primer / probe sequences were used to position primer / probe sequences across exon / exon boundaries, enabling RNA-specific measurements. Furthermore, primers / probes were selected to avoid binding to sequence regions with known single nucleotide polymorphisms (SNPs). In cases where multiple isoforms of the same gene exist, primers were selected to amplify all relevant splice variants or selected splice variants, as appropriate. The specificity of all primer pairs was examined by conventional PCR reactions. After further primer / probe optimization, the primers and probes listed in Table 5 provided the best results. These primers / probes are superior to those known from the prior art, for example, in terms of specificity and amplification efficiency. Because CALM2 was not differentially regulated in the analyzed samples, CALM2 was chosen as a reference gene to normalize the amount of sample RNA. Sample pairs with low RNA content (i.e., CALM2 raw Ct values ​​< 22) due to pre-treatment biopsies or post-treatment resections were excluded.

[0254] [Table 4] TIFF0007762135000006.tif25584

[0255] TaqMan® validation experiments showed that the amplification efficiencies of the target and control were nearly equal, which is preferable for relative quantification of gene expression using the comparative ΔCT method. To perform expression analysis of genes of interest in biological samples, four duplicate assay mixtures were prepared by mixing the respective primers / probes of two specific assays. To separately detect CT values, the assay probes were modified with different fluorescent probes. Each of the four assay mixtures contained 2 μM unmodified forward and reverse primers and 1.2 μM probe. For each reaction, 2.5 μl of total RNA extracted from FFPE sections (see above) was mixed with 2.5 μl of assay mixture, 2.5 μl of enzyme mixture, and 2.5 μl of water in one well of a 96-well optical reaction plate. PCR reactions were measured using a Versant kPCR Cycler (Siemens) or a Light Cycler 480 (Roche) according to the manufacturer's instructions under the appropriate conditions (5 min 50°C, 1 cycle; 20 s 95°C, 1 cycle; 15 s 95°C; 1 min 60°C, 40 cycles).

[0256] Determination of luminal and basal subtypes in the UC cohort by RT-qPCR revealed a similarly wide dynamic range for KRT5 and KRT20 mRNA, with 40-DCT values ​​ranging from 19 to 48. The dynamic range for PD-1 and PD-L1 mRNA expression ranged from 19 to 41 for both mRNA analyses. The dynamic range of FGFR genes is quite distinct within the FGFR family: the dynamic range for FGFR1 ranged from 29 to 37, the dynamic range for FGFR2 from 19 to 39, the dynamic range for FGFR3 from 19 to 43, and the dynamic range for FGFR4 from 19 to 36.

[0257] Figure 4 shows the data distribution of gene expression for luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 as determined by RT-qPCR of FFPE tissues in patients with muscle-invasive bladder cancer (n=61).

[0258] Spearman correlation revealed significant high co-expression of FGFR receptors 2 (p = 0.0008) and 3 (p = 0.0066) within the luminal urothelial carcinoma cell line (KRT20). No significant upregulation of any FGFR genes was observed in basal-like urothelial carcinoma. Furthermore, FGFR2 and FGFR3 expression was significantly associated with low PD-1 (p = 0.02554) and PD-L1 (p = 0.0074) mRNA expression. However, the checkpoint markers PD-1 (p = 0.0004) and PD-L1 (p = 0.0452) showed significant high expression in the basal-like urothelial carcinoma subtype (KRT5). Both PD-1 and PD-L1 mRNA expression are associated with immune cell infiltration into tumor tissue, as previously described (Eckstein et al., Oncotarget 2018).

[0259] Figure 5 shows intergenic Spearman correlations of luminal and basal subtype markers, checkpoint target genes, and FGFR1-4 gene mRNA expression as determined by RT-qPCR in tissues from muscle-invasive bladder cancer patients (n=61).

[0260] mRNA expression analysis of luminal and basal markers, PD-1, PD-L1, and FGFR family, as well as classical and non-classical HLA expression profiling were performed.

[0261] Figure 6 shows intergenic Spearman correlations of HLA gene mRNA expression determined by RT-qPCR of FFPE tissue from muscle-invasive bladder cancer patients (n=61).

[0262] As shown in Figure 6, the intergene correlations of various HLA genes show complex patterns. As an example, HLA-J expression is only moderately correlated with non-classical HLA-G or classical HLA-A or HLA-B / C gene expression, with Spearman correlation coefficients ranging from 0.34, 0.016, and 0.27, respectively. Similarly, other pseudogenes, such as HLA-H, exemplified by the HLA-H exon 1 / 2 assay, are only weakly or moderately correlated with classical and non-classical HLAs, e.g., HLA-G, HLA-A, HLA-B / C, and HLA-J (r = 0.23993, r = 0.2376, r = 0.3373, r = 0.1550). Importantly, there are significant differences in the correlation coefficients of one HLA gene, such as HLA-H exon 1 / 2 versus HLA-H exon 2 / 3, when compared with other HLA gene segments. This represents a differential splicing event that results in intra- and inter-gene interactions of clinical relevance.

[0263] As an example of inter- and intragenic interactions of HLA gene expression that affect disease-specific survival in cancer patients receiving immunotherapy, we analyzed mRNA expression of HLA-G in combination with HLA-A. Both genes were determined by a highly specific assay that defines a unique region in the relatively heterogeneous part of the HLA gene after translation stop in exon 7.

[0264] Figure 7 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from muscle-invasive bladder cancer patients (n=61) stratified by combined HLA-A exon 8, HLA-G exon 8, and HLA-G exon 5 mRNA expression, as quantified by RT-qPCR assay. Relative mRNA expression was determined by the 40-DCT method using CALM2 as the reference gene. Patients whose tumors showed low HLA-G exon 8 mRNA expression (<28.43) and low HLA-G exon 5 mRNA expression (<37.11) had the best survival (gray, solid line). Patients whose tumors showed low HLA-G exon 8 mRNA expression (<28.43) but high HLA-G exon 5 mRNA expression (>37.11) had the second-worst survival (gray, dotted line). Patients whose tumors showed high HLA-G exon 8 mRNA expression (>28.43) and high HLA-A exon 8 mRNA expression (>35.26) showed the second-best survival time (black, dotted line). Patients whose tumors showed high HLA-G exon 8 mRNA expression (>28.43) but low HLA-A exon 8 mRNA expression (<35.26) showed the worst survival time (black, solid line).

[0265] As shown in Figure 7, patients with urothelial carcinoma treated with immuno-oncology drugs after prior chemotherapy failure had the worst survival time if they expressed HLA-G exon 8 but not HLA-A exon 8 (compare the blue and gold curves). These data indicate that the presence of classical HLA can compensate for the otherwise lethal expression of non-classical HLA when patients undergo immuno-oncology ("IO") treatment. This also indicates that immunomodulatory drugs targeting checkpoint inhibition (e.g., anti-PD1 / anti-PDL1) appear to be more effective in tumors with at least partially intact classical HLA function. Furthermore, these data demonstrate the superiority of combining the determination of two or more HLA genes to identify the prognostic value of individual HLA regions, which is a prerequisite for maximizing the efficacy of immunotherapy and reducing the risk of potential hazards associated with the combination of chemotherapy and immunotherapy.

[0266] Figure 8 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from muscle-invasive bladder cancer patients (n=61) stratified by the gene-by-gene combination of HLA-A exon 8 and HLA-G exon 8 mRNA expression, as quantified by RT-qPCR assay. Relative mRNA expression was determined by the 40-DCT method using CALM2 as the reference gene. Patients whose tumors showed high HLA-G exon 8 mRNA expression (>28.43) and high HLA-A exon 8 mRNA expression (>35.26) had the second-best survival (black, dotted line). Patients whose tumors showed high HLA-G exon 8 mRNA expression (>28.43) but low HLA-A exon 8 mRNA expression (<35.26) had the worst survival (black, solid line).

[0267] Figure 9 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from patients (n=61) with muscle-invasive bladder cancer, stratified by the intragenic combination of HLA-G exon 8 and exon 5 mRNA expression, as quantified by RT-qPCR assay. Relative mRNA expression was determined by the 40-DCT method, using CALM2 as the reference gene. Patients whose tumors showed low HLA-G exon 8 mRNA expression (<28.43) and low HLA-G exon 5 mRNA expression (<37.11) had the best survival (gray, solid line). Patients whose tumors showed low HLA-G exon 8 mRNA expression (<28.43) but high HLA-G exon 5 mRNA expression (>37.11) had poorer survival (gray, dotted line).

[0268] As an example of intragenic interactions of HLA gene expression that affect disease-specific survival in cancer patients receiving immunotherapy, we analyzed HLA-G expression in HLA-G exon 8 and HLA-G exon 5 by RT-qPCR. Determining HLA-G exon 8 quantifies the untranslated exon at the 3' end of the gene (after the C-terminus, the cytoplasmic protein tail). This allows for the specific determination of numerous HLA-G splice variants, which may include or exclude, for example, various extracellular alpha domains and / or transmembrane regions as well as the cytoplasmic portion. This type of HLA-G determination is not possible with antibodies at the protein level and represents a highly specific HLA-G assessment. Given the numerous HLA-G splice variants associated with expression of HLA-G exon 5, which resembles the alpha 3 domain, quantifying the combination of two HLA-G mRNA fragments reveals the ability to distinguish different prognostic subgroups of patients with superior or inferior disease-specific survival when considering the time from initiation of immuno-oncology therapy ("IO") to death. Patients with low expression of splice variants containing HLA-G exon 8, but at the same time high levels of fragments containing HLA-G exon 5, are at higher risk of disease-specific death in the advanced chemotherapy-refractory setting, despite initiating IO therapy.

[0269] To demonstrate the superiority of combined HLA diagnosis, we compared the prognostic value of single-gene determinations. As shown in Figure 10, determination of HLA-G exon 8 alone also had a general prognostic value (p=0.0359). However, as shown in Figure 7, there are many patients in the "poor prognosis" group with high single-gene HLA-G exon 8 mRNA expression who also exhibit high HLA-A exon 8 expression (n=20 patients were considered low risk due to high HLA-A exon 8 expression, and n=33 patients were considered "high risk" based on high HLA-G exon 8 mRNA expression). Therefore, nearly two-thirds of patients in the "poor prognosis" group could avoid additional treatment if more accurate tissue diagnosis for the diagnosis of HLA gene combinations were performed.

[0270] Figure 10 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for FFPE tissue from muscle-invasive bladder cancer patients (n=61) stratified by single-gene determination of HLA-G exon 8 and exon 5 mRNA expression alone, as quantified by RT-qPCR assay. Relative mRNA expression was determined by the 40-DCT method using CALM2 as the reference gene. Patients whose tumors showed high HLA-G exon 8 mRNA expression (>28.43) and high HLA-A exon 8 mRNA expression (<28.43) had favorable survival (gray, dotted line). Patients whose tumors showed high HLA-G exon 8 mRNA expression (>28.43) had poorer survival (black, solid line).

[0271] Example 4: Determination of HLA-sense and HLA-antisense mRNA expression levels by reverse transcription (RT) quantitative PCR (RT-qPCR) in a cohort of ovarian cancer patients treated with neoadjuvant therapy Furthermore, the inventors determined whether the combined use of two or more HLA group gene sequences is applicable to other types of tumors other than bladder cancer, such as gynecological cancers, particularly ovarian cancer. Furthermore, the inventors determined whether the combined use of HLA group genes and HLA group antisense expression can be determined to predict cancer outcomes. Furthermore, researchers evaluated whether combinations of HLA sense and antisense can be used to predict response / non-response to treatment regimens other than immuno-oncology treatments, such as chemotherapy and / or hormonal therapy.

[0272] Forty-five newly diagnosed patients with histologically confirmed FIGO stage III-IV epithelial ovarian or peritoneal cancer (hereinafter also referred to as ovarian cancer) who were not suitable candidates for optimal upfront surgery and neoadjuvant chemotherapy were enrolled in the study between September 2004 and December 2007. Other inclusion criteria were age >18 years and hematological, renal, hepatic, and cardiac function suitable for platinum-based chemotherapy. Exclusion criteria were a Karnofsky performance status (KPS) <70%, a history of other malignancies, and contraindications to surgery. The possibility of optimal subtotal surgery was ruled out at baseline by open laparoscopy. After excluding nine patients whose biopsy samples were insufficient for microarray analysis and one patient who was found to be ineligible due to a diagnosis of peritoneal mesothelioma after histological revision, the initial study population of 45 patients was limited to 35. Carboplatin AUC5 and paclitaxel 175 mg / m 2 The standard Q3 regimen was administered over 3 hours every 3 weeks for 6 cycles as neoadjuvant treatment. Three patients aged 75 years or older and one patient with poor performance status (KPS 70%) preferred single-agent carboplatin over combination chemotherapy.

[0273] After surgery, histopathological response was assessed using surgical specimen analysis. To date, no firm histopathological criteria have been established to describe treatment response after neoadjuvant chemotherapy in ovarian cancer. Following literature on response to first-line chemotherapy in ovarian cancer (Le et al. 2007, Sassen et al. 2007) and breast cancer (Ogston et al. 2003), the absence of cancer cells in the surgical specimen was considered a pathological complete response, while the presence of only small clusters (<1 cm) or individual cancer cells, and no visible remnants after surgery, were considered a very good partial response. A pathological partial response was defined as a 30% to 90% reduction in tumor burden at the time of surgery, and stable disease was defined as no reduction in tumor burden or a reduction of less than 30% at surgery compared to the initial diagnostic laparoscopy. Only patients with complete and very good partial responses were considered pathological responders; all other cases were considered pathological non-responders. For survival analysis, the time from initial diagnosis to progression (PFS) or death (OS), or the time between progression and death (PDT), was used for Kaplan-Meier survival estimation and Cox regression analysis. Complete survival data were available for 40 patients. At the end of data collection, the median PFS was 14.7 months, and the median OS was 33.5 months, which is high given the very advanced stage of disease (unresectable FIGO III-IV) at the time of study entry.

[0274] For mRNA detection, collected tissues were snap-frozen and stored in liquid nitrogen until analysis. Approximately 20–100 mg of frozen ovarian tumor tissue was pulverized in liquid nitrogen. RNA was extracted using a commercially available kit (Qiagen), RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and cDNA was synthesized from 1 mg of total RNA using an Invitrogen kit (Invitrogen Corp.) and analyzed on an Affymetrix HG-U133A microarray (Affymetrix Inc., Santa Clara, CA, USA) as described elsewhere (Ihnen et al., 2008).

[0275] For validation purposes, RT-qPCR was applied to total RNA isolated from the same fresh tissue biopsies described above to verify the array data using an independent technical method. Gene-specific TaqMan-based primer / probe sets were used to assess HLA-F or HLA-FAS expression. For detailed analysis of gene expression by RT-qPCR, primers flanking the region of interest and fluorescently labeled probes hybridizing between them were utilized. Target-specific primers and probes were selected using the NCBI primer design tool (www.ncbi.nlm.nih.go). RNA-specific primer / probe sequences were used to position primer / probe sequences across exon / exon boundaries, enabling RNA-specific measurements. Furthermore, primers / probes were selected to avoid binding to sequence regions with known polymorphisms (SNPs). In cases where multiple isoforms of the same gene exist, primers were selected to amplify all relevant or selected splice variants, as appropriate. The specificity of all primer pairs was tested by conventional PCR reactions. After further primer / probe optimization, the primers and probes listed in Table 6 yielded the best results. These primers / probes are superior to primers / probes known from the prior art, for example, in terms of specificity and amplification efficiency. CALM2 was selected as a reference gene to normalize the amount of sample RNA, since CALM2 was not differentially regulated in the analyzed samples. Sample pairs with low RNA content (i.e., CALM2 raw CT value less than 22) were excluded due to pre-treatment biopsies or post-treatment resections.

[0276] [Table 5] TIFF0007762135000008.tif25580

[0277] TaqMan® validation experiments showed that the amplification efficiencies of the target and control were nearly equal, which is preferable for relative quantification of gene expression using the comparative ΔCT method. To perform expression analysis of genes of interest in biological samples, four duplicate assay mixtures were prepared by mixing the respective primers / probes of two specific assays. To separately detect CT values, the assay probes were modified with different fluorescent probes. Each of the four assay mixtures contained 2 μM unmodified forward and reverse primers and 1.2 μM probe. For each reaction, 2.5 μl of total RNA extracted from FFPE sections (see above) was mixed with 2.5 μl of assay mixture, 2.5 μl of enzyme mixture, and 2.5 μl of water in one well of a 96-well optical reaction plate. PCR reactions were measured using a Versant kPCR Cycler (Siemens) or a Light Cycler 480 (Roche) according to the manufacturer's instructions under the appropriate conditions (5 min 50°C, 1 cycle; 20 s 95°C, 1 cycle; 15 s 95°C; 1 min 60°C, 40 cycles).

[0278] Figure 11 shows the data distribution of relative mRNA expression (40-DCT) and antisense HLA-F expression of HLA-F isoforms determined by RT-qPCR. This shows the relative mRNA expression levels of defined sense and antisense regions of HLA genes, as exemplified by HLA-F. The exons of three known HLA-F isoforms, HLA-F1, HLA-F2, and HLA-F3, and the HLA antisense isoforms AS1 and AS2, were determined by RT-qPCR after DNAse digestion of nucleic acid extracts. Interestingly, the expression levels of different HLA-F AS regions varied significantly, with HLA-F AS1 exon 6 expression being the highest, with a median 40-DCT of 37.88 before neoadjuvant chemotherapy. Furthermore, subtractive analysis of isoform comparison revealed particularly high expression of HLA-F2 and HLA-F3 in pretreatment biopsies of ovarian cancer samples.

[0279] HLA-F expression was set in the context of a previously published molecular subtyping study into hormone-dependent and hormone-independent ovarian cancer (Zamagni et al., "Estrogen receptor 1 mRNA is a prognostic factor in ovarian cancer patients treated with neoadjuvant chemotherapy: determination by array and kinetic PCR in fresh tissue biopsies." ERC 2009). For this purpose, mRNA expression of ESR1, HLA-F3, and HLA_F AS1 exon 6 was combined by constructing decision tree models, gene ratios, and linear combinations.

[0280] FIG. 12 shows the data distribution of relative mRNA expression (40-DCT) of ESR1, HLA-F3, and HLA-F AS1 expression determined by RT-qPCR.

[0281] HLA-F3 is a non-classical HLA-binding protein molecule that contains extracellular alpha 1 and alpha 2 domains for forming a peptide-presenting protein groove for antigen presentation, but lacks the alpha 3 domain for interaction with immune cells, such as T cell or natural killer cell activation. Like all known HLA-F isoforms, HLA-F3 also contains a transmembrane domain and is therefore thought to be present on the cell surface for immune cell interaction.

[0282] The predictive value of HLA-F3 mRNA expression was examined by split-testing progression-free survival as an endpoint.

[0283] FIG. 13 shows a split-study of HLA-F3 mRNA expression in pre-treatment biopsy samples of neoadjuvant-treated ovarian cancer patients as determined by RT-qPCR to predict progression-free survival.

[0284] As shown in Figure 13, a cutoff near the median pretreatment HLA-F3 (DCT 34.94) expression separated the neoadjuvant ovarian cancer cohort into two equally sized groups with significantly different median survival times and high HLA-F3 expression, which was associated with prolonged survival (1392 days progression-free survival) versus shortened survival (400 days progression-free survival) at low HLA-F3 expression.

[0285] Figure 14 shows a Kaplan-Meier plot showing progression-free survival (PFS) probability based on stratification by the single gene HLA-F3 alone, as quantified by RT-qPCR assay on fresh tissue from patients with advanced ovarian cancer (n=27). Relative mRNA expression was determined by the 40-DCT method, using CALM2 as the reference gene. Patients whose tumors showed high HLA-F3 mRNA expression (>=34.94) had a favorable survival time (black, solid line). Patients whose tumors showed low HLA-F3 mRNA expression (<34.94) had a worse survival time (black, dashed line).

[0286] Kaplan-Meier analysis demonstrated the significance of HLA-F3 mRNA expression in predicting survival. As shown in Figure 14, patients with high HLA-F3 mRNA expression in primary ovarian cancer tissues had a median progression-free survival of 28.5 months, while patients with low HLA-F3 mRNA expression had a median progression-free survival of 12.5 months.

[0287] Similarly, Kaplan-Meier analysis of overall survival revealed significant survival differences when stratified based on HLA-F3 mRNA. As shown in Figure 14, patients with high HLA-F3 mRNA expression in primary ovarian cancer tissues had a median overall survival of 52.5 months, whereas patients with low HLA-F3 mRNA expression had a median overall survival of 22.9 months.

[0288] Figure 15 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratification by the single gene HLA-F3 alone, as quantified by RT-qPCR assay on fresh tissue from patients with advanced ovarian cancer (n=27). Relative mRNA expression was determined by the 40-DCT method, using CALM2 as the reference gene. Patients whose tumors showed high HLA-F3 mRNA expression (>=34.94) had a favorable survival time (black, solid line). Patients whose tumors showed low HLA-F3 mRNA expression (<34.94) had a worse survival time (black, dashed line).

[0289] When adjusting for clinical parameters such as grade, FIGO stage, and primary site (ovarian vs. peritoneal), mRNA stratification into high and low mRNA expression had an LR Chi of 6.14 (p=0.0132). 2 It remained an independent factor for predicting progression-free survival, with a mean value of 0.22 and a hazard ratio of 0.22, while all other clinical factors were non-significant.

[0290] Figure 15 is based on a multivariate analysis of PFS using a Cox proportional hazards model including grade, FIGO stage, primary site, and HLA-F3 mRNA expression. When adjusting predictions for overall survival for clinical parameters such as grade, FIGO stage, and primary site (ovarian vs. peritoneal), mRNA stratification into high and low mRNA expression had an LR Chi of 3.19. 2 It remained an independent factor for predicting progression-free survival, with a mean value (p=0.0441) and a hazard ratio of 0.31, while all other clinical factors were non-significant.

[0291] Figure 16 shows a multivariate analysis of OS using a Cox proportional hazards model including grade, FIGO stage, primary site, and HLA-F3 mRNA expression.

[0292] As a next step, we place HLA-F3 expression in the context of molecular subtypes by differentiating ESR1 mRNA levels into hormone-dependent and hormone-independent ovarian cancers. ESR1 mRNA stratification using a 40-DCT value of 37.75 distinguished 37% of ovarian cancers that were ERS1-negative and had a median progression-free survival of approximately 15.72 months from ESR1-positive ovarian cancers, which accounted for 63% of all ovarian cancer patients and had a median progression-free survival of 36.47 months (Figure 17).

[0293] FIG. 17 shows a split-screen study of ESR1 and HLA-F3 mRNA expression in pre-treatment biopsy samples of ovarian cancer patients treated with neoadjuvant therapy, as determined by RT-qPCR to predict progression-free survival.

[0294] Figure 18 shows a Kaplan-Meier plot showing progression-free survival (PFS) probability based on stratified ESR1 and HLA-F3 mRNA expression, quantified by RT-qPCR assay in fresh tissue from patients with advanced ovarian cancer (n=27). Relative mRNA expression was determined by the 40-DCT method, using CALM2 as the reference gene. Patients whose tumors showed high ESR1 mRNA expression (>=37.75) and high HLA-F3 mRNA expression (>=34.94) exhibited favorable survival (black, solid line). Patients whose tumors showed high ESR1 mRNA expression (>=37.75) and low HLA-F3 mRNA expression (<34.94) exhibited poorer survival (black, dashed line). Patients with low ESR1 mRNA expression (<37.75) also exhibited a poor prognosis (gray, solid line).

[0295] Kaplan-Meier analysis demonstrated the significance of combining ESR1 and HLA-F3 mRNA expression in predicting progression-free survival. As shown in Figure 18, patients with high ESR1 and high HLA-F3 mRNA expression in primary ovarian cancer tissues had a median progression-free survival of 38.7 months, whereas patients with high ESR1 mRNA expression and low HLA-F3 mRNA expression had a shortened median progression-free survival of 11.6 months. Patients with low ESR1 mRNA expression showed a similar poor prognosis.

[0296] Furthermore, Kaplan-Meier analysis demonstrated the significance of combining ESR1 and HLA-F3 mRNA expression in predicting overall survival. As shown in Figure 18, patients with high ESR1 and high HLA-F3 mRNA expression in primary ovarian cancer tissues had a median overall survival of 38.7 months, whereas patients with high ESR1 mRNA expression and low HLA-F3 mRNA expression had a shortened median progression-free survival of 11.6 months. Patients with low ESR1 mRNA expression also showed a similar poor prognosis.

[0297] Figure 19 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratified ESR1 and HLA-F3 mRNA expression, quantified by RT-qPCR assay in fresh tissue from patients with advanced ovarian cancer (n=27). Relative mRNA expression was determined by the 40-DCT method, using CALM2 as the reference gene. Patients whose tumors showed high ESR1 mRNA expression (>=37.75) and high HLA-F3 mRNA expression (>=34.94) exhibited favorable survival (black, solid line). Patients whose tumors showed high ESR1 mRNA expression (>=37.75) and low HLA-F3 mRNA expression (<34.94) exhibited poorer survival (black, dashed line). Patients with low ESR1 mRNA expression (<37.75) also exhibited a poor prognosis (gray, solid line).

[0298] When adjusting for clinical parameters such as grade, FIGO stage, and primary site (ovarian vs. peritoneal), combined mRNA stratification based on ESR1 and HLA-F3 mRNA expression yielded an LR Chi of 9.53. 2 HLA-F3 mRNA expression remained an independent predictor of progression-free survival at a low level (p=0.0095), while all other clinical factors were non-significant. The hazard ratios for high ESR1 and high HLA-F3 mRNA expression reached 0.097 and 0.152 when compared with high ESR1 and low HLA-F3 or low ESR1 (p=0.0042 and p=0.0136, respectively).

[0299] Figure 20 shows a multivariate analysis of PFS using a Cox proportional hazards model including a combination of grade, FIGO stage, primary site, and ESR1 and HLA-F3 mRNA expression.

[0300] Furthermore, when analyzing overall survival and adjusting for clinical parameters grade, FIGO stage, and primary site (ovarian vs. peritoneal), combined mRNA stratification based on ESR1 and HLA-F3 mRNA expression resulted in an LR Chi of 6.53 (p=0.0383). 2 At this level, HLA-F3 remained an independent predictor of progression-free survival, while all other clinical factors were non-significant. The hazard ratios for high ESR1 and high HLA-F3 mRNA expression reached 0.184 and 0.230 when compared with high ESR1 and low HLA-F3 or low ESR1 (p = 0.0832 and p = 0.0182, respectively).

[0301] Figure 21 shows a multivariate analysis of OS using a Cox proportional hazards model including a combination of grade, FIGO stage, primary site, and ESR1 and HLA-F3 mRNA expression.

[0302] The above data analysis requires adjustment of individual HLA-F3 mRNA levels relative to housekeeping genes to obtain normalized expression levels, expressed as 40-DCT values. Interestingly, the HLA-F3 genomic locus contains an antisense gene, designated HLA-F3 AS, located 3' to exon 8 of the reverse strand, which may be important for gene expression regulation and protein translation of HLA-F3 mRNA. To investigate the relevance of the putative antisense transcript and eliminate the need for housekeeper normalization, we examined the gene ratio of HLA-F3 to HLA-FAS1.

[0303] Figure 22 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratified HLA-F3 and HLA-F AS1 mRNA expression, quantified by RT-qPCR assay in fresh tissues from patients with advanced ovarian cancer (n=27). Instead of relative mRNA expression, housekeeper-free gene ratios are determined by the 40-DCT method using CALM2 as the reference gene. Patients whose tumors showed high HLAF gene ratio expression (>=2.35) showed better survival (black, solid line). Patients whose tumors showed low gene ratios (<2.35) showed worse survival (black, dashed line).

[0304] Kaplan-Meier analysis demonstrated the significance of combining HLA-F3 and HLA-FAS1 mRNA expression to predict progression-free survival, as shown in Figure 22. Patients with high HLA-F3 mRNA expression and simultaneously low levels of HLA-FAS1 mRNA expression in primary ovarian cancer tissues had a median progression-free survival of 38.7 months (gene ratio >= 2.35), whereas patients with low HLA-F3 mRNA expression and high HLA-FAS1 mRNA expression (gene ratio < 2.35) showed a shortened median progression-free survival of 12.6 months.

[0305] Figure 23 shows a Kaplan-Meier plot showing overall survival (OS) probability based on stratified HLA-F3 and HLA-F AS1 mRNA expression, quantified by RT-qPCR assay in fresh tissues from patients with advanced ovarian cancer (n=27). Instead of relative mRNA expression, housekeeper-free gene ratios were determined by the 40-DCT method using CALM2 as the reference gene. Patients whose tumors showed high HLAF gene ratio expression (>=2.35) had better survival (black, solid line). Patients whose tumors showed low gene ratios (<2.35) had worse survival (black, dashed line).

[0306] Kaplan-Meier analysis also demonstrated the significance of the combination of HLA-F3 and HLA-FAS1 in predicting overall survival, as calculated by gene ratio. As shown in Figure 23, patients with high HLA-F gene ratio (>=2.35) expression in primary ovarian cancer tissues had a median overall survival of 61.5 months, while patients with high ESR1 mRNA expression and low HLA-F3 mRNA expression showed a shortened median progression-free survival of 23.1 months.

[0307] When adjusting for clinical parameters including grade, FIGO stage, and primary site (ovarian vs. peritoneal), combined mRNA stratification based on ESR1 and HLA-F3 mRNA expression remained an independent predictor of progression-free survival with an LR Chi2 value of 4.71 (p=0.0301), whereas all other clinical factors except primary site were not significant (p=0.0479). The hazard ratio for high HLAF3 and low HLA-FAS1 mRNA expression reached a hazard ratio of 0.2866 (p=0.0301).

[0308] Figure 24 shows a multivariate analysis of PFS using a Cox proportional hazards model including grade, FIGO stage, primary site, and a combination of HLA-F3 and HLA-FAS1.

[0309] Furthermore, when analyzing overall survival and adjusting for clinical parameters grade, FIGO stage, and primary site (ovarian vs. peritoneal), combined mRNA stratification based on HLA-F3 and HLA-F AS1 mRNA expression resulted in an LR Chi of 8.41 (p=0.0037). 2 At this level, HLA-F3 / low HLA-FAS1 remained an independent predictor of progression-free survival, while all other clinical factors were non-significant. The hazard ratio for high HLA-F3 / low HLA-FAS1 versus low HLA-F3 / high HLA-FAS1 reached a hazard ratio of 0.189 (p=0.0037).

[0310] Figure 25 shows a multivariate analysis of OS using a Cox proportional hazards model including grade, FIGO stage, primary site, and a combination of HLA-F3 and HLA-F AS1.

[0311] Example 5: HLA profiling in advanced chemotherapy-refractory urothelial cancer TUR biopsies and cystectomy specimens from primary tumors refractory to chemotherapy and subsequently treated with first- or second-line immuno-oncology ("IO") therapy with PD-1 and PD-L1 checkpoint inhibitors (i.e., atezolizumab, nivolumab, and pembrolizumab) will be analyzed for HLA expression and correlated with histopathological and molecular parameters as well as response to IO treatment and disease-specific survival after IO.

[0312] Seventy-two newly diagnosed patients with histologically confirmed urothelial carcinoma, including bladder and upper tract urothelial carcinoma, were enrolled in the study between 2016 and 2018. Nivolumab, pembrolizumab, and atezumab were administered as first-, second-, and third-line monotherapy according to approved protocols. All hematoxylin-eosin (HE)-stained tumor tissue sections from the cohort samples were evaluated and classified by two uropathologists according to the UICC TNM classification (2017). Rare histologic variants were classified according to the World Health Organization (WHO) Classification of Genitourinary Tumors (2016). After central histopathological review, 18 tissues were excluded due to insufficient tumor material or non-urothelial carcinoma. Only lymph node tissue was available from five patients and therefore excluded from the primary analysis of the prognostic and / or predictive effect of HLA gene expression (Figure 26; see consort diagram).

[0313] Figure 26 shows the consort diagram for the advanced or metastatic urothelial cancer cohort. After excluding insufficient FFPE blocks and / or lymph node tissue, tissue from 55 patients was available for analysis.

[0314] For mRNA detection, RNA was extracted from FFPE tissues derived from TUR biopsies, cystectomies, and corresponding mapping bladder tissues using a commercially available kit (Xtract, Stratifyer). For each reaction, 2.5 μl of total RNA extracted from FFPE sections was mixed with 2.5 μl of assay mixture, 2.5 μl of enzyme mixture, and 2.5 μl of water in one well of a 96-well optical reaction plate. PCR reactions were performed using a Versant kPCR Cycler (Siemens) or Light Cycler 480 (Roche) according to the manufacturer's instructions under the appropriate conditions: 5 min at 50°C for one cycle; 20 s at 95°C for one cycle; 15 s at 95°C for one cycle; 1 min at 60°C for 40 cycles. Relative mRNA expression was associated with response to IO treatment, as determined based on RECIST criteria assessed at each individual site, and disease-specific survival, as determined from the start of IO treatment until cancer-specific death. Contingency tests using biostatistics JMPSAS 9.0.0 (SAS, Cary, North Carolina, USA) were performed to assess possible differences in response to IO treatment.

[0315] For detailed analysis of gene expression by RT-qPCR, we utilized primers flanking the region of interest and fluorescently labeled probes hybridizing between them. Target-specific primers and probes were selected using the NCBI primer design tool (www.ncbi.nlm.nih.go). RNA-specific primer / probe sequences were used to position the primer / probe sequences across exon / exon boundaries, enabling RNA-specific measurements. Furthermore, primers / probes were selected to avoid binding to sequence regions with known polymorphisms (SNPs). In cases where multiple isoforms of the same gene exist, primers were selected to amplify all relevant or selected splice variants, as appropriate. Primer pair specificity was examined by conventional PCR reactions. After further primer / probe optimization, the primers and probes listed in the table above yielded the best results. These primers / probes are superior to primers / probes known from the prior art, for example, in terms of specificity and amplification efficiency. Because CALM2 was not differentially regulated in the analyzed samples, CALM2 was selected as a reference gene to normalize the amount of sample RNA. TaqMan® validation experiments were performed and showed that the amplification efficiencies of the target and control were approximately equal, which is a prerequisite for relative quantification of gene expression by the comparative ΔCT method.

[0316] Figure 27 shows a Kaplan-Meier plot showing the disease-specific survival (DSS) probability of patients with locally advanced or metastatic bladder cancer (n=55) stratified by HLA-F1 / F2 expression quantified by RT-qPCR assay. Relative mRNA expression is determined by the 40-DCT method using CALM2 as the reference gene.

[0317] As shown in Figure 27, high HLA-F1 / 2 mRNA expression (>=34.63) was significantly associated with better disease-specific survival, with HLA-F1 / F2-positive patients having a 60% survival probability after 2 years, while HLA-F1 / F2-exon-negative patients had a 20% survival probability after 2 years (p=0.0245).

[0318] Figure 28 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-F1 / F2 and HLA-G exon 8 mRNA expression quantified by RT-qPCR assay. Relative mRNA expression is determined by the 40-DCT method using CALM2 as the reference gene.

[0319] As shown in Figure 28, the combination of high HLA-F1 / 2 mRNA expression (>=34.63) and HLA-G exon 8 mRNA expression (>=30.16) improved the predictive value. Interestingly, high HLA-F1 / F2 mRNA expression and low HLA-G mRNA expression were significantly associated with better disease-specific survival (DSS): HLA-F1 / F2-positive / HLA-G exon 8-negative patients had an 80% survival probability after 2 years, whereas HLA-F1 / F2-exon negative patients had a 20% survival probability after 2 years, and HLA-F1 / F2-positive / HLA-G exon 8-positive patients had a 40% survival probability after 2 years (p=0.0245).

[0320] Figure 29 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-F1 / F2 and HLA-B / C exon 8 mRNA expression quantified by RT-qPCR assay. Relative mRNA expression is determined by the 40-DCT method using CALM2 as the reference gene.

[0321] As shown in Figure 29, when high HLA-F1 / 2 mRNA expression (>=34.63) was combined with HLA-B / C Ex8 mRNA expression (>=34.2), high HLA-B / C was significantly associated with better disease-specific survival, with HLA-B / C exon 8 and HLA-F1 / F2-negative patients having a 10% poorer survival probability after 2 years and HLA-B / C-negative and HLA-F1 / F2-positive patients having a 60% favorable survival probability after 2 years (p=0.0071).

[0322] In contrast, when looking only at HLA-B / C exon 8 mRNA expression, no significance could be determined (p=0.2127).

[0323] Figure 30 shows a Kaplan-Meier plot showing disease-specific survival (DSS) probability for muscle-invasive bladder cancer patients with locally advanced or metastatic UBC (n=55) stratified by HLA-B / C exon 8 mRNA expression quantified by RT-qPCR assay. Relative mRNA expression is determined by the 40-DCT method using CALM2 as the reference gene.

[0324] As shown in Figure 30, patients with high HLA-B / C exon 8 mRNA expression (>=34.2) did not have a significantly higher disease-specific survival time than patients with low HLA-B / C expression, with a survival probability of 60% after 2 years, while HLA-B / C exon 8-negative patients had a low survival probability of 40% after 2 years. This indicates that the combination of HLA expression is superior in predicting survival.

Claims

1. 1. A method for determining an individual's HLA pattern of a tumor, comprising: - determining a first expression level of an RNA transcript encoding a first region of a first HLA gene; - determining at least a second expression level of a sense or antisense RNA transcript of at least one second region of at least one second HLA gene; - comparing said determined first and second expression levels to obtain an HLA pattern for the individual; Including, determining the first expression level and the second expression level of the RNA transcript is performed by reverse transcription quantitative PCR and comprises determining whether the first expression level and the second expression level of the RNA transcript are lower or higher than a defined expression threshold for the RNA transcript; Comparing the determined first and second expression levels includes comparative quantification of the first and second expression levels with the defined expression threshold value by a ΔCT method or a ΔΔCT method, the first HLA gene is selected from the group consisting of genes encoding HLA-A, HLA-B, and HLA-C; the second HLA gene is selected from the group consisting of genes encoding HLA-D, HLA-E, HLA-F, HLA-G, HLA-H, and HLA-J; The method further comprises determining an HLA isoform based on the comparison of the first and second expression levels, wherein one of the first region and the second region comprises an exon-exon boundary and the other of the first region and the second region does not comprise an exon-exon boundary, or the first region comprises an exon-exon boundary and the second region comprises an exon-exon boundary.

2. - at least one of said first and second regions encodes a signal peptide region of an HLA group, and / or - at least one of said first and second regions encodes a transmembrane domain of an HLA group, The method of claim 1.

3. - determining whether the individual's HLA pattern is predominantly soluble or membrane-bound based on said comparison of said first and second expression levels. The method of claim 1 or 2, further comprising:

4. 4. The method of any one of claims 1 to 3, further comprising determining one or more further expression levels for one or more further regions of genes encoding HLA groups, wherein said comparison is further based on said determined further expression levels to obtain an HLA pattern of said individual.

5. The method of any one of claims 1 to 4, wherein said comparison comprises forming a ratio of expression levels.

6. A kit for use in the method according to any one of claims 1 to 5, comprising one or more combinations of primer pairs and probes selected from the group consisting of: a forward primer represented by SEQ ID NO: 1, a probe represented by SEQ ID NO: 2, and a reverse primer represented by SEQ ID NO: 3; a forward primer represented by SEQ ID NO: 4, a probe represented by SEQ ID NO: 5, and a reverse primer represented by SEQ ID NO: 6; a forward primer represented by SEQ ID NO: 7, a probe represented by SEQ ID NO: 8, and a reverse primer represented by SEQ ID NO: 9; a forward primer represented by SEQ ID NO: 10, a probe represented by SEQ ID NO: 11, and a reverse primer represented by SEQ ID NO: 12; a forward primer represented by SEQ ID NO: 13, a probe represented by SEQ ID NO: 14, and a reverse primer represented by SEQ ID NO: 15; a forward primer represented by SEQ ID NO: 16, a probe represented by SEQ ID NO: 17, and a reverse primer represented by SEQ ID NO: 18; a forward primer represented by SEQ ID NO: 19, a probe represented by SEQ ID NO: 20, and a reverse primer represented by SEQ ID NO: 21; a forward primer represented by SEQ ID NO: 22, a probe represented by SEQ ID NO: 23, and a reverse primer represented by SEQ ID NO: 24; a forward primer represented by SEQ ID NO: 25, a probe represented by SEQ ID NO: 26, and a reverse primer represented by SEQ ID NO: 27; a forward primer represented by SEQ ID NO: 28, a probe represented by SEQ ID NO: 29, and a reverse primer represented by SEQ ID NO: 30; a forward primer represented by SEQ ID NO: 31, a probe represented by SEQ ID NO: 32, and a reverse primer represented by SEQ ID NO: 33; a forward primer represented by SEQ ID NO: 37, a probe represented by SEQ ID NO: 38, and a reverse primer represented by SEQ ID NO: 39; a forward primer represented by SEQ ID NO: 40, a probe represented by SEQ ID NO: 41, and a reverse primer represented by SEQ ID NO: 42; a forward primer represented by SEQ ID NO: 43, a probe represented by SEQ ID NO: 44, and a reverse primer represented by SEQ ID NO: 45; a forward primer represented by SEQ ID NO: 46, a probe represented by SEQ ID NO: 47, and a reverse primer represented by SEQ ID NO: 48; a forward primer represented by SEQ ID NO: 49, a probe represented by SEQ ID NO: 50, and a reverse primer represented by SEQ ID NO: 51; a forward primer represented by SEQ ID NO: 52, a probe represented by SEQ ID NO: 53, and a reverse primer represented by SEQ ID NO: 54; a forward primer represented by SEQ ID NO: 55, a probe represented by SEQ ID NO: 56, and a reverse primer represented by SEQ ID NO: 57; a forward primer represented by SEQ ID NO: 58, a probe represented by SEQ ID NO: 59, and a reverse primer represented by SEQ ID NO: 60; a forward primer represented by SEQ ID NO: 61, a probe represented by SEQ ID NO: 62, and a reverse primer represented by SEQ ID NO: 63; A forward primer represented by SEQ ID NO: 64, a probe represented by SEQ ID NO: 65, and a reverse primer represented by SEQ ID NO: 66; and A forward primer represented by SEQ ID NO: 67, a probe represented by SEQ ID NO: 68, and a reverse primer represented by SEQ ID NO: 69.

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