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EP-4739796-A1 - A METHOD FOR THE PREPARATION OF ONE OR MORE FORMALIN FIXED PARAFFIN EMBEDDED TUMOR TISSUE SAMPLES FOR USE IN A SUBSEQUENT ANALYSIS

EP4739796A1EP 4739796 A1EP4739796 A1EP 4739796A1EP-4739796-A1

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.

Inventors

  • ZETTERBERG, ANDERS
  • WANG, Xiaoze Li
  • ZETTERBERG, Magnus
  • BIENKO, Magda

Assignees

  • MicroClone Genomics Sweden AB

Dates

Publication Date
20260513
Application Date
20240628

Claims (11)

  1. 1. 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. 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. 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 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%.
  4. 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. 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. 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. 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. 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. 9. The method according to claim 8, wherein the collagenase is collagenase III.
  10. 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. 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.

Description

A method for the preparation of one or more formalin fixed paraffin embedded tumor tissue samples for use in a subsequent analysis Technical field 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). Background art 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). 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. 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. 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. 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. Summary 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. 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: (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