WO2025051952A1

WO2025051952A1 - Method for detection of endometrium carcinoma CN high subtype

Info

Publication number: WO2025051952A1
Application number: PCT/EP2024/074981
Authority: WO
Inventors: Susanne WALZ; Alexander Hennig; Anke Weber; Christoph Schanzenbach; Matthias Preussler
Original assignee: Biotype GmbH
Current assignee: Biotype GmbH
Priority date: 2023-09-09
Filing date: 2024-09-06
Publication date: 2025-03-13
Anticipated expiration: 2026-03-09
Also published as: DE102023124346A1

Abstract

The present invention relates to a method for the detection of an endometrium carcinoma of the molecular subtype copy number high (CN high) by determination of a copy number variation (CNV) of a CNV chromosomal region comprising a panel of analytes. Thus, the present invention provides a new analyte panel suitable for the identification of endometrium carcinoma CN high subtype and a new analyte panel suitable as biomarker for the diagnosis of endometrium carcinoma CN high subtype. The invention further relates to suitable primer and probes for use in said method as well as kits for the detection of CN high endometrium carcinoma.

Description

Method for detection of endometrium carcinoma CN high subtype

Carcinoma of the uterus was first subclassified on the site of origin into tumors of the uterine corpus arising from the endometrium (Endometrial carcinoma, EC, ICD-11 C54.1) and those arising from the cervix (Cervical carcinoma, ICD-11 C53). In 1983, Bokhman [1] recognized two subtypes of EC based on clinical and histo-pathological features: Type I EC were mostly endometroid histotype and lower grade, related to unopposed estrogenic stimulation, and with a favourable prognosis. Type II EC (non-endometroid) were histologically high-grade tumors with clinically aggressive growth behaviour. According to The Cancer Genome Atlas Research Network (TCGA) [2] EC can be divided into four classes (subtypes) based on their molecular genetical profiles which were almost established by next generation sequencing (NGS) technologies:

1. MSI I MMRd hypermutated: Microsatellite instability (MSI) can be found in 25-30% of endometrial carcinomas. In tumors with microsatellite instability, alterations in genes of the DNA repair system (MMRd - mismath repair dysfunctional: Mutations in MLH1 , MSH2, MSH6, PMS1 , PMS2) are present, preventing errors in replication from being corrected. Among other things, this leads to changes in the length of short, repetitive DNA segments known as microsatellites or short tandem repeats (STRs). STRs with repeating units of 1 or 2 nucleotides are frequently used for MSI detection (EP1478781 B1) as surrogate markers for MMRd genes.

2. POLE ultramutated (POLEmut EC): Mutations in the gene POLE are present in 7-10% of EC tumors. Hotspot mutations are found in the exonuclease domain of the POLE gene, which is essential for chromosomal DNA replication and DNA repair. Mutations in POLE result in an ultramutated phenotype, meaning that a large number of other mutations occur throughout the genome. POLE ultramutated EC are copy-number-stable.

3. Copy number high (CN high, or p53abn): 25-30% of tumors show a “copy number high” (CN high) subtype. Large numbers of chromosomal aberrations are found in these tumors, i.e. larger chromosomal segments are either deleted or amplified [also referred to as “somatic copy number plasticity”, “somatic copy number alterations (SCNA)” or “somatic copy number variation” (SCNV)]. In addition, they frequently contain mutations in the tumor suppressor gene TP53 (p53abn - abnormal). Clinically, patient with these tumors have a poor prognosis with a 5 years progression-free survival rate of only 50%.

4. Copy number low (or NSMP - no specific molecular profile): The “copy number low” (CN low) subtype accounts for 15-40% of endometrial carcinomas. Tumors are characterized by the absence of a TP53 mutation but have mutations in a variety of other genes. Mutations in exon 3 of CTNNB1 are associated with a poor prognosis. This group encompasses mostly endometrial atypical hyperplasia/endometroid neoplasia (EAH/EIN histological grading) with high levels of estrogen and progesterone receptor (ER, PR) expression. They frequently persist for years at a precursor stage before progression to invasive carcinomas and may be treated with hormonal therapy. The four groups have prognostic value and represent a promising tool for clinical decision-making regarding adjuvant treatment.

Endometrial abrasions (curettage) or surgical biopsies are the main specimen for histopathological examination (FFPE - formalin-fixed, paraffin-embedded tissue sections and/or immunostaining) or molecular analysis. Despite advances in morphologic and immunohistochemical characterization of EC, histologic subtyping of EC remains subject to considerable interobserver variation, even among experts, and with application of immunohistochemistry (IHC). Therefore, several approaches were conducted to establish and validate Molecular Risk Classifiers (MRC) stemming from the findings of TCGA which were correlated with the early EC type I and II categories or the more detailed FIGO grading scheme recommended by the International Federation of Gynecology and Obstetrics ([3], [4], [5]). Furthermore, clinically applicable surrogate markers for the four molecular subtypes have been developed ([6], [7], [8]). As a result, inter alia the ProMisE Classifier (Proactive Molecular Risk Classifier for Endometrial Cancer) has been established which showed a high degree of interobserver reproducibility [5], However, ProMisE still uses combinations of IHC for MSH6, PSM2, and p53 and focused sequencing on mutations in POLE exons 9-14 (exonuclease domain), but a unified molecular protocol without individually biased errors would be favourable for standardization and laboratory automation. In addition, there is still the need to get more specific molecular markers for early diagnosis, risk stratification of EC, and therapy decision ([9], [10], [11]).

Thus, it is the object of the present invention to provide a unified molecular protocol without individually biased errors that allow for standardization and laboratory automation. It is another object of the present invention to provide more specific molecular analytes (synonym analyte molecular markers), in particular molecular biomarker, for early diagnosis, risk stratification of EC, and for therapy decision. It is another object of the present invention to provide a more specific and limited analyte panel, molecular biomarker panel, for early diagnosis, risk stratification of EC, and therapy decision making. It is the object of the present invention to provide suitable primer and probes for the detection of said analytes for detecting endometrium carcinoma of the copy number high subtype. It is further an object of the present invention to provide instructions on how to create primers and probes for a CNH detection with high specificity and to meet the required sensitivity. It is the object to provide a method for fast detection which is suitable for automation.

In summary, the present invention solves the problems of the prior art with the herein new nonsurrogate molecular diagnostic assay which improves the sensitivity of CN high genotyping (molecular EC type 3). This allows a better prognosis- and therapeutic stratification of the most aggressive endometrial cancers [12]. After extensive development, the herein provided method, in particular assay, preferably multiplex method/assay, is based on a set of new molecular analytes suitable as biomarkers that have been identified by applying bioinformatic analysis of Next-Generation Sequencing (NGS) data (1) and are proven as feasible for the detection of CN high endometrium carcinoma/tumors. Since the biomarkers are preferably detected by relative quantification applying polymerase chain reaction (PCR) technologies it was further necessary to identify reference regions that are genomically stable (or: possess constant, physiological normal copy numbers) in endometrial carcinomas.

The design of a PCR-based multiplex assay uses the cleavable probe technology in combination with a capillary electrophoresis (CE) devise, here MODAPLEX instrument, and an analysis of the results with the delta-Ct method - in which Ct values from instable and stable genomic regions are averaged, respectively, followed by the calculation of difference - to discriminate CN high from non-CN high tumor samples as described in the example.

There is no prior art disclosing or indicating the analyte panel of the present invention comprising a reference that comprises reference sequence encoding within the location of gene ZRANB2 and ZRANB2-AS1 on chromosome 1 , reference sequences encoding within the location of gene TMEM169 on chromosome 2 and reference sequence encoding within the location of gene HCAR1 on chromosome 12, in combination with target sequences encoding within the location of genes COX10, ARHGAP44, ELAC2 and MYOCD, respectively in the cytoband 17p12, and target sequences encoding within the location of genes ZNF555, TLE6, ZNF57 and ZNF77, respectively in the cytoband 19p13.3, for the detection CN high endometrium carcinoma and tumors. Finally, according to the invention, the object of the present invention is solved with the unique and inventive new analyte panel suitable as biomarker for the identification, detection and for diagnosis of endometrium carcinoma CN high subtype. Accordingly, by targeting, labelling, amplification and detection of said biomarkers, the inventive solution is realized. For all herein described embodiments of the method, the reference and the target sequences as defined herein are applicable comprising reference sequence encoding within the location of gene ZRANB2 and ZRANB2-AS1 on chromosome 1 , reference sequence encoding within the location of gene TMEM169 on chromosome 2 and the reference sequence encoding within the location of gene HCAR1 on chromosome 12. Said reference is used to detect the at least one CNV of at least one target sequence encoding within the location of genes COX10, ARHGAP44, ELAC2 and/or MYOCD, respectively in the cytoband 17p12, and target sequences encoding within the location of genes ZNF555, TLE6, ZNF57 and/or ZNF77, respectively in the cytoband 19p13.3.

The object of the invention is solved by the method according to claim 1 , the probe according to claim 22, and the kit according to claim 27. Embodiments of the invention are disclosed in detail in the dependent claims and in the description. All definitions are applicable to all embodiments.

In a first aspect of the present invention a method, preferably a multiplex method, for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high) is provided, wherein at least one copy number variation (CNV) is detected for a CNV chromosomal region comprising at least one cytoband on chromosome 17 and/or chromosome 19, wherein the method comprises the steps: providing a test sample of a subject comprising at least one predetermined target sequence in the CNV chromosomal region, optionally, providing at least a second test sample comprising at least one reference sequence, in particular that is nnegative for CNV (“relative stable” or “affected by CNV in rare cases" as defined herein), optionally determination of the copy number of the at least one or more reference sequences, providing a reference comprising at least one or more reference sequences of a known copy number, preferably the reference is negative for a CNV as defined herein, determining a CNV of at least one predetermined target sequence in the CNV chromosomal region, preferably by amplification or based on suitable probes as defined herein,- evaluating the CNV of the at least one predetermined target sequence in the CNV chromosomal region, preferably for at least one predetermined target sequence in at least one cytoband on chromosome 19 and/ or for at least one predetermined target sequence in at least one cytoband on chromosome 17, more preferably for at least one target sequence in cytoband 19p13.3 and /or 17p12, relative to the copy number of the reference, preferably based on Ct values and delta Ct values as defined herein (Example 1 , Table 5 and Table 6) identification of an endometrium carcinoma of the CN high subtype, wherein the CNV for the CNV chromosomal region is determined CN high, preferably for at least one predetermined target sequence in at least one cytoband on chromosome 19 and/or for at least one predetermined target sequence in at least one cytoband on chromosome 17, more preferably for at least one target sequence in cytoband 19p13.3 and /or 17p12, compared to the copy number of the reference.

The “subject” within the meaning of the present invention is human patient suffering from an endometrium carcinoma. The human patient is of female sex, transgender sex, transgender woman, or a human patient carrying the genetic predisposition for at least one CNV as defined herein of at least one target sequence as defined herein. The “subject” may carry endogenous endometrium, a transplanted endometrium or any transplanted tissue of an endometrium that may carry the genetic predisposition for at least one CNV in the CNV chromosomal region as defined herein.

The ”CNV chromosomal region” is defined by a copy number variation as defined herein, preferably the CNV chromosomal region is a CN high region (copy number variation high) wherein at least one or more target sequence are determined for an altered (increased or reduced) copy number compared to a suitable reference. Thus, within the meaning of the present invention the ”CNV chromosomal region” is a “CN high region”. Preferably, the ”CNV chromosomal region” is a CN high region comprising cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11.2, 19p13.3 and/or 19p13.2 or any combination thereof. The other definitions apply accordingly.

In the method the CNV is determined as CN high as average over all predetermined target sequences in the CNV chromosomal region compared to the copy number of the at least one reference sequence. Preferably the average of CNV is determined for at least one predetermined target sequence in at least one cytoband on chromosome 19 and/ or for at least one predetermined target sequence in at least one cytoband on chromosome 17, more preferably for at least one target sequence in cytoband 19p13.3 and /or 17p12, compared to the average copy number of the reference. The evaluation of CNV and subsequent identification of endometrium carcinoma of the CN high subtype is based on Ct values and delta Ct values (Example 1 , Table 5 and Table 6). Preferably, evaluating the CNV according to the present invention - and in any embodiment of the method - comprises calculating the delta Ct for the corresponding target sequences as defined herein, by subtracting the average Ct value of the reference as defined herein, from the arithmetic mean of the corresponding target sequences (see Table 6).

In an embodiment of the present invention in the method, preferably the multiplex method, the CNV chromosomal region comprises cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11 .2, 19p13.3 and/or 19p13.2 or any combination thereof. Preferably CNV is determined for at least one or more predetermined target sequences in cytoband 17p12 and/or for at least one or more predetermined target sequences 19p13.3, preferably in cytoband 19p13.3 on chromosome 19 and in cytoband 17p12 or chromosome 17. In one embodiment the target sequence may be a target sequence overlapping at least two cytobands.

In another embodiment of the invention, in the step of determining a CNV of at least one predetermined target sequence in the CNV chromosomal region of the method, preferably multiplex method, the CNV is determined for at least one or more predetermined target sequences in at least one cytoband of cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11.2, 19p13.3 and/or 19p13.2 or in any combination of one or more of the aforementioned cytobands.

In another embodiment of the invention, in the step of determining a CNV of at least one or more predetermined target sequences in the CNV chromosomal region of the method, preferably a multiplex method, the CNV is determined for at least one or more predetermined target sequences in cytoband 19p13.3 on chromosome 19 and for at least one or more predetermined target sequences in cytoband 17p12 on chromosome 17.

Preferably in the method of the present invention, preferably in the multiplex method, the at least one CNV in the CNV chromosomal region is detected for at least one or more target sequences within the location of genes COX10, ARHGAP44, ELAC2 and/or MYOCD in the cytoband 17p12 and/or for at least one or more target sequences within the location of genes ZNF555, TLE6, ZNF57 and/or ZNF77 of cytoband 19p13. Preferably, a CNV is detected for a combination of at least two, at least three or for all four genes from the group of COX10, ARHGAP44, ELAC2 and/or MYOCD. Preferably, a CNV is detected for a combination of at least two, at least three or for all four genes from the group of ZNF555, TLE6, ZNF57 and/or ZNF77. More preferably a CNV is detected for any combination of the afore mentioned target sequences.

In another embodiment of the invention, in the method, preferably multiplex method, the reference comprises at least one or more reference sequences is provided with a test sample, in particular with a second test sample, of the same subject - as defined herein - wherein the method comprises further the steps, in particular in addition to the previously describe steps, determining the copy number of at least one or more reference sequences, and Preferably simultaneous determination of the copy number of at least one or more reference sequences and of at least one or more target sequences, evaluating the CNV of the at least one predetermined target sequence in the CNV chromosomal region, relative to the determined copy number of the at least one or more reference sequences of the reference, preferably based on Ct values and delta Ct values as defined herein (Example 1 , Table 5 and Table 6), identification of an endometrium carcinoma of the CN high subtype is determined CN high compared to the determined copy number of the reference.

The test sample may be the same comprising both, at least one or more reference sequences and at least one or more target sequences or two separate test samples are provided comprising either references sequences or target sequences. Preferably, two separate test samples are provided for the reference and for the target sequences, respectively.

In another embodiment of the invention, in the method, preferably multiplex method, the reference is provided as a digital reference independently from the subject comprising the at least one predetermined target sequence and independently from any test sample of any subject. Thus, the reference may be a reference nucleic acid sequence material (synonym: reference material) - measured by performing the method of the present invention - or a digital reference nucleic acid sequence information (synonym: digital reference sequence or digital reference) - which may be provided/uploaded onto the respective device, such as MODAPLEX. The reference may be a digital processed reference sequence or a digitally processed reference.

In a preferred embodiment, the reference is a digital reference or the respective signals representing the released hydrolysis products of the reference. In relation to said reference the released hydrolysis products are detected and the presence of at least one CNV is confirmed. Preferably, in the method of the present invention, preferably multiplex method, the reference - digitally or real - comprises at least one reference sequence on chromosome 1 , and/or at least one reference sequence on chromosome 2 and/or at least one reference sequence on chromosome 12. More preferably, in the method of the present invention the reference - digitally or real - comprises at least one reference in the cytobands 1p32.3, 1 p32.2, 1 p32.1 , 1 p31.3, 1 p31.2, 1 p31.1 , 1 p22.3, 1 p22.2, 1 p22.1 , 1 p21.3, 1 p21.2 and/or 1p21.1 on chromosome 1 , and/or in the cytobands 2q32.1 , 2q32.2, 2q32.3, 2q33.1 , 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1 , 2q36.2, 2q36.3 and/or 2q37.1 on chromosome 2 and/or in the cytobands 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 on chromosome 12 or any combination thereof. In a preferred embodiment of the invention, in the method the reference - digitally or real - comprises at least one reference sequence or more in the cytoband 1p31.3 on chromosome 1 , at least one reference sequence or more in the cytoband 2q35 on chromosome 2 and at least one reference sequence or more in the cytoband 12q24.31 on chromosome 12. Preferably, the reference comprises at least one reference sequence within the location of gene ZRANB2 and at least one reference sequence within the location of gene ZRANB2-AS1 on chromosome 1 , and/or at least one reference sequence within the location of gene TMEM169 on chromosome 2 and/or at least one reference sequence within the location of gene HCAR1 on chromosome 12.

In a preferred embodiment of the present invention, the reference - as defined herein - is negative for a CNV. Preferably, the reference is negative for CNV of at least one or more of the sequences comprising sequences within the location of gene ZRANB2 and ZRANB2-AS1 on chromosome 1 , and/or at least one reference sequence within the location of gene TMEM169 on chromosome 2 and/or at least one reference sequence within the location of gene HCAR1 on chromosome 12. The reference

The detection of the new analytes, in particular of the complete analyte panel, (biomarkers) with a PCR-based method requires the definition of at least one reference sequence, synonym control region, to be able to normalize signals for the target sequences on chromosome 17 and 19 as described herein. The identification of non-correlated controls is done using agglomerative clustering. Independent controls regions have been selected by minimizing either the squared expression or the absolute expression over samples. As many control regions were picked so that at least one of the controls has no chromosomal aberration over all samples in the analyzed dataset. Negative for CNV is based on statistical evaluation that allows to consider a reference as “relative stable” or “affected by CNV in rare cases”. As disclosed in the present invention, based in NGS data (According to The Cancer Genome Atlas Research Network (TCGA) [2] EC) of 232 patients with endometrium carcinoma have been analyzed for the above mentioned reference sequences compared to the target sequences. In contrast to the target sequences, the control regions of the assay are defined by their relatively high genomic stability. Approximately 80% were “negative for a CNV” or “relative stable” within the meaning of the present invention.

Thus, negative for a CNV, in particular (synonym: negative for CN high) means that for at least one reference sequence no copy number variation (CNV) is detected, preferably for at least two reference sequence no CNVs are detected, more preferably for the reference sequences encoding for ZRANB2, ZRANB2-AS1 , TM EM 169 and/or HCAR1 no CNVs are detected.

In a further embodiment of the invention, the method comprises a step of providing suitable primers for amplification of the at least one or more predetermined target sequences in the CNV chromosomal region and optionally suitable primer for amplification of at least one or more reference sequences. Preferably suitable primers for amplification of the at least one or more predetermined target sequences in the cytobands 19p13.3 and/or 17p12 are provided. In one embodiment additionally, suitable primers are provided for the amplification of at least one or more reference sequences. Preferably, suitable primers within the meaning of the present invention are those comprising the technical feature comparable to the primers of table 2. The skilled person is competent to create any suitable primers following the instruction described herein. Most preferably, suitable primer according to the present invention, most preferably for the amplification of the inventive analyte panel, have the sequences of Seq ID No. 1 to Seq ID No. 28. Therefore, another aspect of the present invention is a combination of primer pairs for the amplification of the analyte panel - ZRANB2, ZRANB2- AS1 , TMEM169, HCAR1 , COX10, ARHGAP44, ELAC2, MYOCD, ZNF555, TLE6, ZNF57, ZNF77, wherein the primer have the sequences of Seq ID No. 1 to Seq ID No. 28 (Table 2).

Thus, where a digital reference is used in any embodiment of the present invention, at least one primer for at least one target sequence is provided. In another embodiment - and as presented in the examples herein - a forward primer and a reverse primer is provided for the at least one target sequences. Preferably, suitable primer are provided for the amplification of at least one or more predetermined target sequences in at least one cytoband of cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11.2, 19p13.3 and/or 19p13.2 or in any combination of one or more of the aforementioned cytobands. More preferably, the primers according to the present invention are suitable to amply preferably genes located in cytobands 19p13.3 and/or 17p12 and more preferably for at least one or more target sequences within the location of genes COX10, ARHGAP44, ELAC2 and/or MYOCD in the cytoband 17p12 and/or for at least one or more target sequences within the location of genes ZNF555, TLE6, ZNF57 and/or ZNF77 of cytoband 19p13. The same applies accordingly for suitable primers. According to the present invention suitable primers are provided for the amplification - wherein a reference is determined within the method - of at least one reference sequence on chromosome 1 , and/or at least one reference sequence on chromosome 2 and/or at least one reference sequence on chromosome 12. Preferably, the primers are suitable for the amplification of at least one reference sequence in the cytobands 1 p32.3, 1 p32.2, 1 p32.1 , 1 p31.3, 1 p31.2, 1 p31.1 , 1 p22.3, 1 p22.2, 1 p22.1 , 1 p21 .3, 1 p21.2 and/or 1p21 .1 on chromosome 1 and/or in the cytobands 2q32.1 , 2q32.2, 2q32.3, 2q33.1 , 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1 , 2q36.2, 2q36.3 and/or 2q37.1 on chromosome 2 and/or in the cytobands 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 on chromosome 12 or any combination thereof. More preferably, suitable primer are provided for at least one reference sequence in the cytobands 1 p31.3 on chromosome 1 , 2q35 on chromosome 2 and 12q24.31 on chromosome 12. Preferably, the reference primer are suitable to amply at least one reference sequence within the location of gene ZRANB2 and ZRANB2-AS1 on chromosome 1 , and/or at least one reference sequence within the location of gene TMEM169 on chromosome 2 and/or at least one reference sequence within the location of gene HCAR1 on chromosome 12. According to the present invention any combination or primers for reference sequences as well as for target sequences are suitable to detect an endometrium carcinoma.

In a further embodiment of the invention, the method, in particular a sole amplification method (NAT) of the target sequence and optionally of any reference sequences, further comprises a step of contacting the test sample with the suitable primers, in particular as described herein, specific for the at least one or more target sequences and optionally for the at least one or more reference sequences respectively, hybridization of the at least one primer, preferably at least one primer pair, to its respective complementary sequence on the target sequence and optionally on the reference sequence, preferably the at least one primer, at least one primer of the at least one primer pair, is labelled amplification of the at least one target sequence and optionally reference sequence, separating the amplified target sequences and optionally reference sequences, detection of the amplified target sequences and optionally of the amplified reference sequences, respectively, and evaluating the CNV of the at least one target sequence compared to the copy number of the at least one reference sequence, preferably based on the achieved amplicons.

In this embodiment no probes are necessary. The detection and evaluation is based on labelled amplicons achieved by means of a desired NAT and said primers. Preferably, in the method, in particular sole amplification method, amplification and detection of the at least one or more target sequences and optionally of at least one or more reference sequences, respectively, is performed by real time PCR, end point PCR, digital PCR, geometric multiplexing PCR, digital droplet (or emulsion) PCR (ddPCR) or Next Generation Sequencing (NGS).

In a further embodiment of the invention in the method the at least one CNV for the CNV chromosomal region is determined by the at least one amplified target sequence compared to the at least one reference. The sequences of the amplicons are presented in Table 4. Thus, detection of at least one or more of the amplicons as presented in Table 4 is suitable to identify endometrium carcinoma of CN high subtype. Shorter sequences of said amplicons are also suitable. The design of feasible primers for each of the aforementioned amplification methods are well known to the skilled person.

In another embodiment of the present invention, the method detects endometrium carcinoma of CN high subtype without any amplification of any target or reference sequence but with at least one suitable probe for at least one target sequence and with at least one suitable probe for at least reference sequence. Preferably, said method is a imaging method for any nucleic acid within the test sample.

Thus, in another embodiment of the present invention the method comprises the steps contacting the test sample, in particular a solid biopsy sample, of the subject with at least one oligonucleotide probe, in particular not necessarily cleavable probe (probe), that is specific for the at least one target sequence and with at least one oligonucleotide probe that is specific for the at least one reference sequence, respectively, hybridization of the respective probes to its respective complementary sequences within the test sample, detection of an emitting signal of each hybridized probe, wherein at least one probe for the at least one target sequence and the at least one probe for the at least one reference sequence comprise to each other differentiating fluorophores, evaluating the fluorescence signal for the at least one predetermined target sequence in the CNV chromosomal region relative to the fluorescence signal to the at least one reference sequence, optionally measuring the fluorescence intensity and/or optionally making images of at least one or more layer of the test sample, and identification of an endometrium carcinoma of the CN high subtype, wherein the CNV for the CNV chromosomal region is determined CN high compared to the copy number of the reference. In this embodiment the test sample is a human test sample including the at least one target sequence and at least one reference sequence that are still in a specimen, thus not isolated therefrom. In particular, for the embodiment of the method comprising an imaging method, specimen and primary sample are suitable, such as biopsy from a living body or autopsy dissected post-mortem, a discrete portion of a body fluid or tissue of the subject. The test sample may be from an endometrial abrasions (curettage) or surgical biopsies that are the main specimen for histopathological examination (FFPE - formalin-fixed, paraffin-embedded tissue sections and/or immunostaining) or subsequent molecular analysis.

In this embodiment a multiplex spatial analysis is possible. The method of the present invention is suitable to produces deep contextual data sets that illuminate molecular interactions at subcellular resolution, while preserving the sample tissue. Thus, it is no longer necessary to proceed the tissue, no tissue clearing is necessary. The not necessarily cleavable embodiment of the probe of the present invention may be combined with other means for high resolution e.g. of transcriptomic activity. The present method allows to view DNA and RNA with the required sensitivity, specificity for the detection of endometrium carcinoma, e.g. by means of FISH and other techniques for spatial analysis. One example is the molecular cartography platform of Resolve BioSciences GmbH, DE, that enables to resolve complex biological challenges in areas such as oncology, neuroscience, and infectious disease. In the embodiment of not necessarily cleavable probes all embodiments of target sequences and reference sequence apply accordingly. The sequences of forward and reserve primer are also suitable to create not necessarily cleavable probe for imaging.

In another embodiment of the method, cleavable probes are used and the released hydrolysis products from the respective probe are the signal of interest, in particular a signal of a released hydrolysis product of a cleavable probe. Thus, in another embodiment of the present invention, the method, preferably multiplex method, comprises the steps optionally providing a test sample of a subject comprising at least one predetermined target sequence in the CNV chromosomal region, optionally providing a reference comprising at least one or more reference sequences of a known copy number, contacting the test sample of the subject - as defined herein - with suitable primers - as defined herein - specific for the at least one target sequence and optionally for the at least one reference sequence respectively and contacting the test sample with at least one cleavable oligonucleotide probe (probe), that is specific for the at least one target sequence, respectively, and optionally with at least one cleavable oligonucleotide probe that is specific for the at least one reference sequence, hybridization of the at least one primer to its respective complementary sequence on a same strand of the same target sequence, wherein the cleavable probe hybridizes 3'upstream of the respective primer, and optionally hybridization of the at least one primer to its respective complementary sequence on a same strand of the same reference sequence, wherein the cleavable probe hybridizes 3'upstream of the respective primer, or the reference is a digital reference as described herein, amplification of the at least one target sequence and optionally of the at least one reference sequence, cleavage of the hybridized cleavable probes with a template-dependent 5’ to 3’ nuclease activity to release a cleavable hydrolysis product of the each cleavable probe, separating of the released hydrolysis product(s) and detection of the released hydrolysis product(s). preferably, determining at least one CNV of at least one predetermined target sequence in the CNV chromosomal region by detection of the released hydrolysis product(s), preferably, evaluating the CNV of the at least one predetermined target sequence of the CNV chromosomal region, based on the at least one released hydrolysis product of the specific probe for the at least one predetermined target sequence, relative to the copy number of the reference, in particular based on the at least one released hydrolysis product of the specific probe for the at least one reference sequence, preferably, identification of an endometrium carcinoma of the CN high subtype, wherein the CNV for the CNV chromosomal region is determined CN high compared to the copy number of the reference.

The evaluation of CNV and subsequent identification of endometrium carcinoma of the CN high subtype is based on Ct values and delta Ct values as defined herein (Example 1 , Table 5 and Table 6). This embodiment of the method of the present invention may comprise a step of isolation and/or purification of the one potential nucleic acids comprising target sequences and at least one reference sequence prior providing a test sample.

In a preferred embodiment of the above method, the cleavable hydrolysis product and the released hydrolysis product further comprise at least one modification of at least one nucleotide comprising backbone modifications, none-backbone modifications and/or artificial bases wherein backbone-modification comprises artificial modification at the 2'and/or 4' position of the five-carbon sugar of a nucleotide and non-backbone-modification comprises artificial chemical modification which is coupled to the 5'-end and/or to the 3’-end of the nucleotide.

Examples of cleavable probes for target sequences and for reference sequences comprising modifications according to the present invention are shown in Table 3.

In another embodiment of the above method, the non-backbone-modification comprises spacers which are chemical structures coupled to the 3'- and/or 5'-end of a nucleotide or between two nucleotides and preferably selected from a) alkyl alcohol of (C_n)-OH, wherein n is an integer and at least 3, preferably, 3, 6, 9 or 12 comprising propanyl (Spacer C3), hexanyl (Spacer C6), nonanyl (Spacer C9) and dodecanyl (Spacer C12). b) glycol ether of (C-O-C-C)n-OH wherein n is an integer and at least 1 , preferably comprising triethylene glycol (Spacer 9), tetraethylene glycol (Spacer 12), and hexaethylene glycol (Spacer 18) and/or c) a tetrahydrofuaran derivative containing a methylene group occupied in the 1 position of 2’-deoxyribose.

In another embodiment of the above method, a plurality cleavable probes comprising a plurality of analyte specific 3’-sequences are used, wherein all respective cleavable hydrolysis products comprise the same label coupled to the at least one nucleotide of each cleavable hydrolysis product and each comprise a different linker and/or at least one different modification. Examples of cleavable probes for target sequences and for reference sequences comprising modifications according to the present invention are shown in Table 3.

In another embodiment, the cleavable probe of the present invention, preferably its cleavable hydrolysis product comprises an analyte unspecific 5’-sequence (FLAP) located 5'-upstream from the at least one internal nuclease blocker, and wherein the label is coupled via linker to the 5'-end of the FLAP of the cleavable probe (5 '-linker), or an internal nucleotide 5'-upstream from the at least one internal nuclease blocker.

Preferably, according to the present invention the cleavable hydrolysis product of the cleavable probe comprises at least one modification of at least one nucleotide comprising backbone modifications, none-backbone modifications and/or artificial bases wherein the backbone-modification comprises artificial modification at the 2'and/or 4' position of the five-carbon sugar of a nucleotide and the non-backbone-modification comprises artificial chemical modification which is coupled to the 5'-end and/or to the 3’-end of the nucleotide. In the embodiment of cleavable probes, at least one primer pair is provided for each target, and optionally at least one primer pair for at least one reference sequence. All embodiments of target sequences and reference sequence apply accordingly for any embodiment wherein a cleavable probe is used. Preferably, primers were optimized to have the same melting temperatures, similar GC content, and no repetitive nucleotide motifs as much as possible. Cleavable probes based on the teaching of EP4074839A1 were placed in the proximity of a primer whenever possible to obtain stronger fluorescence signals. In addition, the PCR buffer and polymerase were optimized to obtain the broadest possible linearity of all 14 PCR products of the examples presented herein.

Whereas for the embodiment of not necessarily cleavable probes for detection by means of imaging at least two different labels are used in order to ensure differentiating detection of the reference and the target sequences, in this embodiment only one label is sufficient.

In all embodiments of the present invention directed to cleavable probes, the reference may be a reference nucleic acid sequence material (synonym: reference material) - measured by performing the method of the present invention (“real reference”) - or a digital reference nucleic acid sequence information (synonym: digital reference sequence) - which may be provided/uploaded onto the respective device, such as MODAPLEX. In a preferred embodiment, the reference is a digital reference or the respective signals representing the hydrolysis products thereof. In relation to said reference the released hydrolysis products are detected and the presence of at least one copy number variation is confirmed.

In an embodiment of the present invention, in the method the CNV of the at least one predetermined target sequence is determined by the respective released hydrolysis product compared to the at least one respective, in particular released, reference hydrolysis product of the reference. In another embodiment of the inventive method the respective reference hydrolysis product of the at least one reference is released from at least one reference sequence according to the method of the preceding claims or it is a digital reference hydrolysis product. By means of comparing the CNV of the at least one predetermined target sequence of the CNV chromosomal region, based on the at least one released hydrolysis product of the specific probe for the at least one predetermined target sequence, relative to the copy number of the reference, in particular based on the at least one released hydrolysis product of the specific probe for the at least one reference sequence, is evaluated as defined herein. In another embodiment of the inventive method, the amplification of the at least one target sequence and optionally of the at least one reference sequence, respectively, is performed by real time PCR and separation of the hydrolysis product(s) by capillary electrophoresis. More preferably the method is performed by means of a CE device as defined herein, more preferably by the MODAPLEX device.

Another aspect of the present invention is a specific oligonucleotide probe suitable for hybridization to at least one predetermined target sequence in the CNV chromosomal region comprising cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11 .2, 19p13.3 and/or 19p13.2, preferably for at least one predetermined target sequence in cytoband 19p13.3 and for at least one predetermined target sequence in cytoband 17p12, and/or to at least one reference sequence in the cytobands 1 p31.3 and/or 2q35 and/or 12q24.31 , comprising respectively a 3’-sequence which is reverse complementary to a sequence of the at least one predetermined target sequences or to a sequence of the at least one reference sequences.

In a preferred embodiment of said probes, said 3’-sequences for at least one predetermined target sequence are selected from the forward and/or reverse primers of Table 2 and 3’- sequences for at the least one reference sequence are selected from the forward and/or reverse primers of Table 2. The cleavable probes of Table 3 are also suitable. Starting from the Seq ID No. 29 to 42, the underlined sequences are suitable to transform the specific cleavable probe - which is specific for use in a CE device in combination with a real time PCR - to any suitable embodiment and to a probe for use in a method for detection of CN high endometrium carcinoma by means of imaging, e.g. FISH probe or the like, in particular to adapt said probes to not necessarily cleavable probes for use in a method with detection of CN high endometrium carcinoma by means of imaging, e.g. FisH probe or the like. The skilled person is competent to adapt the underlined sequences to a probe for imaging in a tissue.

One approach for translating the “cleavable probe” or MOPAPLEX assay (probe and primer of Table 2 and Table 3) to an imaging based method would be based on FisH probes that carry a suitable fluorophore on the 5’ end of the oligonucleotide. One skilled in the art can copy the forward primer, probe and/or reverse primer sequence, followed by adding a suitable combination of fluorophores. In case of using the herein presented cleavable probes as a sequence template, one would remove the quencher from the 3’ end, as well as the whole 5’ end modification chemistry as defined herein and replace it with a common FisH fluorophore. For specific examples, one could take the target sequences of the invention and subsequently modify the 5’ end by adding a Cy3 and Cy5 fluorophore for a target- and reference sequence respectively.

It would be also suitable to limit the 14 targets, as used in the present example, to one target on chr17, chr19, each and one reference sequence chr1/2/12 each. This would result in a 5- plex FisH which does not require any cleavable probes or time consuming “stripping” of the sample. The latter is required if one would like to stain more than 5 targets with 5 different fluorophores. Alternatively, it would be suitable to use only one fluorophore for all target sequences and only one fluorophore for all reference sequences since the evaluation of the CNV and identification of CNH is based on the average copy number across the whole detected region, target region compared to reference region. The same applies to any other suitable imaging method.

Preferably the specific oligonucleotide probe of the present invention, in particular is a not necessarily cleavable probe, wherein the at least one probe comprises a detectable label, preferably a fluorophore or one hapten of a specific hapten pair. Thus, a fluorescence signal is detected upon hybridization or after adding another agent to achieve a detectable agent (indirect imaging). Preferably, in a combination of at least one probe for at least one predetermined target sequence and for at least one reference sequence each probe comprise to each other differentiating fluorophores. Such not necessarily cleavable probe, e.g. FISH probes, are well known to the skilled person. However, FISH probes may also be cleavable to increase the multiplex level of the imaging method, e.g. „photocleavable“ or ..selectively chemical cleavable" linkers are combined to release the fluorophore. Thereby the probe remains hybridized to its target sequence whereas it is no longer detectable, not visible. That allows labeling of another sequence with another probe and subsequent layering of a series of images.

In another embodiment, the specific oligonucleotide probe is a cleavable probe, preferably for use in the inventive method and preferably in a multiplex method of the present invention, comprising respectively a 3’-sequence which is reverse complementary to a sequence of the at least one predetermined target sequences within a region being located 3'-downstream of a complementary sequence of an at least one primer, preferably at least of the forward primer, more preferably the at least one primer or forward primer is the corresponding suitable primer for amplification of the respective predetermined target sequence for which the specific probe is provided; the primer and probe combinations as defined herein are suitable and preferred accordingly, a protective group (3'-blocker) at the 3’-end of the analyte specific 3’-sequence inhibiting a primer extension reaction, and at least one internal nuclease blocker at the 5'-region of the hybridizing sequence of the probe conferring resistance to a nuclease activity and structurally dividing the cleavable hydrolysis product from the 3'-downstream cleavable probe and wherein the cleavable hydrolysis product comprises

• at least one nucleotide of the 5'-end of the cleavable probe, and

• a label, preferably a fluorophore, bridged via a linker to the cleavable hydrolysis product.

Another aspect of the present invention is a combination of probes, in particular of one of the described designs of not necessarily cleavable or cleavable probes, wherein each probe is specific for the reference sequence encoding within the location of gene ZRANB2 and ZRANB2- AS1 on chromosome 1 , preferably the probes have the sequences of Seq ID No. 39 and Seq ID No. 40, the reference sequence encoding within the location of gene TMEM169 on chromosome 2, preferably the probes have the sequences of Seq ID No. 41 and Seq ID No. 42, the reference sequence encoding within the location of gene HCAR1 on chromosome 12, , preferably the probes have the sequences of Seq ID No. 37 and Seq ID No. 38, the target sequences encoding within the location of genes COX10, ARHGAP44, ELAC2 and MYOCD respectively in the cytoband 17p12, preferably the probe for COX10 has the sequences of Seq ID No. 29, the probe for ARHGAP44 has the sequences of Seq ID No. 30, the probe for ELAC2 has the sequences of Seq ID No. 31 and the probe for MYOCD has the sequences of Seq ID No. 32, and the target sequences encoding within the location of genes ZNF555, TLE6, ZNF57 and ZNF77, respectively in the cytoband 19p13.3, preferably the probe for ZNF555 has the sequences of Seq ID No. 33, the probe for TLE6 has the sequences of Seq ID No. 34, the probe for ZNF57 has the sequences of Seq ID No. 35 and the probe for ZNF77 has the sequences of Seq ID No. 36.

Preferably the above combination is realized as a combination of cleavable probes, suitable for use in a CE device, preferably for use in a MODAPLEX device. Most preferably, the probes as described in Table 3 are combined according to the present invention. Said probes may comprise the underlined sequences as shown in Table 3 but may have alternative modifications. In an embodiment of the present invention, any combination of the above probes is combined with suitable primers for amplification of said target and reference sequences. Preferably said probes are combined with suitable primers comprising the technical feature comparable to the primers of Table 2, more preferably, suitable primers according to the present invention, most preferably for the amplification of the inventive analyte panel, have the sequences of Seq ID No. 1 to Seq ID No. 28. Therefore, another aspect of the present invention is a combination of primer pairs of Seq ID No. 1 to Seq ID No. 28 with a specific probe or specific probe combination as defined herein, preferably of Seq ID No. 29 to Seq ID No. 42.

Another aspect of the present invention is a specific probe or a specific combination of probes, wherein the at least one probe or combination thereof comprises the underlined sequences of Seq ID No. 29 to Seq ID No. 42. In one embodiment the probes are not necessarily cleavable probes. Alternatively, the at least one probe or combination thereof comprising the underlined sequences of Seq ID No. 29 to Seq ID No. 42 are cleavable probes. Preferably said probes are suitable for use in a CE device, preferably for use in a MODAPLEX device. Different modifications as disclosed herein or in the teaching of EP4074839A1 may be combination with the underlined sequences of Seq ID No. 29 to Seq ID No. 42 to create alternative probes according to the present invention.

A further aspect of the present invention is the use of at least one specific oligonucleotide probe as disclosed herein or any combination thereof disclosed herein, in a method for the detection of an endometrium carcinoma of a copy number high (CN high) subtype according to any one of the preceding embodiments.

A further aspect of the present invention is a kit for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high), wherein at least one copy number variation (CNV) is detected for a CNV chromosomal region comprising at least one cytoband on chromosome 17 and/or chromosome 19, comprising at least one suitable primer or primer pair for the amplification of one or more target sequences within the CNV chromosomal region, and optionally at least one suitable primer or primer pair for the amplification of at least one reference sequence in the chromosomal cytobands 1 p32.3, 1p32.2, 1 p32.1 , 1 p31 .3, 1p31 .2, 1p31 .1 , 1 p22.3, 1p22.2, 1 p22.1 , 1 p21 .3, 1p21.2 and/or 1 p21.1 on chromosome 1 and/or 2q32.1 , 2q32.2, 2q32.3, 2q33.1 , 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1 , 2q36.2, 2q36.3 and/or 2q37.1 on chromosome 2 and/or 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 on chromosome 12, respectively.

The kit for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high), wherein at least one copy number variation (CNV) is detected for a CNV chromosomal region comprising at least one cytoband of chromosome 17 and/or chromosome 19, comprising at least one suitable primer or primer pair for the amplification of one or more target sequences within the CNV chromosomal region, preferably for amplification of at least one or more target sequences selected from locations of genes COX10, ARHGAP44, ELAC2 and/or MYOCD in cytoband 17p12 and/or from locations of genes ZNF555, TLE6, ZNF57 and/or ZNF77 in cytoband 19p13.3, and optionally at least one suitable primer or primer pair for the amplification of at least one reference sequence in the chromosomal cytobands 1 p32.3, 1 p32.2, 1 p32.1 , 1 p31.3, 1 p31.2, 1 p31.1 , 1p22.3, 1p22.2, 1p22.1 , 1p21.3, 1 p21.2 and/or 1 p21.1 of chromosome 1 and/or 2q32.1 , 2q32.2, 2q32.3, 2q33.1 , 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1 , 2q36.2, 2q36.3 and/or 2q37.1 of chromosome 2 and/or 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 of chromosome 12, preferably of at least one reference sequence in the chromosomal cytobands 1 p31.3, 2q35 and 12q24.31 , respectively.

Preferably, the kit or any embodiment thereof comprises the primer or combination of primer pairs comprising the technical feature comparable to the primers of Table 2. Most preferably, the kit comprises suitable primer for the amplification of the inventive analyte panel and have the sequences of Seq ID No. 1 to Seq ID No. 28.

Preferably, the kit as defined herein, further comprises at least one probe or combination of probes as described herein. More precisely, the kit for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high), comprises at least one suitable primer or primer pair for the amplification of one or more target sequences within the CNV chromosomal region, and optionally at least one suitable primer or primer pair for the amplification of at least one reference sequence in the chromosomal cytobands 1 p32.3, 1p32.2, 1 p32.1 , 1 p31 .3, 1p31.2, 1p31.1 , 1 p22.3, 1p22.2, 1 p22.1 , 1 p21.3, 1p21.2 and/or 1 p21.1 on chromosome 1 and/or 2q32.1 , 2q32.2, 2q32.3, 2q33.1 , 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1 , 2q36.2, 2q36.3 and/or 2q37.1 on chromosome 2 and/or 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 on chromosome 12, respectively, and at least one specific probe or a specific combination of probes, wherein the at least one probe or combination thereof comprises the underlined sequences of Seq ID No. 29 to 42, preferably a panel of probes comprising 8 probe for the target sequences and 6 for the reference sequences.

Another aspect of the present invention, is the kit comprising the probes with the sequences of Seq ID No. 29 to Seq ID No. 42 alone or in combination with primers of Seq ID No. 1 to Seq ID No. 28. In another embodiment the kit for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high), comprises at least one specific probe, preferably a specific combination of probes, wherein the at least one probe or combination thereof comprises the underlined sequences of Seq ID No. 29 to Seq ID No. 42, preferably the combination of probes is suitable for the detection of the analyte panel comprising 8 probes for the target sequences and 6 for the reference sequences.

In one embodiment the probes are not necessarily cleavable probes. Preferably said probes are suitable for use in a CE device, preferably for use in a MODAPLEX device. Different modifications as disclosed herein or in the teaching of EP4074839A1 may be combined with the underlined sequences of Seq ID No. 29 to Seq ID No. 42 to create alternative cleavable probes according to the present invention.

The invention is explained in more detail with reference to the figures, without limiting the invention to these embodiments. Showing:

Fig. 1: Target distribution of the Copy Number High Analysis Kit. Assessment of the CNH molecular subtype is conducted by evaluation of CNV on multiple targets on two different instable target regions, located on chromosome 17 (n = 4) and 19 (n = 4) in comparison to multiple almost invariant genomic regions on chromosome 1 , 2 and 12 (n = 2 for each chromosome).

Fig. 2: XY-plot illustrating the Copy Number High Analysis Kit output data of 79 endometrial cancer patient samples that have been analysed. Delta Ct values for chromosome 19 (x-axis) and 17 (y-axis) have been plotted and cut-off values for copy number high subtype determination on both axes implemented as dotted lines. Samples within the black rectangle have been considered as copy number high negative. In turn, samples for which delta Ct values for chromosome 17 and/or chromosome 19 are outside of the black rectangle, a copy number high subtype were assigned. (A) Filled black dots resemble EC patient samples for which a mutated P53 IHC staining pattern has been observed. (B) Filled black dots resemble EC patient samples for which a high degree of CNVs was detected via low coverage whole-genome sequencing.

Fig. 3 Representative Illustration of H&E- and IHC P53 staining of two endometrial cancer specimens which have been assigned as copy number high positive and negative according to the herein presented invention. (A + C) H&E staining has been performed and analysed by board-certified pathologists to confirm the disease of endometrial cancer in both illustrated cases. In line with the inventions result, the CNH positive sample (upper panel, A-B) showed a nucleus accumulation P53 staining pattern in the IHC staining (B). No pathogenic phenotype for P53 was seen in the CNH negative sample (D). P53 resembles the current surrogate biomarker for CNH and the nucleus accumulating phenotype is known to be associated with the respective molecular subtype. Accumulation of P53 in the nucleus is caused by loss-of-function mutations within the TP53 gene. Frequently, TP53 loss- of-function mutations are associated with a loss of the second (wild type) allele.

Fig. 4: Low coverage l/VGS of two endometrial cancer specimens which have been assigned as copy number high positive and negative according to the herein presented invention. (A) Low coverage WGS confirmed the copy number high subtype, which was assigned by the presented invention as well as P53 IHC (Figure 3B). (B) The opposite was seen for the second sample for which no accumulation of CNVs has been observed via low- pass WGS. In line with that, the respective sample was assigned as copy number high negative by the herein presented invention, as well as P53 IHC.

Fig. 5 : cleavable probe according to the present invention (1) and hydrolysis product (3) after hybridization to target DNA. A) A cleavable probe (1) consists of a single stranded oligonucleotide (symbolized by a stretch of “T”s) which sequence specifically hybridizes to a DNA strand of a target nucleic acid (2) and which includes following modifications: A PCR blocker (white up-pointing triangle) at its 3’-end, and a nuclease-cleavable fluorophore which can be released as a hydrolysis product located at its 5’-end upstream of an internal nuclease blocker (white square, (4)). The hydrolysis product possesses at least one target DNA specific nucleotide linked to an internal blocker via a phosphodiester bond (T). Furthermore, it may additionally consist of a modification (white cycle) which does not hybridize to the target nucleic acid and/or a fluorophore (white diamond) that is bridged via a linker (zigzag line) and can be quantified by a detecting unit of a nucleic acid electrophoresis device.

Fig. 6 : Schematic structure of different cleavable probe designs of the present invention. In the schematic view the cleavable probe (dark grey TTT) is hybridized to a target sequence (light grey TTT). (F) represents the label, preferably the fluorophore, that is bridged via a linker (zigzag line) as defined herein. The linker can be an internal linker (c), f) and g) or a 5'-terminal linker (a), b), d), e)). (S) represents a spacer and (M) represents another backbone or nonbackbone modification which does not hybridize to the target sequence. The location of the nuclease blocker is represented by a black T and the 3'blocker (polymerase) is shown. The cleavable hydrolysis product possesses at least one target specific nucleotide (shown) or more (not shown) linked to an internal nuclease blocker via a phosphodiester bond (T). The nuclease blocker is at position -1 downstream from the 5'-end of the hybridizing sequence (shown) but can be at position -2 or -3 or at -1 and -3 (not shown). The shown cleavable probe consist of 10 nucleotides, but 9, 8, 7 or 6 are enough and more than 10 are also within the meaning of the invention.

Definitions

If not defined above, in the following definitions are provide for all aspects and all embodiments of the present invention.

An analyte is generically defined as a constituent of a sample with a measurable property (ISO 18113-1 :2009). Molecular diagnostics of nucleic acids is a collection of techniques used to analyse nucleic acid sequence variations (e. g. genotyping of alleles like single nucleotide polymorphisms, deletion insertion polymorphisms, inversions, translocations between chromosomes, splice variants of RNA, repetitive sequences like short tandem repeats), epigenetic modifications (like CpG methylation or hydroxy-methylations), post-transcriptional variants (e.g. RNA editing and alternative splicing, maturation of RNA by poly-adenylation, ADP-ribosylation, adenine to inosine conversion, ribonuclease degradation etc.) or quantitative alterations of defined DNA (like copy number variants) or RNA (like mRNA in gene expression) sequences. Nucleic acid sequence variants are surrounded by unique stretches of constant nucleic acid sequences which define their position within reference genomes and, thus, their specificity in complex genomes or DNA mixtures of different organisms (e. g. analysis of microbiomes, environmental specimens). Constant DNA stretches up to 35 bp, which are defined herein as addressing sequences, are almost sufficient (depending on the sequence complexity and peculiarities of the detection technology) to define nucleic acid primers and/or probes to analyse the presence of a unique DNA variant within a complex test sample of purified DNA. Taking into account these characteristics, any nucleic acid sequence variation (synonym “variant”, “molecular variation”), epigenetic modification or quantitative nucleic acid alteration together with at least one unique addressing sequence defines a specific molecular genetic analyte (synonym “analyte”). Copy number variations (CNVs) are structural variations within the (human) genome and are the results of deletions or amplifications of chromosomal segments. Consequently, absolute copy numbers of a subset of genes do vary between (healthy) individuals within a population. It is commonly known that CNVs have played and play a key role in the development of new gene variants which, in turn, can increase the overall fitness of a population. In the context of malignant diseases, pathological copy number variations can result in growth/fitness benefits for affected transformed cells, for example when chromosomal segments encoding for tumor suppressor- or proto-oncogenes are deleted (on one or both alleles) or amplified, respectively.

Molecular subtyping of (malignant) diseases, in the context of the present invention, describes the process of clustering tumours from the same entity based on their profile of genetic alterations. The observation of varying responses of cancer patients, suffering from a malignant disease in the same entity, to standard of care gave rise to the concept of molecular subtyping. Molecular subtypes of malignant cancer diseases have been described and established within the last decade when whole-genome/transcriptome sequencing costs have dropped significantly, resulting in an extensive increase in sequencing data sets. In many tumour entities, molecular stratification has a prognostic value for the patients as different molecular subtypes are associated with different progression free- and overall survival rates. Additionally, it has been shown that molecular subtype tailored therapy protocols can improve patients’ therapy outcome. However, patient-individual cancer disease subtype stratification via whole-genome/exome or transcription profile analysis is not feasible in routine diagnostics as it is time consuming and relatively expensive. Because of that, determination of molecular subtypes in cancer based on a much smaller subset of biomarkers is one of the biggest challenges in translational cancer research nowadays.

The copy number high (CNH) molecular subtype in endometrial carcinoma (EC), describes one of multiple discovered and characterized molecular subtypes in EC. CNH is associated with the enrichment of a large number of chromosomal aberrations, which either are caused by deletions or amplifications (also referred as “somatic copy number plasticity”, “somatic copy number alterations (SCNA)” or “somatic copy number variation” (SCNV)]. In the context of endometrial carcinoma, the copy number high subtype is associated with a poor prognosis. Additionally, abnormal tumor suppressor gene 53 (p53) immunohistochemistry (IHC) staining pattern (p53abn) are characteristic for CNH in EC. Accordingly, P53 resembles a surrogate biomarker in molecular diagnostics for CNH determination.

A target sequence (synonyms: target, Target nucleic acid or template sequence or template) is a nucleic acid sequence which includes all information that is required for the detection of a molecular genetic analyte applying a specific technology. This encompasses the molecular variant (sequence and/or quantitative variant), the addressing sequence(s) and all further sequence information which is critical for technical reasons like sequence amplification (e.g. primer binding sites in case of polymerase chain reaction, accessory primer binding sites in case of loop-mediated isothermal amplification) or selective enrichment and/or detection by probe hybridisation. Presently, predetermined target sequences for detection of CN high and reference sequences are distinguished.

Biomarkers are analytes with scientific validity, whereupon ‘scientific validity of an analyte’ means the association of an analyte with a biological condition. Biomarkers are for example used to detect pathogens, to assess environmental or bio-process conditions, for food quality testing, in plant and animal breeding, in forensics, in veterinary and human medicine. In case of in-vitro medical diagnostics they act as indicators of a normal or pathogenic biological process of a human being (or patient), here endometrium carcinoma. They also allow assessment of the pharmacological response to a therapeutic invention. A medical biomarker shows a specific physical trait or measurable biologically produced change in the body that is linked to a disease. The scientific validity of clinical biomarkers must be shown in extensive clinical exploration and validation trials. Furthermore, it should be mentioned that pre-analytic (e.g. patient conditioning, sampling, sample storage and transport, and test sample preparation) and analytic conditions (capabilities of the applied nucleic acid amplification and detection technology) have substantial impact on the scientific validity of the biomarkers, as well as the robustness and reproducibility of a diagnostic assay.

Cytoband according to the present invention is defined as provided by the LICSC Genome Browser (https://genome.ucsc.edu/; University of California, Santa Cruz, US-CA; date of online access 08. September 2023) according to human genome version T2T-CHM13 v2.0/hs1. Table 1 provides the cytobands according to the present invention as defined in the claims. In the following certain suitable coding regions within said preferred cytobands are described. Those are also suitable target sequences according to the present inventions and are encompassed by the present invention: MYH13, MYH8, MYH4, MYH1 , MYH2, MYH3, SCO1 , ADPRM, TMEM220, PIRT, RN7SL601 P, SHISA6, DNAH9, ZNF18, MAP2K4, MIR744, LINC00670, MYOCD, ARHGAP44, MIR1269B, ELAC2, HS3ST3A1 , MIR548H3, COX10, CDRT15, HS3ST3B1 , CDRT7, CDRT8, PMP22, MIR4731 , TEKT3, RN7SL792P, CDRT4, TVP23C, CDRT1 , TRIM16, ZNF286A, TBC1 D26, RNA5SP436, ADORA2B, ZSWIM7, TTC19, NCOR1 , RN7SL442P, PIGL, MIR1288, CENPV, UBB, TRPV2, ZNF287, ZNF624, CCDC144A, RN7SL620P, USP32P1 , KRT16P2, KRT17P1 , TNFRSF13B, MPRIP, RN7SL775P, PLD6, FLCN, COPS3, NT5M, MED9, RASD1 and/or PEMT or any combination of two or more thereof. More preferred are DNAH9, ZNF18, MAP2K4, MIR744, LINC00670, MYOCD, ARHGAP44, MIR1269B, ELAC2, HS3ST3A1 , MIR548H3 and/or COX10 or any combination of two or more thereof. Most preferably and as used in the present experiments are MYOCD, ARHGAP44, ELAC2 and COX10. Further, and TMPRSS9, TIMM13, LMNB2, GADD45B, GNG7, RN7SL121 P, DIRAS1 , SLC39A3, SGTA, THOP1 , ZNF554, ZNF555, ZNF556, ZNF57, ZNF77, TLE6, TLE2, GNA11 , GNA15, S1 PR4, NCLN, CELF5, NFIC, DOHH, FZR1 , MFSD12, C19orf71 , HMG20B, GIPC3, TBXA2R, CACTIN, PIP5K1C, TJP3, APBA3, MRPL54, RAX2, MATK, ZFR2, ATCAY, RN7SL202P, NMRK2, DAPK3, MIR637, EEF2, PIAS4 and/ or ZBTB7A or any combination of two or more thereof are also suitable. More preferred are THOP1 , ZNF554, ZNF555, ZNF556, ZNF57, ZNF77, TLE6 and/or TLE2 any combination of two or more thereof. Most preferably and as used in the present experiments are ZNF555, ZNF57, ZNF77 and TLE6. According to the present invention any combination of at least two or more of the regions encompassing the aforementioned genes have been identified suitable for the identification of a CN high subtype of an endometrium carcinoma. Most preferred are MYOCD, ARHGAP44, ELAC2, COX10 and ZNF555, ZNF57, ZNF77 and TLE6. The CNV chromosomal region according to the present invention encompasses at least one of the aforementioned gene or combinations thereof, most preferably MYOCD, ARHGAP44, ELAC2, COX10 and ZNF555, ZNF57, ZNF77 and TLE6.

Suitable reference sequences are within the cytobands as described in Table 1 and preferably are the reference sequences as described in Table 3. Those references are combined with any target references as described above.

A test sample, in particular human test sample, is defined as the end product of the pre- analytical process which is directly applied in the analytical reaction. It includes the target sequences (used as analyte) of interest in a mixture together with matrix chemicals, the composition and concentration of which depend on the composition of the specimen (see next definition) and the potential purification steps. Matrix chemicals may impair the analysis of the analyte or biomarker (matrix effect). The test sample includes the analyte or biomarker of interest and/or controls (water or nucleic acid free sample buffer as negative control, positive controls with spike-in reference standards, and/or internal amplification control). Test samples must be distinguished from specimen and primary sample. Therefore, any embodiment of the method of the present invention may comprise a step of providing a test sample.

Specimen comprises any conceivable source materials having biological amount including an analyte or biomarker, or an artificial barcode sequence (e.g. used for traceability application). In clinical diagnostic settings, specimen (also referred to as biopsy from a living body or autopsy dissected post-mortem) is a discrete portion of a body fluid or tissue taken for examination, study or analysis of one or more quantities or properties assumed to apply for the whole (ISO 18113-1 :2009 and ISO 21474-1 :2019). Therefore, any embodiment of the method of the present invention may comprise a step of isolation and/or purification of the one potential nucleic acids comprising target sequences and at least one reference sequence prior providing a test sample.

Primary sample (or a subsample of it) is the sample prepared from the specimen for sending to, or as received by, the laboratory and which is intended forexamination (ISO 21474-1 :2019).

Nucleic acid sequencing comprises any technology which allows to encode the primary structure of nucleic acids and is defined in detail in the teaching of EP4074839A1 . The present invention is suitable for combination with such technologies and the applicability of the invention which is disclosed herein, however, is not limited to this compilation of nucleic acid sequencing technologies.

The MODAPLEX device (Hlousek et al. 2012), as used within this invention, is a closed system for real-time multiplex nucleic acid analysis combining two methods i) polymerase chain reaction (PCR) and simultaneous ii) capillary electrophoresis (CE). It is defined in detail in teaching of EP4074839A1. Suitable fluorophores for MODAPLEX devices (for channel 1 and channel 2 of the) are shown below:

However, other fluorophores and combinations for channels 1 and 2 are possible. Other cyanines comprise CY3, CY3.5, CY5, CY5.5, CY7; Alexa 488, rhodamine and derivatives such as Texas Red, R6G, R110, TAMRA, ROX; fluorescein and derivatives such as 5-bromomethyl fluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), 6-carboxylfluorescein (6-FAM), 1 ,2', 4', 1 ,4, -tetra chlorofluorescein (TET), 2',4',5',7',1 ,4-hexa chlorofluorescein (HEX), Lucifer Yellow, IAEDANS, benzophenoxazines, 7-Me 2N-coumarin-4-acetate, 7-OH-4-CH 3- coumarin-3-acetate, 7-NH 2-4-CH 3-coumarin-3-acetate (AMCA), monobromobimane, pyrene trisulfonates, such as Cascade Blue, Oregon Green, and monobromorimethyl- ammoniobimane. Additional examples of fluorescent dyes are provided in, e.g., Johnson I and Spent MY (2010), and the updates thereto, which are each incorporated by reference. Fluorescent dyes are generally readily available from various commercial suppliers including, e.g., Molecular Probes, Inc. (Eugene, LIS-OR) Amersham Biosciences Corp. (Piscataway, US- NJ), Atto-Tec GmbH (Siegen, DE), Dyomics GmbH (Jena, DE), Applied Biosystems (Foster City, LIS-CA) etc. Fluorescein dyes and derivatives thereof are particularly preferred in the methods described herein. The same aforementioned labels, fluorescent dyes, are suitable for not necessarily cleavable probes and for use in the method according to the present invention comprising detection of an endometrium via imaging.

Within the present invention, fluorescence labelled and cleavable oligonucleotide probes have been designed based on the teaching as disclosed in EP4074839A1. The general structure of cleavable probes are depicted in Fig. 5A and embodiments are shown in fig. 6. In combination with the MODAPLEX platform, the tailored cleavable probes resemble a powerful tool allowing the assembly of high-grade multiplex PCR assays. In the present invention, probes as disclosed in EP4074839A1 have been tailored, which are detected in both detection channels, using the whole resolving range of the electrophoresis device and are suitable for the detection of CN high according to the present invention.

Nucleic acid amplification technologies (NAT) refer to enzymatic methods for in-vitro duplication of nucleic acids, in particularly, of target sequences according to the invention. According to the invention, the target sequence is amplified in a repeated duplication reaction, preferably in a duplication reaction of a polymerase chain reaction (PCR), which requires thermic cycles. PCR is well known by the person skilled in the art. RNA can be amplified after conversion into complimentary DNA (cDNA) by a reverse transcriptase. EP0506889B1 , EP0632134B1 , EP1152062B1 , WO2014023318A1 disclose furthermore different technologies for real-time reverse transcriptase PCR to amplify RNA in single reaction tubes. Other NAT proceed isothermally (iNAT). LAMP (loop-mediated isothermal amplification), HDA (helicasedependent amplification), RPA (recombinase polymerase amplification), SI BA (Strand Invasion Based Amplification), RCA (rolling circle amplification) are examples for the isothermal amplification of DNA. Finally, Real-Time NASBA (nucleic acids sequence-based amplification) describes an iNAT for the direct amplification of RNA in combination with a hydrolysis probe (Keigthley et al. 2005). The expert in the field knows of other technologies such as NGS that may be based on NAT. Multiplex NAT or in general multiplex tests (synonym multiplexing approach) describe the ability of an assay to interrogate or detect more than one analyte or biomarker simultaneously in each test sample, ideally within one reaction. Multiplex assays provide time, cost and information content advantages, and therefore allow for higher confidence results than singleplex assays (“monoplex” and “singleplex” are synonyms). In addition, the less hand-on steps reduce the risk of sample mix-up or cross-contamination. Preferably “multiplex” (synonym multiplexing) refers to the detection of multiple different analyte, preferably within different target sequences of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30 or more targets, e.g., at least 50, at least 100, at least 250 or more targets. Preferably at least 20, more preferably at least 30.

Nucleic acid electrophoresis as defined in detail in the teaching of EP4074839A1 and applies here accordingly. Said technologies are applicable according to the present invention to separate nucleic acid amplification products and/or hydrolysis products by the combination of mass and charge within an electric field.

The MODAPLEX device (Hlousek et al. 2012) which has been used within this invention is an automated system that combines a PCR thermocycler together a denaturing capillary electrophoresis (CE) system to allow multiplex amplification of DNA sequence targets and the quantification of DNA analytes or biomarkers, simultaneously. Other examples of CE devices which also support the invention and which have different optical detection units (see definition of fluorophores for MODAPLEX detector characteristics) with one to six channels are Applied Biosystems 3500/3500xL Genetic Analyzers and Applied Biosystems SeqStudio instruments (Thermo Fisher Scientific Inc., Waltham, LIS-MA), Applied Bioystems RapidHIT ID System, Spectrum CE Systems (Promega Corp., Madison, LIS-WI), Beckmann Coulter CEQ™ 8000 Genetic Analysis System (Fullerton, LIS-CA), QIAxcel Advanced System (Qiagen GmbH, Hilden, DE), Nanofor® 05M (Syntol, Moscow, Rll), CEQ8800 DNA Sequencer (Beckman Coulter, Brea, US-CA)and diverse nucleic acid fragment analyzers from Agilent Technologies Inc. (Santa Clara, LIS-CA) like TapeStation, Bioanalyzer, Fragment Analyzer System, ZAG DNA Analyzer System, Femto Pulse System. The cleavable probes are suitable for any of the aforementioned methods.

As used herein, MODAPLEX calibrators are at least one, preferably two or more non- amplifiable size standards detected in capillary electrophoresis in at least one fluorescence channel in a multiplex setup, which are unrelated to the analytes or biomarkers of interest (unrelated or artificial templates and primers). They are comprised by short DNA fragments, 1- 5 nucleotides in length, with a fluorophore attached at the 5’end but also can be attached to an internal nucleotide. They define the range of amplicon detection by the analyzing software of the MODAPLEX instrument. In the described examples of this invention MODAPLEX Size Standard 4 (SST4; Biotype GmbH, DE) was used in a onefold final concentration. It consists of 3 size standards whose signal intensity is constant through PCR cycling. The SST4 also contains an amplification control (AC) calibrator composed of an artificial template (unrelated to human DNA), the corresponding PCR primer pairs and the corresponding probes. The AC plus the 3 non-amplifying size standards run in the 6-FAM channel (blue channel). The calculated length sizes were 114.5 bp, 169.18 bp, and 277.5 bp on the blue channel. The AC’s calculated length is 58.6 bp. However, the apparent length can slightly differ by a maximum of 5 bp without impact on assay analysis. The AC calibrator also represents a templateindependent PCR control and should be added to all sample, negative and positive control wells. Alternatively, the PCR setup can be spiked by calibrators in an appropriate endpoint concentration. In this case other markers must be used as a template-independent PCR control. The regions between the MODAPLEX calibrators can be subdivided into regions for target, allele or profile calling of the amplicons of interest. However, an exact amplicon size calling is not possible.

Migration Time is herein defined as the time that takes a labelled analyte, it being, according to the invention, labelled nucleic acids, to reach the detection point after electrokinetic injection at each of the respective PCR cycles. The MODAPLEX uses “scans” as a unit for migration time, which corresponds to around 150 ms, due to the scanning rate of the detector of around 6.66Hz. Migration times can be converted to migration lengths by doing a linear fit relating the known migration lengths of the calibrator system components with their respective migration times (see above the definition of MODAPLEX calibrator).

FLAP endonucleases (FEN) are structure- and strand-specific endonucleases which cleave the single-stranded DNA- or RNA-sequence of a fork-shaped unpaired 5‘-end (5‘-FLAP) of a DNA double helix. The nuclease preferably cleaves after the first 5’-hybridized nucleotide but is not restricted to this cleavage site. It can also cleave after second, third, fourth and fifth hybridized nucleotide of 5’-flapped nucleotides. Thus, the hydrolysis reaction mainly results in hydrolysis products with the size n+1 but also n+2, n+3, n+4, n+5 and n, n-1 , n-2, n-3, n-4, etc., whereas the minimum fragment size in one. By combination of the modifications described herein the desired cleavage site as defined herein and thereby the activity of the nuclease can be determined and directed. Examples for FEN within the meaning of the invention are based on the teaching of EP4074839A1. Hydrolysis (cleavage) product. At least one 5’-terminal nucleotide of a completely to the target sequence hybridized oligonucleotide which is hydrolyzed by a nuclease, preferably the FEN activity of a DNA-dependent DNA polymerase (e.g. Taq DNA polymerase). The resulting hydrolysis fragment (synonym for “hydrolysis (cleavage) product”) always consists of at least one nucleotide (conventional or non-conventional) with a 3’-OH group and a fluorophore coupled via a linker. Additionally, it can contain further modifications which can also include additional nucleotides if they did not hybridize to the target DNA strand (flapped nucleotides). Prior to hydrolysis by the FEN, the “hydrolysis (cleavage) product” or hydrolysis fragment is part of the probes of alt. a) or c) as defined below. It is hydrolyzed, cleaved or released from the cleavable probes of the present invention of alt. a) or c) after said probes hybridized or annealed to an DNA strand of a target nucleic acid. In a preferred embodiment of the invention described herein, each hydrolysis product exhibits or is characterized by a unique migration pattern resulting a peak (curve) upon separation during a, preferably capillary, electrophoresis, as defined herein.

Specificity of the signal, according to the present invention, means and is defined identically as disclosed in teaching EP4074839A1 in combination with present Fig. 5a (Fig. 1 A in EP4074839A1)..

Oligonucleotide cleavable probes (see also Fig. 5, 6) according to the invention are based on the teaching of EP4074839A1 and comprise an oligonucleotide (preferably with a minimum length of 5 nucleotides), whose sequence specifically hybridizes to a complementary nucleic acid, preferably DNA, strand of a target nucleic acid (Fig. 5. (2)) and further comprise:

A PCR blocker at its 3’-end (synonym protective group), as described herein an internal nuclease blocker as describe herein, and a cleavable hydrolysis product (Fig. 5, (3)) to be released by a nuclease, preferably FEN, as described herein, the cleavable hydrolysis product is located at the 5’-end upstream of the internal nuclease blocker and comprises at its 3'-end at least one target specific (complementary) nucleotide (which is complementary to the strand of the target sequence to which) linked to the internal blocker via a phosphodiester bond. it may additionally comprise at least one modification, described herein, which does not hybridize to the target sequence and/or a label, preferably a fluorophore (e.g. for use with the herein described MODAPLEX Device or Applied Biosystem of Thermo Fisher Scientific), bridged via a linker to the cleavable hydrolysis product, either to the at least one modification or to the at least one target specific (complementary) nucleotide, the label can be quantified by a detecting unit of a nucleic acid electrophoresis device, preferably MODAPLEX device. In one embodiment of the invention the cleavable hydrolysis product does not comprise a label (e.g. for use with CE/MS Systems of Agilent) but remains detectable and quantifiable. In this embodiment the gel comprises an intercalating dye, e.g. SYBR Green.

The function of the cleavable probe is to enable the detection of at least one analyte in a test sample and to provide a cleavable hydrolysis product for the generation of a specific signal. Thus, the released hydrolysis product confirms that the respective cleavable probe hybridized specifically to its complementary sequence on the target and allow the visualization of the specific hybridization trough the migration pattern with peaks (curves) of said hydrolysis products.

Nucleic acid hybridization (syn. hybridization) is defined as the annealing of two complementary single-stranded deoxyribonucleic acid molecules to an anti-parallel doublestranded DNA sequence (non-covalent DNA double helix formation). The two stands are mostly stabilized by hydrogen bonds between corresponding nucleic acid bases (Watson- Crick), London dispersion forces, and the hydrophobic stacking of neighboring base pairs (Altun et al. 2021). The thermodynamics of the process has been extensively studied and the most convenient sequence specific prediction method is based on the nearest-neighbor (NN) model (SantaLucia 1998). Different parameters must be considered for synthetic nucleic acid base (e.g. C5-propynyl derivatives of pyrimidine bases, He and Seela 2002) or backbone modifications like locked nucleic acid (LNA;McTigue et al. 2002).

Backbone Modifications of (synthetic) nucleotides or oligonucleotides or nucleic acid analogues as defined in this invention are based on the teaching of EP4074839A1 (comprise artificial modifications at the 2'and/or 4' position of the five-carbon sugar (ribose or deoxyribose): The backbone of oligonucleotides refers to the internal nucleotide linkage and/or the sugar moiety. In bridged nucleic acids (BNAs) the sugar ring is modified via a bridge or third ring structure. ENA (2'-O,4'-C-ethylene-bridged nucleic acid), BNA3 (2'-O,4'- aminoethylene bridged nucleic acid), and LNA (Locked Nucleic Acid, ribose moiety modified with an extra bridge connecting the 2'-oxygen and 4'-carbon) are well established examples, but others are also available. Other neutral backbones use for example the phosphorodiamidate morpholino oligomer (PMO) or the peptide nucleic acid (PNA) modifications. Backbone modifications that are forming an internal bridge are summarized in a group of “bridging backbone modification” and can be different from those listed herein. The above backbone modifications are known to confer resistance to nucleases and may increase the duplex stability of primers and probes with target nucleic acids. Modifications of the 2’- sugar position with methyl and methoxyethyl groups are also possible. Modifications at the 2’- position are well tolerated in oligonucleotide duplexes. The modification of probes used in the examples of the present invention are described in Table 3.

Feasibility of the internal nuclease blocker LNA according to the invention is shown herein conferring resistance of the hybridized cleavable probe at the defined position to a nuclease activity. Similar or improved resistances is achievable with ENA, BNA3, PMO, PNA, ortho- TINA or para-TINA nucleotides.

Another embodiment of the cleavable probe is, wherein the at least one nuclease blocker is either at position - 1 (downstream) or at position -2 (downstream) from the 5'-end of the hybridizing sequence, in particular hybridizing to its target sequence, of the probe and thereby conferring resistance as defined herein. Examples 8 and 9 of EP4074839A1 show the use of LNA and other blocker as well which are suitable for probes, in particular cleavable probes, of the present invention and their use in the method of the present invention for the detection of endometrium carcinoma.

Another aspect of the present invention is the cleavable probe wherein the cleavable hydrolysis product comprises an analyte unspecific 5’-sequence (FLAP) located 5'-upstream from the at least one internal nuclease blocker, preferably LNA, ENA, BNA3, PMO, PNA, ortho-TINA or para-TINA nucleotides and wherein the label is coupled via linker to the 5'end of the FLAP of the probe (5'-linker), or an internal nucleotide 5'-upstream from the at least one internal nuclease blocker.

The uncleavable probes, in particular for imaging methods, may also comprise FLAPs.

Non-backbone modifications of synthetic nucleotides or oligonucleotides are artificial chemical modifications which are coupled to the ends (5’ or 3’) of an oligonucleotide or to internal nucleotide bases. This can be done during solid-phase synthesis of oligonucleotides at the 5’-end or internally using specifically modified phosphoramidite building blocks or at the 3’-end by starting the cycle oligonucleotide synthesis with specifically modified solid support materials like controlled pore glass (CPG) or macroporous polystyrene (MPPS). Alternatively, reactive chemical groups (e. g. thiol, amino, carboxyl, terminal alkyne) can be introduced during solid-phase synthesis by non-nucleoside phosphoramidites or attached by postsynthetic processing steps (after having finished the automated synthesis and cleavage from the support) to become available for a variety of chemical coupling reactions. One or two nonbridging oxygen atoms of the phosphate group can be replaced by sulfur giving rise to phorothioates (PTO) or phosphorodithioates (diPTO), respectively. In alkyl- or aryl- phosphonates which are uncharged analogues of phosphodiesters, a non-bridging oxygen atom of the phosphate group has been replaced with an alkyl or aryl group. Such none- backbone modification confer resistance, in particular of the nucleotide carrying the at least one modification, to a nuclease activity. The teaching of non-backbone modifications of synthetic nucleotides or oligonucleotides is based on the teaching of EP4074839A1 and applies accordingly to probes, in particular cleavable probes, for use in the present invention. Examples are shown herein.

Spacers as defined in this invention are based on the teaching of EP4074839A1 and are a subgroup of non-backbone modifications and are defined as chemical structures which are coupled to the 3'and/or 5'end of a nucleotide or between two nucleotides (Fig 6). Spacers comprise a) alkyl alcohol of (C_n)-OH, wherein n is an integer and at least 3, preferably, 3, 6, 9 or 12 comprising propanyl (Spacer C3), hexanyl (Spacer C6), nonanyl (Spacer C9) and dodecanyl (Spacer C12), b) glycol ether of (-CH2-O-CH2-CH2)n-OH wherein n is an integer and at least 1 , preferably comprising triethylene glycol (Spacer 9), tetraethylene glycol (Spacer 12), and hexaethylene glycol (Spacer 18) and/or c) a tetrahydrofuaran derivative containing a methylene group occupied in the 1 position of 2’- deoxyribose, also known as abasic furan, abasic spacer or dSpacer.

The above mentioned chemical structures are only some examples representing the most suitable spacers but other spacers are possible and those examples are encompassed by the above definition. The spacer is coupled during the phosphoramidite synthesis and is flanked by two phosphodiester bonds or other bringing backbone modifications. The next 5’ building block can be a conventional nucleotide, a non-conventional nucleotide, an additional spacer or a linker with a fluorophore.

Modified or artificial bases which substitute their natural 2'-deoxynucleoside or ribonucleoside counterparts are a subgroup of non-backbone modifications which increases nucleic acid duplex stability due to internal interaction with other bases via H-bounds. Modified bases comprise 2-amino-deoxyadenosine (2-amino-dA), 5-methyl-deoxycytidine (5-Me-dC), aminoethyl-phenoxazine-deoxycytidine (AP-dC, G-Clamp), C-5 propynyl-deoxycytidine (pdC), and C-5 propynyl-deoxyuridine (pdll). In addition, intercalating nucleic acids like oTINA {orthotwisted intercalating nucleic acid; (S)-1-O-[2-(1-pyrenylethynyl) phenylmethyl] glycerol} and (S)-1-O-(4, 4'-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl) glycerol intercalating pseudonucleotide (IPN), or 3’ minor grove binder (MGB, WO1996032496A2) serves as DNA duplex stabilizers and/or polymerase blockers. The teaching of modified or artificial bases is based on the teaching of EP4074839A1 and applies accordingly to probes, in particular cleavable probes, for use in the present invention. Internal nuclease blocker (synonym internal blocker or nuclease blocker) is defined as a nucleotide having at least one artificial modification which confer resistance of said nucleotide to a nuclease activity, in particular to nucleases. In other words, certain modifications described herein fulfill the function of blocking the nuclease at the position of the modification and thereby defining the cleavage site for the nuclease being 5'upstream from the at least one internal nuclease blocker of the hybridizing sequence. The addition of a second (or more) nuclease blockers downstream from the first does not change the cleavage site but reduces unwanted artefacts in the method described herein. In the event the cleavable hydrolysis product of the invention comprises a FLAP as described herein, a nuclease blocker can be located at the FLAP and a second internal nuclease blocker is located 3' downstream from the at least one hybridizing nucleotide of the 5'end sequence of the cleavable product as described herein (see Fig. 5A). A suitable modification is a backbone modification, more preferably, a modification at 2'-position of the sugar backbone as defined above. Most preferably theses are comprising 2 - O-methyl and 2'-O-methoxyethyl. Non-backbone modifications as defined above, preferably a modification of the phosphate group, wherein one or two non-bridging oxygen atoms of the phosphate group is/are replaced by sulfur, alkyl- and/or aryl-group confers resistance to a nuclease activity, in particular to nucleases. Finally, any artificial modification or combination of two or more of the aforementioned modifications of a nucleotide conferring resistance to a nuclease activity functions as an internal nuclease blocker within the meaning of the invention. Preferably, the internal nuclease blocker is a bridged or intercalating nucleic acid, like LNA, TINA respectively or others described herein or known in the prior art. The teaching of internal nuclease blocker is based on the teaching of EP4074839A1 and applies accordingly to probes, in particular cleavable probes, for use in the present invention.

The at least one internal blocker has different functions. It contributes to the specificity of the signal or it contributes to the stringency of the signal without any impact on the specificity. Dependent on the label, preferably fluorophore, selected for the specific design of the cleavable product of the cleavable probe, within the inventive method, the achieved signal (peak) is distinct and clear (without unwanted artefacts) and therefore specific within the meaning of the invention but may be accompanied by weak or dim lower migrating artefacts depending on the label. In such cases it is preferred to combine at least two or three nuclease blockers within the cleavable hydrolysis product of the cleavable probe. The first will have the position as described herein and shown in Fig. 5A and the second and third may be located immediately downstream of the aforementioned position in Fig. 5A. The internal blocker also facilitates higher multiplexing, by combining it with flapped (mismatched) nucleotides upstream from the cleavage site. For specificity of the signal (see further definitions), the first flapped nucleotide, the position immediately upstream from the cleavage site, contains an internal nuclease blocker. Addition of flapped nucleotides beyond the blocked flapped position, allow stringent control of the size of the cleavable hydrolysis product and its migration. In turn, this enables an increase of the multiplex degree that the cleavable technology can support.

The aim of the at least one modification and/or internal nuclease blocker of the cleavable probe is to ensure that a defined hydrolysis product is released from the cleavable probe by an enzyme exhibiting nuclease activity described herein at a defined cleavage site and is suitable for use in the method of the present invention, which is suitable to combine a plurality of cleavable probes.

Linkers are resulting from coupling reactions: Coupling reactions as defined herein are based on the teaching of EP4074839A1 and are any chemical reactions which are used to introduce a backbone, non-backbone modifications and/or label to the probe, in particular to a nucleotide, and which are compatible with solid-phase phosphoramidite synthesis and/or post-synthetic processing steps (e.g. release from solid support, alkaline deprotection with inorganic bases or amines, HPLC) of oligonucleotides. A subtotal selection of well-established chemistries are reactions of (a) activated organic acids [acid anhydrides, acid chlorides, N- hydroxy-succinimide (NHS)-esters] or isothiocyanates with amino groups, (b) hydrazide or aminooxy groups with aldehyde groups, (c) iodoacetamide or maleimide (2,5-pyrroledione) with thiol groups, (d) cyano-benzothiazoles with cysteine groups, (e) Click Chemistry (azidealkyne cycloaddition; El-Sagheer and Brown, 2012) without and with metal ion catalysis (e. g. cupper ions), and (f) Retro- (or reverse)-Diels-Alder (rDA) reactions (e. g. reaction pairs tetrazine - trans-cyclooctene or tetrazine - norbornene). The different coupling reactions yield chemical structures which are referred to as linkers in this patent application and which are between the (oligo)nucleotide and its modification or between the nucleotide and the label, preferably fluorophore. Linkers which are currently available as reactive phosphoramidite regents are e.g.

2-(3-aminopropyl)-1 ,3-dihydroxypropane phosphoramidite 2,2-di(e-aminopropyl)-1 ,3-dihydroxypropane phosphoramidite

3-amino-1 ,2-dihydroxypropane phosphoramidite 6-amino-1 ,2-dihydroxyhexane phosphoramidite

Additional technologies which are suitable to detected and quantify copy number variations are e.g.:

- Next generation sequencing (NGS): Illumina Next Seq, Thermo Fisher Ion Torrent, Oxford Nanopore - qPCR (quantitative) and dPCR (digital): preferably suitable primer are used for the amplification of the at least one target sequence and at least one reference sequence. For this embodiment no probes, neither cleavable nor uncleavable probes are necessary

- FISH (flourescent in situ hybridization): preferably suitable probes, in particular cleavable and/or uncleavable probes, are used for the detection of the at least one target sequence and at least one reference sequence. For this embodiment no primer are necessary

- Array CGH (comparitive genome analysis): Affimetrix Thermo Fisher GeneChip™ (Santa Clara, LIS-CA), Agilent GenetiSure Cancer CGH+SNP Microarray (Santa Clara, LIS-CA)

- NanoString Technologies nCounter ® (Seattle, LIS-WA)

- DNA Combing or Optical Genome Mapping: Bionano ® Genomics Saphyr (San Diago, LIS-CA), Genomic Vision Fibre Comb® (Bagneux, FR)

- Spartial Biology Imaging or In-situ Sequencing (3D Genomics) or Hi-C (3C, 4C etc.) Technologies: Resolve Biosciences Molecular Cartography™ technology (Monheim am Rhein, DE), Arima Genomics (Carlsbad, LIS-CA): preferably suitable probes, in particular cleavable probes and/or uncleavable probes, are used for the detection of the at least one target sequence and at least one reference sequence. For this embodiment no primer are necessary

Finally, the teaching of the present invention is based on the unique and inventive new analyte panel for the identification and for detection of endometrium carcinoma CN high subtype.

Finally, the teaching of the present invention and the new and inventive analyte panel is suitable as biomarker for the identification and for diagnosis of endometrium carcinoma CN high subtype. Accordingly, by targeting, labelling, amplification and/or detection of said biomarker, the inventive solution is realized. The means for visualization of any signal as proof of detection of said biomarker is variable. Herein at least three different concepts are disclosed

- methods wherein amplification of the target sequences and optionally reference sequences is performed and CN high endometrium carcinoma is detected based on the achieved amplicons.

- methods wherein hybridization of specific labelled probes, in particular uncleavable probes, to the at least one target sequence and to the at least one reference sequence is performed and CN high endometrium carcinoma is detected via imaging due to a fluorescence signal in the test sample or specimen comprising a tissue

- methods wherein hybridization of specific cleavable probes (as disclosed in EP4074839A1) to the at least one target sequence and optionally to the at least one reference sequence and amplification of said sequences are performed, wherein CN high endometrium carcinoma is detected by means of the respective released hydrolysis products. Amplicons are achieved but not relevant for the identification of CN high endometrium carcinoma For all of the above mentioned methods all embodiments of the reference as defined herein and of the target sequences are applicable. Preferably, the analyte panel comprise a reference comprising reference sequence encoding within the location of gene ZRANB2 and ZRANB2- AS1 on chromosome 1 and the reference sequence encoding within the location of gene TMEM169 on chromosome 2 and the reference sequence encoding within the location of gene HCAR1 on chromosome 12. Said reference is used to detect the target sequences encoding within the location of genes COX10, ARHGAP44, ELAC2 and MYOCD, respectively in the cytoband 17p12, and the target sequences encoding within the location of genes ZNF555, TLE6, ZNF57 and ZNF77, respectively in the cytoband 19p13.3. By targeting, labelling, amplifying and detecting of said analyte panel - ZRANB2, ZRANB2-AS1 , TMEM169, HCAR1 , COX10, ARHGAP44, ELAC2, MYOCD, ZNF555, TLE6, ZNF57, ZNF77, as suitable biomarker, CN high endometrium carcinoma is qualitatively identified and preferably quantified.

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[6] Concin N, Matias-Guiu X, Vergote I, Cibula D, Mirza MR, Marnitz S, Ledermann J, Bosse T, Chargari C, Fagotti A, Fotopoulou C, Gonzalez Martin A, Lax S, Lorusso D, Marth C, Morice P, Nout RA, O'Donnell D, Querleu D, Raspollini MR, Sehouli J, Sturdza A, Taylor A, Westermann A, Wimberger P, Colombo N, Planchamp F, Creutzberg CL. ESGO/ESTRO/ESP guidelines for the management of patients with endometrial carcinoma. Int J Gynecol Cancer. 2021 Jan;31 (1):12-39. doi:10.1136/ijgc-2020-002230. Epub 2020 Dec 18. PMID: 33397713. [7] Chang Z, Talukdar S, Mullany SA, Winterhoff B. Molecular characterization of endometrial cancer and therapeutic implications. Curr Opin Obstet Gynecol. 2019 Feb;31 (1):24-30. doi: 10.1097/GCO.0000000000000508. PMID: 30507624.

[8] Jamieson A, Thompson EF, Huvila J, Gilks CB, McAlpine JN. p53abn Endometrial Cancer: understanding the most aggressive endometrial cancers in the era of molecular classification. Int J Gynecol Cancer. 2021 Jun;31(6):907-913. doi: 10.1136/ijgc-2020-002256. Epub 2021 Feb 15. PMID: 33589443.

[9] Huvila J, Pors J, Thompson EF, Gilks CB. Endometrial carcinoma: molecular subtypes, precursors and the role of pathology in early diagnosis. J Pathol. 2021 Apr;253(4):355-365. doi: 10.1002/path.5608. Epub 2021 Feb 6. PMID: 33368243.

[10] Aguilar M, Zhang H, Zhang M, Cantarell B, Sahoo SS, Li HD, Cuevas IC, Lea J, Miller DS, Chen H, Zheng W, Gagan J, Lucas E, Castrillon DH. Serial genomic analysis of endometrium supports the existence of histologically indistinct endometrial cancer precursors. J Pathol. 2021 May;254(1):20-30. doi: 10.1002/path.5628. Epub 2021 Mar 9. PMID: 33506979; PMCID: PMC8252414.

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Recent paper 3D Genomics: Payne AC, Chiang ZD, Reginato PL, Mangiameli SM, Murray EM, Yao CC, Markoulaki S, Earl AS, Labade AS, Jaenisch R, Church GM, Boyden ES, Buenrostro JD, Chen F. In situ genome sequencing resolves DNA sequence and structure in intact biological samples. Science. 2021 Feb 26;371(6532):eaay3446. doi: 10.1126/science.aay3446. Epub 2020 Dec 31. PMID: 33384301 ; PMCID: PMC7962746.

Hi-C Paper: Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009 Oct 9;326(5950):289-93. doi: 10.1126/science.1181369. PMID: 19815776; PMCID: PMC2858594. Example

Material and methods

Human endometrial cancer specimen and patient cohorts

All medical research involving human subjects, including research on identifiable human material and data were performed according to the current ethics standards of The World Medical Associations Declaration of Helsinki (WMA, Ferney-Voltaire, FR), relevant legisation, and with declared consent of the participants.

The herein described invention for the multiplex PCR based identification of CNH positive EC specimens has been developed on artificial template DNA and further optimized and verified on genomic DNA purified from primary EC tissue samples. The latter has been obtained from two different sources, (1) 41 fresh frozen EC samples have been received from Indivumed GmbH (Hamburg, Germany) and (2) 38 FFPE EC samples from the Institute of Pathology at the University Hospital Carl-Gustav-Carus Dresden (Dresden, Germany). The mean ischemia time of the 41 fresh frozen tissue sections obtained was 16 min (min = 2 min, max = 95 min, median = 12 min). Additionally, information concerning age (mean = 68), BMI (mean = 31 ,3) as well as detailed clinical data have been received from Indivumed GmbH. All samples from the Indivumed-cohort have been diagnosed C54.1 (ICD code for malignant disease within the endometrium). TNM classification of the cohort are as follows: 24 out of 41 samples have been assigned to pT1a or pT1 b, 10 samples to pT2 and 7 samples to pT3 pr pT3b. Infiltration of draining lymph nodes has been detected in 6 out of 41 patients (n pN1(a) = 3; n pN2a = 3). For 5 patients, the lymph node status was not clearly determinable (pNX) and in 30 cases no infiltration of tumor cells into the lymph nodes has been seen (pNO). Distant metastasis have been confirmed in 2 out of 41 cases only. All patients from the Indivumed cohort did not received any neoadjuvant chemotherapy (= systemic therapy prior surgery).

Very limited patient and sample data has been received for the 38 FFPE EC specimens from the Institute of Pathology at the University Hospital Carl-Gutav-Carus Dresden. However, the endometrial cancer disease has been confirmed for all 38 patient samples by board-certified pathologists. The FFPE EC samples from which gDNA has been isolated are between 3 and 10 years old and have been stored at room temperature.

DNA purification from human EC FFPE samples

The 41 fresh frozen EC samples obtained from Indivumed GmbH have been further processed in the Institute of Pathology at the University Hospital Carl-Gustav-Carus Dresden. Briefly, fresh frozen EC samples have been fixated in formaldehyde, dehydrated, and embedded in paraffin using standard protocols. The FFPE samples from both cohorts, Indivumed and the Institute of Pathology of the University Hospital Carl-Gustav-Carus Dresden, have been than evaluated in terms of their individual tumor content (TC). This has been done by hematoxylin & eosin staining of 4 pM microtome FFPE sections. Subsequently, starting material for DNA isolation has been collected directly from the FFPE tissue samples from tumor regions exclusively using scalpel. DNA isolation has been conducted with the QIAamp DNA Micro Kit (Qiagen GmbH, Hilden, Germany) following the manufacturers' instructions. The DNA purification protocol includes a proteinase K digestion step and is based on DNA binding to a silica-matrix. The DNA concentration was determined using a QuBit Fluorometer (ThermoFisher Scientific Inc., Waltham, USA) and the QuBit dsDNA BR Assay-Kit (ThermoFisher Scientific Inc., Waltham, USA).

Design and synthesis of PCR primers and cleavable probes

Primers and cleavable probes have been generated within the respective analyte target- & reference sequence regions on chromosome 1, 2 & 12 (stable reference regions) and chromosome 17 & 19 (instable target regions) using Geneious 10.2.6 (Biomatters Ltd., Auckland, NZ) (Fig. 1 , Table 1). All oligonucleotides are summarized in Table 2 and Table 3. All oligonucleotides were obtained in HPLC-purified quality from commercial manufactures. Amplicon lengths vary between 75 nt and 103 nt ( Table 4).

Table 1 - Target region and reference region definition referred to human genome assembly T2T-CHM13 v2.0/hs1 (GenBank Assembly GCA_009914755.4, by Jan 24, 2022: https://www.ncbi.nlm.nih.ciov/datasets/aenome/GCF 009914755.1 A-

Target- and control regions, comprising target and reference sequences, are cytoband- overlapping. Cytobands highlighted with bold letters resemble to binding sites of the respective primers and probes. However, the assay would also work for primers/probes that bind to the neighboring cytobands.

Target sequences in chromosomal regions have been identified and subsequently predetermined as target sequences for the CNH determination with the help of extensive bioinformatical analysis of public available sequencing data set (whole genome sequencing) of 232 endometrial cancer patients from the TCGA-UCEC data base. Two genomic regions which are exclusively highly instable in the respective EC CNH positive sub-cohort have been found in the chromosomal regions 17p12 and 19p13.3. Within the EC patient cohort, all samples are either CNV affected in chromosomal region 17p12, 19p13.3 or both. In the patient cohort analyzed, deletions on the aforementioned chromosomal regions are present in a significant fraction, while amplifications are rare. Subsequently, four molecular genetic analytes have been established on both chromosomal region cytobands, covering the target sequences of genes MYOCD, ARHGAP44 and COX10 on chromosome 17 and the target sequences of genes ZNF77, ZNF57, TLE6, ZNF555 on chromosome 19. In the herein described invention, suitable PCR primers and suitable oligonucleotide probes as defined herein have been generated, allowing the amplification of the respective predetermined target sequences. Together with reference sequences, identification of the CNH subtype is feasible with high accuracy.

Reference within the meaning of the invention comprises reference chromosomal regions (reference regions) and therein at least one reference sequence or more which have been identified analogous to the aforementioned target regions. Within the same EC sequencing data set, genomic regions have been identified, that are relatively stable in terms of the presence of CNV. In total three reference regions have been combined to form the inventions reference and are located on the cytobands 1 p31.3, 2q35 and 12q24.31. None of the three reference regions have been found to be 100% stable in all CNH positive samples within the 232 patient samples analysed. CNVs in one of the three control regions have been seen in 7.33%, 7.76% and 3.02% for Chr1 , Chr12 and Chr2, respectively. Two CNV affected target regions have been seen in 3.45% (Chr1 + Chr12), 0.86% (Chr2 +Chr12) and 0.43% (Chr1 + Chr2) cases. Within the cohort, none of the data sets showed CNV in all three control regions within one individual patient sample. The absence of CNV in all control regions within one sample has been detected in 77.16% of the cohort. Overall, all three reference regions in combination are required to form a suitable reference region.

TaWe 2 - Primers used in rite examples. Enlisted are all oligonucleotides that are included in the copy number high detection assay. Annealing temperature has been calculated using the IDT QljgpAnalYZ§{ tool (ffeXsMS (monovalent cations) = 70 mM. Mg*- Cone (bivalent cations) = 3 mM). Oligonucleotide target site position has been defined based on the T2T-CHM13 v2.0/hs1 (Jan 2022) genome & the UCSC genome browser

(htps:fgenoTO.ucsc.edu/index.html).

Table 3 - Overview of all cleavable probes used in the examples. Enlisted are all probes (based on the teaching of EP4O74839A1) that are included in the copy number high detection assay. Annealing temperature has been calculated using the IDT oligcAnataer tool (Na^Conc. (monovalent cations) = 70 mM Mg** Cone, (bivalent cations) = 3 mM) Oligonucleotide target site position has been defined based on the T2T- CHM13 <20 genome & the UCSC genome browser (htps:fgenorne.ucsc.edirtndex.htrnl). Locked nucleotide acids (LNAs) have been indicated with a “+^s (e.g. +T).

The underlined sequences are suitable for adaption of the above specific deavab e probes to any suitable embodiment and to not necessarily cleavable probes for use in a method with detection of CN high endometrium carcinoma by means of imaging, e.g. HsH probe or the like.

Table 2 - Overview Copy Number High Detection Assay Amplicons.

Polymerase chain reaction (PCR) protocol for the MODAPLEX 2.0 system: PCR was performed in a final volume of 25 pL and contained one-fold MODAPLEX Buffer 11 (Biotype GmbH, Dresden, Germany; critical components in final concentrations of 20 mM Tris HCI buffer pH 8.8 at 25 °C, 50 nM KOI, 0.2 mM dNTPs and 3,0 mM MgCI₂), 2.0 units Hot Start Taq DNA Polymerase (Biotechrabbit, Berlin, Germany), oligonucleotides (Table 2, Table 3) in an individually optimized concentration (0.05 - 0.4 pM), MODAPLEX calibrator SST4 (Biotype GmbH, Dresden, Germany) and 10 ng of human genomic DNA as template input. Prior initiation of the PCR, multiwell plates were sealed with Aluminum Sealing Film (Biotype), spun using a Bench-top centrifuge with plate adaptor and the sealing removed gently. Additionally, 40 pL of mineral oil (Merck - SIGMA-Aldrich, Darmstadt, DE) were applied to each well, before starting the PCR.

PCR cycling combined with capillary electrophoresis using the MODAPLEX 2.0 system: The MODAPLEX 2.0 (Biotype GmbH, Dresden, Germany) is a modular device consisting of a PCR thermocycler, an automatic sampler, a capillary gel electrophoresis device and a fluorescence detector with two analysis channels (US7445893, US7081339, US7674582, US8182995; Hlousek et al. 2012). The MODAPLEX 2.0 was completely operated with reagents of the manufacturer and according to its instructions.

The PCR was set up with the chemicals of the manufacturer, wherein the final concentrations of PCR primers and probes corresponded to those described by standard PCR. The PCR program comprised of 2 min of hot start activation at 98 °C, followed by 2 cycles of 45 s at 60 °C, 45 s at 73 °C and 5 s at 98 °C, 4 cycles of 45 s at 59.5 °C, 45 s at 72.5 °C and 5 s at 98 °C, 10 cycles of 45 s at 59.0 °C, 45 s at 72.0 °C and 5 s at 98 °C, and 24 cycles, including 12 injections for CE separation, of 45 s at 58.5 °C, 220 s at 71.5 °C and 10 s at 96 °C. Real time analysis by capillary gel electrophoresis was carried out 12 times in the elongation phase at 72 °C by electro kinetic injection (10.000 V, 15 s) from 17th to 39th cycle (every second cycle). The detection unit records fluorescence, expressed in relative fluorescence units (RFU), and provides an electropherogram as an output based on the migration time recorded. Real-time PCR Amplification curves are built from all electropherograms recorded at different PCR cycles. Non-amplifying internal calibrators (see aforementioned MODAPLEX size standard 4), detected in the blue channel, are used in each assay to calculate migration lengths of cleavable probe hydrolysis fragments. The Materials and methods were used for all patient samples analyzed within the presented example of the present invention.

P53 IHC staining and evaluation

Determination of the copy number high subtype in endometrial cancer was performed via IHC staining of the surrogate marker P53, which harbors genomic mutations in around 90% of CNH positive cases. IHC staining of FFPE endometrial cancer tissue sections has been conducted using standard protocols, which include a de-paraffinization step in xylene, sample rehydration with different dilutions of ethanol, multiple washing in 1x TBST buffer, a quenching step of endogenous peroxidases in methanol supplemented with H2O2, a boiling step in sodium citrate buffer, blocking, anti-P53 antibody incubation and a detection step with horseradish peroxidase. P53 IHC stainings of all 79 EC patients have been evaluated and interpreted by board-certified pathologists from the Institute of Pathology at the University Hospital Carl- Gustav-Carus Dresden. Staining patterns were classified into three different groups, (1) single cell, (2) nucleus accumulated and (3) loss. The single cell pattern resembles the phenotype which can be found in tissues without any alterations in the TP53 gene (= non-pathogenic phenotype). Nucleus accumulations, however, are typical for loss-of-function mutations which result in an increased translation and a reduced ubiquitinoylation of P53 (= TP53 mutated). The deletion of both TP53 alleles can result in a negative P53 IHC staining (= pathogenic phenotype).

Low coverage whole-genome sequencing of endometrial cancer specimens

Besides P53 IHC, low coverage (0.9x- <1.3x) whole-genome sequencing was performed with genomic DNA from 47 out of 79 FFPE EC tissue samples (Genewiz/Azenta, Leipzig, Germany) as an additional method for the copy number high molecular subtype determination. Low coverage sequencing allows to detect copy number variations of larger genomic regions with a resolution of about 10 kb. Genome-wide copy numbers of all samples were used for a hierarchical clustering. All samples with strong genomic aberrations fall into one cluster and were defined as copy number high samples according to Nature 497, 67-73, 2013 (https://doi.Org/10.1038/nature12325, “Erratum: Integrated genomic characterization of endometrial carcinoma”). Another aspect of the present invention is a combination of amplicons, in particular achieved by the method according to the present invention, preferably by any method or embodiment thereof that comprises an amplification stept, wherein the amplicons have the sequences of Seq ID No. 43, Seq ID No. 44, Seq ID No. 45, Seq ID No. 46, Seq ID No. 47, Seq ID No. 48, Seq ID No. 49, Seq ID No. 50, Seq ID No. 51 , Seq ID No. 52, Seq ID No. 53, Seq ID No. 54, Seq ID No. 55 and/or Seq ID No. 56. In particular any combination of the sequences are part of the invention. Preferably, the combination of all of the aforementioned sequences. Alternatively, a combination of amplicons is also part of the invention, wherein any of the amplicons has not the identical sequence but a homology of at least 70%, 75 %, 80 %, at least 90 %, at least 95 %, 96, 97, 98, 99 % to the respective Sed ID No.

Example 1 - Comparison of the copy number high determination of 79 human endometrial cancer specimens using IHC staining of P53, low-coverage sequencing and the multiplex PCR based MODAPLEX copy number high detection assay.

Genomic DNA isolated from 79 human EC tissue samples has been analyzed using the herein presented invention on the MODAPLEX 2.0 platform. Next, primer efficiency correction has been conducted for all Ct values (raw data summarized in Table 5). Resulting primer efficiency corrected Ct values have been averaged (arithmetic mean) for targets on 17p12, 19p13.3 and the reference sequences, respectively. Subsequently, the delta-Ct for 17p12 and 19p13.3 was calculated by subtracting the average Ct value of the control regions from the 17p12 and 19p13.3 arithmetic mean (see Table 6). Plotting of the delta Ct values obtained for 17p12 and 19p13.3 resulted in the identification of one larger cluster and a subset of samples with delta Ct values greater for chromosome 17 and/or chromosome 19 compared to the main cluster (not shown). For one case, the delta Ct value for chromosome 19 was lower than for the population of samples within the main cluster. IHC P53 staining results have been implemented in the plotted delta Ct values, showing that 14 out of 22 P53 mutated (according to IHC staining pattern) EC samples are located outside of the main delta Ct17/19 cluster and 8 inside (Fig. 2 A). In return, 55 out of 57 samples harboring a wild type associated pattern in the P53 IHC staining are locate within the main cluster of the delta chromosome 17/19 plot. Two from the 57 samples with an unsuspicious P53 staining pattern had high delta Ct values for chromosome 17 or 19 respectively. This data led to the assumption that the main cluster within the delta chromosome 17/19 diagram might resemble the population of copy number high negative samples. Table 5 - Summary of primer efficiency corrected Ct values from 79 EC patient derived genomic DNA samples analysed witii the herein presented invention. In total 14 genomic targets have been analysed via simultaneous PCR amplification in one reaction. Raw Ct values data have been corrected based on previous determined primer efficiencies. Targets can be grouped into targets on the instable regions on chromosome 17 and 19, as well as stable control on chromosome 1, 2 and 12.

Table 3- Summary of all delta Ct values for chromosome 19 and 17. Delta Ct values have been calculated by subtracting the average Ct value of the control regions from the 17p12 and 19p13.3 arithmetic mean.

Table 6 continued

Low coverage whole genome sequencing has been performed to determine the copy number high status using an independent precise molecular biological method. Genomes of all samples with particularly high or low delta Ct values for chromosome 17 and/or 19 (n = 16) as well as 31 samples from the potential copy number high negative cluster were sequenced (low coverage WGS). Sequencing data analysis revealed that 16 out of 18 copy number high positive samples (according to low coverage WGS) are located outside of the plotted main cluster in the delta Ct17/19 diagram, which previously has been assumed to be the copy number high negative population (Fig. 2 B). For two samples with a characteristic accumulation of CNV of larger chromosomal fragments, delta Ct values have not been particularly higher or lower compared to the main cluster within the plot. For all samples (n = 29) for which no copy number high genotype has been detected via low coverage WGS, subtyping was in line with the invention’s results.

Results of IHC staining, WGS and multiplex PCR of a representative example of a copy number high positive and negative EC sample are illustrated in Fig. 3.

Based on the data obtained from the analysis of the 79 patient samples, as well as the determination of the assays limit of blank and precision, delta Ct cut-off values have been defined for chromosome 17 (-0.49 & -1.3) and 19 (0.75 & 1.73), which allow to assign the subtype of copy number high in endometrial cancer based on PCR data generated with the herein presented primer and probes of the invention.

The experimental data further have been statistically analyzed via confusion matrix calculation. Comparison with the IHC data, resulted in an inventions sensitivity, specificity, and accuracy of 64%, 96% and 87% respectively. However, since WGS revealed multiple false positive and negative IHC staining outcomes, confusion matrix analysis additionally has been performed implementing the WGS data set. As a result, the inventions sensitivity (89%), specificity (100%) and accuracy (96%) are comparable to WGS methods.

Based on the given primary tissue sample set, the herein presented invention outperformed IHC staining of P53 which by now is considered as a surrogate marker for copy number high in endometrial cancer. Additionally, the invention's sensitivity, specificity, and accuracy were comparable to WGS, which resembles a precise method for CNH detection, but which can take at least several days or weeks and requires up to 20-50 times more input material of genomic DNA.

Claims

1. Method for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high), wherein at least one copy number variation (CNV) is detected for a CNV chromosomal region comprising at least one cytoband on chromosome 17 and/or chromosome 19, wherein the method comprises the steps providing a test sample of a subject comprising at least one predetermined target sequence in the CNV chromosomal region, providing a reference comprising at least one or more reference sequences of a known copy number, determining a CNV of at least one predetermined target sequence in the CNV chromosomal region, evaluating the CNV of the at least one predetermined target sequence in the CNV chromosomal region relative to the copy number of the reference, identification of an endometrium carcinoma of the CN high subtype, wherein the CNV for the CNV chromosomal region is determined CN high compared to the copy number of the reference.

2. The method according to claim 1, wherein the CNV chromosomal region comprises cytobands 17p13.3, 17p13.2, 17p13.1, 17p12, 17p11.2, 19p13.3 and/or 19p13.2 or any combination thereof.

3. The method according to claim 1 or 2, wherein in the step of determining a CNV of at least one predetermined target sequence in the CNV chromosomal region, the CNV is determined for at least one or more predetermined target sequences in at least one cytoband of cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11.2, 19p13.3 and/or 19p13.2 or in any combination of one or more of the aforementioned cytobands.

4. The method according to any one of the claims 1 to 3, wherein in the step of determining a CNV of at least one or more predetermined target sequences in the CNV chromosomal region, the CNV is determined for at least one or more predetermined target sequences in cytoband 19p13.3 on chromosome 19 and for at least one or more predetermined target sequences in cytoband 17p12 on chromosome 17.

5. The method according to any one of the claims 1 to 4, wherein the at least one CNV in the CNV chromosomal region is detected for at least one or more target sequences within the location of genes COX10, ARHGAP44, ELAC2 and/or MYOCD in the cytoband 17p12 and/or for at least one or more target sequences within the location of genes ZNF555, TLE6, ZNF57 and/or ZNF77 of cytoband 19p13.

6. Method according to any one of the claims 1 to 5, wherein the reference comprising at least one or more reference sequences is provided with a test sample of the same subject wherein the method comprises further the steps determining the copy number of at least one or more reference sequences, evaluating the CNV of the at least one predetermined target sequence in the CNV chromosomal region, relative to the determined copy number of the at least one or more reference sequences of the reference, identification of an endometrium carcinoma of the CN high subtype is determined CN high compared to the determined copy number of the reference.

7. Method according to any one of the claims 1 to 6, wherein the reference is provided as a digital reference independently from the subject comprising the at least one predetermined target sequence and independently from any test sample of any subject.

8. The method according to any one of the claims 1 to 7, wherein the reference comprises at least one reference sequence on chromosome 1, and/or at least one reference sequence on chromosome 2 and/or at least one reference sequence on chromosome 12.

9. The method according to any one of the claims 1 to 8, wherein the reference comprises at least one reference in the cytobands 1p32.3, 1p32.2, 1p32.1 , 1p31.3, 1p31.2,

1 p31.1 , 1p22.3, 1p22.2, 1 p22.1 , 1 p21.3, 1p21.2 and/or 1 p21.1 on chromosome 1 and/or in the cytobands 2q32.1 , 2q32.2, 2q32.3, 2q33.1, 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1, 2q36.2, 2q36.3 and/or 2q37.1 on chromosome 2 and/or in the cytobands 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 on chromosome 12 or any combination thereof.

10. The method according to any one of the claims 1 to 9, wherein the reference comprises at least one reference sequence in the cytoband 1p31.3 on chromosome 1 , at least one reference sequence in the cytoband 2q35 on chromosome 2 and at least one reference sequence in the cytoband 12q24.31 on chromosome 12.

11 . The method according to any one of the claims 1 to 10, wherein the reference comprises at least one reference sequence within the location of gene ZRANB2 and at least one reference sequence within the location of gene ZRANB2-AS1 on chromosome 1 , and/or at least one reference sequence within the location of gene TMEM169 on chromosome 2 and/or at least one reference sequence within the location of gene HCAR1 on chromosome 12.

12. The method according to any one of the claims 1 to 11 , wherein the reference is negative for a CNV.

13. The method according to any one of the claims 1 to 12, wherein the method comprises a step of providing suitable primers for amplification of the at least one or more predetermined target sequences in the CNV chromosomal region and optionally suitable primer for amplification of at least one or more reference sequences.

14. The method according to any one of the claims 1 to 13, wherein the method further comprises a step of contacting the test sample with the suitable primers specific for the at least one or more target sequences and optionally for the at least one or more reference sequences respectively, hybridization of the at least one primer to its respective complementary sequence on the target sequence and optionally on the reference sequence, amplification of the at least one target sequence and optionally reference sequence, separating the amplified target sequences and optionally reference sequences, detection of the amplified target sequences and optionally of the amplified reference sequences, respectively, and evaluating the CNV of the at least one target sequence compared to the copy number of the at least one reference sequence.

15. The method according to any one of the claims 1 to 14, wherein amplification and detection of the at least one or more target sequences and optionally of at least one or more reference sequences, respectively, is performed by real time PCR, end point PCR, digital PCR, geometric multiplexing PCR, digital droplet (or emulsion) PCR (ddPCR) or Next Generation Sequencing (NGS).

16. The method according to any one of the claims 1 to 15, wherein the at least one CNV for the CNV chromosomal region is determined by the at least one amplified target sequence compared to the at least one reference.

17. The method according to any one of the claims 1 to 12, wherein the method comprises the steps contacting the test sample of the subject with at least one oligonucleotide probe (probe), that is specific for the at least one target sequence and with at least one oligonucleotide probe that is specific for the at least one reference sequence, respectively, hybridization of the respective probes to its respective complementary sequences within the test sample, detection of an emitting signal of each hybridized probe, wherein at least one probe for the at least one target sequence and the at least one probe for the at least one reference sequence comprise to each other differentiating fluorophores, evaluating the fluorescence signal for the at least one predetermined target sequence in the CNV chromosomal region relative to the fluorescence signal to the at least one reference sequence, and identification of an endometrium carcinoma of the CN high subtype, wherein the CNV for the CNV chromosomal region is determined CN high compared to the copy number of the reference.

18. The method according to any one of the claims 1 to 13, wherein the method comprises the steps contacting the test sample of the subject with suitable primers specific for the at least one target sequence and optionally for the at least one reference sequence respectively and contacting the test sample with at least one cleavable oligonucleotide probe (probe), that is specific for the at least one target sequence, respectively, and optionally with at least one cleavable oligonucleotide probe that is specific for the at least one reference sequence, hybridization of the at least one primer to its respective complementary sequence on a same strand of the same target sequence, wherein the cleavable probe hybridizes 3'upstream of the respective primer, and optionally hybridization of the at least one primer to its respective complementary sequence on a same strand of the same reference sequence, wherein the cleavable probe hybridizes 3'upstream of the respective primer, amplification of the at least one target sequence and optionally of the at least one reference sequence, cleavage of the hybridized cleavable probes with a template-dependent 5' to 3' nuclease activity to release a cleavable hydrolysis product of the each cleavable probe, separating of the released hydrolysis product(s) and detection of the released hydrolysis product(s).

19. The method according to claim 18, wherein the CNV of the at least one predetermined target sequence is determined by the respective released hydrolysis product compared to the at least one respective reference hydrolysis product of the reference.

20. The method according to claim 19, wherein the respective reference hydrolysis product of the at least one reference is released from at least one reference sequence according to the method of the preceding claims or it is a digital reference hydrolysis product.

21. The method according to any one of the claims 18 to 20, wherein amplification of the at least one target sequence and optionally of the at least one reference sequence, respectively, is performed by real time PCR and separation of the hydrolysis product(s) by capillary electrophoresis.

22. A specific oligonucleotide probe suitable for hybridization to at least one predetermined target sequence in the CNV chromosomal region comprising cytobands 17p13.3, 17p13.2, 17p13.1 , 17p12, 17p11.2, 19p13.3 and/or 19p13.2, preferably for at least one predetermined target sequence in cytoband 19p13.3 and for at least one predetermined target sequence in cytoband 17p12, and/or to at least one reference sequence in the cytobands 1p31.3 and/or 2q35 and/or 12q24.31 , comprising respectively, a 3’-sequence which is reverse complementary to a sequence of the at least one predetermined target sequences or to a sequence of the at least one reference sequences.

23. The specific oligonucleotide probe of claim 22, wherein the at least one probe comprises a detectable label, preferably a fluorophore or one hapten of a specific hapten pair.

24. The specific oligonucleotide probe of claim 22, wherein the probe is a cleavable probe comprising respectively a 3’-sequence which is reverse complementary to a sequence of the at least one predetermined target sequences within a region being located 3'-downstream of a complementary sequence of an at least one primer, a protective group (3'-blocker) at the 3’-end of the analyte specific 3’-sequence inhibiting a primer extension reaction, and at least one internal nuclease blocker at the 5'-region of the hybridizing sequence of the probe conferring resistance to a nuclease activity and structurally dividing the cleavable hydrolysis product from the 3'-downstream cleavable probe and wherein the cleavable hydrolysis product comprises

• at least one nucleotide of the 5'-end of the cleavable probe, and

25. A combination of probes according to any one of the preceding claims, wherein each probe is specific for the reference sequence encoding within the location of gene ZRANB2 and at least one reference sequence within the location of gene ZRANB2-AS1 on chromosome 1 , the reference sequence encoding within the location of gene TMEM169 on chromosome 2, the reference sequence encoding within the location of gene HCAR1 on chromosome 12, the target sequences encoding within the location of genes COX10, ARHGAP44, ELAC2 and MYOCD respectively in the cytoband 17p12, and the target sequences encoding within the location of genes ZNF555, TLE6, ZNF57 and ZNF77, respectively in the cytoband 19p13.3.

26. A kit for the detection of an endometrium carcinoma of a molecular subtype copy number high (CN high), wherein at least one copy number variation (CNV) is detected for a CNV chromosomal region comprising at least one cytoband on chromosome 17 and/or chromosome 19, comprising at least one suitable primer or primer pair for the amplification of one or more target sequences within the CNV chromosomal region, and optionally at least one suitable primer or primer pair for the amplification of at least one reference sequence in the chromosomal cytobands 1p32.3, 1p32.2, 1p32.1, 1p31.3, 1p31.2, 1p31.1, 1p22.3, 1p22.2, 1p22.1, 1p21.3, 1p21.2 and/or 1 p21.1 on chromosome 1 and/or 2q32.1, 2q32.2, 2q32.3, 2q33.1 , 2q33.2, 2q33.3, 2q34, 2q35, 2q36.1 , 2q36.2, 2q36.3 and/or 2q37.1 on chromosome 2 and/or 12q24.13, 12q24.21 , 12q24.22, 12q24.23, 12q24.31 , 12q24.32 and/or 12q24.33 on chromosome 12, respectively.

27. The kit according to claim 26, further comprising at least one probe or combination of probes according to any one of the claims 21 or 22 to 25.

28. A combination of amplicons, achieved by the method of any of the proceedings claims, wherein the amplicons have the sequences of Seq ID No. 43, Seq ID No. 44, Seq ID No. 45, Seq ID No. 46, Seq ID No. 47, Seq ID No. 48, Seq ID No. 49, Seq ID No. 50, Seq ID No. 51, Seq ID No. 52, Seq ID No. 53, Seq ID No. 54, Seq ID No. 55 and Seq ID No. 56.