Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

The study was approved by the Ethics Committee of the Medical University of Graz (reference number 20-066), Austria. Tissue samples were obtained from two cases of surgically resected primary invasive breast cancers after the patients had given written informed consent.

1. Tissue fixation

NOTE: Tissue fixation is performed either with standard buffered formalin solution (SBFS), defined as 10 % formalin solution containing a mass fraction of 3.7 % (corresponding to a volume fraction of 4 %) formaldehyde buffered to pH 6.8 to pH 7.2 according to CEN/TS 16827-1:2015 or the non-cross-linking, formalin-free tissue fixation system (also referred to as the alternate fixation solution below) consisting of a fixative and a stabilizer solution at room temperature (RT).

1. Cut the breast cancer tissue samples with a sterile scalpel into two halves each 4 mm thick to ensure adequate tissue quantity. Use a maximum size 4 x 15 x 15 mm for NCFPE or SBFS tissue fixation.
2. Place each half of the tumor specimen in a standard tissue cassette and submerge it in SBFS or the alternative fixation solution for 24 h with a volume per volume ratio (v/v) of 4 parts fixative and one part tissue (4 mL/1 g tissue (ideally 10 mL/1 g)).
3. Transfer the alternatively-fixed samples into the stabilizer solution for 2-72 h by opening the container, turning the tissue cassette by 180° and submerging it.
4. Place the tissue cassettes with the SBFS-fixed samples in 250 mL 70 % ethanol for 30 min at RT to remove residual SBFS.
   NOTE: This step is important to avoid SBFS contamination of non-cross-linking fixative-treated samples in the automated tissues processor, which would destroy the beneficial properties of non-cross-linking fixatives for preservation of biomolecules.
   CAUTION: SBFS is a hazardous solution, causes skin irritation, serious eye damage, may cause an allergic skin reaction, is suspected of causing genetic defects, may cause cancer, respiratory irritation, drowsiness and causes damages to organs. The alternative fixative is toxic by inhalation, in contact with skin and if swallowed, irritating to eyes and skin, danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed. Inhalation and physical contact should be avoided for both fixation solutions. Wear suitable protective clothing, gloves, and eye and face protection. Work in a fume hood.
5. Proceed to tissue processing, embedding and tissue sectioning (sections 2 and 3).

2. Tissue processing and embedding

1. Perform stepwise dehydration of tissue cassettes with the fixed tissue samples in 70 % (2 x 15 min), 80 % (30 min), 90 % (60 min), and 99 % (2 x 60 min) ethanol, followed by xylene (2 x 60 min). Then infiltrate with paraffin in an automated tissue processor (4 x 45 min).
2. Using forceps, place the dehydrated and paraffin-infiltrated samples (alternatively and SBFS-fixed) in metal molds.
3. Embed the samples in 5 – 10 mL molten paraffin (solidification point 56-58 °C) using a paraffin embedding instrument.
4. Place the molds onto a cooling plate at -5 °C for 30 min.
5. Recover the paraffin-embedded tissue blocks from the molds.
6. Store the resulting blocks (alternatively-fixed, paraffin-embedded (NCFPE) and SBFS-fixed paraffin-embedded (FFPE)) in the dark at 4 °C until use.

3. Tissue sectioning and slide preparation for FISH

NOTE: FFPE sections are directly used for FISH, while slides with NCFPE sections are post-fixed in SBFS for 24 h before FISH procedure.

1. Insert a single-use blade into the microtome and adjust the microtome to 10 µm.
2. Take the first block from the cooling plate and insert it into the instrument.
3. Trim the block until an even surface is achieved. Clean the microtome using a brush and adjust it to 3 µm. Discard the first 2-3 sections.
4. Cut 3 µm sections of NCFPE and FFPE blocks and place them in a cold water bath (ultrapure water).
   NOTE: To obtain better photographs without overlapping nuclei, here, images of 2 µm sections instead of 3-5 µm as recommended in the manufacturer´s instructions are shown.
5. Pick up each floating section with a fine paint brush and a needle onto a coated microscope slide for adhesion of the tissue sections.
6. Plunge this slide into a warm (40 °C) water bath and let the section stretch completely.
7. Put the section back onto the slide by carefully pulling the slide out of the water and avoid wrinkles.
8. Let all sections dry at at room temperature for 1 h.
9. Heat the slides at 70 °C for 30 min in an incubator to melt the paraffin.
10. Put the slides horizontally in a glass staining jar.
11. Rehydrate the sections stepwise at RT in the staining jars filled with 250 mL each of the following liquids: 100 % xylene 2 x 15 min, 96 % ethanol 2 x 15 min, 90 % ethanol 2 min, 80 % ethanol 2 min, 70 % ethanol 2 min, 50 % ethanol 2 min, and distilled water 2 x 10 min.
    CAUTION: Perform deparaffinization and rehydration steps in a fume hood, do not inhale toxic volatilized solvents.
    NOTE: Depending on the sections and tissue, it may be necessary to test different incubation times in advance.

4. Post-fixation of NCFPE specimens

1. Perform post-fixation of NCFPE slides by putting them into a new staining jar filled with SBFS for 24 h at RT. Place the jar in a fume hood during the 24 h post-fixation step.
2. Remove excess SBFS by washing with phosphate buffered saline (3 x 10 min) and distilled water (2 x 10 min).

5. Fluorescence in situ hybridization (FISH)

NOTE: Here FISH is performed using the commercially available HER2/CEN17 dual color probe Kit according to the manufacturer's instructions. Do not allow the tissue sections to dry during the hybridization and washing steps. Do not allow the DNA probe and 4', 6-diamidino-2-phenylindole (DAPI) DNA counterstain solutions to be exposed to light for a longer period. Perform these steps in the dark using lightproof containers. Depending on the age and the fixation step of the sample material, increase or decrease the denaturing and wash temperatures to obtain better hybridization results.

1. Place a staining jar containing 250 mL of heat pre-treatment solution citric in a 98 °C water bath.
2. Place the rehydrated FFPE and post-fixed NCFPE slides into the jar and incubate for 15 min. Do not use more than six slides per staining jar.
3. Wash the slides in distilled water for 3 min at RT in a new staining jar.
4. For proteolysis, place the slides into a temperature controlled hybridization system at 37 °C.
   1. Immediately, apply the-ready-to-use Pepsin solution (2mg/ml) dropwise (~ 30 µl per drop) to the tissue sections until it is completely covered. Incubate for 9 min at 37 °C in the hybridization system.
      NOTE: An incubation time of 5-15 min is recommended. The optimum time for proteolysis should be ascertained in advance.
   2. Place the slides into a jar containing wash buffer SSC and incubate for 5 min.
   3. Rinse the slides in distilled water for 1 min at RT.
   4. Perform dehydration of the sections in the following graded alcohols: 50 %, 70 %, 90 % and 100 % each for 1 min. Air dry the sections.
   5. For hybridization, pipette 10 µL of HER2/CEN17 probe onto the dried tissue sections, cover each sample with a coverslip and seal it with rubber cement.
      NOTE: 10 µL of probe is recommended per 22 x 22 mm tissue area. A gentle warming of the probe makes the pipetting process easier. Avoid bubbles and long exposure of the probe to the light.
   6. Co-denature probe and specimen DNA in the hybridization system at 75 °C for 10 min.
   7. Set the hybridization system to 37 °C and hybridize overnight.
   8. Carefully remove the rubber cement with forceps for post-hybridization and detection.
   9. Incubate the slides in a staining jar containing 37 °C pre-warmed 1 x wash buffer A for 5 min. Remove the coverslips.
   10. Wash the slides using pre-warmed 1 x wash buffer A twice for 5 min at 37 °C.
   11. Dehydrate the slides in 70 %, 90 % and 100 % ethanol, 1 min each.
   12. Dry the samples in air and protect from light.
   13. For staining, apply 30 µL DAPI-solution to the hybridized area avoiding bubbles. Cover the sections with a coverslip.
   14. Incubate for 15 min protected from light.
   15. Carefully remove excess DAPI-solution by gently pressing the slide between filter papers.
   16. Store the slides at 2 – 8 °C in the dark for up to 2 weeks until evaluation by confocal microscopy.

6. Confocal imaging and scanning of FISH slides

1. Perform confocal imaging and scanning of the slides on a confocal digital scanner equipped with a 40x/1.2 NA objective, quad band filters (DAPI/FITC/TRITC/Cy5) and a 5.5Mpx, 16 bit, cooled scientific complementary metal oxide semiconductor (CMOS) camera.
2. Acquire the images with filters for green (excitation: 503 nm, emission: 528 nm) and orange (excitation: 547 nm, emission: 572 nm) channels.

7. Interpretation

NOTE: The HER2/CEN17 probe is a mixture of a red fluorochrome-labelled CEN17 probe specific for the alpha satellite centromeric region of chromosome 17 (D17Z1) and a green fluorochrome-labelled HER2 probe specific for the HER2 gene (probe location 17q12). Tumor specimens with a HER2:CEN17 ratio ≤2.0 per nucleus are scored as normal, whereas those with a HER2:CEN17 ratio ≥2.0 are scored as amplified¹⁷.

1. Perform quality analysis of the images with the open source CaseViewer 2.1²⁰.
   1. Evaluate at least 20 cells that are located in two different regions of the invasive component of the carcinoma as defined by a pathologist. Include clearly distinguishable well distributed nuclei in the counting area. Exclude from counting tissue regions at the boundary and retracted or squeezed areas.

8. RNA Quality

1. RNA Extraction
   1. Ensure that all instruments and tools are RNAse free. Use a RNAse-removing solution.
   2. Cut 10 to 20 sections (5 µm thick) of NCFPE or FFPE samples using a microtome following the protocol until step 3.8.
   3. Extract RNA from NCFPE samples using the RNA isolation kit (see the Table of Materials).Extract RNA from FFPE samples by using an FFPE RNA isolation kit.
      NOTE: Extraction of RNA from non-cross-linking fixatives requires an isolation method adapted to the fixation method. Do not use any other RNA isolation method (e.g. Trizol, RNeasy FFPE, etc. for NCFPE specimen).
   4. Determine the RNA concentration and purity using a spectrophotometer. The ratio of the absorbance at 260 nm and 280 nm (A260/280) should be ~ 2.0.
   5. Store the isolated RNA at -70 °C until further analysis.
2. Perform a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) real-time reverse transcription PCR using primers to generate amplicons of different lengths^8,9.
   1. Use a cDNA reverse transcription kit according to manufacturer´s instructions for cDNA synthesis. Use 500 ng RNA of each sample. Store cDNA at -20 °C until further use.
   2. Prepare the PCR master mix according to manufacturer´s instructions.
   3. Pipette the PCR master mix in triplicates in a 384-well reaction plate. Add 4 µL of 1:16 diluted cDNA (PCR template). Perform the PCR according to manufacturer´s instructions.
      NOTE: For human GAPDH, the following primers were used: GAPDH forward: 5´-CCACATCGCTCAGACACCAT-3´; GAPDH reverse: 71 bp 5´-ACCAGGCGCCCAATACG-3´, 153 bp 5´-GTAAACCATGTAGTTGAGGTC-3´, 200 bp 5´-TTGACGGTGCCATGGAATTT-3´, 277 bp 5´-ACTTGATTTTGGAGGGATCT-3´, 323 bp 5´-AAGACGCCAGTGGACTCCA-3´, and 530 bp 5´-ACGATACCAAAGTTGTCATG-3´.
   4. Analyze the results using a software applicable to the PCR machine. Nonspecific products must be excluded based on melting curve analysis²¹.