Using this method, HER2 status testing can be conducted in a simplified manner for more accurately identifying the patients who are eligible for trastuzumab therapy and potentially leading to the improvement of breast cancer patient care in the future. Tissue samples were routinely processed for paraffin-embedding after fixing with an alcoholic formalin fixative. A protocol was established so that the entire assay procedure consisting of baking, deparaffinization, pretreatment, hybridization, stringency wash, signal detection, and counterstaining was completed as a one-step fully automated assay.
Deparaffinized tissue sections were pretreated with a combination of heat treatment with Reaction Buffer Tris-based pH 7. Pretreatment conditions were chosen for optimal signal to noise ratio and tissue morphology preservation for the xenograft control slides as well as clinical case tissue slides.
Reaction Buffer was used for the washing steps during immunological detection.
Brightfield double in situ hybridization BDISH signal detection scheme with a sequential in situ hybridization method. Then, chromosome 17 centromere CEN 17 signal was detected with a DNP-labeled CEN 17 oligoprobe hybridization followed by fast red and naphthol phosphate reaction signal detection system. Excess mounting media was removed from the slides by gently pressing the slides against paper towels. Different coverslipping methods were also evaluated for preserving the fast red staining during the assay development.
Scoring occurred at different sites and at different occasions using different microscopes. Each individual observer evaluated the set of slides at their own pace and judgement. No scores were provided by the observers when the staining quality was deemed not adequate. Concordance data of FISH scores vs. The consensus among observers was defined as the agreement of three or more observers on a given observation. Because of the size difference and color contrast of black dots for HER2 gene and red dots for CEN 17, they could be visually separated even when red and black signals were co-localized in the nuclei of MCF7 tumor cells arrowheads, Figure 2E.
CEN 17 signal is detected as red dots that are slightly larger than silver black dots C, D. One successful way to preserve the fast red staining was the use of a toluene-based Cytoseal 60 mounting medium placed onto completely dried tissue sections prior to coverslipping with glass coverslips. The most common method of coverslipping tissue sections stained with fast red is the use of an aqueous mounting medium.
However, this method did not produce crisp fast red staining for quantitative analyses of BDISH signals data not shown.
Individual concordance ranges were between All discordant cases, that we defined it even by one observer disagreement between the BDISH and FISH scores, were re-examined for possible causes of conflicting results and there were 9 discordant cases. All nine 9 discordant cases of the BDISH slides presented at least some degree of the genotypic heterogeneity of tumor cell populations. Breast cancers with obvious tumor cell heterogeneity are shown as examples in Figure 4. Three 3 of 9 discordant cases demonstrated the segregated tumor cell heterogeneity while the other 6 cases showed various degrees of the variegated tumor cell heterogeneity.
The heterogeneity of breast tumor cell populations.
The heterogeneity of breast cancer cells was demonstrated with brightfield double in situ hybridization BDISH stained tissue sections. In general, there were 2 types of the tumor cell heterogeneity: 1 variegated tumor populations of, as an example, non-amplified blue asterisk and amplified yellow asterisk HER2 gene cells in the same area A and 2 segregated tumor populations of, as an example, discrete B and clustered C HER2 gene cells in different areas.
Accurate HER2 status testing is important for identifying breast cancer patients who may benefit from receiving trastuzumab therapy. Two potential solutions for improving the standardization of HER2 status testing include: 1 automating the entire process for slide staining [ 36 ] and slide reading [ 36 — 39 ] and 2 consolidating the HER2 testing process within experienced laboratories and pathologists that perform large numbers of HER2 tests [ 15 ].
An evaluator of FISH slides must have access to specialized fluorescence microscopy in a dark room. Furthermore, digital images of the FISH slide need to be captured with a sensitive camera system for each patient case for the HER2 gene status record. Therefore, it is desirable to automate a tissue-based HER2 gene status test that can be observed with a regular brightfield microscope and that produces stained slides that can be archived.
While the concept of multi-color brightfield ISH applications was published in 's [ 40 , 41 ], it was a recent achievement to visualize HER2 and CEN 17 targets within the same nuclei of tissue sections with a manual dual brightfield ISH application [ 42 ]. The advantages of the BDISH application for HER2 gene and CEN 17 presented in the current study are: 1 the automation of the ISH application; 2 the visualization of both HER2 gene and CEN 17 targets in the nuclei of the same cell; 3 the generation of discrete HER2 gene signals; 4 the ability to reproducibly detect endogenous HER2 and CEN 17 signals in the stromal tissues and lymphocytes as a reliable internal assay control; 5 the ability to visualise signal with brightfield microscopy with non-oil immersion lenses; and 6 the capability to permanently archive the slides.
For CEN 17 ISH, we have optimized a new detection system with an alkaline phosphatase-conjugated antibody and fast red chromogen and naphthol phosphate substrate reaction. Because fast red precipitate is soluble in organic solvents, in general, aqueous mounting medium is used for coverslipping.
However, the standard coverslipping method with aqueous mounting medium on wet tissue sections did not produce tissue sections with high resolution and therefore detailed tissue structure could not be observed data not shown. Incomplete drying resulted in faint red background staining particularly around the fast red precipitate sites with this method.
Interestingly, the use of aqueous mounting medium onto the dried tissue slides produced yellowish background staining on tissue sections and this method did not produce satisfactory results data not shown.
Amplified HER2 genes are located not only on chromosome 17, but also are translocated on other chromosomes [ 29 ]. As both probes are labeled with the same DNP hapten, our first concern was to determine if detecting specific signal for each probe was feasible.
Thus, the SISH detection and the fast red detection can be combined to perform a sequential double ISH assay with 2 probes labeled with the same hapten. Because the sequential BDISH application uses 2 specific stringency conditions based on the length and sequences of 2 probes, it is not necessary to design 2 probes that require the same stringency for co-hybridization, like double color FISH assays.
There were 9 discordant cases 9. A similar observation was reported with an international HER2 testing proficiency study [ 15 ]. They also stated "equivocal cases are difficult to interpret, even highly experienced and validated laboratories" [ 15 ]. It has been speculated that genomic and phenotypic heterogeneity of tumor cells is the main reason for the inconsistency of HER2 testing results [ 44 ].
Assessment of HER2 gene status can be conducted without the use of a specialized fluorescence microscope and the time required for completing HER2 gene status assessment can be shortened significantly. Furthermore, this application has the potential to be used for other gene targets, any combination of a gene and its chromosome centromere, and tissue section-based gene assessment tests including gene translocation studies.
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