Material and Method: Immunohistochemisty and silver enhanced in situ hybridization of 73 invasive breast carcinoma results were evaluated manually to determine HER2/neu status. Later, silver enhanced in situ hybridization and immunohistochemistry results were reevaluated with Ventana Image Analysis System. Afterwards correlation of both methods with image analysis system and manuel interpretation were calculated.

Result: All cases were score 2 with immunohistochemistry. With image analysis system, 5 cases were score 1, 56 cases were score 2 and 12 cases were score 3. When in situ hybridization results were reevaluated with image analysis system, 6 cases were discordant compared with manual interpretation.

Conclusion: The correlation rate of immunohistochemistry interpretation results between manuel method and image analysis system was %76; but silver enhanced in situ hybridization interpretation results between manuel method and image analysis system were %91 concordant and it was statistically significant (k= 0.832 and p<0.001).

Immunohistochemistry (IHC) is a widely used and relatively inexpensive test. However, the subjectiveness of the evaluation process and the variability between observers make it difficult to standardize the method. The fixation and maintenance conditions of the samples also need to be optimized to obtain standard results.

In situ hybridization methods are based on the use of DNA probes to visualize and detect the copy number of the HER2/neu gene and chromosome 17 (Kr-17, CHR 17) in various ways using DNA probes. DNA probes are marked with fluorescence for fluorescent in situ hybridization (FISH), and a chromogen that can be visualized with the light microscope for chromogenic in situ hybridization (CISH) ². DNA probes marked with silver are used in the silver-enhanced in situ hybridization (SISH) method. The HER2/neu gene copy number and Kr-17 copy number are determined separately in invasive tumoral cells and the ratio calculated. The sections are evaluated with the light microscope for cases investigated with SISH and the signals can be archived, similar to CISH and IHC, as they do not fade. SISH is a quantitative method like FISH and CISH and there is a high degree of concordance between evaluations in these methods. SISH also fits the recommendation of ASCO/CAP guidelines that the method should be 95% consistent with FISH ³.

Pathologist-assisted computer-mediated visual image analysis systems developed in recent years aim to eliminate the subjectiveness derived from manual evaluation and also make tele-consultation possible. Any field on the preparation can be examined in detail, making it easier to correctly evaluate tumor morphology and various tumor markers ^4-7.

We then re-evaluated the ICH and SISH results of these cases using the Ventana Image Analysis System (VIAS, Ventana Medical Systems, AZ, USA). We compared the degree of conformance between manual and computer-mediated visual analysis system for both methods (ICH and SISH). Cases where the primary tumors were evaluated at our department were fixated for 6-12 hours in 10% buffered neutral formaline and embedded in paraffin blocks following routine procedures.

Afterwards, sections 5 μm thick were obtained and stained routinely with hematoxylin-eosin and immunohistochemically for HER2/neu.

HER2/neu (CB11, Ventana&Pathway) scoring was performed by evaluating membrane staining, using the ASCO/CAP (American Society of Clinical Oncology/ College of American Pathologists) 2007 recommendations ⁹. Accordingly;

- N egative IHC staining for the HER2/neu protein, 0 or 1+: No staining or weak, incomplete membranous staining in a certain percentage of tumor cells.

- Significant IHC staining for the HER2/neu protein, 2+: Weak or non-homogenous complete membranous staining in at least 10% of the tumor cells.

- Positive IHC staining for the HER2/neu protein, 3+s: Homogenous, dense complete membranous staining in more than 30% of the invasive tumoral cells.

SISH staining was also performed, after sections 4 μm thick prepared from the best block were obtained onto slides with adhesive to study and stained with the BenchMark automatic preparation stainer (Ventana Medical Systems AZ, USA). The SISH protocol consisted of deparaffinization citrate addition, incubation with ISH protease, addition of the HER2/neu DNA or Kr-17 probe and incubation for hybridization, incubation with Solver C, using hematoxylin as a counterstain and incubation with bluing following counterstaining. The protease duration and incubation duration with DNA probes was optimized for each material to protect tissue morphology and make signals visible. SISH results were evaluated according to the producer’s guidelines on the light microscope (x20, x40 lens) with the the semiquantitative method (method 1) or the quantitative method (method 2 or method 2a). The HER2/neu and Kr-17 slide adequacy was determined before using this method. The mean HER2/neu and Kr-17 signal number was determined semiquantitatively on method-1 and the ratio determined. The ratio was evaluated as follows:

1. HER/Chr17 < 1.4: Negative for HER2/neu gene amplification.

2. 1.4 ≤ HER/Chr17 ≤ 4: Borderline for HER2/neu gene amplification

3. HER/Chr17 > 4: Positive for HER2/neu gene amplification.

Method 2 was used when the results were borderline with method 1. Method 2 is a quantitative method. Once the adequacy of the slide was confirmed, HER2/neu signals were counted in 20 cells in a suitable target area within the invasive tumoral area followed by counting the Kr-17 signals in 20 cells and the ratio of the HER2/neu signals to Kr-17 signals was calculated. The assumption was that gene amplification was not present if this ratio was less than 1.8 while it was present if higher than 2.2. Method 2a was used if the ratio was ≥1.8 and ≤2.2 and the HER2/neu and Kr-17 signal was counted in another 20 cells in a suitable adjacent area in addition to the 20 cells in method 2 (a total of 40 cells). Similarly, gene amplification is not present if this ratio is less than 1.8 and present if it is more than 2.2. Cases where the the ratio was ≥1.8 and ≤2.2 were accepted as borderline and controversial.

The SISH results and ICH results for all these cases obtained by manual evaluation and the required number of different microscopic images obtained taking the same fields as the basis were again evaluated with the VIAS.

Statistical Analysis: The significance of the conformance between the VIAS and manual evaluation of SISH results and the VIAS SISH and IHC results were determined using the Kappa coefficient. A p<0.05 meant that the results were significant.

There was no statistically significant conformance between IHC results evaluated by VIAS and SISH results evaluated by VIAS (k= 0.040 and p= 0.263) (Table I). The rate of consistency between the VIAS evaluation and manual evaluation of SISH results was 91% and these evaluation results were consistent in a statistically significant manner (k= 0.832 and p<0.001) (Table II) The HER2/neu score was 2 in all cases when the IHC was evaluated manually and we were therefore unable to evaluate the statistical consistency rate between the manual and VIAS IHC evaluation and similarly manual IHC and SISH evaluation. The consistency rate between manual HER2/neu IHC evaluation and VIAS evaluation results was 76%.

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Table I: Cross-table of IHC and SISH results evaluated by VIAS

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Table II: Cross-table of VIAS and manual evaluation of SISH results

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Figure 1: VIAS photographs from various areas of the case that was immunohistochemically evaluated as HER2/neu score 2.

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Figure 2: VIAS evaluation of the absence of HER-2 gene amplification by SISH in the same case. There is staining with a normal pattern as 1-2 spots representing HER-2 in the tumor cells.

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Figure 3: Kr-17 SISH preparations of the same case show staining with a normal pattern as 1-2 black spots in tumor cells. However, these have not been counted by the device and the cells where Kr-17 spots will be counted have been selected by the pathologist.

VIAS has been found to successfully select invasive tumoral areas in all cases. However, the system also makes it possible for the pathologist to exclude any areas if there are any suspicions about a particular area. Some authors see computer-mediated systems as a method that will increase the success of IHC evaluation ^5,7,9. It has also been reported that inter-observer differences in evaluation due to tumor heterogenousness can be eliminated with these methods ⁷. However, the disadvantage of visual image analysis systems is their high cost which precludes daily practical use at all sites ⁵.

The consistency between manual and VIAS evaluation of SISH results was statistically significant, possibly due to SISH being an objective evaluation method. There was also only 1 case that was different as regards positivity/negativity between manual and computer-mediated evaluation of SISH results. All other results were borderline cases. This supports the notion that both are current methods for HER2/neu evaluation.

There is no statistically significant consistency between the VIAS-evaluated SISH results and IHC results. This is probably due to the difficulty in standardizing tissue followup and fixation that can lead to IHC staining differences.

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