A method for the preparation of one or more formalin fixed paraffin embedded tumor tissue samples for use in a subsequent analysis

Abstract

The disclosure relates to a method for the preparation of one or more formalin fixed paraffin embedded (FFPE) tumor tissue sample(s) for use in a subsequent analysis, such as in fluorescent in-situ hybridization, comprising the steps of: (a) deparaffinizing the sample; (b) heat treating the sample; (c) optionally permeabilizing the sample; (d) optionally acid treating the sample; (e) enzyme treating the sample, by adding at least one proteolytic enzyme to the sample; (f) formamide treating the sample; (g) optionally re-fixating the sample; (h) hybridizing the sample; thereby obtaining a tumor tissue sample having an essentially intact morphology prepared for subsequent FISH analysis.

Description

[0001] A method for the preparation of one or more formalin fixed paraffin embedded tumor tissue samples for use in a subsequent analysis

[0002] Technical field

[0003] The present disclosure relates to a method for the preparation of one or more formalin fixed paraffin embedded tumor tissue samples for use in a subsequent analysis. More specifically, the disclosure relates to a method for the preparation of one or more formalin fixed paraffin embedded tumor tissue sample for use in a subsequent analysis as defined in the introductory part of the independent claim(s).

[0004] Background art

[0005] There are many publications in the literature on methods for the isolation of DNA, RNA, and proteins from formalin-fixed, paraffin-embedded (FFPE) tissue. Common to these methods is that they strive for maximum yield of DNA, RNA or protein for subsequent chemical analysis. To achieve this, the tissue must be destroyed. Such methods are therefore not applicable for microscopic in situ analysis, which requires intact tissue structure and well-preserved cells and nuclei (well-preserved morphology).

[0006] There are also quite a few publications concerning immunohistochemistry (IHC) for protein analysis in situ ("antigen retrieval"). Although the tissue architecture is usually well enough preserved to be able to localize specific proteins to cell and tissue structures, these methods are still too destructive to the chromatin and therefore give too weak and highly variable FISH signals to allow sufficiently accurate high-resolution genetic analyses.

[0007] CN-A-107828862 discloses a pretreatment method for fluorescence in-situ hybridization (FISH) detection of paraffin-embedded sections of lymphoma tissue using pepsin as a part of an enzyme treatment. CN-A-103382499 discloses a method for sample pretreatment for reducing autofluorescence interference in FISH, including use of e.g., sodium thiocyanate, collagenase type IV and pepsin in different steps. CN-A-115261368 discloses a ready-to-use pepsin working fluid for pretreatment enzymatic digestion of FISH FFPE samples. CN-A-105018598 discloses pretreatment fluids for FFPE samples, wherein FISH autofluorescence is reported to be reduced for liver cancer samples. Also, Leong et al. (Pathology. 2010 Jan; 42(1):77-81. “Citraconic anhydride: a new antigen retrieval solution") discloses the use of citraconic anhydride at elevated temperatures for immunohistochemistry. Teng et al. (Virchows Arch. 2017 May;470(5):567-573. doi: 10.1007 / s00428-017-2097-z. Epub 2017 Feb 25., "A new method for real-time evaluation of pepsin digestion of paraffin-embedded tissue sections, prior to fluorescence in situ hybridization") discloses a method for determining optimal time of enzyme treatment.

[0008] Chin et al (Mol Pathol. 2003 Oct;56(5):275-9. doi: 10.1136 / mp.56.5.275. "A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumor samples") discloses what may be regarded as the latest standard method for heat treatment of FFPE samples. With the aim of preparing FFPE samples for FISH, it is disclosed that dewaxed tissue sections were incubated in lOmM citric acid buffer at 80 degrees C for 30 minutes to two hours, followed by a short pepsin digestion (1-5 mg / ml). Pretreated tissues were co-denatured with DNA probes at 80 degrees C for 10 minutes, followed by hybridization at 37 degrees C for 48-72 hours.

[0009] A problem with the solutions of the prior art is that they are typically too destructive for the sample tissue causing artificial variability within the sample, and / or they do not provide a tissue sample that enables subsequent analysis with a sufficient resolution. There is thus a need for improved methods solving these problems.

[0010] Summary

[0011] It is therefore an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages in the prior art and solve at least one of the above-mentioned problems.

[0012] According to a first aspect there is provided a method for the preparation of one or more formalin fixed paraffin embedded tumor tissue samples for use in a subsequent analysis, such as in fluorescent in-situ hybridization (FISH), comprising the steps of:

[0013] (a) deparaffinizing the sample;

[0014] (b) heat treating the sample by (i) adding citrate and exposing the sample to an increased temperature of about 80 °C at a duration of about 45 to 120 minutes, (ii) adding glycerol at a concentration of 10% to 30% to the sample and exposing the sample to an increased temperature in the interval of about 90-95 °C at a duration of about 10-30 minutes, and (iii) adding dithiotreitol (DTT) and ethylene diamine tetraacetate (EDTA) to the sample and exposing the sample to an increased temperature of about 80 °C for 10-30 min, wherein steps (i)-(iii) can be performed in any order;

[0015] (c) optionally permeabilizing the sample;

[0016] (d) optionally acid treating the sample;

[0017] (e) enzyme treating the sample, by adding at least one proteolytic enzyme to the sample;

[0018] (f) formamide treating the sample;

[0019] (g) optionally re-fixating the sample; hybridizing the sample, thereby obtaining a tumor tissue sample having an essentially intact morphology prepared for subsequent FISH analysis. Typically, said sample exhibits at least 5 %, at least 10%, at least 20%, at least 50 % at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more increase of a fluorescent gene signal as compared to a sample exposed to a standard preparation.

[0020] Thus, the present inventors have focused on developing and combining various "gentle" methods, which do not change the morphology, but which at the same time break the methylene bridges between protein and DNA in the chromatin in the most efficient way possible, which were formed during the formalin fixation, and which prevent FISH - penetration of the probe through the cell and binding to the gene in the chromatin. This creates clearly improved prerequisites for the FISH technology in the form of strong (and nonvariable) gene-specific signals in the morphologically well-preserved cell nuclei. Thus, the method substantially enhances the gene specific signal in a FISH staining without affecting tissue architecture or cellular morphology.

[0021] By the term "in association with" is in the context of the heat treatment steps (b) (i), (ii) and (iii) meant that the steps are performed directly before or after each other, so that all heat treatment steps are performed in one block. However, the order of the steps can vary depending on the specific embodiment. Step (b)(ii) of adding glycerol during heat treatment has the effect of stabilizing the tissue, which thereby tolerates a higher temperature.

[0022] Step (b)(iii) of adding DTT-EDTA during heat treatment has the effect of reducing disulphide and electrostatic interaction, thereby facilitating subsequent analysis of the sample.

[0023] In some embodiments, the method further comprises an enzyme treatment performed as part of step (e), wherein pepsin is added at a concentration of 0,01 to 0,1 mg / ml in combination with at least one additional enzyme having the ability to increase the effective concentration of pepsin.

[0024] The step of adding pepsin at a low concentration (thereby limiting or completely avoiding negative effects on morphology) together with at least one additional enzyme during enzyme treatment has the effect of increasing the concentration window of pepsin, thereby providing an up to 10-fold increased positive effect of pepsin. Thus, by adding pepsin at a low concentration, negative effects on morphology are limited or completely avoided.

[0025] In some embodiments, the method further comprises a refixation performed as part of step (g), comprising an alkaline treatment in order to stabilize the sample, followed by (i) a first fixation by adding methanol and acetic acid, and (ii) a second fixation by adding formalin for 5 to 20 min at a concentration of about 1%.

[0026] The step of re-fixating the sample has the effect of stabilizing chromatin in a decondensed state, with decreased interaction between protein and DNA, thereby exposing more of the DNA in the chromatin, making it accessible for the FISH probe. A further improved effect is obtained if three steps are combined: (1) increased pH (to reduce number of bonds between DNA and histone), (2) fixation of DNA in methanol - acetic acid (to fixate DNA in a partially decondensed form), and (3) stabilising chromatin partially with low concentration of formalin (in order to cope with any subsequent FISH colouring (which is performed at a high temperature) without being destructed).

[0027] According to some embodiments, step (b) (ii ) is performed before step (b)(i) of adding citrate to the sample, thereby obtaining an improved effect. According to some embodiments, step (b)(iii) is performed after step (b)(i) of adding citrate to the sample, thereby obtaining an improved effect.

[0028] According to some embodiments, citraconic anhydride is added at a concentration of about 0.01% to the sample during the heat treatment of step (i) and / or step (ii), and exposing the sample to an increased temperature and a duration in accordance with step (i) and / or (ii).

[0029] According to some embodiments, citraconic anhydride is added at a concentration in the interval of 0,002-0,01% and SDS is added at a concentration of about 0.2% during the heat treatment of step (i) and / or step (ii), and exposing the sample to an increased temperature and a duration in accordance with step (i) and / or (ii).

[0030] By adding citraconic anhydride (CA) according to this disclosure, CA "opens up" hidden DNA- protein complexes that become available for the gene specific FISH probe. By lowering CA concentration, three effects were obtained: (1) the cells remain on the glass, (2) good cell morphology and (3) increased FISH signal. The most preferred effect (most reproducible results) is obtained if CA is performed in early steps of heat treatment (Gly90 and CB80). If performed after DTT-EDTA, the sample may de-attach from glass and decrease FISH signal.

[0031] According to some embodiments, the heat-treated sample is exposed to an enzyme treatment, wherein pepsin is added at a concentration of about 0,05 mg / ml in combination with a collagenase. Also, since a low dose of pepsin is used, the pepsin treatment could potentially be repeated should one want the effect of the pepsin treatment to be increased and / or if insufficient results are obtained after the first pepsin treatment.

[0032] The combination of pepsin and collagenase considerably increased the "concentration window", so that pepsin had a positive effect at a 10-fold lower concentration, without any negative effect on morphology.

[0033] According to some embodiments, the collagenase is collagenase III.

[0034] According to some embodiments, formamide (FA) treatment is performed after the heat treatment and the enzyme treatment, and before the optional refixation.

[0035] This has the effect of breaking hydrophobic interactions to facilitate the charged water-soluble FISH-probe to penetrate compact protein structures surrounding the chromatin. An improved effect is observed if FA performed after heat and enzyme treatment, but before refixation, since, heat and enzyme treatment has been observed to make the FFPE tissue sample sensitive for FA treatment.

[0036] According to some embodiments, 1, 2 or 3 or of the optional steps (c), (d) and (g) are performed.

[0037] Thus, in its most specific definition, all optional steps of the method are performed, thereby obtaining all associated benefits of the disclosed method.

[0038] In summary, the method of the present disclosure is well suited for applications related to counting of copy number abnormalities, for which standards and demands become increasingly higher, thereby requiring an advanced, quantitative FISH analysis. By increasing the signal (as is evident from e.g. figure 3, showing a very high signal-to-noise ratio), and maintaining the morphology of the sample, as provided by the method of the present disclosure, these objects can be achieved. The resulting effect is that cancer diagnostics is developed and improved for the benefit of patients.

[0039] Moreover, the embodiments and features of the invention have at least the following advantageous effects:

[0040] - The step of adding glycerol during heat treatment has the effect of stabilizing the tissue, which thereby tolerates a higher temperature.

[0041] - The step of adding DTT-EDTA during heat treatment has the effect of reducing disulphide and electrostatic interaction, thereby facilitating subsequent analysis of the sample.

[0042] - The inventors made the interesting discovery that the strength of the FISH signal increased if the formamide (FA) treatment was performed after the heat treatment and the enzyme treatment (as described above), but before the refixation. Thus, it appears that these pretreatments sensitize the FFPE tissue to the subsequent FA treatment. Formamide treatment as such is known

[0043] - The purpose of the refixation is to stabilize the chromatin in a maximally decondensed form with reduced interaction between protein and DNA. Thereby, more of the DNA in the chromatin would be exposed and available for binding by the FISH probe. Further, by combining different sample treatment steps a clearly improved signal to noise ratio is obtained, as can be seen from figure 1-3, where the individual contribution of each single treatment step is shown (figure 1-2) as well as the resulting signal when all steps are combined.

[0044] Thus, the present inventors have focused on developing and combining various relatively gentle methods, which do not change the morphology, but which at the same time break the methylene bridges between protein and DNA in the chromatin in the most efficient way possible, which were formed during the formalin fixation, and which prevent FISH-penetration of the probe through the cell and binding to the gene in the chromatin. This creates clearly improved prerequisites for the FISH technology in the form of strong (and non-variable) genespecific signals in the morphologically well-preserved cell nuclei. Thus, the method substantially enhances the gene specific signal in a FISH staining without affecting tissue architecture or cellular morphology.

[0045] In summary, the method of the present disclosure is well suited for applications related to counting of copy number abnormalities, for which standards and demands become increasingly higher, thereby requiring an advanced, quantitative FISH analysis. By increasing the signal (as is evident from e.g. figure 3, showing a very high signal-to-noise ratio), and maintaining the morphology of the sample, as provided by the method of the present disclosure, these objects can be achieved. The resulting effect is that cancer diagnostics is developed and improved for the benefit of patients.

[0046] The present disclosure will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments of the disclosure by way of illustration only. Those skilled in the art understand from guidance in the detailed description that changes and modifications may be made within the scope of the disclosure.

[0047] Hence, it is to be understood that the herein disclosed disclosure is not limited to the particular component parts of the device described or steps of the methods described since such device and method may vary. It is also to be understood that the terminology used herein is for purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings does not exclude other elements or steps.

[0048] Definitions

[0049] By the term "formalin fixed paraffin samples (FFPE)" is meant samples that have been preserved and prepared for subsequent analysis by formalin fixation and paraffin embedment. A skilled person in this technology would be aware of this technology. Typically, such samples originate from expected or known tumor tissue of a human or animal subject.

[0050] By the term "gene signal compared to a sample exposed to a standard preparation" is meant a quantified and comparable signal of a gene and / or nucleic acid probe, e.g., as measured by FISH technology.

[0051] By the term "essentially intact morphology" is meant that the morphology of the tissue of the sample is essentially intact as a result of the pretreatment steps, thereby allowing measurement and analysis of the intact tissue.

[0052] The shortages used in this disclosure have the following meanings: "CB80" refers to citrate buffer at about 80C, "Enz" refers to enzyme treatment, "Gly 90" refers to glycerol treatment at about 90 C, "FA" refers to formamide treatment, "DTT-EDTA" refers to dithiotreitol- ethylene diamine tetraacetate, "Re-FIX" refers to refixation and "CA-SDS" refers to citraconic anhydride - sodium dodecyl sulfate.

[0053] Brief descriptions of the drawings

[0054] Figure 1 discloses the gene signal over local background for FFPE sections, by showing the relative effect (% increase) of each individual per-hybridization condition (mean-range), i.e., CB 80 Enz, Gly 90, FA, DTT-EDTA, Re-FIX and CA-SDS.

[0055] Figure 2 discloses the gene signal over local background for FFPE sections, by showing the accumulated effect of successive additions of different pre-hybridization conditions, i.e., CB 80 NoEnz, CB 80 Enz, Gly90, DTT-EDTA, RE-Fix, CA-SDS and FA. Figure 3 discloses FISH results for a clinical FFPE sample, showing gene specific probe binding vs non-specific probe binding in the cell nucleus and background outside the cell nucleus (so called "black" background).

[0056] Detailed description

[0057] The above objects, as well as additional objects, features and advantages of the present disclosure, will be more fully appreciated by reference to the following illustrative and nonlimiting detailed description of example embodiments of the present disclosure, when taken in conjunction with the accompanying drawings and examples. The disclosure may, however, be embodied in other forms and should not be construed as limited to the herein disclosed embodiments. The disclosed embodiments are provided to fully convey the scope of the disclosure to the skilled person.

[0058] Standard method

[0059] In the development of the results presented in this disclosure, the inventors have started from the current standard method for FISH analysis of paraffin sections (FFPE): (Chin et al., 2003 (Mol Pathol. 2003 Oct;56(5):275-9. doi: 10.1136 / mp.56.5.275.), “ simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumor samples").

[0060] Improved method according to the present disclosure

[0061] The present disclosure describes a new method that radically improves the possibilities of using fluorescence in situ hybridization methodology (FISH), for specific staining of individual genes, in formalin-fixed and paraffin-embedded histopathological tumor material (FFPE), which is routinely used in clinical cancer diagnostics.

[0062] The method also allows the use of advanced FISH technology on archival histopathological material, which has been stored in paraffin blocks for many decades. As such material can be linked to patient databases, new biobanks worldwide open up for unique retrospective cancer genetic research studies with long clinical follow-up time. The method is based on an extensive chemical and enzymological study, where the effect of various pretreatments of the FFPE material, prior to subsequent FISH staining, was analyzed quantitatively. The focus has been to identify and combine "mild" pretreatment methods, which amplify the FISH signal in the cell nucleus without damaging the architecture of the tissue or the morphology of the individual cells.

[0063] A large number of experimental conditions were tested. Among most of the conditions where an effect was expected, the inventors found a negative or no effect. However, the inventors found six different pretreatment conditions that showed a limited but significant effect (fig. 1).

[0064] These six pretreatment conditions were found to have an additive or in some cases synergistic effect. Despite the limited positive effect exhibited by each of these six pretreatment conditions, their combination provided a dramatic enhancement of the FISH signal above the local background (figs 2 and 3).

[0065] The first aspect of this disclosure shows a method for the preparation of one or more formalin fixed paraffin embedded FFPE tumor tissue samples for use in a subsequent analysis, such as in fluorescent in-situ hybridization (FISH), comprising the steps of:

[0066] (a) deparaffinizing the sample;

[0067] (b) heat treating the sample by (i) adding citrate and exposing the sample to an increased temperature of about 80 °C at a duration of about 45 to 120 minutes, (ii) adding glycerol at a concentration of 10% to 30% to the sample and exposing the sample to an increased temperature in the interval of about 90-95 °C at a duration of about 10-30 minutes, and (iii) adding dithiotreitol (DTT) and ethylene diamine tetraacetate (EDTA) to the sample and exposing the sample to an increased temperature of about 80 °C for 10-30 min, wherein steps (i)-(iii) can be performed in any order;

[0068] (c) optionally permeabilizing the sample;

[0069] (d) optionally acid treating the sample;

[0070] (e) enzyme treating the sample, by adding at least one proteolytic enzyme to the sample; (f) formamide treating the sample;

[0071] (g) optionally re-fixating the sample;

[0072] (h) hybridizing the sample; thereby obtaining a tumor tissue sample having an essentially intact morphology prepared for subsequent FISH analysis. Typically, said sample exhibits at least 5 %, at least 10%, at least 20%, at least 50 % at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500% or more increase of a fluorescent gene signal as compared to a sample exposed to a standard preparation.

[0073] Tumor tissues

[0074] Samples to be used in the methods of the present disclosure typically originate from a known or expected tumor tissue, with the purpose of analyzing or measuring the presence, growth and / or status of a cancer related tumor. Examples of tissues from which the samples may originate are breast, prostate, colorectal, lung, cervical, endometrial, urinary bladder and pancreas.

[0075] Deparaffinization (or dewaxing)

[0076] The first step of the method according to the present disclosure relates to the deparaffinization (or dewaxing) of a FFPE sample. The first step is performed by any technique for removing paraffin or wax from the sample as known in the art.

[0077] Heat treatment

[0078] As a second step of the method according to the present disclosure, heat treatment is performed in one or more sub-steps.

[0079] According to the standard method of heat treatment (see above), which breaks some of the methylene bridges formed during the formalin fixation, the paraffin section is heated to 80 degrees for 45 to 120 minutes in citrate buffer (CB 80, Fig 1 and 2). If one applies a higher temperature, both the morphology of the cell nucleus and the DNA in the chromatin are damaged, which leads to a weak or no FISH signal. (1) High temperature in the presence of glycerol.

[0080] With the aim of making the heat treatment more efficient than the standard method, i.e. breaking more methylene bridges, but at the same time not damaging the DNA or the morphology of the cell nucleus, the inventors tested a large number of different heat treatments at a higher temperature for a limited time and in the presence of different chemicals. The inventors then found that the addition of 10 to 30 % glycerol, typically about 20% glycerol, had a protective effect against high temperature. The inventors were now able to go up to 90 degrees for 10 minutes (Gly90, figs 1 and 2), which resulted in an enhancement of the FISH signal without deterioration of the nuclear morphology. It was also observed that the Gly90 treatment had the most beneficial effect if it was performed before the standard treatment (CB80).

[0081] (2) disulfide bridges and electrostatic bonds.

[0082] In addition to methylene bridges, which are formed during formalin fixation, disulfide bridges, which are formed during oxidation, and electrostatic bonds via calcium and magnesium ions also contribute to creating macromolecular complexes, which prevent the penetration of the FISH probe into the tissue section and binding to DNA. The inventors have also been able to show that heat treatment in the presence of Dithiothreitol (DTT, a thiolated sugar alcohol, which is used as a reducing agent in biochemistry to break sulfur bridges in proteins) and EDTA (Ethylene Diamine Tetra Acetate, which has the property of binding divalent positive ions to itself), further enhances the FISH signal without affecting the morphology of the cell nucleus (DTT-EDTA, figs 1 and 2). It was also observed that the DTT-EDTA treatment had the most beneficial effect if it was performed after the standard treatment (CB80).

[0083] (3) citraconic anhydride (see Leong et al., Pathology. 2010 Jan; 42(1): 77-81. "Citraconic anhydride: a new antigen retrieval solution").

[0084] Another way to make the heat treatment more efficient, without damaging the DNA in the chromatin and the morphology of the cell nucleus, is to use citraconic anhydride (CA), which was previously used in protein analysis with immunohistochemistry, IHC (see Leong et al., above). CA interferes with the amino groups of lysine and arginine in such a way that epitopes, which were hidden in hydrophobic protein complexes, can now be exposed to the antibodies and visualized by IHC. Correspondingly, one can imagine that CA could "open" hidden DNA- protein complexes, which thereby become accessible to the gene-specific FISH probe. The reason why some positive CA results have not been presented before in the FISH technology may be that (a) CA was not tested for FISH at all, or (b) that too high a concentration and temperature were used, 0.05% and 98 °C respectively, as with IHC. Under such experimental conditions, the FISH technique appears not to work. Most of the tumor tissue detaches from the slide during FISH staining and no gene-specific FISH signal is seen in the few cell nuclei that may remain on the slide. When the present inventors drastically reduced the CA concentration to one-tenth (0.005%) and at the same time kept the temperature in the range of 80 to 90 °C, it was surprisingly found that the CA treatment worked in three respects: (1) the cells remained on the glass, (2) good cell morphology was achieved, and at the same time (3) an amplified FISH signal was observed. Furthermore, it was found that the timing of CA treatment was critical for the outcome. The most reproducible results were obtained if the CA treatment was performed during the early stage of heat treatment (steps Gly90 and / or CB80). If performed during or after the DTT-EDTA step, the specimen often detached from the glass and the FISH signal was degraded.

[0085] In embodiments, both glycerol addition and DTT / EDTA addition steps are included in the heat treatment.

[0086] In embodiments, the step of glycerol addition is performed before the step of adding citrate to the sample.

[0087] In embodiments, the step of DTT / EDTA addition is performed after the step of adding citrate to the sample.

[0088] In embodiments, citraconic anhydride CA is added at a concentration of about 0.01% to the sample during the heat treatment of adding citrate) and / or adding glycerol and exposing the sample to an increased temperature in accordance with the step of adding citrate and / or adding glycerol, respectively.

[0089] In embodiments, citraconic anhydride CA is added at a concentration in the interval of 0,002- 0,01% and SDS is added at a concentration of about 0.2% during the heat treatment of the step of adding citrate and / or adding glycerol and exposing the sample to an increased temperature in accordance with step of adding citrate and / or adding glycerol, respectively.

[0090] Sample permeabilization

[0091] As a third, optional, step of the method of the present disclosure, the sample is permeabilised in Tween, Triton X, NP40 or any other similar detergent.

[0092] Acid treatment

[0093] As a fourth, optional, step of the method of the present disclosure, the sample is acid treated, e.g., using HCI at pH 1-2 for 5 minutes.

[0094] Enzymatic treatment

[0095] As a fifth step of the method of the present disclosure, the sample undergoes enzymatic treatment in one or more sub-steps exposing the sample for one or more enzymes.

[0096] The general problem with enzyme treatments is the narrow range in enzyme concentration and treatment time that produces the desired effect or habituation damage. One way to try to optimize enzyme treatment with respect to treatment time is described below (Teng et al., Virchows Arch. 2017). But even with this method, unfortunately, you often see areas in the same preparation that are under-digested (no FISH signal) and areas that are over-digested (destroyed tissue with destroyed cell morphology). This is unsatisfactory, especially as the inventors were interested in examining the clonal genetic variation within the entire heterogeneous tumor tissue. A large number of different enzymes, both exopeptidases and endopeptidases, were therefore unconditionally tested in varying concentrations and treatment times, partly individually and partly in different combinations. This proved to be a successful approach that outweighed the extensive work effort. Surprisingly, the inventors found that pepsin could be used at a 20- to 100-fold lower concentration than is normally used (standard method, as disclosed in Chin et al., 2003), if the pepsin digestion was combined with collagenase. Strong FISH signal and good morphology could now be seen over the entire specimen with only slight variation within different areas of the tumor tissue. Most of the time, low-concentration pepsin followed by collagenase is sufficient, but in a small number of cases, the best result was obtained after the combination of low-concentration pepsin followed by collagenase and then another round of low-concentration pepsin.

[0097] In embodiments, an enzyme treatment is performed, wherein pepsin is added at a concentration of about 0,01 to 0,1 mg / ml, typically about 0,05 mg / ml in combination with at least one additional enzyme having the ability to increase the effective concentration of pepsin.

[0098] In embodiments, the heat-treated sample is exposed to an enzyme treatment, wherein pepsin is added at a concentration of about 0,01 to 0,1 mg / ml, typically about 0,05 mg / ml in combination with a collagenase.

[0099] In embodiments, the collagenase is collagenase III.

[0100] Formamide treatment

[0101] As a sixth step of the method of the present disclosure, the sample is formamide treated.

[0102] Formamide (FA) interferes with and destabilizes hydrophobic bonds in macromolecular complexes. Within the FISH methodology, FA has long been used to destabilize the hydrogen bonds between the two chains of the DNA molecule. This is for purely practical reasons to be able to perform the FISH staining at a lower temperature, 50 °C instead of 90 °C. However, no positive effect on the FISH signal itself is seen. The question raised by the inventors, however, was whether FA could interfere with protein-protein bonds or protein-DNA bonds in formalin- fixed paraffin sections (FFPE) in such a way that the penetration of the FISH probe through the cell and into the chromatin was facilitated and thus resulted in a stronger FISH-signal. The inventors found that FA alone had no detectable effect. However, the inventors made the interesting discovery that the strength of the FISH signal increased if the FA treatment was performed after the heat treatment and the enzyme treatment (as described above), but before the refixation. Thus, it appears that these pretreatments sensitize the FFPE tissue to the subsequent FA treatment. Typically, the FA treatment is performed at room temperature for more than 2 hours, and for practical reasons often overnight. In embodiments, formamide treatment is performed after the heat treatment and the optional enzyme treatment, and before the optional refixation.

[0103] Re-fixation

[0104] As a seventh, optional, step of the method of the present disclosure, the sample is re-fixated.

[0105] In embodiments, a refixation is performed, comprising an alkaline treatment in order to stabilize the sample, followed by (i) a first fixation by adding methanol and acetic acid, and (ii) a second fixation by adding formalin at a concentration of about 1%.

[0106] The purpose of the refixation is to stabilize the chromatin in a maximally decondensed form with reduced interaction between protein and DNA. Thereby, more of the DNA in the chromatin would be exposed and available for binding by the FISH probe. The inventors have succeeded beyond expectations by combining three fundamentally different physical-chemical treatments. First a treatment of the tissue preparation in elevated pH (about pH 9-10 for 5 minutes) to reduce the number of bonds between DNA and histone, then with methanolacetic acid fix the DNA in this now partially decondensed form, and finally stabilize the chromatin partially with low concentration of formalin, to withstand the subsequent FISH- coloring, which takes place at a high temperature, without being destroyed.

[0107] Sample hybridization

[0108] As a final step of the method of the present disclosure, the sample is hybridized. Any standard FISH procedure can be used, such as iFISH.

[0109] Subsequent analysis

[0110] After the pretreatment steps, the sample may undergo further analysis, typically by fluorescent in situ hybridization (FISH).

[0111] The person skilled in the art realizes that the present disclosure is not limited to the preferred embodiments described above. The person skilled in the art further realizes that modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed disclosure, from a study of the drawings, the disclosure, and the appended claims. The aspects of the present disclosure will now be further explained by way of the following detailed examples.

[0112] EXAMPLES

[0113] Example 1 - Experimental protocol

[0114] DAY 1

[0115] 1. Deparaffinization

[0116] A standard procedure in pathology to remove all paraffin. All deparaffinization steps below are performed in a fume hood.

[0117] - Heat FFPE slide @60°C, lh

[0118] - Xylene, 3min

[0119] - Xylene, 3min

[0120] - ETOH 100%, 3 min

[0121] - ETOH 100%, 3 min

[0122] - ETOH 85%, 2min

[0123] - ETOH 70%, 2min

[0124] - ETOH 50%, 2min

[0125] Wash in Nuclease free water, 3min'

[0126] 2. Heat Treatments

[0127] During formalin fixation methylene bridges are formed which crosslinks protein to DNA. The idea behind the heat treatment is to break the methylene bridges and thereby convert highly condensed chromatin structures into a more de-condensed state.

[0128] The heat-treatments are performed in 3 subseguent steps (1-3 below).

[0129] 2.1 Step 1 (Gly90

[0130] High temperature in the presence of glycerol that protects cell structures

[0131] 90 °C, for 10 min in 20% glycerol in O.lx SSC pH 7,5 with or without (CA) citraconic anhydride (0.01 %} Step 2 (DTT-EDTA)

[0132] DTT and EDTA at high PH to reduce disulfide and electrostatic crosslinks in chromatin

[0133] - DTT lOmM, EDTA 1 mM, Tris-CI lOOmM, pH9 at 80°C for 20min Step 3 (CB80)

[0134] A standard procedure used in immune-histochemistry (antigen retrieval), now (according to the present disclosure) with low concentration of citraconic anhydride (CA) (0.002- 0.04%)

[0135] Perform the Sodium citrate citraconic anhydride (CA) (0.002-0.04%) buffer step in a water bath

[0136] Warm to 80°C for 45min in 0.01M Sodium citrate buffer pH 6.0 + tween 0.05% (the solution should be 80°C from start)

[0137] Cool down the slide to RT (room temp) for 30min (still inside the sodium citrate buffer) ermeabilization

[0138] -wash 2 x 5 min in lxPBS / 0.05% Triton X-100, RT, while gently rotating

[0139] -incubate in lxPBS / 0.5% Triton X-100 for 20 min RT

[0140] -wash 2 x 5 min in lxPBS / 0.05% Triton X-100 RT (while gently rotating) cid treatment rinse briefly in 0.1M HCI at RT incubate in 0.1M HCI 5 min at RT rinse 3 x quickly with lxPBS / 0.05% Triton X-100 at RT

[0141] - wash 2 x 5 min in lxPBS / 0.05% Triton X-100 at RT (while gently rotating)

[0142] - wash in lxPBS, 5 min at RT 5. Enzyme treatment (Enz)

[0143] The purpose of using proteolytic enzymes to is to break protein-protein and protein-DNA interactions to facilitate the FISH-probe to penetrate compact protein structures surrounding the chromatin. The disadvantage of using proteolytic enzymes is the narrow enzyme concentration window that exists between the positive effects on probe penetration and increased FISH signal and the negative effects on the morphology due to over-digestion resulting in tissue destruction. The majority of a large number of different proteolytic enzymes tested showed this narrow concentration window. However, it was found, to the inventor's surprise, that the combination of pepsin and collagenase (Enz) considerably increased the concentration window in the sense that pepsin now had a positive effect at a 10-fold lower concentration than normally used. At this low concentration of pepsin no significant negative effect on morphology was seen.

[0144] Draw line with Hydrophobic pen

[0145] Mix 20pl of Pepsin (2mg / ml) and 80pl of 0.1M HCI in 700pl H2O (final cone.: 0.05mg / ml pepsin and 0.01M of HCI), add lOOpI of mixture to each section. 37°C, 10 min.

[0146] - O.IX SSC wash 2',2x,

[0147] Mix 400pl collagenase III in 400pl H2O, add lOOpI to section, 37°C, 20 min.

[0148] 0.1X SSC wash 2 min 2x

[0149] 6. Formamide overnight (FA o / n)

[0150] The purpose of this treatment is to break hydrophobic interactions to facilitate the charged water-soluble FISH-probe to penetrate compact protein structures surrounding the chromatin. rinse with 50% FA / 2xSSC / 50mM sodium phosphate buffer pre-warmed to RT (you need --to put it at RT at least 2 hours before; ideally use a surface thermometer to monitor if the solution has reached RT) put in fresh 50% FA / 2xSSC / phosphate buffer solution and incubate at RT overnight (o / n) DAY 2

[0151] 7. Re-fixation (Re-FIX)

[0152] The purpose of this treatment is to stabilize the chromatin in a decondensed state (decreased interaction between DNA and protein) exposing more of the DNA for the FISH-probe.

[0153] - Tris-cl pH9 lOmin re-fixation step 1 Methanol: Acetic acid 3:1. -20°C, 30min re-fixation step 2 1% PFA, pH9, lOmin

[0154] - wash 5' with 125 mM Glycine / lxPBS, RT

[0155] - wash 3 x 5' in lxPBS, RT, while gently rotating

[0156] - go through ethanol series: o 70% EtOH, 2 min RT o 85% EtOH, 2 min RT o 100% EtOH, Imin RT o 100% EtOH, 2 min RT let the slide air dry RT (~30 min)

[0157] 8. Hybridization: (according to the ifish protocol Nature Communication)

[0158] * Article https: / / doi.org / 10.1038 / s41467-019-09616-w

[0159] Table 1. o Add 200 pl of the ifish dna pre-hybridization, cover with parafilm, at 37°C, lh o dilute lpl of 0.5nM probe stock to 9pl of the ifish_dna hybridization, and vortex in the DNA low bind eppi-tube. Final cone, is 0.06nM. o Add 20-30 p.1 of the hybridization mix (0.06nM probe each), cover with the coverslip or lOOpI in chamber, sealed with glue o Denature in 8' at 88 °C. o Incubate at 37 °C for 24 hours of hybridization.

[0160] DAY3

[0161] Warm the water bath to 60°C

[0162] Pre-warm an aliquot of 0.2xSSC / 0.2% Tween to 60°C in a falcon tube

[0163] Gently remove the coverslips in 2xSSC / 0.2% Tween

[0164] - 2XSCC / 0.2% Tween wash, 2 x RT pre-warmed 0.2xSSC / 0.2% Tween, Wash 2 x at 60°C for 7 min

[0165] - 4xSSC / 0.2% Tween wash lx, RT

[0166] Rinse with 2xSSC, RT, the rest of samples in ifish ma wash buffer during other samples photobleaching.

[0167] Rinse twice with the ifish ma wash buffer, RT, the rest of samples in ifish rna wash

[0168] - Thaw the 1:50 (2uM) dilution of the labelled oligo, Prepare the hybridization mix at 1:100 with the ifish rna hybridization buffer (final 20nM / oligo)

[0169] Table 2.

[0170] Pipette 100 pl of the hybridization mix and covered with parafilm / or coverslip

[0171] Seal the chamber and hybridize in darkness at 30°C for overnight

[0172] DAY4

[0173] Wash away the parafilm / coverslips with ifish_rna wash buffer Wash with the ifish_rna wash buffer, at 30°C for 1 h in darkness

[0174] Prepare lpl of stock(lpg / pl) in 1ml ifish_rna washing buffer, final con. 1 ng / pl

[0175] Hoechst in the ifish_rna wash buffer, Wash for 30' with the Hoechst / ifish_rna wash buffer solution at 30°C

[0176] Wash briefly a couple of times with 2xSSC

[0177] Mount with the prolong Gold overnight and image

Claims

CLAIMS1. A method for the preparation of one or more formalin fixed paraffin embedded (FFPE) tumor tissue sample(s) for use in a subsequent analysis, such as in fluorescent in-situ hybridization (FISH), comprising the steps of:(a) deparaffinizing the sample;(b) heat treating the sample by (i) adding citrate and exposing the sample to an increased temperature of about 80 °C at a duration of about 45 to 120 minutes, (ii) adding glycerol at a concentration of 10% to 30% to the sample and exposing the sample to an increased temperature in the interval of about 90-95 °C at a duration of about 10-30 minutes, and (iii) adding dithiotreitol (DTT) and ethylene diamine tetraacetate (EDTA) to the sample and exposing the sample to an increased temperature of about 80 °C for 10-30 min, wherein steps (i)-(iii) can be performed in any order;(c) optionally permeabilizing the sample;(d) optionally acid treating the sample;(e) enzyme treating the sample, by adding at least one proteolytic enzyme to the sample;(f) formamide treating the sample;(g) optionally re-fixating the sample;(h) hybridizing the sample; thereby obtaining a tumor tissue sample having an essentially intact morphology prepared for subsequent fluorescent in-situ hybridization (FISH) analysis.

2. The method according to claim 1, wherein the method further comprises an enzyme treatment performed as part of step (e), wherein pepsin is added at a concentration of 0,01 to 0,1 mg / ml in combination with at least one additional enzyme having the ability to increase the effective concentration of pepsin.

3. The method according to claim 1 or 2, wherein the method further comprises a refixation performed as part of step (g), comprising an alkaline treatment in order to stabilizethe sample, followed by (i) a first fixation by adding methanol and acetic acid, and (ii) a second fixation by adding formalin for 5 to 20 min at a concentration of about 1%.

4. The method according to any of the preceding claims, wherein, in the heat treatment of step (b), step (ii) is performed before step (i).

5. The method according to any of the preceding claims, wherein, in the heat treatment of step (b), step (iii) is performed after step (i).

6. The method according to any of the preceding claims, wherein citraconic anhydride (CA) is added at a concentration of about 0.01% to the sample during the heat treatment of step (i) and / or step (ii).

7. The method according to any of the preceding claims, wherein citraconic anhydride (CA) is added at a concentration in the interval of 0,002-0,01% and SDS is added at a concentration of about 0.2% during the heat treatment of step (i) and / or step (ii).

8. The method according to any of the preceding claims, wherein the heat-treated sample is exposed to an enzyme treatment, wherein pepsin is added at a concentration of about 0,05 mg / ml in combination with a collagenase.

9. The method according to claim 8, wherein the collagenase is collagenase III.

10. The method according to any of the preceding claims, wherein formamide treatment is performed after the heat treatment and the enzyme treatment, and before the optional refixation.

11. The method according to any of the preceding claims, wherein 1, 2 or 3 of the optional steps (c), (d) and (g) are performed.

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