Methods for detecting chromosome aberrations

Abstract

The present invention relates to a method for identifying chromosome aberrations, in particular chromosome structural and / or numerical aberrations, and preferably chromosome structural aberrations, by detecting chromosomes and / or DNA regions in a biological sample, preferably in one or more cells and / or in one or more cell nuclei, using in situ hybridization.

Description

[0001] This application is a divisional application of Chinese patent application 201680034524.X, whose application date is June 23, 2016, whose applicant is Zito Vision Co., Ltd., and whose invention name is “Method for detecting chromosome aberrations”. Technical Field

[0002] The present invention relates to the technical field of methods for detecting chromosome anomalies or chromosome aberrations.

[0003] Specifically, the present invention relates to a method for detecting chromosome aberrations by in situ hybridization. In addition, the present invention relates to a composition suitable for detecting chromosome aberrations and its use according to the present invention. Another object of the present invention is to provide the use of a site-specific hybridization probe marked with a detection marker. Finally, an object of the present invention is to provide a kit for detecting chromosome aberrations. Background Art

[0004] Many neoplastic diseases are based on chromosome structural and numerical variations, such as translocations, inversions, segmental duplications, deletions, insertions, duplications, aneuploidies, and amplifications. Detection of these changes as predictive, prognostic, or differential diagnostic indicators is usually performed by in situ hybridization (ISH).

[0005] In situ hybridization is based on the hybridization or pairing of complementary bases of nucleic acid single strands, especially DNA single strands, so that specific nucleic acid sequences in samples, especially tissues or cell preparations, can be detected. For this purpose, synthetically produced probes that are directly or indirectly labeled are hybridized to nucleic acid single strands of the sample and then detected.

[0006] For detection purposes, fluorescently labeled nucleic acid fragments or fluorescently labeled hybridization probes can be used (fluorescent ISH (FISH)). In addition, antigen-labeled probes, in particular hapten-labeled probes, can be used, which are then visualized by a color reaction using antibodies, so that light microscopy analysis can be performed (bright field ISH (BrISH)), chromogenic ISH (CISH), silver ISH (SISH)).

[0007] The advantage of FISH is that multiple genomic regions can be detected simultaneously, so that distinguish from each other.For this purpose, for different genomic regions or to its special nucleic acid fragment, be marked or coupled by different fluorescent dyes, in each case, every kind of fluorescent dye is different from each other in adsorption spectrum and / or emission spectrum.If this multicolor probe comprising the different single probes of separation is used for metaphase chromosome preparation or interphase cell nuclear preparation, each color can be described respectively by using specific microscope filter, it will import the light wavelength range of accurate definition into preparation to excite dye, and will also import the light wavelength range of accurate definition emitted by dye into evaluator (being called single bandpass filter group).In addition, also exist and allow to describe different fluorescent dyes simultaneously and the filter and filter group of describing multiple nucleic acid fragments thus.In the case of two different fluorescent dyes, for example, a kind of is called dual-bandpass filter group.

[0008] However, since only one label can be used for each genomic region detected by each specific probe, clear limitations are set for simultaneous description. In addition, the absorption and emission ranges of dyes are usually close to each other, so that they can not be separated from each other by a microscope filter set. For these reasons, usually only two colors (orange / red and green) or three colors (orange / red and green at the same time or with blue nuclear countercolor (DAPI)) are analyzed simultaneously in FISH. The roughly the same limitations that can be described with FISH are also applicable to BrISH. Here, the state of the art is to use two kinds of haptens that are usually selected from biotin, dinitrobenzene (DNP) and digoxin groups, and two kinds of antibody-coupled enzymes, usually alkaline phosphatase and peroxidase.

[0009] The limitations of the simultaneous description or analysis have a decisive influence on the composition (composition) or coordination of the probes used to detect chromosome structural and numerical aberrations, which can be used to diagnose tumors whose cells are in the interphase, only using so-called site-specific probes, where sometimes so-called repeat sequence probes are also used to detect chromosome numerical mutations.

[0010] Site-specific probes are considered to be probes that process selected DNA fragments in chromosomes, and are referred to as gene-specific probes or "single copy" probes, and the DNA fragments are usually single genes or adjacent genes, with a total size of up to about 1000 kb. Repeat-specific probes are probes that process repetitive sequences and therefore process regions of multiple 1000 kb in size. For example, these probes also include centromere probes or α-satellite probes.

[0011] Regarding the detection of translocation and inversion, there are basically two decisive technologies and potential probe compositions or probe compositions: on the one hand, the occurrence of fusion signals (called dual-color dual fusion method) (WO 02 / 093130A2) and on the other hand the principle of separation of fusion signals (called dual-color-break-separation or dual-color separation method). In the representation and derived signal patterns of the following two principles, it must be noted that normal cells are usually diploid, that is, each allele is repeated. Since usually only one of the two alleles is affected by the abnormality, in various cases, the normal signal of the allele that is not affected by the abnormality is also visible together with the abnormal signal. For better understanding, the signal image of the normal signal will not always be clearly described below.

[0012] In the two-color two-fusion method, the region of the first breakpoint of the chromosome is located on the proximal and distal sides of the nucleic acid fragment of the same color (e.g., orange), and the region of the second breakpoint, i.e., the reciprocal translocation partner, is located on the proximal and distal sides of the nucleic acid fragment of the second color (e.g., green). In this regard, the normal situation, i.e., the absence of chromosome breaks in the regions of the two translocation partners, is characterized by green and spatially separated orange signals.

[0013] In the case of mutual translocation, the break occurs in the breaking point region of two translocation partners, and the proximal region of a translocation partner merges with the distal region of another translocation partner, and vice versa. Because different colored signals often overlap, two green / orange signal pairs (also referred to as fusion signals) occur. The shortcoming of this probe technology is that fusion signals only occur when the breaking point of two translocation partners is located in the region of the nucleic acid fragment of the corresponding mark. In the case where translocation only involves one of two partners, fusion signals will not occur. In this regard, only additional signals are formed, which have the color of the signal of the characteristic of the partner affected by translocation. This means that if the breaking point of translocation is located in the region covered by nucleic acid labeled with green fluorescent dye, additional green signals will occur. The shortcoming of this probe composition is to use only when two breaking point regions of identical translocation or inversion are labeled with two colors, so only specific translocation or inversion can be detected.

[0014] In the case of the two-color-break-separation method, the region of the breakpoint is located on the proximal and distal sides of the differently labeled or colored nucleic acid fragments (e.g., distal orange, proximal green). In this regard, the normal situation, i.e., the absence of chromosome breaks in this region, is characterized by a fusion signal. In abnormal situations, i.e., if chromosome breaks occur between the probe fragments, the signals are almost impossible to separate from each other spatially. Therefore, the difference between the normal situation and the abnormal situation is characterized by the distance between the different colored signals. Statements about the involvement of translocation partners are not suitable for this method. It only allows conclusions on the occurrence of specific chromosome rearrangements. The disadvantage of this probe composition is that only a single breakpoint region is used, and therefore only two colors are used to detect a specific translocation or inversion.

[0015] As far as deletions, aneuploidies and amplifications are concerned, usually only one decisive technique is used with locus-specific probes and potential probe compositions. However, in some cases, locus-specific probes are also supplemented with so-called repeat-specific probes, such as centromere probes or alpha satellite probes: The principle of detecting the gain or loss of signal resulting from the occurrence of deletions, aneuploidies and amplifications is usually carried out with the so-called two-color probe method. In this principle, as well as in the principles to be described below and the signal images derived therefrom, it must be noted that normal cells are usually diploid, i.e. each allele is present in duplicate.

[0016] In the dual-color probe method, two different chromosomal regions are labeled with differently labeled or colored nucleic acid fragments (e.g., genomic region 1 or target region 1 is colored orange, and genomic region 2 or target region 2 is colored green). Normal conditions, i.e., no gain or loss of these regions, are characterized by two orange and two green signals. In abnormal conditions, i.e., when gain or loss of genomic regions in target regions 1 and / or 2 occurs, fewer green and / or orange signals can be seen in the case of loss, and more signals can be seen in the case of gain. In the case of strong gene amplification or genomic region amplification, many additional signals can be seen, which can also be depicted as clusters.

[0017] Therefore, using the above-mentioned standard methods and standard compositions and using two standard signals or two standard colors, each method can only detect one (possible) abnormality in the case of structural variations and a maximum of two (possible) abnormalities in the case of chromosome number variations.

[0018] In addition to the two two-color applications listed above, three-color, rarely four-color, and even fewer five-color probes are also used for FISH analysis. According to definition, the method of using at least three different ligands or fluorescent dyes (without countercolor) to label probes is a multicolor FISH method (mFISH method). In this regard, not only the significantly stronger standard fluorescent colors orange / red and green are used, but also available weaker colors such as gold or gold / yellow or gold, red or blue are used. Therefore, usually, for example, four-color FISH probes are only used for the detection of deletion or amplification, because here, it is usually possible to lag behind the repetitive sequence for amplifying the color intensity of blue and gold, for example, in the case of the labeling of probes. These methods are described in EP 1 035 215 B1, WO 2007 / 028031A1 and EP 0 549 709B1, etc.

[0019] In addition, in the prior art, triple FISH methods are described, which are used to address the detection of different translocation events, which can cluster with each other in a chromosome region (i.e. involve different genes, which are located near each other). In this regard, in each case, only two colors are analyzed for the evaluation of the corresponding signal pattern, wherein a single abnormality is detected in each case, and the third color does not work. In each case, three different site-specific probes are used, which are labeled with different markers.

[0020] As far as bright field (BrISH) using more than two colors is concerned, in the prior art in this respect, it is described to perform chromogenic triple in situ hybridization, which, in general, aims at the detection of three repeated chromosome regions. According to the current state of the art, translocations can only be detected by BrISH when two haptens and thus two dyes are used. In patent application WO2012 / 150022 A1, a method is described, which discloses the use of three different probes by the BrISH method, using three labels biotin, digoxigenin and DNP, which result in three different colors, for detecting inversions.

[0021] WO 2005 / 111235 A2 describes a method comprising using three colors for detection purposes. However, the chromosome region marked by the third marker of the probe is not directly affected by the change, so in the case of a change in chromosome structure, the first fusion signal is eliminated, so that a new split signal and a new fusion signal occur. The method uses probes that are each marked with only one marker. In addition, by this method, only a possible translocation predetermined by the probe can be detected.

[0022] WO 02 / 093130 A3 discloses a method for detecting chromosome translocations using two or more four labels or dyes flanking distally and proximally of the two breakpoints involved in the translocation. The method does not have the possibility to detect more than one translocation in the breakpoint region flanked by probes.

[0023] In summary, it can therefore be seen that with the methods known from the prior art it is not possible to effectively detect multiple chromosomal aberrations present in a cell or tissue simultaneously or synchronously by in situ hybridization.

[0024] It is currently known that only in the case of methods for detecting whole chromosomes, such as chromosome arms, with a size of about 50 Mbp to 250 Mbp or larger chromosome regions in humans, the so-called "whole chromosome painting probes" (WCP) or "partial chromosome painting probes" (PCP) are used to simultaneously assign (simultaneous assignment) or assign (assignment at the same time) defined DNA or chromosome regions within the scope of in situ hybridization. Using basic techniques such as mFISH (multiplex FISH), SKY-FISH (spectral karyotyping), multicolor FISH, COBRA-FISH (combined binary ratio labeling FISH) or 24-color FISH, about four to seven different fluorescent dyes can be used to mark and distinguish a total of twenty-four different "chromosome painting probes". In a similar method, the chromosome-arm-specific probes of all chromosomes can be marked in different ways, the so-called 42-color FISH. However, the above method is only applicable to cells in metaphase. The above method is possible because only the genome / chromosome regions in metaphase can be evaluated with the probes used therein, at least essentially the chromosome material does not overlap. The analysis of interphase cells, which is usually required for the analysis of the genetic material of solid tumors, is not possible using this method. Furthermore, since they involve large areas, the probes used in this respect can be detected relatively easily. The evaluation of these analyses cannot be carried out manually, i.e. by observing the signals on a fluorescence microscope, but only in a computer-based manner, using suitable evaluation software.

[0025] Related to chromosome structural and numerical mutations or abnormalities, in particular, methods and probe compositions known in the prior art, methods and probe compositions for BrISH and FISH related to tumor or cancer diseases, are associated with certain disadvantages. For example, there are no site-specific probe and method compositions that allow reliable, simple and rapid detection and discrimination of a variety of potentially different chromosome structural and / or numerical mutations or abnormalities.

[0026] Particularly in the case of chromosomal structural variants, the simultaneous analysis or analysis at the same time of multiple different chromosomal mutations that are independent of one another, i.e. without any exchange of chromosomal material or with one another, is completely impossible or can only be performed with extreme difficulty, in particular in the case of basic fragmentation-segregation methods.

[0027] Therefore, the present invention is based on the task of providing a method or composition suitable for the detection and analysis of chromosome mutations or chromosome aberrations, which avoids the disadvantages of the prior art as described above, at least avoids them to a large extent or at least mitigates them.

[0028] In particular, the invention is based on the task of providing a method which makes it possible to reliably and simultaneously detect a plurality of chromosomal aberrations which are different from one another, in particular chromosomal aberrations which are independent of one another (i.e. not correlated with one another), in particular in one method. In the same way, the invention is based on the task of providing a method which, in addition, makes it possible to assign chromosomal aberrations to specific chromosomal regions or DNA regions. Summary of the invention

[0029] In order to achieve the above-mentioned object, the invention proposes a method according to claim 1; in this respect, further advantageous embodiments are the object of the dependent claims.

[0030] Furthermore, the object of the present invention is to provide a composition for detecting chromosomal aberrations according to the independent claims in this regard, or a composition for the preventive or therapeutic treatment or diagnosis or prognosis of a disease associated with chromosomal aberrations.

[0031] Another object of the present invention is to provide a use of the composition of the present invention according to the independent claim in this aspect.

[0032] Furthermore, according to the independent claim in this respect, the invention relates to the use of at least two, in particular at least three, preferably at least four site-specific hybridization probes, which probes are different from each other.

[0033] Furthermore, according to the independent claim in this respect, it is an object of the present invention to provide the use of at least one site-specific hybridization probe labeled with at least two detection labels.

[0034] Finally, it is an object of the present invention to provide a kit or a kit component or a kit for detecting chromosomal aberrations; furthermore, advantageous properties are the object of the dependent claims in this regard.

[0035] It should be understood that in the following, specific configurations, embodiments, etc. described in conjunction with only one aspect of the present invention may also be applied similarly with reference to other aspects of the present invention without explicit mention.

[0036] Furthermore, it must be noted that for all relevant or percentage quantity information, in particular weight-related quantity information, the person skilled in the art selects this information within the scope of the present invention in such a way that the total amount of the individual ingredients, active substances, additives or auxiliary substances etc. always amounts to 100% or 100 wt-%. However, this is obvious to the person skilled in the art.

[0037] Furthermore, a person skilled in the art may deviate from the following numerical values, ranges or quantity information depending on the application or individual circumstances without departing from the scope of the present invention.

[0038] Furthermore, the present invention is equally applicable to the use of standardized or explicitly indicated determination methods, or alternatively, the use of determination methods well known to those skilled in the art, which can fundamentally determine or establish all parameter information shown below, etc.

[0039] In order to accomplish the above-mentioned task, according to a first aspect of the present invention, the present invention proposes a method for detecting chromosome aberrations, in particular chromosome structural and / or numerical aberrations, preferably chromosome structural aberrations, by in situ hybridization and by detecting chromosome regions and / or DNA regions in a biological sample, preferably in one or more cells and / or one or more cell nuclei (cell nucleus / nuclei), wherein the in situ hybridization is performed in the form of interphase / in situ hybridization, wherein the in situ hybridization is performed with at least four mutually different site-specific hybridization probes, each probe being labeled with a first detection marker, wherein in particular for generating at least one mixed signal, at least one of the site-specific hybridization probes is labeled with at least one additional detection marker, the additional detection marker being different from the first detection marker, a signal image being generated based on the corresponding site-specific hybridization probes, and wherein the signal image is used to identify the existing chromosome aberrations and / or assign them to chromosome regions and / or DNA regions.

[0040] In other words, the present invention is based on the basic principle of targeted generation of mixed signals in the signal image generated by interphase / in situ hybridization, allowing simultaneous detection or detection at the same time of multiple chromosomal aberrations and their assignment to the detected chromosomal regions or DNA regions, wherein the multiple chromosomal aberrations are different from each other in the biological sample, in particular independent of each other, i.e. not chromosomal aberrations of each other.

[0041] Therefore, in particular, within the scope of the present invention, the chromosomal aberrations are chromosomal aberrations that are independent of each other. That is, according to the present invention, it can be provided that the chromosomal aberrations are independent of each other. Likewise, it can be provided that the chromosomal aberrations are not mutual.

[0042] According to a specific embodiment of the present invention, it is further preferred that the chromosomal aberration is not connected or associated with mutual or interdependent chromosomal aberrations.

[0043] The present invention is associated in a non-limiting manner with the various advantages and features discussed below and should be evaluated as indicative of the patentability of the invention.

[0044] In the scope of the present invention, a method for detecting chromosome aberrations can be provided in a completely surprising manner, within the scope of interphase / in situ hybridization, which allows to clearly detect multiple possible chromosome structures or number aberrations on the one hand, and on the other hand, in a single hybridization method, it allows to clearly distinguish these chromosome aberrations or to clearly distribute the detected chromosome aberrations to specific chromosome regions or DNA regions. In particular, the chromosome aberrations can be chromosome aberrations that are independent of each other, and they are not interdependent and are not mutual. This has been impossible so far in the prior art, particularly within the scope of interphase / in situ hybridization.

[0045] Thus, using the method according to the invention, samples, in particular biological samples to be detected for chromosomal aberrations, such as tissue fragments, in particular tumor tissues, can be analyzed in a significantly faster and more efficient manner, because a single sample can be tested simultaneously, in other words, in one method, multiple chromosomal aberrations that are different from each other can be tested simultaneously. In addition, any chromosomal aberration detected can be assigned to a defined or specific DNA region or chromosomal region.

[0046] In addition, using the method of the present invention, the amount of sample required for detecting chromosome aberrations can be significantly reduced. This is particularly advantageous for removing the background of tissue for examination purposes, especially in connection with the diagnosis or identification or further analysis of cancer diseases, which is usually performed by fine needle aspiration biopsy, while only a limited amount of sample is allowed to be taken, while open biopsies, which also require the use of larger amounts of tissue, are also being performed less and less.

[0047] Furthermore, due to the high efficiency of the method of the present invention, the amount of materials required for in situ hybridization, such as enzymes, fluorescent dyes, etc. (some of which may be cost-intensive) is reduced, so the method is also economically and ecologically advantageous.

[0048] In order to better understand the present invention, the core terms and names of the method according to the present invention are defined as follows:

[0049] According to the present invention, the term chromosome aberrations, also referred to synonymously as chromosome anomalies, is particularly understood to mean chromosome structural and numerical aberrations. In the case of chromosome structural aberrations, there are changes in the structure of the chromosome, which are therefore also referred to as chromosome mutations. In particular, this may involve inversions, translocations, deletions, segmental duplications, insertions, repetitions or amplifications. On the contrary, chromosome number aberrations lead to changes in chromosome quantity. Synonymously, the term genome mutation is used. In the case of chromosome number aberrations or genome mutations, these may particularly relate to aneuploidy or polyploidy. The method according to the present invention is particularly suitable for chromosome structural aberration detection.

[0050] The in situ hybridization used according to the invention is based on the hybridization or pairing of complementary bases of nucleic acid single strands, in particular DNA single strands, making it possible to detect specific nucleic acid sequences in samples such as tissue or cell preparations. Within the scope of in situ hybridization, a synthetically produced, in particular site-specific hybridization probe, directly or indirectly labeled, hybridizes with a nucleic acid single strand of the sample and is subsequently detected.

[0051] Basically, in situ hybridization can occur or be performed at different stages of the cell cycle of the cell or cell nucleus to be tested, and this process is performed in metaphase when the chromosomes are in a condensed state, or in interphase when the chromosomes are in a condensed state, it has been established. Depending on the goal or purpose of the in situ hybridization, it is not always possible to perform it on condensed chromosomes in metaphase, especially in detecting chromosomal aberrations in solid tumor cells. According to the present invention, it is therefore provided to perform in situ hybridization on cells or cell nuclei in interphase.

[0052] In the scope of the present invention, site-specific hybridization probe is understood to be a probe complementary to genetic material in a certain chromosome region or DNA region or DNA region of a specific chromosome region or DNA material or a sample to be tested. Usually, the hybridization probe used according to the present invention is based on nucleic acid or nucleic acid fragment, and can be specifically combined or hybridized with a chromosome region to be tested or a DNA region. The chromosome region to be tested or the DNA region can have a variable length. In particular, the chromosome region to be tested or the DNA region can be provided to contain all or part of a single (single) or single (individual) gene. Similarly, the chromosome region to be tested or the DNA region can also be provided to contain all or part of a plurality of genes, preferably adjacent genes, preferably two genes.

[0053] As for the configuration of the hybridization probe, according to the present invention, in particular, it can be provided that the site-specific hybridization probe is based on multiple, in particular multiple nucleic acid fragments (synonymically also probe fragments), all of which are referred to as site-specific hybridization probes. In addition, although less preferred, it is possible that the site-specific hybridization probe is based on only a single nucleic acid fragment or is formed by a single nucleic acid fragment.

[0054] Within the scope of the present invention, detection label refers to the material or substance that is coupled to nucleic acid or nucleic acid fragment for determination or detection purpose. The selection of suitable detection label is within the basic ability of those skilled in the art, and no further explanation is required at this point. The nucleic acid fragments marked with detection label, combined or hybridized with the DNA fragment or chromosome fragment to be determined or detected by in situ hybridization, can be detected by the method of detection label suitable for direct or indirect use known to those skilled in the art, for example, by fluorescence microscopy, or especially after enzyme reaction or visualization by enzyme reaction dye substrate, by bright field microscopy. In particular, within the scope of in situ hybridization, the detection label at the site-specific hybridization probe produces a signal image, which serves as the basis for checking the possible chromosome aberration of the sample.

[0055] Furthermore, the term "detection marker" as used according to the present invention hereinafter relates to the kind or type of detection marker, and not to the number of detection marker molecules, i.e., such as "at least one detection marker" refers to a certain type of detection marker or a specific choice of detection markers. Thus, the term "multiple detection markers" also relates to the selection of different types of detection markers, which are different from each other, and not to the number of detection marker molecules used. It is obvious to the person skilled in the art that within the scope of labeled hybridization probes, these are usually coupled to more than one detection marker molecule.

[0056] Therefore, the site-specific hybridization probes used according to the invention, in particular probe fragments or nucleic acid fragments, specifically hybridize with selected DNA regions or chromosomal regions of the genetic material in the sample and, based on the coupled detection label, generate a signal image within the scope of in situ hybridization. Within the scope of the present invention, based on site-specific hybridization probes labeled with detection labels, the signal image is understood to be the sum of all signals generated by in situ hybridization.

[0057] In this regard, it is surprisingly found that within the scope of the present invention, a good detectable mixed signal can be produced by marking the site-specific hybridization probe with at least two different detection labels in the signal image, which can be well distinguished from other signals, and the signal allows the chromosome aberration that occurs in the tested DNA region or chromosome region to be distributed. Therefore, a mixed signal in the sense of the present invention is a signal produced by at least two and multiple detection labels that are different from each other and are located on the site-specific hybridization probe. Since the mixed signal is produced by at least two, particularly multiple detection labels of the site-specific hybridization probe, even in the case of chromosome aberration, these are also visible in the signal image of in situ hybridization, and even continue to exist in the case of chromosome aberration. The possible embodiments or configurations of the site-specific hybridization probe for producing the mixed signal will be described in detail below.

[0058] The preferred embodiments of the method according to the present invention are described in detail below:

[0059] According to a first embodiment of the invention, it can be provided that the first detection label of the site-specific hybridization probes used is identical in each case. According to a second, equally preferred embodiment of the invention, it can be provided that the site-specific hybridization probes used are labeled with first detection labels that are different from each other in each case.

[0060] In other words, according to the invention it can be provided that the site-specific hybridization probes used - simply as an example and not limitation - have the same fluorescent dye or the same hapten, for example as first detection label.

[0061] Likewise, the site-specific hybridization probes used may have as first detection labels different fluorescent dyes from one another, different haptens from one another, etc., in each case again as an example and not as a limitation. Labeling with different first detection labels has proven to be advantageous, in particular for the detection of chromosomal aberrations following chromosomal breaks (e.g. translocations or inversions).

[0062] As for the detection of chromosome aberrations, according to the present invention, further according to the present invention, preferably at least one, in particular a plurality of chromosome aberrations that are different from each other, are detected and / or determined from a plurality of possible chromosome aberrations in a sample.

[0063] Likewise, it can be provided that among a plurality of possible chromosomal aberrations, in particular at least two, in particular a plurality of chromosomal aberrations that are different from one another are detected in the sample simultaneously.

[0064] According to another preferred embodiment of the present invention, the method according to the present invention can be provided as a multiplex method for simultaneously detecting a plurality of mutually different chromosomal aberrations.

[0065] Therefore, a particular advantage of the method according to the invention compared to the methods known from the prior art for detecting chromosomal aberrations by in situ hybridization - already explained above - lies in the fact that now, in particular based on samples of interphase cells or cell nuclei, it is possible to simultaneously or simultaneously detect a plurality of possible chromosomal aberrations in a single hybridization method and to assign these abnormalities to specific DNA regions or chromosomal regions.

[0066] In this regard, in particular, chromosome aberrations can be identified in the signal image by at least one mixed signal, in particular a plurality of mixed signals, and assigned to the chromosome region and / or DNA region to be detected. Figure 7 , in which the detection of chromosomal aberrations in amplified form according to the invention can be seen. Figure 7 , four different chromosomal regions were examined using four hybridization probes that were different from one another, three of which were labeled with at least one additional detection marker for generating a specific hybrid signal (see Figure 7 a). On the basis of three markers in four chromosomal regions with a specific mixed signal, in each case the "clusters" generated by amplification of the detected chromosomal region in the signal image can be assigned to the hybridization probe or the detected chromosomal region (see Figure 7 b).

[0067] According to the invention, it can therefore be provided in particular that further site-specific hybridization probes are labeled with at least one further detection marker in such a way that the site-specific hybridization probes labeled with the at least one further detection marker generate mixed signals in the signal image that differ from one another in each case.

[0068] Likewise, it can be provided that - in particular for the detection of chromosomal aberrations which are not caused by chromosome breaks (e.g. amplifications or deletions) - further site-specific hybridization probes are labeled with at least one further detection marker in such a way that a mixed signal for a chromosomal region and / or a DNA region is generated in the signal image by each site-specific hybridization probe labeled with at least one further detection marker. Thus, within the scope of the present invention, it is preferred that the individual regions detected by the site-specific hybridization probes can be assigned to a specific signal, in particular a mixed signal, within the signal image.

[0069] Furthermore, according to the invention, preferably, if the additional site-specific hybridization probes are labeled with at least one additional detection label, a specific mixed signal is used to assign each chromosomal aberration to be detected in the signal image to the detected chromosomal region and / or DNA region.

[0070] As regards the number of site-specific hybridization probes which are labeled with at least one additional detection marker, this number is variable and depends in particular on the number of chromosomal aberrations to be detected or examined:

[0071] According to the present invention, it is preferred that at least two, in particular at least three, preferably at least four, preferably at least five, particularly preferably at least six, and very particularly preferably at least seven additional site-specific hybridization probes are labeled with at least one detection marker that is different from the first detection marker. Similarly, by at least two, in particular at least three, preferably at least four, preferably at least five, particularly preferably at least six, and very particularly preferably at least seven additional site-specific hybridization probes, a mixed signal for a chromosome region and / or a DNA region can be provided in the signal image.

[0072] The method of the above process is particularly suitable for detecting chromosomal aberrations (such as amplifications or deletions) that are not caused by chromosome breakage. Based on the increase in the number of hybridization probes labeled with at least one additional detection marker, preferably multiple detection markers, the probes generate a single mixed signal, and in each case, the number of chromosomal aberrations to be detected or determined at the same time will also increase.

[0073] According to another specific embodiment of the present invention, chromosomal aberrations caused by chromosomal breaks (such as translocations or inversions) can also be detected and assigned to specific chromosomal regions or DNA regions:

[0074] According to this embodiment of the present invention, two site-specific hybridization probes can be provided, each of which is located on the side of the chromosome fragment (particularly the breakpoint region), wherein the site-specific hybridization probes located on the side of the chromosome fragment (particularly the breakpoint region) are labeled with detection markers different from each other, so that a fusion signal is generated in the signal image in each case by the site-specific hybridization probes located on the side of the chromosome fragment (particularly the breakpoint region), particularly in the absence of chromosome aberrations.

[0075] In this regard, "flank" preferably refers to the hybridization of the specific end of the hybridization probe closest to the chromosome fragment or breakpoint region to a base at a distance of 0 to 1 Mbp from the chromosome fragment or breakpoint region, in particular a distance of 0 to 500 kb, preferably a distance of 0 to 100 kb, preferably a distance of 0 to 10,000 bp, and particularly preferably a distance of 0 to 1,000 bp.

[0076] In other words, according to this embodiment of the present invention, it is provided that the DNA region or chromosome region to be detected is located at the distal end and the proximal end of the chromosome relative to a specific selected chromosome segment, in particular the potential breakpoint region. In this regard, it can be provided in particular that at least one additional hybridization probe labeled with a detection marker different from the first detection marker is used to detect the chromosome segment located at the distal end or at the proximal end.

[0077] In the scope of the present invention, the breakpoint region is understood to be a chromosomal region that may be affected by chromosomal breakage. As a result of chromosomal breakage, chromosomal aberrations based on structural rearrangements, particularly translocations or inversions, may occur. Chromosomal aberrations are known to many diseases, wherein in each case, the abnormality is disease-specific and based on chromosomal breakage, particularly translocations or inversions. Using the method according to the present invention, multiple, particularly known breakpoint regions can be examined in the genetic material of a sample, particularly a tissue sample to obtain the presence of chromosomal aberrations.

[0078] In this embodiment according to the invention, a fusion signal is therefore generated by the two site-specific hybridization probes flanking the breakpoint region, the two detection labels based on the first and second site-specific hybridization probes being in each case different from one another, in particular in the absence of chromosomal aberrations.

[0079] In contrast to the mixed signals generated by different detection labels of site-specific hybridization probes already described above, the fusion signal is generated by site-specific hybridization probes, which are different from each other and which are present in close proximity to each other, hybridizing to genetic material or DNA in the sample.

[0080] In other words, within the scope of the method according to the invention, a fusion signal is generated, in the case where there are no chromosomal aberrations at the flanking breakpoints, the site-specific hybridization probe used hybridizes in the immediate vicinity with the genetic material or DNA in the sample. In contrast, if there are chromosomal aberrations in this region, the site-specific hybridization probe used is no longer able to bind in the immediate vicinity due to the structural rearrangement of the DNA fragments. Instead of a fusion signal, two separate signals (synonymous "split signals") that are preferably different from each other are detected in the signal pattern.

[0081] In addition, hybridization probes labeled with at least one additional detection marker different from the first detection marker generate mixed signals based on the first detection marker and at least one additional detection marker of the corresponding hybridization probe. Therefore, in the absence of chromosomal aberrations, these can form mixed signals and fusion signals in the signal map with the second hybridization probes on the sides of the chromosome fragments, especially the breakpoint region. In the case of chromosomal aberrations, on the contrary, based on the chromosome fragments in normal cells or normal cell nuclei, especially the hybridization probes on the sides of the breakpoint region, one of which is labeled with at least one additional detection marker, a single signal and a single signal accompanied by a mixed signal are generated in the signal image.

[0082] Within the scope of the present invention, the chromosomal aberration defined by the mixed signal can therefore be assigned to the breakpoint region or the detected chromosomal region or DNA region based on the site-specific hybridization probe labeled with at least one further detection label.

[0083] Therefore, according to the invention, it is preferred that in each case the hybridization probes labeled with at least one further detection marker which is different from the first detection marker together with the second site-specific hybridization probes flanking the chromosome fragment, in particular the breakpoint region, generate mixed signals and fusion signals in the signal image, in particular in the absence of chromosome aberrations.

[0084] Furthermore, it can be provided that the hybridization probe labeled with at least one further detection marker different from the first detection marker and the second site-specific hybridization probe flanking the chromosome fragment, in particular the breakpoint region, generate a single signal in the signal image in each case. In particular, the hybridization probe labeled with at least one further detection marker also generates a mixed signal in the signal image, in particular in the presence of chromosome aberrations.

[0085] In particular, within the scope of the method according to the invention it can be provided that in the signal image a chromosomal aberration is assigned to a detected chromosomal region and / or DNA region and / or chromosomal fragment, in particular a breakpoint region, by means of a mixed signal.

[0086] According to this preferred embodiment of the method of the invention, a series of potential breakpoint regions in a single sample can therefore be simultaneously depicted or marked by interphase / in situ hybridization. In the case where no chromosomal aberration occurs at the marked breakpoint, a fusion signal is generated in the signal image, while the presence of a single signal or a separate signal indicates the presence of a chromosomal aberration. Based on the additionally generated mixed signal, the abnormal single signal or separate signal can be assigned to a specific hybridization probe and thus to a specific breakpoint region (see Figure 1 ).

[0087] In particular, within the scope of the present invention, it is provided that at least six, preferably at least eight, preferably at least ten, particularly preferably at least twelve, even more preferably at least fourteen different site-specific hybridization probes are used, wherein in each case two site-specific hybridization probes flank the chromosome fragment, in particular the breakpoint region, and that up to twenty-four different site-specific hybridization probes are used, wherein in each case two site-specific hybridization probes flank the chromosome fragment, in particular the breakpoint region.

[0088] In addition, according to the present invention, preferably, if an additional site-specific hybridization probe is labeled with at least one additional detection marker different from the first detection marker, the chromosome fragments on each side, in particular the breakpoint region and / or each chromosome region and / or DNA region to be detected can be identified and / or assigned in the signal image using fusion signals and mixed signals.

[0089] In this respect, it is particularly preferred according to the invention that if in each case a first breakpoint region to be detected is flanked by two hybridization probes, which have only one detection label, this breakpoint region is only visible in the signal image by a fused signal or two individual signals or separate signals. In contrast, by labeling at least one of the two flanking hybridization probes with at least one further detection label in the signal image, a specific or separate mixed signal is assigned to each further breakpoint region to be detected, so that in total a plurality of chromosome regions or DNA regions, in particular breakpoint regions, can be depicted in a distinguishable manner in the signal image.

[0090] According to a particularly preferred embodiment of the method according to the invention, it can be provided that

[0091] (a) a first site-specific hybridization probe labeled with a detection marker A and a second site-specific hybridization probe labeled with a detection marker B are located on the side of a first chromosome fragment (particularly a first breakpoint region) and generate a fusion signal AB in a signal image generated by in situ hybridization,

[0092] (b) 2 to 12 additional site-specific hybridization probes are located on the sides of up to six additional chromosome fragments, in particular the breakpoint region, wherein in each case one of the two site-specific hybridization probes located on the sides of the chromosome fragment (in particular the breakpoint region) is labeled with a detection label A, and in each case one of the two site-specific hybridization probes located on the sides of the chromosome fragment (in particular the breakpoint region) is labeled with a detection label B, such that the site-specific hybridization probes located on the sides of the chromosome fragment (in particular the breakpoint region) generate a fusion signal AB in the signal image generated by in situ hybridization, and

[0093] (c) at least one, preferably a plurality of site-specific hybridization probes are labeled with at least one additional detection label X, so that the site-specific hybridization probes labeled with at least one additional detection label produce a fusion signal and a mixed signal AB / X in the signal image generated by in situ hybridization,

[0094] wherein the fusion signal and the mixed signal AB / X generated by in situ hybridization become mixed signals A / X and / or B / X in the case of chromosome aberration, and / or

[0095] In the case of chromosomal aberration, in the signal image generated by in situ hybridization, the fusion signal AB is transformed into a single signal A and / or B, so that using the signal image generated by in situ hybridization, the chromosomal aberration is assigned to the chromosomal region and / or DNA region and / or chromosomal fragment, in particular the breakpoint region, which is flanked by two site-specific hybridization probes.

[0096] In addition, the detection mark X provided in the above embodiment can be formed by a single detection mark, especially the detection mark X1.

[0097] Furthermore, it can be provided that the detection marker X is formed of a plurality of detection markers that are different from each other, preferably selected from the detection markers X 1 , X 2 , ... and / or X n , wherein the index "n" represents a natural integer from 1 to 20, in particular from 1 to 10, preferably from 1 to 5. In this regard, a detection marker X may also be provided 1 , X 2 , ... and / or X n Used to generate mixed signals different from each other, especially specific mixed signals with different proportions.

[0098] According to another embodiment, the detection mark X may also be formed by a plurality of detection marks different from each other, preferably selected from the detection mark X 1 , X 2 , X 3 , X 4 , X 5 and / or X 6 In this regard, preferably, the detection marker X 1 , X 2 , X 3 , X 4 , X 5 and / or X 6 Used to generate mixed signals different from each other, especially specific mixed signals with different proportions.

[0099] Therefore, within the scope of the present invention, surprisingly, multiple mixed signals can be generated even based on several individual detection labels. In order to generate mixed signals, the site-specific hybridization probe with at least one additional detection label is labeled with multiple detection labels that are different from each other to generate mixed signals.

[0100] In particular, according to the invention, it is provided that, in order to generate a mixed signal within the label range of the site-specific hybridization probe, a plurality of, in particular two to six, mutually different detection labels in different (quantity) ratios are used in each case. As an example - which is by no means limiting - the site-specific hybridization probe can have three further detection labels - in addition to the first detection label, wherein in this respect, reference is made to the three further detection labels, the total of the first detection label is 20%, the total of the second detection label is 60% and the total of the third detection label is 20%.

[0101] According to another embodiment of the present invention, it is also possible to provide

[0102] (a) a first site-specific hybridization probe labeled with a detection marker A and a second site-specific hybridization probe labeled with a detection marker B are located on the side of a first chromosome fragment (particularly a first breakpoint region) and generate a fusion signal AB in a signal image generated by in situ hybridization,

[0103] (b) a third site-specific hybridization probe labeled with detection marker A and a fourth site-specific hybridization probe labeled with detection marker B are located on the side of the second chromosome fragment (particularly the second breakpoint region) and generate a fusion signal AB in a signal image generated by in situ hybridization, and

[0104] (c) Using another detection marker X 1 labeling the third site-specific hybridization probe and / or the fourth site-specific hybridization probe, and generating a fusion signal and a mixed signal AB / X in a signal image generated by in situ hybridization 1 ,

[0105] wherein in the signal image, a chromosomal aberration of a first chromosome segment, in particular a first breakpoint region, is identified by individual signals A and / or B and / or wherein a mixed signal A / X is identified by 1 and / or B / X 1 A chromosomal aberration is identified and / or assigned to a second chromosome segment, in particular a second breakpoint region.

[0106] The method with the step of analyzing chromosome aberrations in the signal image generated by in situ hybridization has proven to be effective, in particular if in a first step, a fusion signal generated by a first detection marker is detected and / or analyzed, and in a subsequent step, in the event of the occurrence of a single signal, the mixed signal and its assignment to the chromosome region and / or DNA region being tested is detected and / or analyzed.

[0107] In particular, this can include the following steps: first, during the analysis of the signal image, a filter is used, with the help of which only the signal generated by the first detection marker, i.e. the fusion signal or potentially the individual signals, is visible. Since the chromosomal aberration to be further analyzed is only present when an individual signal appears in the signal image, based on this mixed signal - combined with the position of the fusion signal or the individual signals in the signal image - the chromosomal aberration can be assigned to a specific chromosomal region or DNA region, in particular by using a different filter, in particular a filter suitable for describing the mixed signal.

[0108] In addition, according to a specific embodiment of the method according to the invention, the detection of the signal image can be performed by computer-assisted analysis. It is particularly advantageous if, for the generation of mixed signals, the hybridization probe is labeled with more than at least one additional detection marker, preferably at least two additional, preferably a plurality of additional detection markers in different or defined ratios to each other. Computer-assisted analysis can also distinguish mixed signals based on the measurement of potential color components or colors, which cannot be distinguished from each other by the naked eye when observed under a fluorescence microscope.

[0109] According to the invention, therefore, it is particularly provided that the detection of translocations or inversions is performed using up to twenty-four site-specific hybridization probes, wherein in each case two site-specific hybridization probes are located on the distal and proximal sides of the respective breakpoint region, and the individual site-specific hybridization probes of these probe pairs are simultaneously labeled with an additional detection marker. Thus, preferably, up to twelve different breakpoint regions can be examined by a method, using up to twelve different break-separation methods, to describe up to twelve translocations or inversions. The detection of specific translocations is performed by separation of the recognition probe pairs or separation of the fusion signals of the probe pairs and using the respective mixed colors or mixed signals.

[0110] Translocations and inversions can also be detected using up to twenty-four site-specific hybridization probes, wherein in each case two probes flank the corresponding breakpoint region at the distal and proximal ends, and these probe pairs are labeled in each case with the same markers A and B, wherein in each case one probe is labeled with marker A and the other probe is labeled with marker B, and the individual probes of these probe pairs are simultaneously labeled with an additional detection marker X. Detection of specific translocations and / or inversions is performed by changing the specific fusion signal and mixed signal AB / X to a new separate mixed signal A / X and / or B / X in the case of a chromosomal aberration. In this regard, in the case of a probe pair, it is also possible not to use an additional marker X, so that in the case of a basic abnormality, the usual individual signals A and / or B occur only on this probe pair.

[0111] Thus, for the first time, in the first analysis of a plurality of different potentially detectable structural chromosomal mutations, using only signals A and B, using a specific filter system, for example, a double filter for signals A and B, which only makes visible signals A and B and not the additional marker X, it is possible to first quickly state whether a breakaway separation of the fusion signal AB has occurred and, in general, whether a translocation or an inversion is present. Only in the case of the occurrence of individual signals A and / or B is the mixed signal evaluated, accompanied by the marker X, and thus an unambiguous assignment of a potential translocation is made.

[0112] In the following, further features or embodiment possibilities of the method according to the invention will also be described, which apply analogously to all possible embodiments of the method according to the invention described above:

[0113] With regard to the detection of a single chromosome region or DNA region with a single site-specific hybridization probe in each case, within the scope of the present invention, it preferably has a length of less than 5 Mbp, in particular less than 2 Mbp, preferably less than 1 Mbp, preferably less than 750 kbp, particularly preferably less than 500 kbp. Likewise, it can be provided that the chromosome region and / or DNA region detected by the single site-specific hybridization probe has a length of at least 500 bp, in particular at least 1 kbp, preferably at least 5 kbp, preferably at least 10 kbp. Finally, it can also be provided that the length of the chromosome region and / or DNA region detected by the single site-specific hybridization probe is in the range between 500 bp and 5 Mbp, in particular in the range of 1 kbp to 2 Mbp, preferably in the range of 5 kbp to 1 Mbp, preferably in the range of 10 kbp to 750 kbp, particularly preferably in the range of 10 kbp to 500 kbp.

[0114] Furthermore, with regard to further embodiments of site-specific hybridization probes used according to the invention, these are preferably in the form of nucleic acid fragments, in particular polynucleotides, modified polynucleotides, modified nucleic acid fragments, oligonucleotides and / or oligonucleotides. Specifically, with regard to modified nucleic acid fragments, these may in particular be locked nucleic acids (LNA) or peptide nucleic acids (PNA).

[0115] According to the first embodiment of the present invention, it can also be provided that, in each case, the site-specific hybridization probe is formed by a single nucleic acid fragment covering the chromosome region and / or DNA region to be detected.

[0116] According to another and further preferred embodiment of the present invention, it can also be provided that, in each case, the site-specific hybridization probe is formed by a plurality of nucleic acid fragments ("probe fragments") covering the chromosome region and / or DNA region to be detected. In this respect, it is further preferred that the length of the individual nucleic acid fragments ("probe fragments") of the site-specific hybridization probe is in the range of 5 to 2,000 bp, in particular in the range of 10 to 1,500 bp, preferably in the range of 50 to 1,000 bp.

[0117] Furthermore, as for generating a mixed signal by using a site-specific hybridization probe having at least one additional detection label different from the first detection label, for achieving the goal of generating a mixed signal, it can be performed in different ways:

[0118] According to a first embodiment of the invention, in this respect it can be provided that, in order to generate a mixed signal, the nucleic acid fragment of the site-specific hybridization probe ("probe fragment") is labeled together with the first detection marker with a further detection marker that is different from the first detection marker. The hybridization probe labeled in this way thus generates a mixed signal based only on two detection markers that are different from each other.

[0119] Furthermore, it can be provided that according to another embodiment of the invention - in particular in the context of increasing the bandwidth of a specific mixed signal or increasing the number of specific mixed signals, i.e. signals that can be distinguished from one another - in order to generate a mixed signal, the nucleic acid fragments of the site-specific hybridization probes ("probe fragments") are labeled together with the first detection marker with a plurality, in particular two to twenty, preferably two to ten, preferably two to six detection markers that are different from the first detection marker. In this respect, it can be provided in particular that the detection markers are used in amounts that differ from one another.

[0120] According to the invention, it can therefore be provided that, in order to generate a mixed signal within the label range of the site-specific hybridization probe, a plurality of detection labels that are different from one another are used in each case in different ratios to one another. For example - and this is by no means limiting - the site-specific hybridization probe can have three further detection labels - in addition to the first detection label - wherein, with reference to the three further detection labels, the total of the first detection label is 20%, the total of the second detection label is 60%, and the total of the third detection label is 20%.

[0121] According to this embodiment, it can therefore be provided that the hybridization probe has at least two, preferably a plurality of detection labels that are different from each other and the first detection label. By using different detection labels in different ratios, the number of specific mixed signals can be increased overall, in particular signals that can be distinguished from each other, which in turn makes it possible to detect more detectable chromosome aberrations.

[0122] Within the scope of the labeling of site-specific hybridization probes, it can be provided according to the invention that, furthermore, essentially, the individual nucleic acid fragments of the hybridization-specific probe are only labeled with one detection label. In this respect, this can be the first detection label and / or any further detection label (see Figure 2 I)).

[0123] To state it differently, therefore, according to the invention, a first portion of the nucleic acid fragments of the site-specific hybridization probe is labeled only with a first detection label, and in each case, another portion of the nucleic acid fragments of the site-specific hybridization probe is labeled with an additional detection label that is different from the first detection label. According to one embodiment of the invention, the presence of detection labels that are different from one another on nucleic acid fragments of different hybridization-specific probes can generate a mixed signal (see Figure 2 I)).

[0124] Furthermore, it can be provided that the individual nucleic acid fragments of the hybridization-specific probe are labeled with a plurality of detection labels that are different from one another, in particular wherein the first detection label and / or any further detection labels may be involved (see Figure 2 II)).

[0125] According to this embodiment of the present invention, it can be provided that detection labels that form a mixed signal and are different from each other are present on the same nucleic acid fragment of the hybridization-specific probe, or are present together on the nucleic acid fragment of the hybridization-specific probe (referred to as a "mixed probe") (see Figure 2 II)).

[0126] Furthermore, it can be provided that, within the scope of the present invention, the above-mentioned two embodiments are combined to produce a mixed signal, i.e., a part of the nucleic acid fragments of the site-specific hybridization probe forming the mixed signal is labeled with only one detection label, and another or multiple other parts of the site-specific hybridization probe are labeled with at least two different detection labels.

[0127] Furthermore, mixed signals are generated if there are spacings of up to 3 Mbp, in particular up to 2.5 Mbp, preferably up to 2 Mbp, preferably up to 1 Mbp, particularly preferably up to 500 kb, even more preferably up to 200 kb between the individual hybridized nucleic acid fragments of the site-specific hybridization probe labeled with at least one additional detection marker. Stated differently, according to the invention, mixed signals are generated if there are "gaps" of up to 3 Mbp, in particular up to 2.5 Mbp, preferably up to 2 Mbp, preferably up to 1 Mbp, particularly preferably up to 500 kb, even more preferably up to 200 kb between the individual hybridized nucleic acid fragments of the site-specific hybridization probe labeled with at least one additional detection marker in the hybridized state (see Figure 2 III)).

[0128] Thus, in particular, mixed signals can be generated by using "mixed labeling" of individual probes. In particular, in order to generate a mixed signal that is specific for a chromosome region or a genomic segment, optionally, i) all fragments of the probe or optionally only individual fragments of the probe are labeled with a plurality of labels or II) the same fragments are labeled in each case with different labels or III) alternating fragments are labeled with different labels, so that here, too, ultimately only one mixed signal is visible or detectable (see Figure 2 I to III).

[0129] In this respect, mixed markings and mixed signals can also be generated in the sense of the method according to the invention if individual segments or all segments mentioned above under I) to III) overlap only partially.

[0130] Mixed labeling in the sense of the method according to the invention can also be produced if individual fragments or groups of fragments labeled with at least one labeled probe, and other individual fragments or groups of fragments labeled with at least one additional labeled probe, have an interval of 2 Mbp, optionally 1 Mbp, optionally 500 kb, optionally 200 kb.

[0131] If two or more probes with identical or almost identical sequences are used, i.e. two or more probes are directed to the same specific chromosomal region or the same genomic fragment, but are labeled with different labels, wherein the probes may also be only 95%, optionally 90%, optionally 80%, optionally 70%, optionally 60%, optionally 50% identical, wherein the differences are due to sequence variations of essentially similar sequences or only due to partial overlap of the individual regions of the probes, mixed labeling and mixed signals can therefore also be produced in the sense of the method according to the invention.

[0132] The selection of an appropriate detection label is within the basic capabilities of those skilled in the art and occurs as a function of the method used to perform the in situ hybridization.In general, direct or indirect labeling of the hybridization probe can be performed by selecting an appropriate detection label.

[0133] In the context of the present invention, if the detection label is selected from dyes; dye substrates; chemiluminescent dyes, in particular acridinium; radioisotopes; spin labels; enzymes, in particular alkaline phosphatase, horseradish peroxidase, soybean peroxidase and / or β-galactosidase; haptens, in particular digoxigenin, biotin, 2,4-dinitrophenol, 5(6)-carboxyfluorescein, rhodamine, bromodeoxyuridine, acetylaminofluorene, trinitrophenol, trinitrophenol derivatives, estradiol and / or 2,4-dinitrophenol; the amount Better results can be obtained by using subdots; beads; aminohexene; pyrene; and / or fluorescent dyes, especially fluorescein, fluorescein derivatives, 5(6)-carboxyfluorescein, coumarin, coumarin derivatives, rhodamine, rhodamine derivatives, tetramethylrhodamine, Lissamine, Texas Red, AMCA, TRITC, IR dyes, Alexa dyes, Dyomics dyes, phycoerythrin, Cascade Blue, Oregon Green 488, Pacific Blue and / or Rhodamine Green.

[0134] As far as in situ hybridization is performed, it can be performed in different ways.

[0135] In particular, according to a first preferred embodiment of the in situ hybridization, it can be provided that it is performed by direct labeling of the hybridization probes, in particular by fluorescence in situ hybridization (FISH).

[0136] Likewise, it can be provided that the hybridization probes for in situ hybridization are labeled with fluorescent dyes (in particular emitting in the visible, infrared and / or ultraviolet range, preferably in the green, orange / red, red, gold and / or blue range).

[0137] According to a further preferred embodiment of the in situ hybridization, it can likewise be provided that it is carried out by indirect labeling of the hybridization probes, in particular by bright field in situ hybridization (BrISH).

[0138] Furthermore, according to the invention, it can be provided that the in situ hybridization is carried out using a hapten, in particular biotin, digoxigenin and / or DNP-labeled hybridization probe, followed by detection by antibody-coupled alkaline phosphatase, antibody-coupled peroxidase and / or antibody-coupled β-galactosidase.

[0139] As far as analyzing fluorescence-based in situ hybridization is concerned, it is preferably performed using specific single or multiple filter sets, which in particular allow targeted delineation of fused and mixed signals.

[0140] Furthermore, it is advantageous to generate a mixed signal, in particular on the basis of at least one further detection marker, in particular on the basis of at least two further, preferably a plurality of further detection markers having different or defined ratios with respect to one another, which is evaluated by means of a computer-assisted analysis. Furthermore, it can also be provided that, in particular by means of a computer-assisted analysis, a superposition image is obtained which allows a joint depiction of signal images of different single or multiple filter sets.

[0141] Fundamentally, a variety of different types of chromosome aberrations can be detected using the method according to the invention. In particular, the method according to the invention can be used to detect translocations, inversions, segmental duplications, deletions, insertions, duplications, aneuploidy and amplifications, particularly translocations and / or inversions.

[0142] Within the scope of the method according to the invention, it can be provided in this respect that the chromosomal aberration is associated with a disease, in particular malignancies, preferably carcinomas, sarcomas and / or leukemias.

[0143] The genes for potential chromosomal aberrations to be tested are preferably selected from the group consisting of: ALK, ROS1, RET, NRG1, NTRK1, CARS, EML4, FGFR2, FGFR3, KIF5B, TGF, BCR, ABL, ALK, BCL2, BCL6, BIRC3, CCND1, EGR1, ETV6, FGFR1, FGFR3, IGH, KMT2A, MYC, PML, RARA, RUNX1, RUNX1T1, EWSR1, CHOP, FUS, COL1A1, DDIT3, JAZF1, NR4A3, FOXO1, FUS, P AX3, PAX7, PDGFB, SS18, TFE3, USP6, WT1, HER2 / ERBB2, FGFR1, ALK, CCND1, CDK4, CD274, PDCD1LG2, EGR1, EGFR, ESR1, ETV1, FGF3,4, 19. FGFR2, FGFR3, FHIT(RCC), KRAS, MDM2, MDM4, MET, MYB, MYC, MYCN, PIK3CA, PTEN, SMARCB1, SOX2, TERT, TOP2A, TP53, TYMS and / or VHL.

[0144] Particularly good results can be achieved within the scope of the method according to the invention if the method according to the invention is used for detecting inversions and / or translocations:

[0145] Within the scope of a preferred embodiment of the present invention, the method according to the invention is used for the detection of different translocations and / or inversions, in particular in lung tumors, wherein in particular the genes ALK, ROS1, RET, NRG1, NTRK1, CARS, EML4, FGFR2, FGFR3, KIF5B and / or TGF are affected.

[0146] Furthermore, it can be provided that the method according to the invention is used for the detection of different translocations and / or inversions, in particular in lymphomas and leukemias, wherein in particular the genes BCR, ABL, ALK, BCL2, BCL6, BIRC3, CCND1, EGR1, ETV6, FGFR1, FGFR3, IGH, KMT2A, MYC, PML, RARA, RUNX1 and / or RUNX1T1 are affected.

[0147] According to another preferred embodiment of the present invention, the method described in the present invention is used for the detection of different translocations and / or inversions, in particular in sarcomas, wherein in particular the genes EWSR1, CHOP, FUS, COL1A1, DDIT3, JAZF1, NR4A3, FOXO1, FUS, PAX3, PAX7, PDGFB, SS18, TFE3, USP6 and / or WT1 are affected.

[0148] According to the invention, it can also be provided that the method according to the invention is used for detecting inversions and / or translocations, wherein in particular the genes ALK and ROS1 are affected.

[0149] Furthermore, within the scope of the present invention, excellent results can be achieved if the method according to the invention is used for the detection of amplifications and / or deletions:

[0150] In order to detect amplification or deletion, up to twenty-four different site-specific probes can be used, each of which processes a corresponding genomic region, and in which different probes are labeled with different markers in different combinations and ratios. Based on the mixed signals generated in the signal image, different site-specific probes can be clearly distinguished. Therefore, up to twenty-four different amplification events and / or deletion events of the genomic region in question can be examined using a single method. Specific amplification or deletion detection is performed by counting different mixed signals or mixed colors.

[0151] Preferably, the method according to the invention is used to detect different amplifications and deletions, in particular in breast, colon and lung tumors, wherein in particular the genes HER2 / ERBB2, FGFR1, ALK, CCND1, CDK4, CD274, PDCD1LG2, EGR1, EGFR, ESR1, ETV1, FGF3,4,19, FGFR2, FGFR3, FHIT (RCC), KRAS, MDM2, MDM4, MET, MYB, MYC, MYCN, PIK3CA, PTEN, SMARCB1, SOX2, TERT, TOP2A, TP53, TYMS and / or VHL are affected.

[0152] Generally speaking, within the scope of the present invention, it is therefore surprisingly found that when using a site-specific probe with a mixed label, mixed signals can be well detected in a manner that allows assessment, and these signals allow clear identification of the chromosome regions affected by abnormality. Therefore, the method according to the present invention allows the first detection of a plurality of different chromosome structures and / or number mutations in a surprising manner. It is impossible in the prior art.

[0153] According to the second aspect of the present invention, another object of the present invention is to provide a composition for detecting chromosome aberrations, especially chromosome structural and / or numerical aberrations, preferably chromosome structural aberrations, by in situ hybridization, especially by detecting chromosome regions and / or DNA regions in a biological sample, preferably in one or more cells and / or one or more cell nuclei (cell nucleus / nuclei), especially by a method according to one of the preceding claims, wherein the composition comprises at least two, especially at least three, preferably at least four site-specific hybridization probes, which are different from each other and are all labeled with a first detection marker, and wherein according to the corresponding site-specific hybridization probe, at least one of the site-specific hybridization probes is labeled with at least one additional detection marker, and the additional detection marker is different from the first detection marker.

[0154] Likewise, according to this aspect of the invention, it is an object of the invention to provide a composition for the prophylactic and / or therapeutic treatment and / or for the diagnosis and / or prognosis of diseases associated with chromosomal aberrations, in particular malignancies, preferably carcinomas, sarcomas and / or leukemias, particularly preferably lung tumors, lymphomas, leukemias, sarcomas, breast cancer and / or colon cancer, wherein the composition comprises at least two, in particular at least three, preferably at least four site-specific hybridization probes that are different from each other and are each labeled with a first detection marker, and wherein at least one of the site-specific hybridization probes is labeled with at least one further detection marker, which is different from the first detection marker, depending on the respective site-specific hybridization probe.

[0155] In this respect, it can in particular be provided that the composition according to the invention is intended or used for carrying out a method as described above.

[0156] For further details of this aspect of the invention, reference may be made to the above description regarding other aspects according to the invention, which similarly also applies to this aspect of the invention.

[0157] In addition, according to the third aspect of the present invention, an object of the present invention is to provide a use of a composition, in particular a composition as described above, for detecting chromosome aberrations, in particular chromosome structural and / or numerical aberrations, preferably chromosome structural aberrations, by in situ hybridization, in particular by detecting chromosome regions and / or DNA regions in a biological sample, preferably in one or more cells and / or one or more cell nuclei (cell nucleus / nuclei), in particular by the method as described above.

[0158] For further details of this aspect of the invention, reference may be made to the above description regarding other aspects according to the invention, which similarly also applies to this aspect of the invention.

[0159] According to the fourth aspect of the present invention, another object of the present invention is to provide at least two, in particular at least three, preferably at least four site-specific hybridization probes that are different from each other and labeled with a first detection marker for use in detecting chromosome aberrations, in particular chromosome structural and / or numerical aberrations, preferably chromosome structural aberrations, by in situ hybridization, in particular by detecting chromosome regions and / or DNA regions in biological samples, preferably in one or more cells and / or one or more cell nuclei (cell nucleus / nuclei), in particular by the method as described above, wherein according to the corresponding site-specific hybridization probe, at least one of the site-specific hybridization probes is labeled with at least one additional detection marker, and the additional detection marker is different from the first detection marker.

[0160] Likewise, according to this aspect of the invention, an object of the present invention is to provide at least two, in particular at least three, preferably at least four site-specific hybridization probes for use in the diagnosis and / or prognosis of diseases associated with chromosomal aberrations, in particular malignancies, preferably carcinomas, sarcomas and / or leukemias, particularly preferably lung tumors, lymphomas, leukemias, sarcomas, breast cancer and / or colon cancer, each of the site-specific hybridization probes being labeled with a first detection marker, wherein, according to the respective site-specific hybridization probe, preferably within the scope of the previously described method according to the invention, at least one of the site-specific hybridization probes is labeled with at least one additional detection marker, which is different from the first detection marker.

[0161] For further details of this aspect of the invention, reference may be made to the above description regarding other aspects according to the invention, which similarly also applies to this aspect of the invention.

[0162] In addition, according to the fifth aspect of the present invention, another object of the present invention is to provide at least one site-specific hybridization probe labeled with at least two detection labels, and at least one, especially at least two, preferably at least three additional site-specific hybridization probes that are different from each other, and each additional site-specific hybridization probe has at least a first detection label, for use in detecting chromosome aberrations, especially chromosome structural and / or numerical aberrations, preferably chromosome structural aberrations, by in situ hybridization, especially by detecting chromosome regions and / or DNA regions in biological samples, preferably in one or more cells and / or one or more cell nuclei (cell nucleus / nuclei), especially by the method as described above.

[0163] For further details of this aspect of the invention, reference may be made to the above description regarding other aspects according to the invention, which similarly also applies to this aspect of the invention.

[0164] Finally, according to the sixth aspect of the present invention, an object of the present invention is to provide a kit or kit component or kit set for detecting chromosome aberrations, especially chromosome structural and / or numerical aberrations, preferably chromosome structural aberrations, by in situ hybridization, especially by detecting chromosome regions and / or DNA regions in a biological sample, preferably in one or more cells and / or one or more cell nuclei (cell nucleus / nuclei), comprising at least two, especially at least three, preferably at least four site-specific hybridization probes, which are different from each other and are all labeled with a first detection label, wherein at least one of the site-specific hybridization probes is labeled with at least one additional detection label, and according to the corresponding site-specific hybridization probe, the additional detection label is different from the first detection label, in particular, wherein the kit is intended and / or used to implement the above method.

[0165] In this context, it is particularly provided that at least two, in particular at least three, preferably at least four site-specific hybridization probes which are different from one another are present in a common composition, in particular in a composition as described above.

[0166] Likewise, it can be provided that at least two, in particular at least three, preferably at least four site-specific hybridization probes which are different from one another are present separately from one another in separate compositions.

[0167] For further details of this aspect of the invention, reference may be made to the above description regarding other aspects according to the invention, which similarly also applies to this aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0168] BRIEF DESCRIPTION OF THE DRAWINGS

[0169] Hereinafter, the present invention will be described in more detail using drawings and examples. As shown in the drawings:

[0170] Figure 1 : Schematic representation of a method according to the invention for detecting two translocations.

[0171] Figure 2 : A schematic diagram of a method for using multiple markers to depict mixed markers and mixed signals according to the present invention.

[0172] Figure 3: Schematic diagram of the signal image using a quadruple FISH probe "Zytolight SPEC ALK&ROS1 Break Apart Dual-Mix NG-FISH Probe" produced by ZytoVision Co., Ltd.

[0173] Figure 4 : Schematic diagram of the signal image using a quadruple FISH probe "Zytolight SPEC ALK&ROS1 Break Apart single-Mix NG-FISH probe" produced by ZytoVision Co., Ltd.

[0174] Figure 5 : Schematic diagram of the signal image using a six-fold FISH probe "Zytolight SPEC ALK&ROS1&RET Break Apart single-Mix NG-FISH probe" produced by ZytoVision Co., Ltd.

[0175] Figure 6 : Schematic diagram of the signal image using a six-fold FISH probe "Zytolight SPEC ALK&ROS1&RET Break Apart single-Mix II NG-FISH probe" produced by ZytoVision Co., Ltd.

[0176] Figure 7 : A schematic diagram of a method for detecting four numerical distortions according to the present invention.

[0177] Figure 8 : Schematic diagram of a method for detecting seven translocations or amplifications according to the present invention.

[0178] Fig. 9 : FISH analysis for detecting translocation of the ROS1 region in 6q22 using the quadruple FISH probe "Zytolight SPEC ALK&ROS1 Break Apart single-Mix NG-FISH Probe" from ZytoVision Inc.

[0179] Fig.10 : CISH analysis for detecting translocation of the ALK region in 2p23 using the quadruple CISH probe "ZytoDot SPEC ALK&ROS1 Break Apart single-MIX NG-FISH Probe" from ZytoVision Inc.

[0180] Fig.11:Using ZytoVision's five-fold probe "Zytolight SPEC ERBB2, EGFR, FGFR1, MET&SOX2 FiveCheck TM FISH analysis using the "NG-FISH probe" to detect amplification of the ERBB2 region.

[0181] Detailed description with drawings

[0182] Figure 1 A schematic diagram of a method for detecting two translocations according to the present invention using four probes and three markers, wherein one probe is simultaneously labeled with two markers, is shown. It shows signal images in the case of normal cells and in the case of cell translocations in the ALK gene in 2p23 or the ROS1 gene in 6q22.

[0183] In each case, the two breakpoint regions (ALK and ROS1) were flanked by markers A and B of the quadruple ISH probe, respectively, resulting in fusion signal AB. In addition, the ALK breakpoint region was also flanked by marker C on one side, resulting in a hybrid marker A / C.

[0184] In interphase of normal cells (no ALK or ROS1 abnormalities), the ROS1 gene locus is marked with a fusion signal AB, and the ALK gene locus is marked with a fusion signal AB, which is accompanied by a mixed signal A / C. In interphase of cells affected by ALK translocation, the ALK gene affected by the translocation is marked with a single signal of marker B and a mixed signal A / C separated from the former, and the ROS1 gene affected by the translocation is marked with a single signal of marker A and a single signal of marker B.

[0185] Figure 2 Shown is a schematic diagram of a method for representing mixed labeling and mixed signal according to the present invention using multiple marks. For the sake of clarity, only two kinds of marks are listed. If under the following circumstances, a mixed signal specific to a locus-specific probe and therefore specific to a chromosome region or a genome fragment can be produced: I) probe fragments are marked with different marks separately, and / or II) all fragments of probe or optionally only a single fragment of probe is marked with multiple marks, and / or III) alternate fragments are marked with different marks, so finally only mixed signal is visible or detectable here. In this respect, according to I) to III), all or only single fragments can be superimposed or overlapped (not shown), and if according to I) to III) described single fragment or fragment group have an interval of up to 2Mbp, for example, in the "gap" shown, mixed labeling may also be produced.

[0186] Figure 3Schematic diagram of a signal image using the corresponding quadruple FISH probe "Zytolight SPEC ALK&ROS1 Break Apart Dual-Mix NG-FISH probe" from ZytoVision Inc. The probe consists of a green labeled polynucleotide (absorbing at 503 nm and emitting at 528 nm) targeting sequences located proximal to the ALK breakpoint region in 2p23 and to sequences located proximal to the ROS1 breakpoint region in 6q22, an orange labeled polynucleotide (absorbing at 547 nm and emitting at 572 nm) targeting sequences located distal to the ALK breakpoint region in 2p23 and to sequences located distal to the ROS1 breakpoint region in 6q22, and a blue labeled polynucleotide (absorbing at 426 nm and emitting at 480 nm) targeting sequences located distally and proximal to the ALK breakpoint region in region 2p23.

[0187] When using the appropriate filter set, the hybridization signal of the non-rearranged ALK gene appears as a green-orange fluorescent fusion signal, which is composed of green / blue and orange / blue fluorescent mixed signals. The hybridization signal of the non-rearranged ROS1 gene appears as a green-orange fluorescent fusion signal.

[0188] In interphase of normal cells (no ALK or ROS1 abnormalities), four green-orange fusion signals appear when using the appropriate green-orange dual bandpass filter set, two blue signals appear when using the appropriate single bandpass filter set, and two green-orange fusion signals and two green-orange / blue fusion signals and mixed signals appear when using the appropriate triple bandpass filter set (see Figure 3 a).

[0189] The 2p23 locus affected by ALK translocations is characterized by a single green / blue mixed signal and a single orange / blue mixed signal (see Figure 3 b).

[0190] The 6q22 locus affected by ROS1 translocation is characterized by a single green signal and a single orange signal (see Figure 3 c).

[0191] When using a suitable dual bandpass filter set for the green and orange signals, the green signal and the orange signal separated therefrom, it is therefore firstly only permitted to state that essentially an ALK or ROS1 translocation is present. A diagnostically potentially relevant distinction between an ALK or ROS1 translocation can then be made with the inclusion of the blue fluorescence signal. If the individual green signal and the blue signal (green / blue mixed signal) overlap, or if the individual orange signal and the blue signal (orange / blue mixed signal) overlap, an ALK translocation is indicated. If the individual green and orange signals do not overlap with the blue signal, a ROS1 translocation is indicated.

[0192] Figure 4 A schematic diagram of a signal image using the corresponding quadruple FISH probe "Zytolight SPECALK&ROS1 Break Apart single-Mix NG-FISH probe" from ZytoVision is shown. The probe consists of a green labeled polynucleotide (absorbing at 503 nm and emitting at 528 nm) targeting a sequence located proximal to the ALK breakpoint region in 2p23 and a sequence located proximal to the ROS1 breakpoint region in 6q22, an orange labeled polynucleotide (absorbing at 547 nm and emitting at 572 nm) targeting a sequence located distal to the ALK breakpoint region in 2p23 and a sequence located distal to the ROS1 breakpoint region in 6q22, and a blue labeled polynucleotide (absorbing at 426 nm and emitting at 480 nm) targeting a sequence located distal to the ALK breakpoint region in region 2p23.

[0193] When using the appropriate filter set, the hybridization signal of the non-rearranged ALK gene appears as a green-orange fluorescent fusion signal, which is composed of a mixed signal of green and orange / blue fluorescent signals. The hybridization signal of the non-rearranged ROS1 gene appears as a green-orange fluorescent fusion signal.

[0194] In interphase of normal cells (no ALK or ROS1 abnormalities), four green-orange fusion signals appeared when using the appropriate green-orange dual bandpass filter set, two blue signals appeared when using the appropriate single bandpass filter set, and two green-orange fusion signals and two green-orange / blue fusion signals and mixed signals appeared when using the appropriate triple bandpass filter set (see Figure 4 a).

[0195] The 2p23 locus affected by ALK translocation is characterized by a single green signal and a single mixed orange / blue signal (see Figure 4 b).

[0196] The 6q22 locus affected by ROS1 translocation is characterized by a single green signal and a single orange signal (see Figure 4 c).

[0197] When using a suitable dual bandpass filter set for the green and orange signals, the green signal and the orange signal separated therefrom, it is therefore firstly only permitted to state that essentially an ALK or ROS1 translocation is present. A diagnostically potentially relevant distinction between an ALK or ROS1 translocation can then be made with the inclusion of the blue fluorescence signal. If the individual orange signal and the blue signal (orange / blue mixed signal) overlap, an ALK translocation is indicated. If the individual orange signal and the blue signal do not overlap, a ROS1 translocation is indicated.

[0198] Figure 5 Schematic diagram showing a signal image using the corresponding six-fold FISH probe “Zytolight SPECALK & ROS1 and RET Break Apart Dual-Mix NG FISH Probe” from ZytoVision. The probe consists of a green labeled polynucleotide (absorbing at 503 nm and emitting at 528 nm) that targets sequences proximal to the ALK breakpoint region in 2p23, sequences proximal to the ROS1 breakpoint region in 6q22, and sequences proximal to the RET breakpoint region in 10q11, an orange labeled polynucleotide (absorbing at 547 nm and emitting at 572 nm) that targets sequences distal to the ALK breakpoint region in 2p23, sequences distal to the ROS1 breakpoint region in 6q22, and sequences distal to the RET breakpoint region in 10q11, and a blue labeled polynucleotide (absorbing at 426 nm and emitting at 480 nm) that targets sequences distal to the ALK breakpoint region in region 2p23, and sequences proximal to the RET breakpoint region in 10q11.

[0199] When using the appropriate filter set, the hybridization signal of the non-rearranged ALK gene appears as a green-orange fluorescent fusion signal, consisting of green and orange / blue fluorescent mixed signals. The hybridization signal of the non-rearranged RET gene appears as a green-orange fluorescent fusion signal, consisting of a green / blue mixed signal and an orange signal. The hybridization signal of the non-rearranged ROS1 gene appears as a green-orange fluorescent fusion signal.

[0200] In interphase of normal cells (no ALK, ROS1, or RET abnormalities), six green-orange fusion signals appeared when using the appropriate green-orange dual-bandpass filter set, four blue signals appeared when using the appropriate single-bandpass filter set, and two green-orange fusion signals, two green-orange / blue fusion signals and mixed signals, and two green / blue-orange fusion signals and mixed signals appeared when using the appropriate triple-bandpass filter set (see Figure 5 a).

[0201] The 2p23 locus affected by ALK translocation is characterized by a single green signal and a single orange / blue mixed signal (see Figure 5 b).

[0202] The 6q22 locus affected by ROS1 translocation is characterized by a single green signal and a single orange signal (see Figure 5 c).

[0203] The 10q11 locus affected by RET translocations is characterized by a single orange signal and a single mixed green / blue signal (see Figure 5 d).

[0204] When using a suitable dual bandpass filter set for green and orange signals, green signals and orange signals separated therefrom, it is first permitted to state that essentially ALK, ROS1 or RET translocation is present. A diagnostically potentially relevant distinction between ALK, ROS1 or RET translocations can then be made with the inclusion of the blue fluorescent signal. If the individual orange signal blue signal (orange / blue mixed signal) overlaps, an ALK translocation is indicated. If the individual green signal blue signal (green / blue mixed signal) overlaps, a RET translocation is indicated. If neither the individual orange signal nor the individual green signal overlaps the blue signal, a ROS1 translocation is indicated.

[0205] Figure 6Schematic diagram showing a signal image using the corresponding six-fold FISH probe “ZytolightSPEC ALK&ROS1 and RET Break Apart Dual-Mix II NG-FISH Probe” from ZytoVision Inc. The probe consists of a green labeled polynucleotide (absorbing at 503 nm and emitting at 528 nm) that targets sequences proximal to the ALK breakpoint region in 2p23, sequences proximal to the ROS1 breakpoint region in 6q22, and sequences proximal to the RET breakpoint region in 10q11, a red labeled polynucleotide (absorbing at 580 nm and emitting at 599 nm) that targets sequences distal to the ALK breakpoint region in 2p23, sequences distal to the ROS1 breakpoint region in 6q22, and sequences distal to the RET breakpoint region in 10q11, a blue labeled polynucleotide (absorbing at 426 nm and emitting at 480 nm) that targets sequences distal to the ALK breakpoint region in region 2p23, and a gold labeled polynucleotide (absorbing at 532 nm and emitting at 553 nm) that targets sequences proximal to the RTE breakpoint region in region 10q11.

[0206] When using the appropriate filter set, the hybridization signal of the non-rearranged ALK gene appears as a green-red fluorescent fusion signal, consisting of green and red / blue fluorescent mixed signals. The hybridization signal of the non-rearranged RET gene appears as a green-red fluorescent fusion signal, consisting of a green / gold-yellow mixed signal and a red signal. The hybridization signal of the non-rearranged ROS1 gene appears as a green-red fluorescent fusion signal.

[0207] In interphase of normal cells (no ALK, ROS1, or RET abnormalities), six green-red fusion signals appear when using the appropriate green-red dual bandpass filter set, two blue signals appear when using the appropriate single bandpass filter set, and two gold-yellow signals appear when using the appropriate single bandpass filter set (see Figure 6 a).

[0208] The 2p23 locus affected by ALK translocation is characterized by a single green signal and a single mixed red / blue signal (see Figure 6 b).

[0209] The 6q22 locus affected by ROS1 translocation is characterized by a single green signal and a single red signal (see Figure 6 c).

[0210] The 10q11 locus affected by RET translocations is characterized by a single red signal and a single green / gold-yellow mixed signal (see Figure 6 d).

[0211] When using a suitable dual-bandpass filter set for green and red signals, green signals and separate red signals, it is therefore first permitted to state that essentially ALK, ROS1 or RET translocations are present. A diagnostically potentially relevant distinction between ALK, ROS1 or RET translocations can then be made with the inclusion of blue or gold-yellow fluorescent signals. If a single red signal blue signal (red / blue mixed signal) overlaps, an ALK translocation is indicated. If a single green signal gold-yellow signal (green / gold-yellow mixed signal) overlaps, a RET translocation is indicated. If neither a single red signal nor a single green signal overlaps a blue or gold-yellow signal, a ROS1 translocation is indicated.

[0212] Figure 7 A schematic diagram of a method for detecting four numerical aberrations using four probes and four markers according to the present invention is shown, wherein three probes are simultaneously labeled with two markers, wherein in each case the markers used for combination are different from each other in the case of these three probes. It shows signal images in the case of normal cells and in the case of cell amplification of the MET gene in 7q31. The region 17q11.2-q12 of the ERBB2 gene is covered with marker A, the region 7p12 of the EGFR gene is covered with marker A and an additional marker D, so that a mixed marker A / D appears, the region 8p11.23-p11.22 of the FGFR1 gene is covered with marker A and an additional marker C, so that a mixed marker A / C appears, and the region 7q31 of the MET gene is covered with marker A and an additional marker B, so that a mixed marker A / B appears.

[0213] In the interphase of normal cells (no ERBB2, EGFR, FGFR1 or MET number aberrations), all sites are marked with the signal of marker A. The colocalization of the signal of marker A with the signal of marker B results in mixed marker A / B and marks the MET gene site. Therefore, mixed marker A / C marks the FGFR1 gene site, and mixed marker A / D marks the EGFR gene site. The ERBB2 gene site is characterized by the absence of colocalization with another marker. In the interphase of cells with MET gene amplification, the signal of marker A, which is colocalized with the signal of marker B, increases, so the signal of mixed marker A / B increases.

[0214] Figure 8A schematic diagram of a method for detecting seven translocations or amplifications using 14 probes and five markers according to the present invention is shown, wherein in each case one of the probes is simultaneously labeled with two markers and the amount ratio distinguishes the two markers of each probe. The probes are each located on the side of a breakpoint region ("breakpoint") or an amplification region ("amplification"), and another region on the same chromosome (e.g., a centromere region), such as Figure 8 As shown in the upper left and upper right parts. The two probes of the chromosome are labeled with two different markers and one identical marker (e.g., probe 1: 25% green, 75% blue and probe 2: 25% yellow and 75% blue). Therefore, each of the two probes located on the side of the breakpoint region produces a fusion signal and a mixed signal from the three markers in cells not affected by the translocation. Therefore, the two probes respectively locate the amplified region and the other region on the same chromosome, thereby producing separate mixed signals in cells not affected by the amplification (unless the distance between the two probes is so small that a fusion signal and a mixed signal are produced). Different mixed signals are produced by changing the amount ratio between the two markers marked by the probes, so that multiple probes can be marked in a distinguishable manner using the same marker (e.g., in the example shown, four probes for breakpoint regions 1 to 4: 25% to 75%; 50% to 50%; 75% to 25% and 100% to 0%).

[0215] In the interphase of normal cells (no translocation), the breakpoint region of the gene is marked by mixed signals AB and CB, which combine to produce an A / B / C fusion signal. In the interphase of normal cells (no amplification), the amplified region of the gene is marked with mixed signal AB, and other regions on the same chromosome, such as the centromere region, are marked with mixed signal CB. In the interphase of cells affected by translocation, the genes affected by translocation are marked by a separate signal of marker AB and a mixed signal CB separated from the latter. In the interphase of cells affected by amplification, the genes affected by amplification are marked by a recurring mixed signal (e.g., AB) of the marker pair.

[0216] Fig. 9FISH analysis for detecting translocations of the ROS1 region in 6q22 using the quadruple FISH probe "Zytolight SPEC ALK&ROS1Break Apart Single-Mix NG-FISH Probe" from ZytoVision is shown. The probe consists of a green labeled polynucleotide (absorbing at 503 nm and emitting at 528 nm) targeting sequences proximal to the ALK breakpoint region in 2p23 and proximal to the ROS1 breakpoint region in 6q22, an orange labeled polynucleotide (absorbing at 547 nm and emitting at 572 nm) targeting sequences distal to the ALK breakpoint region in 2p23 and proximal to the ROS1 breakpoint region in 6q22, and a blue labeled polynucleotide (absorbing at 426 nm and emitting at 480 nm) targeting sequences proximal to the ROS1 breakpoint region in region 6q22.

[0217] When using appropriate filter sets, hybridization signals for non-rearranged ROS1 and / or ALK genes appear as a green-orange fluorescent fusion signal and, for rearranged ROS1 and / or ALK genes, as separate green signals and separate orange signals (see Fig. 9 A, which shows green and orange fluorescent signals). In this regard, the ROS1-specific green signal colocalizes with the blue fluorescent signal (see Fig. 9 B, which shows a blue fluorescent signal), so that the non-rearranged ROS1 gene consists of orange and green / blue fluorescent mixed signals. The hybridization signal of the non-rearranged ALK gene appears as a green-orange fluorescent fusion signal, without a mixed signal of the blue fluorescent signal. The 6q22 locus affected by the ROS1 translocation is characterized by a single green signal and a single orange signal ( Fig. 9 Arrows in A and C). In this regard, the green signal alone overlaps with the blue signal. This green / blue mixed signal indicates that the gene affected by the translocation is ROS1, not ALK (see Fig. 9 C, which shows blue, green, and orange fluorescence signals). Using appropriate filter sets, the signal images can be easily seen.

[0218] Fig.10CISH analysis for detecting translocation of the ALK region in 2p23 using the quadruple CISH probe "ZytoDot SPEC ALK&ROS1Break Apart Single-MIX NG-FISH Probe" from ZytoVision is shown. The probe consists of a digoxigenin-labeled polynucleotide targeting a sequence located proximal to the ALK breakpoint region in 2p23 and a sequence located proximal to the ROS1 breakpoint region in 6q22, a DNP-labeled polynucleotide targeting a sequence located distal to the ALK breakpoint region in 2p23 and a sequence located distal to the ROS1 breakpoint region in 6q22, and a biotin-labeled polynucleotide targeting a sequence located distal to the ROS1 breakpoint region in region 6q22. Detection of the label is performed by a primary (unlabeled) antibody (anti-DIG / anti-DNP / anti-BIO), which is followed by an enzymatic reaction of a secondary polymerase-conjugated antibody (HRP polymer / AP polymer / β-GAL), and a substrate (AP-RED / HRP-GREEN / β-GAL-BLUE), which results in the formation of strong, permanent, red (R), green (G), and blue (B) signals that can be depicted by light microscopy, e.g., using a 40x dry lens.

[0219] Fig.11 The five-fold FISH probe "Zytolight SPEC ERBB2, EGFR, FGFR1, MET & SOX2 FiveCheck" from ZytoVision is shown. TM "NG-FISH probe" FISH analysis for detecting ERBB2 region amplification. The probe consists of a green labeled polynucleotide (absorbing at 503nm and emitting at 528nm) targeting the region 17q11.2-q12 of the ERBB2 gene, the region 7p12 of the EGFR gene, the region 8p11.23-p11.22 of the FGFR1 gene, the region 7q31 of the MET gene, and the region 3q26.3-q27 of the SOX2 gene, as well as a blue labeled polynucleotide (absorbing at 426nm and emitting at 480nm) targeting the regions of the EGFR gene and the SOX2 gene, a gold-yellow labeled polynucleotide (absorbing at 532nm and emitting at 553nm) targeting the region of the FGFR1 gene, and a red labeled polynucleotide (absorbing at 580nm and emitting at 599nm) targeting the regions of the MET gene and the SOX2 gene.

[0220] When a suitable single bandpass filter bank is used, nine individual green signals and one green signal cluster are visible, which occupies the surface area of ​​multiple individual green signals (see Fig.11 A), four blue signals (see Fig.11 B), two gold-yellow signals (see Fig.11 C) and four red signals (see Fig.11 D).

[0221] Image superposition (see Fig.11 E) shows that single green signals and clusters of green signals are not co-localized with signals of different colors (arrows). Single green signals are non-amplified ERBB2 genes; clusters of green signals identify ERBB2 gene amplification. Co-localized green / blue mixed signals identify two copies of the EGFR gene, co-localized green / gold-yellow mixed signals identify two copies of the FGFR1 gene, co-localized green / red mixed signals identify two copies of the MET gene, and co-localized green / blue / red mixed signals identify two copies of the SOX2 gene (see Fig.11 E). DETAILED DESCRIPTION

[0222] In order to further illustrate the properties of the method according to the present invention, the following in situ hybridization was further performed:

[0223] Example 1: Using the five-fold FISH probe "SPEC ERBB2, EGFR, FGFR1, MET & SOX2 FiveCheck" from ZytoVision Inc. TM FISH analysis of multiple numerical aberrations in different cell types using NG-FISH probes

[0224] FISH was performed on sections of formalin-fixed paraffin-embedded (FFPE) lung and breast cancer tumor preparations with 3 to 5 μm thickness, without and with previously diagnosed ERBB2 gene amplification, which were applied to coated glass mounts and baked at 58°C overnight.

[0225] In order to remove paraffin, the preparation was first heated on a hot plate at 70°C for 10 minutes, then incubated twice in 100% xylene at room temperature (RT), for 10 minutes each time. Subsequently, the preparation was rehydrated by a descending ethanol series (once for 5 minutes, RT, in 96%, 96%, 90%, 70% denatured ethanol) and incubated in ultrapure water (twice, two minutes each time, RT). In order to permeabilize the cells, they were then heat-pretreated at 98°C for 15 minutes in a heat pretreatment solution citric acid (ZytoVision GmbH), followed by further incubation twice in ultrapure water at room temperature for 2 minutes each time. Proteolytic pretreatment was performed by dropping a pepsin solution (Pepsin Solution, ZytoVision GmbH) onto the preparation and then incubating in a humidity chamber at 37°C for 25 minutes. After a subsequent incubation in washing buffer SSC (ZytoVision Gmb) for 5 minutes, the preparations were dehydrated (one minute each, in each case at RT, in ultrapure water, 70%, 90%, 96% ethanol). After the preparations were air-dried, 10 μl of the FISH probe Zytolight SPEC ERBB2, EGFR, FGFR1, MET&SOX2 FiveCheck TMNG-FISH probe (ZytoVision GmbH) were applied directly to the sections by pipette.

[0226] The probe is a mixture based on 5 site-specific hybridization probes, wherein the mixture consists of green labeled polynucleotides (absorbed at 503nm and emitted at 528nm), which are directed to the region 17q11.2-q12 of the ERBB2 gene, the region 7p12 of the EGFR gene, the region 8p11.23-p11.22 of the FGFR1 gene, the region 7q31 of the MET gene, and the region 3q26.3-q27 of the SOX2 gene, and blue labeled polynucleotides (absorbed at 426nm and emitted at 480nm), which are directed to the regions of the EGFR gene and the SOX2 gene, gold-yellow labeled polynucleotides (absorbed at 532nm and emitted at 553nm) directed to the FGFR1 gene region, and red labeled polynucleotides (absorbed at 580nm and emitted at 599nm) directed to the regions of the MET gene and the SOX2 gene. Subsequently, a glass cover was placed, bubbles were removed, and the edges were sealed with Fixogum (Marabu). After denaturation of the preparation on a hot plate at 75°C for 10 minutes, hybridization was performed in a preheated humidity chamber at 37°C, overnight (approximately 16 hours) in a heating oven.

[0227] After hybridization, the Fixogum was removed and the preparation was incubated for 3 minutes at 37°C in a washing buffer (1x WashBuffer A, ZytoVision GmbH) in a glass cuvette. After removing the cover glass, the preparation was washed twice at 37°C in a washing buffer (1x WashBuffer A, ZytoVision GmbH) for 5 minutes each time. Subsequently, the preparation was dehydrated and dried in an ascending ethanol series for one minute each time (in each case, RT, in 70%, 90%, 96%), wherein the preparation was protected from direct light. After using an anti-stain (20 μl DAPI DuraTect Solution (ZytoVision GmbH)), the cover glass was covered, bubbles were removed, and the preparation was incubated at room temperature for at least 30 minutes, protected from light.

[0228] Subsequently, evaluation was performed using a fluorescence microscope (Axio Scope.A1, illumination unit HXP 120V, Carl ZeissMicroscopy GmbH) using appropriate filter sets (Sp.Green HC mFISH filter set; Sp.Red HC mFISH filter set; Sp.Aqua HC mFISH filter set; ZyGold HC mFISH filter set (all AHFAnalysentechnik AG)).

[0229] In this regard, when using the green filter, in each case 10 green signals were found in the nuclei in preparations without ERBB2 amplification (or more green signals in case of ERBB2 amplification, see Fig.11 A). Using the ZyGold filter, two gold-yellow signals are visible in each cell nucleus, whose spatial positions coincide with those of the two green signals in each case (e.g. Fig.11 C). Using a red filter, four red signals are visible in each cell nucleus, whose spatial positions coincide with the spatial positions of the four green signals in each case (see Fig.11 D). Using the light green filter, four light green signals are visible in each cell nucleus, whose spatial positions coincide with the spatial positions of the four green signals in each case and, in the case of two signals, also coincide with the spatial positions of the two red signals in each case (see Figure 5A). Fig.11D). The signal images can be interpreted as follows: two green signals in the absence of spatially consistent signals of another color identify two ERBB2 gene copies in diploid cells. Two green signals in spatially consistent relationship with two light green signals identify two EGFR gene copies, two green signals in spatially consistent relationship with two gold-yellow signals identify two FGFR1 gene copies, two green signals in spatially consistent relationship with two red signals identify two MET gene copies, and two green signals in spatially consistent relationship with two red and two light green signals identify two SOX2 gene copies.

[0230] In the nuclei of the preparations with ERBB2 amplification, a signal pattern comparable to that described above was found, except that in addition to the nine green signals, a green signal cluster or signal pattern consisting of approximately fifteen signals was observed, which were so compact that they could not be separated (see Fig.11 A). This green signal image does not co-localize with signals of different colors, thus identifying ERBB2 gene amplification.

[0231] Example 2: FISH analysis of translocation of ALK and ROS1 regions in different cell types using the quadruple FISH probe "Zytolight SPEC ALK&ROS1 Break Apart Single-Mix NG-FISH probe" of ZytoVision Inc.

[0232] In formalin-fixed paraffin-embedded (FFPE) Hela cell lines with a thickness of 3 to 5 μm ( CCL-2 TM ), HCC78 (provided by Professor Schildos of Göttingen) and H3122 (provided by Professor Schildos of Göttingen),

[0233] It was applied to a coated glass substrate and baked overnight at 58°C

[0234] To remove paraffin, the preparation was first heated on a hot plate at 70°C for 10 minutes, then incubated twice in 100% xylene at room temperature (RT) for 10 minutes each time. Subsequently, the preparation was rehydrated by a descending ethanol series (once for 5 minutes, RT, in 96%, 96%, 90%, 70% denatured ethanol) and incubated in ultrapure water (twice, two minutes each time, RT). To permeabilize the cells, they were then heat pretreated at 98°C for 15 minutes in a heat pretreatment solution citric acid (ZytoVision GmbH), followed by further incubation twice in ultrapure water at room temperature for 2 minutes each time. Proteolytic pretreatment was performed by dropping a pepsin solution (Pepsin Solution, ZytoVision GmbH) onto the preparation and then incubating in a humidity chamber at 37°C for 15 minutes. After a subsequent incubation in washing buffer SSC (ZytoVision Gmb) for 5 minutes, the preparation was dehydrated (one minute each, in each case at RT, in ultrapure water, 70%, 90%, 96% ethanol). After the preparation was air-dried, 10 μl of the FISH probe Zytolight SPEC ALK&ROS1 Break Apart Single-MixNG-FISH Probe (ZytoVision GmbH) was applied directly to the section by pipette.

[0235] The probe is a mixture based on four site-specific hybridization probes, wherein the mixture consists of a green labeled polynucleotide (absorbed at 503 nm and emitted at 528 nm) for sequences located proximal to the ALK breakpoint region in 2p23 and for sequences located proximal to the ROS1 breakpoint region in 6q22, an orange labeled polynucleotide (absorbed at 547 nm and emitted at 572 nm) for sequences located distal to the ALK breakpoint region in 2p23 and for sequences located distal to the ROS1 breakpoint region in 6q22, and a blue labeled polynucleotide (absorbed at 426 nm and emitted at 480 nm) for sequences located proximal to the ROS1 breakpoint region in region 6q22. Subsequently, a glass cover was placed, air bubbles were removed, and the edges were sealed with Fixogum (Marabu). After denaturing the preparation on a hot plate at 75°C for 10 minutes, hybridization was performed in a preheated humidity chamber at 37°C, overnight in a heating oven (approximately 16 hours).

[0236] After hybridization, Fixogum was removed and the preparation was incubated in wash buffer (1x WashBuffer A, ZytoVision GmbH) in a glass cuvette for 3 minutes at 37°C.

[0237] After removing the cover glass, wash twice at 37°C in washing buffer (1x Wash Buffer A, ZytoVision GmbH) for 5 minutes each. Subsequently, the preparation is dehydrated in an ascending ethanol series (one minute, in each case, RT, in 70%, 90%, 96% ethanol) and air-dried, wherein the sample is protected from direct light. After applying the counterstain (20 μl DAPI DuraTect Solution (ZytoVision GmbH)), the cover glass is covered, bubbles are removed, and the preparation is incubated at room temperature for at least 30 minutes, protected from light.

[0238] Subsequently, evaluation was performed using a fluorescence microscope (Axio Scope.A1, illumination unit HXP 120V, Carl ZeissMicroscopy GmbH) using appropriate filter sets (Dualband green / orange-red filter set, AHF Analysentechnik; Sp. Aqua HC mFISH filter set, AHF Analysentechnik).

[0239] In this regard, when using the orange / green dual filter, six orange / green fusion signals were found in each case in the nuclei of the HeLa cell line, in the majority of the analyzed nuclei; no individual green and / or orange signals were observed. Using the light green filter, three light green signals were visible in each nucleus in each case, and their spatial positions were consistent with those of the fusion signals. In agreement with the literature, the signal images were interpreted as three copies of the ALK gene and three copies of the ROS1 gene. No ALK or ROS1 translocation was present.

[0240] In the nuclei of the cell line H3122, in which a translocation of the ALK gene has been described in the literature, seven orange / green fusion signals and one orange signal were found in most of the analyzed nuclei when using the orange / green dual filter. When using the light green filter, two light green signals were visible in each nucleus, and in each case their spatial position was consistent with that of the two fusion signals. The signal pattern was interpreted in agreement with the literature as six copies of the ALK gene, one of which was affected by the translocation, and two copies of the ROS1 gene.

[0241] In the nuclei of the cell line HCC78, for which a translocation of the ROS1 gene is described in the literature, in most of the analyzed nuclei, four orange / green fusion signals were found when using an orange / green dual filter, in each case two individual orange signals and two individual green signals, i.e. separate from the other green signals. When using a light green filter, four light green signals were visible in each nucleus, in each case their spatial position being consistent with two fusion signals and two individual green signals. The signal pattern was interpreted in agreement with the literature as four copies of the ROS1 gene, two of which were affected by the translocation, and two copies of the ALK gene.

[0242] Example 3: FISH analysis for detecting translocations in the ROS1 region at 6q22 using the quadruple FISH probe "Zytolight SPECALK & ROS1 Break Apart Single-Mix NG-FISH Probe" from ZytoVision Inc.

[0243] FISH analysis for detecting translocations of the ROS1 region in 6q22 using the quadruple FISH probe "Zytolight SPEC ALK&ROS1Break Apart SingleMix NG-FISH Probe" from ZytoVision Inc. The probe is a mixture based on four site-specific hybridization probes, wherein the mixture consists of a green labeled polynucleotide (absorbing at 503 nm and emitting at 528 nm) targeting sequences located proximal to the ALK breakpoint region in 2p23 and to sequences located proximal to the ROS1 breakpoint region in 6q22, an orange labeled polynucleotide (absorbing at 547 nm and emitting at 572 nm) targeting sequences located distal to the ALK breakpoint region in 2p23 and to sequences located distal to the ROS1 breakpoint region in 6q22, and a blue labeled polynucleotide (absorbing at 426 nm and emitting at 480 nm) targeting sequences located proximal to the ROS1 breakpoint region in region 6q22.

[0244] When using the appropriate filter set, the hybridization signal of the non-rearranged ROS1 and / or ALK gene appears as a green-orange fluorescent fusion signal, and for the rearranged ROS1 and / or ALK gene, appears as a separate green and separate orange signal. In this regard, the ROS1-specific green signal is co-localized with the blue fluorescent signal, so that the non-rearranged ROS1 gene consists of orange and green / blue fluorescent mixed signals. The hybridization signal of the non-rearranged ALK gene appears as a green-orange fluorescent fusion signal, and the mixed signal without the blue fluorescent signal ( Fig. 9A). The 6q22 locus affected by ROS1 translocation is represented by independent green signals and a single orange signal ( Fig. 9 A (arrow).

[0245] At this point, the separate green signal overlaps with the blue signal (blue signal Fig. 9 B). This green / blue mixed signal indicates ROS1, but not ALK, as the gene affected by the translocation. Using an appropriate filter set can make the signal image easier to see.

[0246] Example 4: CISH analysis for detecting translocations of the ALK region in 2p23 using the quadruple CISH probe "ZytoDot SPECALK & ROS1 Break Apart Single-MIX NG-FISH probe" from ZytoVision Inc.

[0247] In addition, CISH analysis for detecting translocations of the ALK region in 2p23 was performed using a quadruple CISH probe "ZytoDot SPEC ALK&ROS1Break Apart SingleMIX NG-FISH Probe" by ZytoVision Inc. This probe is a mixture based on four site-specific hybridization probes, wherein the mixture consists of a digoxigenin-labeled polynucleotide targeting a sequence located proximal to the ALK breakpoint region in 2p23 and a sequence located proximal to the ROS1 breakpoint region in 6q22, a DNP-labeled polynucleotide targeting a sequence located distal to the ALK breakpoint region in 2p23 and a sequence located distal to the ROS1 breakpoint region in 6q22, and a biotin-labeled polynucleotide targeting a sequence located distal to the ROS1 breakpoint region in region 6q22. Detection of the label is performed by a primary (unlabeled) antibody (anti-DIG / anti-DNP / anti-BIO), which is followed by an enzymatic reaction of a secondary polymerase-conjugated antibody (HRP polymer / AP polymer / β-GAL), and a substrate (AP-RED / HRP-GREEN / β-GAL-BLUE), which results in the formation of strong, permanent, red, green, and blue signals that can be depicted by light microscopy, e.g., using a 40x dry lens.

[0248] Diploid or disomic nuclei without rearrangement or translocation of the ALK or ROS1 gene show two signals, each consisting of a red signal and a green signal, which are so close together that they cannot be separated or partially overlap or mix, and are specific for the two copies of the ALK gene ( Fig.10). In addition, in each case, two signals were visible, each consisting of a red signal, a green signal, and a blue signal, which were so close that they could not be separated or partially overlapped or mixed, and were specific for the two copies of the ROS1 gene.

[0249] Diploid or disomic nuclei with ALK gene rearrangement or translocation but not ROS1 allele rearrangement or translocation show red-green signal, which is specific for the non-rearranged ALK allele ( Fig.10 ). In addition, they show a single green signal and a single red signal separated from the former, which are specific for the rearranged ALK allele ( Fig.10 , arrows "G" = green, "R" = red). In addition, two red-green-blue signals are visible, which are specific for the two copies of the ROS1 gene.

[0250] Diploid or disomic nuclei with ROS1 gene rearrangement or translocation but without ALK allele rearrangement or translocation show red-green-blue signals, which are specific for the non-rearranged ROS1 allele. In addition, they show a single green signal and a single red-blue signal separated from the former for the rearranged ROS1 allele, as well as two red-green signals, which are specific for the two copies of the ALK gene.

[0251] Example 5: Using the five-fold FISH probe "Zytolight SPEC ERBB2, EGFR, FGFR1, MET & SOX2 FiveCheck" from ZytoVision TM FISH analysis of ERBB2 region amplification using "NG-FISH probe"

[0252] Finally, the fivefold FISH probes "Zytolight SPEC ERBB2, EGFR, FGFR1, MET & SOX2 FiveCheck" from ZytoVision were used. TMThe NG-FISH probe is a FISH assay for detecting ERBB2 region amplification. The probe is a mixture based on 5 site-specific hybridization probes, wherein the mixture consists of green labeled polynucleotides (absorbing at 503 nm and emitting at 528 nm) targeting regions 17q11.2-q12 of the ERBB2 gene, region 7p12 of the EGFR gene, region 8p11.23-p11.22 of the FGFR1 gene, region 7q31 of the MET gene, and region 3q26.3-q27 of the SOX2 gene, as well as blue labeled polynucleotides (absorbing at 426 nm and emitting at 480 nm) targeting regions of the EGFR gene and SOX2 gene, gold-yellow labeled polynucleotides (absorbing at 532 nm and emitting at 553 nm) targeting regions of the FGFR1 gene, and red labeled polynucleotides (absorbing at 580 nm and emitting at 599 nm) targeting regions of the MET gene and SOX2 gene.

[0253] When using an appropriate single bandpass filter bank, nine individual green signals and one green signal cluster can be seen that occupies the surface area of ​​multiple individual green signals, as well as four blue signals, two gold-yellow signals, and four red signals.

[0254] Overlay of the images shows that single green signals as well as clusters of green signals do not co-localize with signals of other colors. Single green signals refer to the non-amplified ERBB2 gene; clusters of green signals identify ERBB2 gene amplification. Co-localizing green / blue mixed signals identify two copies of the EGFR gene, co-localizing green / gold-yellow mixed signals identify two copies of the FGFR1 gene, co-localizing green / red mixed signals identify two copies of the MET gene, and co-localizing green / blue / red mixed signals identify two copies of the SOX2 gene.

[0255] Further aspects of the invention

[0256] The first aspect:According to the first aspect, the present invention relates to a method for detecting (and distinguishing) (multiple) chromosome structural and / or numerical aberrations based on directly or indirectly labeled nucleic acid fragments (probes) to detect multiple different chromosome regions or DNA regions in a cell, characterized in that 4 to 24 site-specific probes are each labeled with one of 1 to 24 different markers, and at least one site-specific probe is also simultaneously labeled with at least one additional marker (and a maximum of six additional markers), so that a mixed signal is generated by these mixed markers, wherein optionally, single probes with the same mixed markers can be distinguished by different proportions of the various markers, with the aid of the different mixed signals they may produce, so that the abnormal signal image in the case of chromosome aberration can be clearly located to the affected site, that is, the mixed signal image can be used to identify the site affected by the abnormality, wherein a mixed signal can be generated if all fragments of the probe or optionally only a single fragment a) are labeled with multiple markers and / or b) the same fragments are labeled with different markers and / or c) alternating fragments are labeled with different markers, wherein the above-mentioned fragments can also be superimposed in the genomic region or can also have a distance of up to 2 Mbp.

[0257] The second aspect: According to On the other hand, the present invention relates to a method according to the first aspect, wherein at least two, optionally three, optionally four, optionally five, optionally six, optionally seven, optionally eight, optionally nine, and optionally ten site-specific probes are simultaneously labeled with at least one additional label and up to six additional labels, so that a mixed signal is generated by these mixed labels.

[0258] The third aspect: According to another aspect, the invention relates to a method according to the first aspect, wherein at least 5 to 24, optionally 6 to 24, optionally 7 to 24 and optionally 8 to 24 site-specific probes are each labeled with one of 1 to 24 different labels.

[0259] The fourth aspect: According to On the other hand, the present invention relates to a method for detecting chromosome structural aberrations based on directly or indirectly labeled nucleic acid fragments (probes) to detect multiple different chromosome regions or DNA regions in cells, characterized in that

[0260] a first probe (probe 1) labeled with marker A and a second probe (probe 2) labeled with marker B flanking breakpoint region 1, the probes forming a fusion signal AB, and,

[0261] According to the same principle, 2 to 12 further probes (probes 3 to 14) flank 1 to 6 further breakpoint regions (breakpoint regions 2-7) and in each case also form a fusion signal AB, and,

[0262] Further probes, but at least one of the above probes is simultaneously labeled with a further label, in particular selected from labels C to F, or optionally with labels that differ from one another in ratio, so as to form a specific fusion signal and a mixed signal AB / X, wherein,

[0263] a) marker C and / or marker D and / or marker E and / or marker F may have different ratios in each case, and b) the specific fusion signal and the mixed signal AB / X change at the chromosomal aberration to form a new, individual mixed signal A / X or B / X, and c) optionally, in addition, the specific fusion signal becomes a new individual signal A or B (if no additional marker X is used for the probe pair) and d) based on these changed signal patterns, the affected breakpoint region can be clearly detected.

[0264] The fifth aspect: According to On the other hand, the present invention relates to a method for detecting chromosome structural aberrations based on directly or indirectly labeled nucleic acid fragments (probes) to detect multiple different chromosome regions or DNA regions in cells, characterized in that

[0265] A first probe (probe 1) labeled with marker A and a second probe (probe 2) labeled with marker B flanking breakpoint region 1, the probes forming a fusion signal AB, and

[0266] The third probe (probe 3) labeled with marker A and the fourth probe (probe 4) labeled with marker B located on the side of the breakpoint region 2 also simultaneously form a fusion signal AB, and probes 3 and / or 4 are simultaneously labeled with another marker C, thereby forming a fusion signal AB / C, wherein

[0267] The above signals change at the site of chromosome aberration to form separate signals A and / or B (breakpoint 1) or fusion signals A / C and / or B / C (breakpoint 2).

[0268] The sixth aspect: According to On the other hand, the invention relates to a method according to one of the preceding aspects, wherein firstly, only signals A and / or B are considered, based on the use of suitable filters, marker A and / or marker B are used, and only when there is an abnormal signal image of A and / or B, additional markers C to F are considered to clearly determine the affected chromosomal region.

[0269] The seventh aspect: According to On the other hand, the present invention relates to a method according to one of the preceding aspects, wherein the genomic region detected by the site-specific probe is less than 5 Mbp, optionally less than 2 Mbp, optionally less than 1 Mbp, optionally less than 750 kb, optionally less than 500 kb, optionally less than 250 kb, optionally less than 100 kb, optionally less than 10 kb, and optionally less than 1 kb.

[0270] The eighth aspect: According to On the other hand, the invention relates to a method according to one of the preceding aspects, wherein instead of mixed labeling of the probes, multiple identical probes with different labeling are used, wherein optionally, identical probes are considered to be identical even if they are at least 90%, optionally at least 80%, optionally at least 70%, optionally at least 60%, and optionally at least 50% identical.

[0271] Ninth aspect: According to another aspect, the present invention relates to a method according to one of the preceding aspects, wherein preferably chromosomal aberrations can be detected in malignancies, preferably in carcinomas, preferably in sarcomas, preferentially in leukemias.

[0272] The tenth aspect: According to another aspect, the invention relates to a method according to one of the preceding aspects, wherein the probe is a polynucleotide, a modified polynucleotide or a modified nucleic acid fragment or an oligonucleotide or a modified oligonucleotide.

[0273] The eleventh aspect: According to another aspect, the present invention relates to a method according to one of the preceding aspects, wherein the label is selected from a dye, a dye substrate, a chemiluminescent dye (e.g. an acridinium), a radioisotope, a spin label, an enzyme (e.g. alkaline phosphatase, horseradish peroxidase, soybean peroxidase and / or β-galactosidase), a hapten (e.g. digoxigenin, biotin, 5(6) carboxyfluorescein, rhodamine, bromodeoxyuridine, acetylaminofluorene, trinitrophenol, trinitrophenol derivatives, estradiol and / or DNP), quantum dots, beads, aminohexene, pyrene and a fluorescent dye (e.g. fluorescein, fluorescein derivatives, 5(6)-carboxyfluorescein, coumarin, coumarin derivatives, rhodamine, rhodamine derivatives, tetramethylrhodamine, lissamine, Texas Red, AMCA, TRITC, IR dyes, Alexa dyes, Dyomics dyes, phycoerythrin, Oregon Green 488, Pacific Blue and / or Rhodamine Green).

[0274] The twelfth aspect: According to On the other hand, the invention relates to a method according to one of the preceding aspects, wherein the method is performed by a FISH method using directly built-in fluorescent dyes for the entire visible, infrared and ultraviolet radiation range, preferably for the green, orange / red, red, gold and blue radiation range.

[0275] Thirteenth aspect: According to In another aspect, the invention relates to a method according to one of the preceding aspects, wherein the method is performed by the BrISH method using biotin, digoxigenin and DNP combined with antibody-coupled alkaline phosphatase, antibody-coupled peroxidase and antibody-coupled β-galactosidase.

[0276] Fourteenth aspect: On the other hand, the present invention relates to a method according to one of the preceding aspects, wherein the genes ALK, ROS1, RET, NRG1, NTRK1, CARS, EML4, FGFR2, FGFR3, KIF5B, TGF, BCR, ABL, ALK, BCL2, BCL6, BIRC3, CCND1, EGR1, ETV6, FGFR1, FGFR3, IGH, KMT2A, MYC, PML, RARA, RUNX1, RUNX1T1, EWSR1, CHOP, FUS, COL1A1, DDIT3, JAZF1, NR4A3, FOXO1, Chromosomal aberrations of FUS, PAX3, PAX7, PDGFB, SS18, TFE3, USP6, WT1, HER2 / ERBB2, FGFR1, ALK, CCND1, CDK4, CD274, PDCD1LG2, EGR1, EGFR, ESR1, ETV1, FGF3,4,19, FGFR2, FGFR3, FHIT(RCC), KRAS, MDM2, MDM4, MET, MYB, MYC, MYCN, PIK3CA, PTEN, SMARCB1, SOX2, TERT, TOP2A, TP53, TYMS, and / or VHL.

[0277] The fifteenth aspect: According to In another aspect, the present invention relates to a preparation for detecting and distinguishing chromosome structural and / or numerical aberrations based on directly or indirectly labeled nucleic acid fragments (probes) for detecting multiple different chromosome regions or DNA regions in cells,

[0278] wherein 4 to 24 locus-specific probes are each labeled with one of 1 to 24 different labels, and

[0279] At least one site-specific probe is also simultaneously labeled with at least one additional marker (and a maximum of six additional markers) such that a mixed signal is generated by these mixed markers, wherein optionally, individual probes with the same mixed markers can be distinguished by means of different proportions of the various markers, with the aid of the different mixed signals they may generate, so that the abnormal signal image in the case of chromosomal aberrations can be clearly localized to the affected site and / or the site affected by the abnormality can be identified using the mixed signal image, wherein a mixed signal can be generated if all fragments of the probe or optionally only a single fragment a) are labeled with multiple markers and / or b) the same fragments are labeled with different markers and / or c) alternating fragments are labeled with different markers, wherein the above-mentioned fragments can also be superimposed in the genomic region or can also have a distance of up to 2 Mbp.

[0280] The sixteenth aspect:According to another aspect, the present invention relates to a preparation according to the fifteenth aspect, wherein the probe is configured according to the first to thirteenth aspects.

Claims

1. A composition for detecting at least two chromosomal aberrations independently of each other by in situ hybridization, wherein the composition comprises at least four site-specific hybridization probes, in each case the probes are different from each other and are labeled with a first detection label, and at least one of the site-specific hybridization probes is labeled with an additional detection label, which is different from the first detection label according to the corresponding site-specific hybridization probe, and wherein the hybridization probe is directly labeled with the detection label, and wherein the detection label is a fluorescent dye, wherein the at least four site-specific hybridization probes include first and third site-specific hybridization probes labeled with detection marker A, and second and fourth site-specific hybridization probes labeled with detection marker B, wherein the first and second site-specific hybridization probes are configured to be located on the side of the chromosome fragment and to generate a fusion signal AB in a signal image generated by in situ hybridization, wherein the third and fourth site-specific hybridization probes are configured to flank the chromosome fragment and produce a fusion signal AB in a signal image generated by in situ hybridization, and At least one of the site-specific hybridization probes is labeled with at least one additional detection label X, so that the site-specific hybridization probe labeled with at least one additional detection label generates a fusion signal and a mixed signal AB / X in the signal image generated by in situ hybridization.

2. The composition according to claim 1, wherein the detection label is a material or substance coupled to the nucleic acid or nucleic acid fragment in the probe.

3. A composition for the diagnosis and / or prognosis of a disease associated with chromosomal aberrations, wherein the disease is selected from the group consisting of: malignant tumors, wherein in each case, the composition comprises at least four site-specific hybridization probes which are different from each other and which are labeled with a first detection label, and wherein at least one of the site-specific hybridization probes is labeled with at least one additional detection label which is different from the first detection label of the corresponding site-specific hybridization probe, and wherein the hybridization probe is directly labeled with the detection label, and wherein the detection label is a fluorescent dye, wherein the at least four site-specific hybridization probes include first and third site-specific hybridization probes labeled with detection marker A, and second and fourth site-specific hybridization probes labeled with detection marker B, wherein the first and second site-specific hybridization probes are configured to be located on the side of the chromosome fragment and to generate a fusion signal AB in a signal image generated by in situ hybridization, wherein the third and fourth site-specific hybridization probes are configured to flank the chromosome fragment and produce a fusion signal AB in a signal image generated by in situ hybridization, and At least one of the site-specific hybridization probes is labeled with at least one additional detection label X, so that the site-specific hybridization probe labeled with at least one additional detection label generates a fusion signal and a mixed signal AB / X in the signal image generated by in situ hybridization.

4. The composition according to claim 3, wherein the detection label is a material or substance coupled to the nucleic acid or nucleic acid fragment in the probe.

5. The composition according to any one of claims 3-4, wherein the malignancy is selected from carcinoma, sarcoma and / or leukemia.

6. A kit for detecting at least two chromosomal aberrations independently of each other by in situ hybridization, comprising at least four site-specific hybridization probes different from each other, each of the site-specific hybridization probes being labeled with a first detection label, wherein at least one of the site-specific hybridization probes is labeled with at least one additional detection label, the additional detection label being different from the first detection label according to the corresponding hybridization probe, and wherein the hybridization probe is directly labeled with the detection label, and wherein the detection label is a fluorescent dye, wherein the at least four site-specific hybridization probes include first and third site-specific hybridization probes labeled with detection marker A, and second and fourth site-specific hybridization probes labeled with detection marker B, wherein the first and second site-specific hybridization probes are configured to be located on the side of the chromosome fragment and to generate a fusion signal AB in a signal image generated by in situ hybridization, wherein the third and fourth site-specific hybridization probes are configured to flank the chromosome fragment and produce a fusion signal AB in a signal image generated by in situ hybridization, and At least one of the site-specific hybridization probes is labeled with at least one additional detection label X, so that the site-specific hybridization probe labeled with at least one additional detection label generates a fusion signal and a mixed signal AB / X in the signal image generated by in situ hybridization.

7. The kit according to claim 6, wherein the detection label is a material or substance coupled to the nucleic acid or nucleic acid fragment in the probe.

8. The kit according to claim 7, wherein the at least four site-specific hybridization probes that are different from each other are present in a common composition.

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