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

The overall goal of this laboratory procedure is to simultaneously define, quantify and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers by means of high-throughput silver in situ hybridization. This method can help address key choosing breast cancer translation and research such as the study of HER2 gene amplification heterogeneity, a large cohort of cases retrieved from pathology archives. The main advantage of this technique is that it allows for high-throughput HER2 schizoanalysis across different topographic areas of multiple and heterogeneously processed breast cancers.

Generally, individuals new to in situ hybridization will struggle because retrospective studies typically require the analysis of archival samples that underwent different fixation, processing and storage procedure. We first had the idea for this method when we decided to reclassify all breast cancers from our institutional database since 2003. Demonstrating this procedure will be Giulia Ercoli, a technician from our laboratory.

Begin this protocol with selection of patients and tissue specimens as described in the text protocol. Create and open a new spreadsheet file, incorporating the records of the distinct areas of each case. Include separate columns for case ID, block ID, area ID, tissue type, topography, morphology, biomarker status and all other immunohistochemical features available.

Create a TMA project. Define the PMA recipient blocks by navigating to file, new, block. Click on the first field to fill and type in the dimensions of the paraffin block.

Next create a tissue array project by navigating to file, new, tissue array. Click the import icon, then choose the template file of the recipient block previously created. Click next.

Then choose one millimeter as the spot size by clicking the round button. Choose 500 microns as the spot spacing, which is the space between two centers of neighboring spots. Then click on the calculator icon to calculate the total number of spots created, which should be 231.

Then click on the button define to create grids and subgrids. Delete columns eight and 15 and the central line. Maintain three spots of line one for orientation.

The final map should encompass a total number of 183 spots. Define the list of tissue types by navigating to file, new, list of tissue types. Then define the project by navigating to file, new, booster project.

Next prepare the acceptor blocks. Fill cleansed metal molds with elasticized paraffin at 65 degrees Celsius. Leave the paraffin blocks to cool at film temperature overnight inside of the metal molds.

The next day, extract the paraffin blocks from the metal molds. Construct the TMA using an automated or semi-automated arrayer by first turning on the arrayer and then inserting the accepter blocks on the arrayer carousel. Define the starting position onto each recipient block by using the arrow keys to move the laser light and align it with the top left edge on the paraffin of the recipient block.

Click next and repeat for the vertical alignment. Click next again to automatically create a hole in the previously defined coordinate of the recipient block. Empty the punch.

Position the donor block for sampling and remove a tissue core from the previously annotated area of interest. Insert the donor tissue core into the hole in the recipient block. Then remove the donor block from the arrayer.

Repeat these steps until the TMA is completed. Put the tissue side of the newly generated TMA block on a sterile blank slide and place them in the lab oven at 45 degrees Celsius for five minutes. Gently press the block on the slide for five seconds to flatten the tissue cores.

Repeat once for a total of two times. Next cover the TMA tissue surface with a thin layer of elasticized paraffin at 65 degrees Celsius. Leave the TMA block at room temperature for two hours.

Finally, transfer the TMA block at four degrees Celsius for 24 hours before performing histochemical and immunohistochemical analyses as described in the text protocol. Place the TMA slides to analyze at 60 degrees Celsius for 10 minutes. Then start the immunostainer instrument and software.

At the home view, click the protocols button and then click create/edit protocols. Select the dual ISH cocktail procedure to modify the basic template. Flag baking in the box list and set the temperature at 63 degrees Celsius for 20 minutes.

Then flag deparaffinization. Next flag cell conditioning in the box list. Then flag cell conditioning CC2 and set the temperature at 86 degrees Celsius and the incubation time at four minutes.

Flag mild CC2 for eight minutes, standard for 12 minutes and extended for eight minutes. Flag ISH-Protease three in the box list and set the incubation time at 16 minutes. Select the probe's HER2 dinitrophenyl and chromosome 17 digoxigenin.

Incubate for 16 minutes and then set the denaturation for 20 minutes at 80 degrees Celsius. Set the probe's incubation time at six hours. Then set the SISH multimer incubation time at 32 minutes.

Set the silver chromate and incubation time at four minutes. Next set the red multimer incubation time at 24 minutes. Set the red chromate and incubation time at eight minutes.

Then flag counterstaining in the box list, select hematoxylin two and set the incubation time at eight minutes. Flag post counterstaining in the box list, select bluing reagent and set the incubation time at four minutes. Finally, perform schizoanalysis for HER2.

Assess the HER2 gene amplification as positive if the HER2 chromosome enumeration probe 17 ratio is greater than or equal to 2.0 with an average HER2 copy number greater than or equal to 4.0 signals per cell. Assess as negative if the HER2 chromosome enumeration probe 17 ratio is less than 2.0 with an average HER2 copy number less than 4.0 signals per cell. Shown here are representative micrographs showing the silver in situ hybridization analysis of a HER2 heterogeneous breast cancer.

In this paradigmatic example, two distinct areas of high-grade invasive breast cancer of no special type show different results in terms of HER2 gene amplification, visualized as black dots, compared to the centromeric enumeration probe 17, visualized as red dots. In particular, one area belonging to the tumor core and showing cytokeratin seven irregular staining pattern was HER2 amplified, while a single spot front the invasive front displayed a wild-type HER2 pattern. After watching this video, you should have a good understanding of how to perform readable schizoanalysis of tissue marked arrays incorporating multiple and heterogeneously processed breast cancers.

Once mastered, this technique can be done in two working days from the case selection. Though this method is optimized for breast cancer, it can be translated to other types of cancer in which HER2 heterogeneity is critically relevant such as gastric cancer. The implications of this technique extend toward the simple assessment of HER2 because it is designed for the comprehensive mapping of multiple topographic areas in larger series of breast cancers.

While attending this procedure, it's important to avoid any crack on the TMA block. Even a minimal crack in the paraffin infers the quality, reproducibility and the overall clarity of the in situ hybridization. Following this procedure, MALDI mass spectrometry imaging can be performed in order to answer additional questions on the proteomics of breast cancer, particularly in terms of intra and inter-tumor heterogeneity.

Don't forget that working with TMA punches and hot paraffin wax can be hazardous and precautions such as wearing disposable nitrile gloves should always be taken while performing this procedure.