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   Sixty-nine invasive ductal carcinoma specimens that had been submitted to the Department of Pathology, King Chulalongkorn Memorial Hospital for HER2 DISH assay during 2012-2013 were included in the study. Fifty-eight cases were institutional cases while the remaining eleven cases were specimens received from other institutes.

   The morphological grading was available for 55 institutional cases (6 grade 1, 29 grade 2, and 20 grade 3). The HER2 IHC score using the ASCO/CAP guidelines⁵ was available for all 58 institutional cases (32 cases with 2+, 26 cases with 3+).

   The 4 micron-thick sections were prepared from formalin-fixed paraffin-embedded tissue of invasive carcinoma specimens. Sections were then stained with Ventana Benchmark XT system (Roche Diagnostics), using an Inform HER2 Dual ISH DNA Probe Cocktail (Ventana Medical System). The HER2 signals were visualized with an UltraView SISH DNP Detection Kit (Ventana Medical Systems) while the chromosome 17 centromere (CEP17) signals were visualized with an UltraView Red ISH DIG Detection kit (Ventana Medical Systems).

   One pathologist (SS) inspected the DISH slides and digitally captured 10-20 images of the invasive components at the x600 magnification, using Olympus DP22 digital camera for microscopes (Olympus Optical CO. LTD., Japan). Two investigators (TA and TC), without the knowledge of HER2 IHC status, independently counted the signals of 20 tumor cells with well-visualized signals, and calculated the HER2/CEP17 ratio and HER2/nucleus ratio. Interpretation of the HER2 gene copy number was made according to the ASCO/CAP guidelines as shown in Table 1.⁵ The results were then compared between the two investigators.

Table 1: Interpretation Guideline of HER2 ISH (ASCO/CAP 2013).

Statistical analysis

   We constructed a scatter diagram from the HER2/CEP17 ratios of both investigators and made a comparison. The Spearman’s correlation coefficient (Rho) was calculated using SPSS statistic 20 (IBM Corporation, New York, USA). The Rho of 0 to 1 indicates a positive correlation and -1 to 0 indicates negative correlation. The strength of correlation coefficients is as follows: 0 and 1 indicate zero and perfect correlation respectively; ±0.1 to ±0.3 indicate weak correlation; ±0.4 to ±0.6 indicate moderate correlation; and ±0.7 to ±0.9 indicate strong correlation. A p-value was calculated using p-value of < 0.05 as the cut off point for statistical significance.

   The agreement of both investigators (amplified vs. non-amplified) was compared using ĸ statistic. The ĸ statistic of 0 to 0.4 indicates poor agreement, 0.4 to 0.6 indicates moderate agreement, 0.6 to 0.8 indicates good agreement and 0.8 to 1 indicates excellent agreement.

   Two cases were considered uncountable. One example showed only CEP17 signals without HER2 signals and was regarded as uncountable by both investigators. Another case showed strong CEP17 signals with faint and small HER2 signals in only a small portion of the tumor cells. This case was regarded as uncountable by investigator 1 but countable by the other (HER2/CEP17 = 0.76, non-amplified). These two cases were excluded from analysis. There were 5 disagreed cases as shown in Table 2. All of them are considered non-amplified by investigator 1 and amplified by investigator 2. Table 3 shows the correlation between the IHC score and DISH HER2 status, excluding the 5 discordant cases and 2 failed DISH cases. The tumors with IHC score 2+ were 14% HER2 amplified by DISH method while the tumors with IHC score 3+ were 77% HER2 amplified by DISH method. Agreement of both investigators for HER2 gene diagnosis was 92.75 % (ĸ = 0.848), reflecting an excellent agreement. The HER2/CEP17 ratio of investigator 1 and 2 ranged from 0.8 to 13.46 and 0.76 to 11.63 respectively. 26 cases were interpreted as HER2-amplified by investigator 1, while investigator 2 interpreted 31 cases as amplified. None of the cases was in the equivocal category or showed heterogeneous HER2 gene copy number. Representative images of HER2-amplified and nonamplified are depicted in Figure 1 and 2, respectively.

Table 2: Disagreed Cases.

Table 3: Correlation of HER2 IHC score and DISH status of all concordance cases.

Figure 1: Invasive breast cancer with HER2 amplified by DISH method.

Figure 2: Invasive breast cancer with HER2 non-amplified by DISH method.

Figure 3

   The HER2/CEP17 ratios obtained from both investigators were compared. The scatter diagrams revealed a strong correlation between the two (Rho = 0.883, p < 0.001) (Figure 3). The case by case comparison is shown in (Figure 4). All 5 discordant cases have HER2/CEP17 ratios of less than 3 and most of them fall in the equivocal range of the previous ASCO guideline.⁹ The HER2 count and CEP17 count were compared separately. Both HER2 and CEP17 counts from two investigators show strong correlation (HER2 count: Rho = 0.955, p < 0.001; CEP17 count: Rho = 0.779, p < 0.001). The scatter diagrams are shown in Figure 5 and 6.

Figure 4: HER2 DISH results assessed independently by two investigators (disagreed cases marked with red arrow heads).

Figure 5: Invasive breast cancer with HER2 amplified by DISH method.

Figure 6: Invasive breast cancer with HER2 amplified by DISH method.

   Discovery of HER2 and its implication in breast cancer is one of the examples of molecular oncology in which fundamental biological knowledge has been transformed into a practical application and has saved lives. To be able to fully appreciate the importance of this knowledge, HER2 status must be established in virtually all breast cancer cases. Accessibility to the HER2 tests is dependent on overcoming technical difficulty, logistical issues, interpretation difficulty, and cost. HER2 immunohistochemistry (IHC) has been used widely at an acceptable cost and has proven to be a great screening tool for determining HER2 status.¹⁰ When HER2 IHC status is inconclusive (2+), assessment of HER2 gene copy number is mandatory and the FISH method has been regarded as the gold standard for this purpose for many years. FISH technique, however, requires the costly fluorescent microscope for visualization. HER2 DISH assay that can assess the gene copy number under an ordinary microscope has emerged as an alternative tool to FISH. Multiple studies validating the HER2 DISH assay with FISH have shown promising results.^7,8 Comparison between DISH and FISH methods is shown in Table 4. We can expect the shift from the FISH assay to DISH in the near future.

   Determination of the HER2 gene copy number is typically done by manual counting⁵, and interpersonal and even intrapersonal reproducibility may have an impact on the testing result. To address this issue, two observers were asked to independently assess the captured DISH images in this study. Very high correlation was found in the result obtained from the two investigators, and this implies that all tumor samples enrolled were reasonably homogenous in terms of HER2 status. Previous evaluations of intratumoral heterogeneity in breast cancer have also demonstrated similar results.^11,12 Having said that, we do acknowledge the increasingly recognized cases of breast cancers with heterogeneous HER2 copy number^13,14 but such an example was not found in our relatively small series of cases.

Table 4: Comparison between DISH and FISH methods for HER2 gene assessment.

   The ASCO 2013 set the cut-off for HER2/CEP17 ratio at 2.0 for a tumor to be assigned as being ‘HER2 amplified’. As expected, cases with discrepancy in the results in our cohort had a HER2/CEP17 ratio around that level (negative for or low level of amplification). Multiple studies have shown that the response to Trastuzumab therapy correlates highly with the level of amplification, with a significant response noted when HER2/CEP17 > 6.^6,15 Therefore, the discrepancy cases in our series are unlikely to have a huge impact on the patients’ long term survival.

   The DISH method for determining the HER2 gene copy number is highly reliable, with high inter-observer reproducibility. Only a few discrepancy cases fell into the range of negative or low level of HER2 gene amplification. The high inter-observer reproducibility also indicates that, in terms of HER2 status, most of the invasive breast carcinomas are fairly homogeneous throughout the lesion.