‘348 International Journal of Surgical Pathology Vol. 18 No. 4 October 2005, (3+) by IHC and 1 showed weak positivity (2+) by IHC. There was only 1 discordant result between FISH and CISH™; this particular case was a ductal carcinoma scored as 2+ by IHC, nonamplified by FISH, and low-level amplified by CISH™ with dis- omy of chromosome 17 (Table 3). ‘Comparison of IHC with CISH™ for HER2 Status, According to Tumor Type Data are summarized in Table 2. Lobular Carcinoma The 2 lobular carcinoma cases with score 2+ by IHC did not show amplification by CISH™. The other 3 cases were negative for HER2 analysis by THC and CISH™, including 1 case of HER2 deletion (chromosome 17 polysomy not associated with in- creased copies of HER? gene). Ductal Carcinoma Ten of 12 ductal carcinomas with 3+ by THC dis- played high-level amplification by CISH™. Of the remaining 2 cases, 1 displayed low-levels of HER2 amplification and aneusomy for chromosome 17 {and considered as not true amplification); the other 1 was negative by CISH™, Two of 21 cases with a 2+ score by IHC showed low levels of HER2 amplification and disomy for chromosome 17; therefore, they were considered as ‘truly amplified. Two cases scored as 2+ by IHC dis- played #7ER2 low-level amplification and aneusomy for chromosome 17 and were not considered as true amplification. Seventeen of the 21 cases were nega- tive by CISH™. ‘The remaining 9 cases were negative by IHC and did not show gene amplification by CISH™ (Table 2) Pleomorphic Lobular Carcinoma One pleomorphic lobular carcinoma showed a 2+ score by IHC. This case displayed HER2 amplifica- tion. The remaining case was negative for IHC and cIsH™, ‘Table 3. Correlation Between HER-2 Amplification, by FISH and CISH in Ductal Carcinoma Micropapillary Carcinoma From the 6 mictopapillary carcinoma cases eval- uated, 1 showed a 3+ score by IHC and high-level HER2 amplification (Table 2). Four cases were 2+ by IHC for HER2. Two of them showed high-level HER2 amplification, 1 case displayed low-level HER2 amplification and aneusomy for chromosome 17 (no true amplification), and 1 was not amplified by CISH™. The remaining case showed negativity for IHC and CISH™. The correlation between HER2 gene amplification and protein overexpression is shown in Table 4 Discussion HER? analysis in tumor specimens, which is essential for starting trastuzumab (Herceptin®) treatment, has been examined by different methods [4,5,14,18-20] In clinical practice, IHC evaluation is the most widely used, and along with FISH, they are the 2 methodolo- gies currently approved by the FDA [6]. IHC on FFPE tissues has shown many discrepant results due to antigenic alterations related to fixa~ tion procedures [3], differences in sensitivities and specificities of the commercially available antibodies [9,21-25}, and interobserver variability {21] ‘Although no true gold standard has been estab- lished, FISH is considered a reference method be- cause it has provided more consistent results that correlate with prognosis [12] and response to ther- apy {7,8,26]. However, itis not available in most lab- oratories and requires special facilities and expertise, CISH™ has been introduced as a practical alter- native to FISH in detecting HER2 amplification in FEPE breast carcinoma samples [14]. Several arti- cles have pointed out the advantages of CISH™ over FISH in the assessment of HER2 amplification, ‘These advantages include: (1) hybridization can be visualized with an ordinary microscope, eliminating the need for a fluorescence microscope: (2) the sig- nal intensity does not decrease over time; (3) the histology is easier to analyze; and (4) it is less ex- pensive than FISH [9,14,18,19] ‘Table 4, Comparison Between HER-2 Amplification by CISH and HER-2 Overexpression by IHC FISH cist ‘Amplification 10 11 No amplification 2 2 Total 2 32 mc cist one 2 a “Amplification ° 5 rn Noamplificaion 4 23 2CISH™ for HER2 Status in Breast Cancer @ LiNing-Tet al. 349 In this study we have tested the applicability of CISH™ for the determination of the HER2 starus in archived surgical samples. As quality control, a sub- set of our CISH™ cases was compared with FDA-ap- proved FISH performed and evaluated indepen- dently. CISH™ and FISH gave the same results in 31 of 32 cases (96.9%). Previous reports have found a good agreement between CISH™ and FISH within a range of 83.8% to 100% [9,14,19,27,28]. ‘Whether chromosome 17 polysomy can substan- tially affect HER2 protein expression and conse- quently alter the correlations between hybridiza- tion-based tests and THC still needs to be determined. In fact, some results indicate that FISH HER2 total number correlates better with overex- pression than do HER2 numbers corrected for chro- mosome 17 [13.29,30]. Moreover, it has been ar- gued that chromosome 17 polysomy adds relevant, information in only a minority of cases (9,31,32). We decided to evaluate chromosome 17 polysomy in all the CISH™ cases because this cor- rection is part of the FISH assay approved for the se lection of candidates to trastuzumab. Without this, correction only 27 of the 32 cases (84.4%) would have been concordant with FISB. Furthermore, a cutoff of 6 HER2 signals has been proposed to define amplification without the need to correct the results for chromosome 17 polysomy [3]. This is based on the observation that chromo- some 17 polysomy is infrequent above 5 copies in breast carcinomas [13,34]. Nevertheless, in our study this cutoff was not sufficient to detect true amplification, Chromosome 17 assessment was re- quired particularly in the group with 6 to 10 copies of HER2 allowing us to reclassify 4 of 6 low-amplified cases as nonamplified and to corroborate the diag- nosis of low-level amplification in 2 cases associated with chromosome 17 disomy. No variations in the diagnosis of the high-level amplified group occurred due to the polysomy correction. Hence, our results, underscore the need to apply the chromosome 17 correction as a minimum to al the low-amplified re- sults by CISH™ and suggest that it can be omitted in the majority of the rest of the cases. In routine prac- tice, reducing the number of cases that need to be corrected for chromosome 17 should further reduce costs and time of processing significantly. Other than the chromosome 17 correction, many technical factors can contribute to the minor CISH™-FISH discrepancies observed earlier. Exper- tise and optimization of the technique according to the conditions of each laboratory should help in re- ducing differences. In our experience, slight techni- cal adjustments to the manufacturer's protocol were needed, commonly regarding times and tempera- tures, In some cases the DAB chromogen produced excessive background precipitation that eventually could lead to misinterpretations. Fast Red chro- mogen can be an alternative to DAB; for that pur- pose an alkaline phosphatase-labeled probe should be used instead of the original horseradish-peroxi- dase-labeled probe. Our cases that were negative by IHC were also negative for HER2 gene amplification (100% agree- ment) including 1 case of HER2 deletion (a lobular carcinoma with chromosome 17 polysomy not as- sociated with increased copies of HER2 gene). Using the same A0485 antibody (DAKO) for THC, Zhao et al [9] obtained results with a similar agreement. The lower potential false-negative results of the A0485 antibody compared to other antibodies commonly used such as CB11 or TAB250 [9,14] suggest that this antibody could be better suited for the initial screening of candidates for trastuzumab therapy. On the contrary, cases scored as 2+ by IHC showed a low concordance with HER2 amplification. by CISH™ (5 of 28; 17.86%). These results are within the range of 12% to 36% reported in com- parative FISH-IHC studies (23,32,35,36], and the results of Zhao et al [9], who also compared the 0485 antibody to CISH™. The low specificity of IHC in the 2+ scored category has been attributed to chromosome 17 polysomy [35], up-regulation of transcription, or protein accumulation [20,31] However, in most 2+ cases that lack gene amplifica- tion, chromosome 17 polysomy does not seem to be the cause of overexpression because they do not ex- hibit a significant increase in the chromosome 17 number when compared to 0-1+ cases [34]. Fur- thermore, no increase in mRNA has been observed in the majority of weakly positive cases by IHC in absence of HER2 amplification [32]. Therefore, al- though overexpression unrelated to amplification exists [37], most of these 2+ cases are likely false positives due to THC technical artifacts. In our series, 11 of 13 cases that were scored as 3+ by IHC showed HER2 amplification by CISH™ (84.61% agreement). The same concordance was found in the study of Zhao et al [9]. In contrast to the 2+ group, overexpression without amplification seems to be due to chromosome 17 polysomy in a subset of 3+ cases [34,38]. Accordingly, chromo- some 17 polysomy was detected in 1 of the 2 dis- ‘crepant cases. Factors such as single-copy overex- pression at the transcriptional level [3,5,32] or CISH™ technical limitations might be the cause of the discordance in the other case. Interestingly, sim- ilar correlations in the 3+ category have been de- scribed with FISH (between 75% and 100%) [23,32,35]. One study even found a low 49% FISH-‘350 _ International Journal of Surgical Pathology Vol. 13 No. 4 October 2005 (3+) THC concordance [22], and the authors sug- gested that FISH should be performed to confirm all the 3+ cases. Importantly, although standardized scoring crite- ria were used in the evaluation of the IHC results, the commonly accepted CISH™ scoring criteria [14] are not comparable in stringency, While the IHC test requires that a minimum of 10% of tumor cells be reactive 10 be considered positive, CISH™ scoring according to Tanner et al {14} requires that more than 50% of tumor cells show an increase in gene copies to be considered amplified. It is conceivable that this difference could account for some of the discordant results observed between CISH™ and IHC. Increased IHC-FISH dissociations have been de- scribed in invasive lobular carcinoma [22] and duc- tal carcinoma in situ [31], suggesting that mecha- nisms other than amplification might lead to overexpression in different histologic settings. In our study, owing to the small number of cases, we could not corroborate the increased discrepancy be- tween overexpression and amplification described in lobular carcinomas. Consistent with previous reports that correlated overexpression and amplification by different methodologies [9,12,23,31,32,35,36,39], our study found a good concordance between CISH™ and IHC in the group of the 0-1+ and 3+ scored cases and a poor concordance with the 2+ group. It has been shown that most of the 2+ scores and possibly some 3+ by IHC of FEPE specimens are not reliable and that confirmation with amplification is necessary. In our experience, CISH™ is a sensitive and spe- cific instrument in the evaluation of HER2 status. To detect amplification as defined by the FDA-ap- proved FISH test, chromosome 17 correction should be applied to all low-level amplified cases. Unlike FISH, CISH™ allows an easy histologic interpreta- tion, which can be helpful, for example, in cases where foci of invasive carcinomas are obscured among extensive areas of an in situ component (the expression of HER2 in ductal carcinoma in stu does not have the same prognostic or predictive implica~ tions as in invasive carcinomas [40,41]}). Although not approved by the FDA, CISHT™ is a technique that can be easily implemented and offers advantages over the sole THC evaluation because it offers important information on the gene amplifica~ tion status. Furthermore, it can help discriminate whether a positive IHC staining is due to gene am- plification or to other factors. Therefore, a more complete HER2 status analysis combining molecular and histopathologic findings can be done by imple- menting CISH™ in a surgical pathology laboratory. Along with IHC, CISH™ can provide very important information to be used in the management of breast cancer and in clinical tals, References 1. 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