Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Summary

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Abstract

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Introduction

HER2 is a proto-oncogene that is overexpressed and amplified in 15 - 30% of all invasive breast cancers^1,2. HER2 overexpression is inferred by the presence of >10% cells with strong membrane immunohistochemical (IHC) staining (3+), while the gene amplification can be assessed when either the HER2/centromere ratio is ≥2 or the gene copy number is ≥6, on counting at least 20 cells by in situ hybridization (ISH)³.

Intra-tumor genetic heterogeneity has been widely described in breast cancers, being a potentially adverse contributor to biomarkers evaluation and treatments response⁴. According to the College of American Pathologists (CAP), HER2 heterogeneity exists if HER2 is amplified in >5% and <50% of infiltrating tumor cells⁵. Regrettably, the actual incidence of HER2 spatial heterogeneity in breast cancers remains a subject of controversy among pathologists, with some authors maintaining that it is an exceedingly rare event, and others suggesting that up to 40% of cases are HER2-heterogeneous^{1,5,6,7,8,9,10}. Despite the biological mechanisms that underpin this condition are not yet fully clarified, the prognostic and clinical impacts of intra-tumor HER2 heterogeneity are crucial for breast cancer patients¹¹.

Recently, bright-field molecular techniques, such as chromogenic ISH (CISH) and silver ISH (SISH), have emerged as reliable methods to detect HER2 heterogeneity in FFPE tissues, with some advantages compared to fluorescent ISH (FISH)¹². Regrettably, the bulk analysis of single cases remains impractical in large-cohort research studies. Several groups have suggested that the combination of histochemistry, IHC, and ISH with TMA technologies could represent a valuable strategy in the study of cancer biology^13,14,15,16. With this widely adopted method, tissue samples from different patients can be analyzed concurrently, minimizing the tissue and reagents employed and thereby fostering the uniform analysis of a large series of cases¹⁴. However, no protocols are available for the simultaneous high-throughput molecular characterization of multiple tissue samples that underwent different processing in terms of reagents, fixation times, and conservation methods employed, such as archival blocks.

Given the prognostic and clinical implications of HER2 spatial heterogeneity in breast cancers, we developed an integrated molecular platform to assess it in large series of heterogeneously processed cases. Here, we portray the laboratory strategies to generate and analyze the intra-tumor heterogeneity of HER2 amplification in high-yield TMAs of breast cancer by means of SISH. The following protocol has been developed for tumors measuring >5 mm (>pT1b according to the TNM 2017)¹⁷. For smaller lesions, we recommend performing the analysis on full-face serial sections. Our procedure allows for the simultaneous IHC and SISH analysis of up to 30 breast cancers, encompassing a mean of 6 distinct areas (range 4 - 8) for each case. Altogether, 180 tissue cores of 1 mm in diameter, with 500 µm between the cores, and 2 mm between the grid and edges will be generated for each TMA block.

Protocol

This study was approved by the Institutional Review Boards from IRCCS Ca' Granda Foundation, Policlinico Hospital, Milan, Italy.

1. Selection of Patients and Tissue Specimens

1. Retrieve the archival slides of all cases to be analyzed, including all the available hematoxylin and eosin (H&E) and IHC slides from the original diagnosis and, if present, one H&E of matched non-neoplastic breast tissue (e.g., surgical margin).
2. Perform case reviews.
   NOTE: For this task, at least two pathologists with experience in breast pathology should discuss the diagnostic slides and resolve disagreements collegially. Use either a conventional microscope or telepathology tools (favor the latter in multicentric studies). If any, exclude all discordant cases.
   1. Perform the histological re-classification of all cases according to the latest edition of the World Health Organization (WHO) classification of tumors of the breast¹⁸.
   2. Ensure that the normal tissue for each case, if present in the archival clinical samples, comprises at least one non-neoplastic terminal ductal-lobular unit.
   3. Identify all neoplastic areas that show heterogeneous cytological, architectural, or IHC features using a bright-field microscope (to be jointly performed by a pathologist and the technician(s) who will construct the TMAs).
3. Highlight the discrete topographic areas with a marker on the glass slide or with the proper tool on a digital slide software (Figure 1A).
4. Based on the selected slides, retrieve the corresponding FFPE blocks from the archives.
   NOTE: To optimize the TMA construction, to avoid tissue depletion, and to preserve the TMA punch structural integrity, cases with <1 mm-thick residual tissue should be excluded.

2. Design and Construct TMAs Based on the Kononen Technique

1. Create and open a new spreadsheet file incorporating the records of the distinct areas of each case. Include the following data on separate columns, as exemplified in Table 1: Case ID, Block ID, Area ID, tissue type (e.g., normal tissue, invasive component histotype, in situ component histotype), topography (e.g., tumor core, invasive front), morphology (e.g., nuclear grade, architecture, mitoses, cytological features), biomarkers status (i.e., estrogen receptor (ER), progesterone receptor (PR), Ki67, and HER2), and all other IHC features available.
   1. Save the document.
2. Create a TMA project.
   1. Install and open the TMA designer software.
   2. Define the TMA recipient block(s): access through the menu File > New > Block or by using CTRL+ B.
   3. Click in the first field to fill or press the Tab key, and type in dimensions of the paraffin block: width 35 (mm), height 20 (mm). Type "2" as the "margin" value (i.e., distance from paraffin edge in mm) (Figure 1B). Save the data by clicking the proper button. The new template of recipient block is now saved and ready to be used in new tissue array templates.
   4. Create a tissue array project: access to the menu File > New > Tissue Array or by using CTRL+ T.
   5. Fill in the name of the Tissue array, comments, and author, then click "next." Click the "import" icon, choose the template file of recipient block previously created, and click "next."
   6. Choose 1 mm as the spot size by clicking the round button. Choose 500 µm as spot spacing which is the space between two centers of neighboring spots. Click on the calculator icon to calculate the total number of spots created, which should be 231. Click "next."
   7. Define the spot numbering mode by clicking once on it. Click on the button "define" to create grids and sub-grids. Delete the central line (line 6) and the columns 8 and 15. Maintain 3 spots of line 1 for orientation. The final map should encompass a total number of 183 spots, as represented in Figure 2.
   8. Define the list of tissue types: access through the menu File > New > List of tissue types or by using CTRL+ L.
   9. Insert the tissue descriptions previously defined. Press down arrow or click on the + button to add a line.
   10. Define the project: access through the menu File > New > Booster project or by using CTRL+ P.
   11. Fill in the name of the tissue array, comments, and author, then click "next." Import the spreadsheet file, list of tissue types, and the tissue array model previously created by clicking the proper icons.
   12. Click "select all" and define the number of spots for each tissue type; click on the "match" button to apply settings. A table listing the total number of cores that will be sampled and transferred to recipient blocks in this project will appear. Save the project and close the program.
3. Prepare the acceptor block(s) (Figure 1B).
   1. Fill cleansed metal molds with elasticized paraffin at 65 °C.
   2. Leave the paraffin blocks to cool at room temperature overnight inside of the metal molds.
   3. Extract the paraffin blocks from the metal molds. If cracks occur, discard the block and repeat steps 2.3.1-2.3.3.
4. Construct the TMA using an automated or semi-automated arrayer¹⁹.
   1. Retrieve all selected FFPE blocks and the corresponding H&E sections with annotations.
   2. Turn on the arrayer and insert the acceptor block(s) on the arrayer carousel.
   3. Open the arrayer control station software, click on "New Tissue Array", and load the project previously created (Figure 1C).
   4. Click "Continue" and select the position of the donor block(s) from the menu.
   5. Define the starting position onto each recipient block: use the arrow keys to move the laser light and align it with the top-left edge on the paraffin of the recipient block.
   6. Click "next" and repeat for the vertical alignment.
   7. Click "next" to automatically create a hole in the previously defined coordinate of the recipient (acceptor) block.
   8. Empty the punch.
   9. Position the donor block for sampling and remove a tissue core from the previously annotated area of interest.
   10. Insert the donor tissue core into the hole in the recipient (acceptor) block.
   11. Remove the donor block from the arrayer.
   12. Repeat steps 2.4.5-2.4.11 until the TMA is completed.
5. Put the tissue side of the newly generated TMA block on a sterile blank slide and place them in the lab oven at 45 °C for 5 min.
6. Gently press the block on the slide for 5 s to flatten the tissue cores. Repeat once (for a total of 2 times).
7. Remove the TMA block together with the slide from the oven, flip them, and gently detach the block from the slide.
8. Cover the TMA tissue surface with a thin layer of elasticized paraffin at 65 °C.
9. Leave the TMA block at room temperature for 2 h.
10. Transfer the TMA block at 4 °C for 24 h.
    NOTE: The TMA block can be stored at 4 °C until exhaustion.

3. Histochemical and Immunohistochemical Analyses

1. Cut TMA sections.
   1. Place the TMA acceptor block(s) with the paraffin-side down on a -10 °C surface for 10 min to chill the paraffin.
   2. Fill a floatation bath with ultrapure water and set heat to 38 °C.
   3. Place a new blade on a rotary microtome and insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane.
   4. Approach the block carefully with the blade and cut a few thin sections to ensure the positioning is correct; adjust if necessary.
   5. Cut 3 µm-thick sections.
   6. Pick up the sections using tweezers and float them on the water bath surface.
   7. Pick the sections out the water bath on regular and IHC/ISH glass slides.
   8. Place the slides on a rack and store them in the oven at 45 °C overnight.
      NOTE: Once prepared, the TMA sections should be stained within 24 h.
2. Perform histochemical staining (H&E) using an autostainer.
   1. Insert the slides in the autostainer rack and place them at 60 °C for 10 min.
   2. Open the load drawer of the autostainer and insert the rack with the TMA slides in one of the load stations with a standard H&E clip facing outwards.
   3. Once the drawer is closed, the clip will be automatically recognized by the system and the following protocol will start: oven station at 37 °C (6 min), xylene (2 min), xylene (2 min), ethanol 100% (2 min), ethanol 100% (2 min), ethanol 95% (2 min), ethanol 95% (2 min), distilled water (4 min), Carazzi's hematoxylin (9 min), low-pressure running water (2 min), eosin 1% (1 min), low-pressure running water (2 min), ethanol 95% (20 s), ethanol 95% (20 s), ethanol 95% (20 s), ethanol 95% (20 s), ethanol 100% (15 s), ethanol 100% (15 s), xylene (30 s), xylene (30 s).
   4. Unload the racks from the drawer and mount the slide with a cover slip.
      1. Collect a drop of mount with a pipette and put it on an outer edge of the cover slide.
      2. Place the wet side of the cover slip on the slide and let the mount dry for 30 min.
3. Perform IHC staining for ER, PR, Ki67, and HER2 using an automated immunostainer.
   NOTE: Before beginning a staining run, start up the system, and check the consumables reagent bottles (Table of Materials) and waste containers.
   1. Place the TMA slides to analyze at 60 °C for 10 min.
   2. Start the immunostainer instrument and software.
   3. At the Home View, click the Protocols button, and then click Create/Edit Protocols.
   4. Select the standard DAB (3, 3'-diaminobenzidine) IHC procedure to modify the basic template.
   5. Flag "Deparaffinization" in the box list and set the Medium Temperature at 72 °C.
   6. Flag the cc1 unmasking solution, set the Medium Temperature at 95 °C, and the incubation time at 36 min.
   7. Flag the Primary Antibody box, insert the ID of the ER inline dispenser (the instrument software will recognize the dispenser on the reagent carousel), and set the incubation time at 16 min.
   8. Flag the Counterstaining box, select Hematoxylin I, and set the incubation time at 12 min.
   9. Click the "Save As" button and name the protocol.
   10. Repeat the steps 3.3.3-3.3.9 for the remaining antibodies using the following primary antibodies incubation times: PR 16 min, Ki67 16 min, and HER2 12 min. Use pre-diluted ready-to-use (RTU) antibodies.
   11. At the Home View, click the Create Label button.
   12. Click the Protocols button and add the ER, PR, Ki67, and HER2 protocols for each of the TMA to analyze.
   13. Click the Close /Print button.
   14. As the label template appears, enter the TMA IDs for the label.
   15. Click the Print button to print the labels and then apply them on the TMA slides.
   16. Click the instrument icon and set the instrument startup mode as "Ready".
   17. Open the hood that covers the reagents carousel, place the detection system, and primary antibodies, then close the hood.
   18. Click the front button on a slide drawer to open it, load the TMA slides, and close the drawer by clicking the same button.
   19. Set the instrument startup mode as "Running" and follow the instructions.
   20. At the end of the run, unload the TMA slides by pressing the Open All Drawers with Completed Slides button on the instrument Slide Control and rinse them in warm soapy water to remove any remaining reagents.
   21. Wash the slides under low-pressure cold running water, dehydrate the TMA slides, and apply a glass coverslip to each slide.
4. Perform IHC analysis of ER, PR, and HER2 following the American Society of Clinical Oncology (ASCO)/CAP guidelines, and of Ki67 according to the recommendations of the International Ki67 in Breast Cancer working group (Table 2).
   NOTE: This analysis should be performed separately in the discrete tissue spots by a pathologist with an experience in breast pathology.
   1. Assess the ER expression as positive if ≥ 1% of tumor cell nuclei are immunoreactive^20,21,22,23.
   2. Assess the PR expression as positive if ≥ 1% of tumor cell nuclei are immunoreactive^20,21,22,23.
   3. Assess the HER2 expression as: a) score 3+ if circumferential membrane staining that is complete and intense, b) score 2+ if circumferential membrane staining is incomplete and/or weak/moderate and within > 10% of the invasive tumor cells or complete and circumferential membrane staining is intense and within ≤ 10% of the invasive tumor cells, c) score 1+ if incomplete membrane staining is faint/barely perceptible and within > 10% of the invasive tumor cells, d) negative if no staining is present or membrane staining is incomplete and faint/barely perceptible and within ≤ 10% of the invasive tumor cells^3,24.
   4. Assess the Ki67 index as the percentage of cells with nuclear staining in at least 1,000 neoplastic cells randomly selected over 10 high-power fields (magnification, 400X)²⁵.

4. SISH Analysis of HER2

1. Cut TMA sections (repeat step 3.1).
2. Perform SISH for HER2 using an automated immunostainer.
   1. Place the TMA slides to analyze at 60 °C for 10 min.
   2. Start the immunostainer instrument and software.
   3. At the Home View, click the Protocols button, and then click Create/Edit Protocols.
   4. Select the "U Dual ISH CKT" procedure to modify the basic template.
   5. Flag "Drying" in the box list and set the temperature at 63 °C for 20 min.
   6. Flag "Cell Conditioning" in the box list, then "Cell Conditioning CC2", and set the temperature at 86 °C and incubation time at 4 min.
   7. Flag CC2 Mild (Cycle 1), Standard (Cycle 2), and Extended (Cycle 3) for 8 min, 12 min, and 8 min, respectively.
   8. Flag "ISH-Protease 3" in the box list and set the incubation time at 16 min.
   9. Select the probes HER2DNP and CHR17DIG and set the incubation time at 6 h.
   10. Set the washing at 76 °C for 8 min.
   11. Set the SISH multimer (SIL ISH DNP HRP) incubation time at 32 min.
   12. Set the silver chromogen (SIL ISH DNP CHRC) incubation time at 4 min.
   13. Set the red multimer (RED ISH DIG AP) incubation time at 24 min.
   14. Set the red chromogen (RED ISH DIG FR) incubation time at 8 min.
   15. Flag "Counterstaining" in the box list, select "HEMATOXYLIN II", and set the incubation time at 8 min.
   16. Flag "Post-Counterstaining" in the box list, select "BLUING REAGENT", and set the incubation time at 4 min.
   17. Repeat steps from 3.3.11-3.3.15 (select modified U Dual ISH CKT protocol).
   18. Open the hood that covers the reagents carousel, place the washing and the ISH detection systems, then close the hood.
   19. Repeat steps from 3.3.18-3.3.21.
3. Perform SISH analysis for HER2: assess the HER2 gene amplification as positive if the HER2/CEP17 ratio is ≥2.0 with an average HER2 copy number ≥4.0 signals per cell, and negative if the HER2/CEP17 ratio is <2.0 with an average HER2 copy number <4.0 signals/cell^3,24.
   NOTE: Similar to IHC, this analysis should be performed separately in the discrete tissue spots by a pathologist with experience in breast pathology.

Results

Overall, 444 invasive breast cancers were incorporated in 15 TMAs specifically optimized for ISH analyses. Among the 2,664 spots sampled, 2,651 (99.5%) were representative of the previously selected areas and therefore considered amenable for subsequent analyses. Intra-tumor heterogeneity was determined by means of IHC and SIH, with a particular focus on the heterogeneous distributions of HER2-positive clones in the distinct topographic areas of the tumors. Table 3 depict...

Discussion

Here, we have detailed the laboratory strategies to perform SISH analyses of the HER2 gene and its corresponding centromere in high-yield TMAs of heterogeneously processed breast cancers. This method is cost-effective and can be carried out in most laboratories for the study of HER2 gene amplification heterogeneity in large cohorts of breast cancers retrieved form pathology archives.

Due to the clinical importance of HER2 testing in breast cancer and the challenges generated ...

Materials

Name	Company	Catalog Number	Comments
Surgipath Paraplast	Leica Biosystems, Wetzlar, Germany, EU	39601006	Tissue embedding medium, 56 °C melting point
Eosin Y  1% water solution	Bio Optica, Milan, Italy, EU	510002	Eosin yellowish, water-soluble
Carazzi’s hematoxylin	Bio Optica, Milan, Italy, EU	506012	Alum hematoxylin ripened using potassium iodate
Diamond quality	Laboindustria, Arzergrande, Italy, EU	33533	26x76 mm microscope slides
Leica CV Mount	Leica Biosystems, Wetzlar, Germany, EU	14046430011	Mounting medium, with no monomers, based on polymers of butylmethacrylate in xylene
FLEX IHC microscope slides	Agilent Technologies (Dako),  Santa Clara, CA, USA	K8020	Coated microscope slides for adhesion of FFPE for use in IHC
BenchMark ULTRA	Ventana medical system, Tucson, AZ, USA	N750-BMKU-FS	Slide staining system
CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody	Ventana medical system, Tucson, AZ, USA	790-4324	Primary antibody, ready-to-use
CONFIRM anti-Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody	Ventana medical system, Tucson, AZ, USA	790-2223	Primary antibody, ready-to-use
CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody	Ventana medical system, Tucson, AZ, USA	790-4286	Primary antibody, ready-to-use
PATHWAY HER2 (4B5) Rabbit Monoclonal Primary Antibody	Ventana medical system, Tucson, AZ, USA	790-4493	Primary antibody, ready-to-use
ultraView Universal DAB Detection Kit	Ventana medical system, Tucson, AZ, USA	760-500	Indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies
INFORM HER2 Dual ISH DNA Probe Cocktail	Ventana medical system, Tucson, AZ, USA	780-4422	INFORM HER2 Dual ISH assay - Dual color in situ hybridization FDA approved automated assay for determining HER2 gene status in breast cancer patients
ultraView Silver ISH DNP Detection Kit	Ventana medical system, Tucson, AZ, USA	800-098
ultraView Red ISH DIG Detection Kit	Ventana medical system, Tucson, AZ, USA	800-505
ISH Protease 3	Ventana medical system, Tucson, AZ, USA	780-4149	Used in the ISH process to remove protein that surrounds the target DNA sequences of interest
Hematoxylin	Ventana medical system, Tucson, AZ, USA	760-2021	Modified Gill's hematoxylin counterstain reagent
Hematoxylin II Counterstaining	Ventana medical system, Tucson, AZ, USA	790-2208	Modified Meyer's hematoxylin counterstain reagent
Bluing reagent	Ventana medical system, Tucson, AZ, USA	760-2037	Aqueous solution of buffered lithium carbonate for bluing hematoxylin stained sections on glass slides
HybReady	Ventana medical system, Tucson, AZ, USA	780-4409	Formamide-based buffer for ISH assays
EZ Prep (10x)	Ventana medical system, Tucson, AZ, USA	950-102	Concentrate solution for paraffin removal from tissue samples during IHC and ISH reactions, and to dilute 1:10.
SSC Buffer (10X)	Ventana medical system, Tucson, AZ, USA	950-110	Sodium Chloride Sodium Citrate buffer solution is used for stringency washes and to rinse slides between staining steps and provide a stable aqueous environment for the in situ hybridization reactions. Dilute 1:5.
ULTRA LCS	Ventana medical system, Tucson, AZ, USA	650-210	Prediluted (ready-to-use) coverslip solution used as a barrier between the aqueous reagents and the air to prevent evaporation in the IHC and ISH reactions
Reaction Buffer (10x)	Ventana medical system, Tucson, AZ, USA	950-300	Tris based buffer solution (pH 7.6 ± 0.2) to rinse slides between staining steps during IHC and ISH. Dilute 1:10.
ULTRA Cell Conditioning (ULTRA CC2)	Ventana medical system, Tucson, AZ, USA	950-223	Pretreatment steps in the processing of tissue samples during IHC and ISH. Ready to use.
ULTRA Cell Conditioning (ULTRA CC1)	Ventana medical system, Tucson, AZ, USA	950-224
ultraView Silver Wash II	Ventana medical system, Tucson, AZ, USA	780-003	Ready-to-use solution to rinse slides between IHC and ISH staining steps
Microtome	Leica Biosystems, Wetzlar, Germany, EU	RM 2255	Automated rotary microtome
Multistainer	Leica Biosystems, Wetzlar, Germany, EU	ST 5020	Workstation for automated staining and coverslipping
Minicore 1	Alphelys, Plaisir, France, EU	00-MICO-1	Semi-automatic arrayer for TMA contruction with TMA Designer 2 embedded software
Aperio ScanScope CS2	Leica Biosystems, Wetzlar, Germany, EU	K080254	Image capture device - digital pathology scanner
Tissue-Tek III Uni-Cassette	Sakura Finetek Europe B.V	4135	Cassette
Tissue-Tek Paraform Standard Base Mold	Sakura Finetek Europe B.V	7055	Stainless-Steel base metal mold