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Year : 2012  |  Volume : 55  |  Issue : 2  |  Page : 175-179
HER 2 status in invasive breast cancer: Immunohistochemistry, fluorescence in-situ hybridization and chromogenic in-situ hybridization

Division of Molecular Pathology, Department of Pathology, Tata Memorial Hospital and Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Mumbai, India

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Date of Web Publication3-Jul-2012


Introduction : HER2/neu gene status in breast cancers can be evaluated by targeting protein and gene - immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH). Recent studies have shown chromogenic in-situ hybridization (CISH) as a relatively cheaper alternative. Materials and Methods : Forty-three nonconsecutive, randomly selected primary invasive breast cancer cases were evaluated for c-erbB-2 (HER2 protein) by IHC and gene amplification by FISH and CISH. Results of each of the same were compared. Results : CISH showed approximately 90% and 100% concordance for IHC negative and positive cases, respectively; while approximately 94.4% and 91% concordance with FISH amplified and non-amplified cases, respectively. Conclusion : This study showed feasibility of incorporation of CISH as a low cost option in routine management of breast carcinoma in the Indian setting. Secondly, reconfirmation of IHC negative and positive cases can be done by CISH.

Keywords: Breast cancer, chromogenic in-situ hybridization, fluorescence in-situ hybridization, HER2/neu, immunohistochemistry

How to cite this article:
Shirsat HS, Epari S, Shet T, Bagal R, Hawaldar R, Desai SB. HER 2 status in invasive breast cancer: Immunohistochemistry, fluorescence in-situ hybridization and chromogenic in-situ hybridization. Indian J Pathol Microbiol 2012;55:175-9

How to cite this URL:
Shirsat HS, Epari S, Shet T, Bagal R, Hawaldar R, Desai SB. HER 2 status in invasive breast cancer: Immunohistochemistry, fluorescence in-situ hybridization and chromogenic in-situ hybridization. Indian J Pathol Microbiol [serial online] 2012 [cited 2023 Jan 30];55:175-9. Available from:

   Introduction Top

Evaluation of HER2/neu gene status is an important prognostic and predictive biomarker in breast cancer management. [1],[2],[3] It is estimated that 20% of the current HER2 testing may be inaccurate. [4] HER2 testing can be done by targeting protein and gene. The most widely used methods to detect HER2 amplification are immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH), both are amenable on formalin-fixed paraffin-embedded (FFPE) specimens. The former is relatively inexpensive but technically less specific while the latter is specific but expensive. [5]

Chromogenic in-situ hybridization (CISH) is a cost-effective amalgamation of the aforementioned two techniques with a reasonable accuracy. [6],[7],[8],[9] The present study has been undertaken to evaluate the feasibility of introduction of CISH in the routine diagnostic surgical or molecular pathology laboratory and comparison with IHC and FISH for determination of HER2/neu status.

   Materials and Methods Top

The study protocol was approved by institutional review board, TMH. Forty-three nonconsecutive, randomly selected primary invasive breast cancer cases were evaluated for HER2 protein and gene amplification in the Divisions of Immunohistochemistry and Molecular Pathology, Department of Pathology, TMH & ACTREC, by IHC, FISH, and CISH. Twelve of 43 cases were referrals. The in-house cases were adequately fixed in 10% neutral-buffered formalin for a minimum of 12-16 h for excision specimens and an hour for biopsy specimens. Sections were obtained from the same block for all three - IHC, CISH and FISH tests. Three to four micron thick tissue sections were obtained on double-coated poly-L-lysine (PLS) slides for CISH and FISH; while on single-coated PLS slides for IHC.

Chromogenic In-situ Hybridization

Single color SPoT-Light HER2 CISH polymer detection kit (Zymed, Invitrogen, USA) comprising HER2 double-stranded DNA probe labeled with digoxigenin was used. The hybridization results were visualized using Nikon bright field microscope under 40×, 60×, and oil immersion objectives. Only the areas showing invasive component were studied. Necrotic areas, overlapping nuclei, and nuclei with weak signal intensity were avoided. The results were interpreted as per the guidelines by Tanner et al.[6] i.e. > 5 signals per nucleus were considered as amplified. In cases where signal count was 5 to 6, additional 30 tumor cells were counted. If the test failed due to sections washing off, excess paraffin or improper protease digestion, the assay was repeated. If the results were uninterpretable, the test was considered failed. The normal ductal cells, lymphocytes and stromal cells served as the internal control, showing two signals in each nucleus [Figure 2]a and b.
Figure 1: (IHC): Representative photomicrographs of cases with immunohistochemical evaluation for c-erb B2. Eleven cases were negative (a ×400; score 0 and b ×200; score1+), twenty-one equivocal (c ×400; score 2+) and eleven were positive (d ×400; score 3+)

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Figure 2: (CISH ×400): Normal native duct epithelium (a) and lymphocytes (b) showing an average of two signals per nucleus as represented by brown colored dots within the nucleus. 2C and D – Representative photomicrographs of cases showing non-amplification for HER2 by CISH, one of the case showing an average of 2 signals per nucleus (c), while the other case represented in (d) shows average of 3– 4 signals per nucleus

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Immunohistochemical Evaluation

HER2 protein/c-erbB-2 expression by IHC using 3B5 monoclonal antibody (Immunotech, Marseilles, France) was first validated against the US FDA approved HercepTest (DAKO Corp, Carpinteria, Calif.) in 1:10 dilution. Antigen retrieval was done by microwave method. The slides were interpreted using ASCO/CAP guidelines. [10]

Fluorescence In-situ Hybridization

FISH was performed using the US FDA approved PathVysion (Abbott Molecular Inc., Des Plaines, IL) HER2 DNA dual-colored Probe Kit and the slides were interpreted as previously described by Panjwani et al. [11] ProbeChek control slides were run simultaneously with the test cases. The slides were then stored in the dark at -20 o C.

IHC, FISH and CISH results were interpreted independently and blinded manner by the three pathologists.

   Results Top

A total of 43 cases were subjected for evaluation of HER2 protein by immunohistochemistry and HER2 gene evaluation by FISH and CISH.


Eleven were negative (score 0 and 1), 21 equivocal (score 2) and 11 were positive (score 3+) as per the ASCO guidelines [Figure 1]a to d. [10]

Chromogenic In-situ Hybridization

In 39 cases, 100 nuclei were counted. In three cases, it was not possible to assess 100 tumor nuclei due to inadequate tumor tissue availability; at least 50 nuclei were counted. One case showed too faint signals to evaluate and were seen in less than 30% of nuclei, thus the case was excluded from the statistical evaluation. As per the scoring criteria by Tanner et al.,[6] 20/42 cases showed amplification (> 5 signals per nucleus [Figure 3]a,b and [Figure 4]a,b) and 22/42 were non-amplified (≤ 5 signals per nucleus) [Figure 2] c,d.
Figure 3: (CISH a,b ×400): Photomicrographs showing HER2 gene amplification by CISH in the form of multiple signals with average of more than 6 signals per nucleus (as shown by arrows)

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Figure 4: (CISH a,b ×400): Photomicrographs showing HER2 gene amplifi cation by CISH in the form of large gene clumps in the nucleus

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Fluorescence In-situ Hybridization

Minimum of 40 tumor cells were counted in all 43 cases. As per the ASCO guidelines, 18 were amplified (ratio >2.2) and 23 were non-amplified (ratio <1.8). Two cases were equivocal (ratio 1.8-2.2).

Comparison between IHC and CISH

All the 11 IHC 3+ cases were CISH amplified (100% [Figure 5]a, c). Out of the 21 IHC 2+ cases, 8 (38.09%) were amplified and 13 (61.90%) were non-amplified by CISH. Of the 11 IHC negative (score 0/1+), one case failed, 9 cases (81.82%) were non-amplified while one (9.09%) was amplified by CISH.

Concordance between IHC negative (score 0/1+) cases with CISH non-amplified cases was 90.90%, that between IHC 3+ cases with CISH amplified was 100% while 61.90% (13/21) of IHC 2+ cases were CISH non-amplified [Table 1].
Figure 5: Photomicrographs of an HER-2 gene amplifi ed case shown by IHC (a ×400; score 3+), FISH (b ×1000; amplifi cation seen by large gene clusters as represented by red clumps) and CISH (c ×400; amplifi cation seen by large gene clusters as represented by brown clumps within the nuclei)

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Table 1: Correlation between CISH and IHC

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Combining the IHC negative (score 0/1+) cases with equivocal (score 2+) cases and on comparing this group with CISH negative group, the concordance was 70.96% [Table 2], while the concordance between the combined IHC 3+ and 2+ group and CISH positive was 90% [Table 3].
Table 2: Comparison of CISH and IHC after combining IHC Negative (0 and1+) and Score 2+ Cases

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Table 3: Comparison of CISH and IHC after combining Score 2+ and Score 3+ Cases

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Comparison between FISH and CISH [Table 4]

Of the 18 FISH amplified cases, 17 cases showed amplification and one showed no amplification by CISH [Figure 5]b, c. The latter, which was detected as amplified by FISH, showed an average of 5 signals by CISH falling short of the stipulated cutoff of 6 for amplification. Of the 23 FISH non-amplified cases, 21 were non-amplified and two were amplified by CISH. The two cases which were interpreted as amplified by CISH in fact had polysomy 17, as demonstrated on FISH. Of the two equivocal FISH cases, one showed low level of amplification with CISH and the other case had an average of 3-5 signals [Figure 6]a, b and c.
Figure 6: Photomicrographs of a case, where HER 2 was equivocal (score 2) by IHC (a ×400). Not amplified by FISH, however showed polysomy 17 (b ×1000) and showed low amplifi cation by CISH (c ×400; variable population with admixture of cells with 4– 6 signals)

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Table 4: Correlation between CISH and FISH for the detection of HER2 gene (n = 43)

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To facilitate statistical analysis, we compared CISH non-amplified cases with combined FISH equivocal cases and non-amplified cases as shown in [Table 5]. The concordance for the amplified group was 94.44% and for non-amplified was 87.50%.
Table 5: Correlation between CISH and FISH after combining equivocal and non-amplifi ed groups of FISH

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   Discussion Top

CISH is a cost effective amalgamation of the positive features of both IHC and FISH; allowing gene evaluation using bright field microscopy with simultaneous morphological assessment. [6],[7],[8],[9] The slides can be archived and no elaborate training is needed for interpretation.

In the present study, we evaluated invasive breast cancer cases (with known HER2 status on IHC and FISH) for the feasibility of CISH. We deliberately chose to include non-consecutive cases in order to have an optimum representation of different IHC groups (negative: score 0/1+, equivocal: 2+, and positive: 3+ cases).

Of the 43 cases, results of CISH could be interpreted in 42 of them (97.67%) suggesting its feasibility for incorporation in the routine practice. CISH was 100% concordant with the IHC 3+ cases while it had a concordance of 90% for IHC negative cases [Table 1]. Our results are in keeping with those of previous study by Vijver et al.,[12] in which overall concordance of 92% in IHC negative (score 0/1+) cases and a concordance of 91% in IHC 3+ cases was documented.

There was no amplification in 61.90% of IHC 2+ cases on CISH [Table 1], which is in keeping with the literature that most of the IHC score 2+ cases are not gene amplified. [13] A single IHC negative case showed low amplification with CISH. Based solely on IHC result, this case would have been excluded from the potential candidature for anti-HER2 treatment. IHC is a protein assay. Any variation from the standard tissue processing could lead to degradation of the proteins or down regulation of the post-transcriptional or translational events may lead to low protein expression; while DNA is more resistant, hence pre-analytical factors have less effect on the test result. CISH detects amplification at the gene level. It has been documented that about 10% cases are spuriously score 3+ IHC positive. This positivity is attributed to excess antigen retrieval, polysomy 17, over-expression at the mRNA transcriptional level or beyond without actual gene amplification. However, we found that all the IHC 3+ cases in the present study were CISH amplified [Table 1].

Of 21 IHC 2+ cases of the present study, 8 (38.09%), cases showed gene amplification; which is slightly higher than the incidence reported in different studies in the literature. [12],[13] The reasons for this appear to be to underscoring of HER2 on IHC, either because of subjective interpretative variation in reporting, variable pre-analytical conditions, imperfect IHC methodology etc. Thomson et al[14] showed poor inter-observer agreement in the scoring of IHC 1+ and 2+ cases in their study. In addition, a significant proportion of cases in the present study were referred from elsewhere, where various aspects of processing like nature, period of tissue fixation, method of processing, temperature of paraffin embedding etc., lead to improper preservation of antigenicity, thereby affecting the IHC results. [5],[11]

Hence a gene assay is required to determine the actual candidates for targeted therapy in this group. According to the ASCO 2007 guidelines, all the indeterminate cases must be subjected to FISH confirmatory test. [10] However, since FISH is an expensive test, it would be rewarding if a more economical test can substitute FISH; and still accurately determine the gene status of the indeterminate cases. Recent studies have shown CISH as a reasonable alternative. [6] In the present study, of the total 18 FISH amplified cases, 17 also showed amplification with CISH [Table 4] and [Table 5]. A single case which failed to show amplification had more than 50% of cells showing an average of 5 signals, just short of the required cutoff value of 6. This difference could be due to the difference in the scoring system of FISH, which is reported as a ratio of gene signal with chromosome enumeration probe signal; while CISH used in the study is single-colored assay, where average number of signals is reported. Two cases which showed amplification by CISH were non-amplified on FISH, in addition to having polysomy. The polysomy 17 in these cases were the cause of erroneous CISH results. This is the major drawback of single color CISH, which can be overcome by using dual-color probe kits. Of the two equivocal FISH cases, one case showed low level of amplification with CISH while the other was non-amplified.

The main utility of gene assay is to resolve the equivocal IHC 2+ cases. Here, in the present study, we compared the results of CISH and FISH in detection of the gene status of the IHC 2+ cases. FISH detected amplification in 8 cases. CISH could successfully detect amplification in 7 of these cases. The overall concordance between the two tests, FISH and CISH, was approximately 94.4 and 91% for amplified and non-amplified cases, respectively [Table 4], which is slightly lower as compared to other studies. [13],[15] However, in the present study, the limitation is the sample size, which is relatively smaller. However, taking into consideration of the vagaries of IHC related to pre-analytical and inter-observer variations, CISH, being economical than FISH, can be effectively used to reconfirm IHC results and substitute FISH in IHC negative and 3+ cases.

   Conclusions Top

The study shows the feasibility of CISH to assess HER2 gene status in routine surgical or molecular pathology laboratory, especially in Indian setting as a low cost option. Secondly, reconfirmation of HER2 status in IHC negative cases can be reliably done on CISH, and may not require the use of FISH. In IHC 2+ and 3+ cases, CISH can serve as a useful adjunct. However, in single color CISH assay, polysomy would still pose a problem. Since polysomy is infrequent, single color CISH can still be used for detecting potential candidates for the anti-HER2 therapy. Now, with the availability of dual-color CISH detection kits these issues can be very well addressed in a larger study.

   Acknowledgments Top

The project was funded by our institutional intramural grant (project 441).

We acknowledge Ms. Savita Rajpurohit, Ms. Sampada Gursale, Mr. Mahendra Palkar and Ms. Rekha Thorat for their technical contribution.

   References Top

1.Walker R. Use and assessment of diagnostic and predictive markers in breast pathology. Curr Diagn Pathol 2007;13:126-34.  Back to cited text no. 1
2.Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, et al. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 2000;124:966-78.  Back to cited text no. 2
3.Ross JS, Fletcher JA, Bloom KJ, Linette GP, Stec J, Symmans WF, et al. Targeted therapy in breast cancer: The HER-2/neu gene and protein. Mol Cell Proteomics 2004;3:379-98.  Back to cited text no. 3
4.Tsuda H. HER-2 (c-erbB-2) Test update: Present status and problems. Breast Cancer 2006;13:236-48.  Back to cited text no. 4
5.Tawfik OW, Kimbler BF, Davis M, Donahue JK, Persons DL, Fan F, et al. Comparison of immunohistochemistry by automated cellular imaging system (ACIS) versus fluorescence in-situ hybridization in the evaluation of HER-2/neu expression in primary breast carcinoma. Histopathology 2006;48:258-67.  Back to cited text no. 5
6.Tanner M, Gancberg D, Leo A, Larsimont D, Rouas G, Picarrt MJ, et al. Chromogenic in situ hybridization- A practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. Am J Clin Pathol 2000;157:1467-72.  Back to cited text no. 6
7.Isola J, Tanner M, Forsyth A, Cooke TG, Watters AD, Bartlett JM. Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ Hybridization. Clin Cancer Res 2004;10:4793-8.  Back to cited text no. 7
8.Hanna WM, Kwok K. Chromogenic in situ hybridization: A viable alternative to fluorescent in situ hybridization in the HER2 testing algorithm. Mod Pathol 2006;19:481-7.  Back to cited text no. 8
9.Palma S, Collins N, Faulkes C, Ping B, Ferns G, Haagsma B, et al. Chromogenic in situ hybridisation should be an accepted method in the routine diagnostic evaluation of HER2 status in breast cancer. J Clin Pathol 2007;60:1067-8.  Back to cited text no. 9
10.Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists Guideline recommendations for Human Epidermal Growth Factor Receptor 2 testing in breast cancer. J Clin Oncol 2007;25:118-45.  Back to cited text no. 10
11.Panjwani P, Epari S, Karpate A, Shirsat H, Rajsekharan P, Basak R, et al. Assessment of HER-2/neu status in breast cancer using fluorescence in situ hybridization and immunohistochemistry: Experience of a tertiary cancer referral centre in India. Indian J Med Res 2010;132:287-94.  Back to cited text no. 11
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12.van de Vijver M, Bilous M, Hanna W, Hofmann M, Kristel P, Penault-Llorca F, et al. Chromogenic in situ hybridisation for the assessment of HER2 status in breast cancer: An international validation ring study. Breast Cancer Res 2007;9:R68.  Back to cited text no. 12
13.Pothos A, Plastira K, Plastiras A Vlachodimitropoulos D, Goutas N, Angelopoulou R. Comparison of chromogenic In situ hybridisation with fluorescence in situ hybridisation and immunohistochemistry for the assessment of Her-2/neu oncogene in archival material of breast carcinoma. Acta Histochem. Cytochem 2008;41:59-64.  Back to cited text no. 13
14.Thomson TA, Hayes MM, Spinelli JJ, Hilland E, Sawrenko C, Phillips D, et al. HER-2/neu in breast cancer: Interobserver variability and performance of immunohistochemistry with four antibodies compared with fluorescence in situ hybridization. Mod Pathol 2001;14:1079-86.  Back to cited text no. 14
15.Saez A, Andrew FJ, Segui MA, Bare ML, Fernandez S, Dinares C, et al. HER2 gene amplification by Chromogenic in situ hybridization (CISH) compared with Fluorescence in situ hybridization (FISH) in breast cancer-A study of two hundred cases. Breast 2005;15:519-27.  Back to cited text no. 15

Correspondence Address:
Sridhar Epari
Department of Pathology, Tata Memorial Hospital and Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Mumbai, Maharashtra
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Source of Support: The project was funded by our institutional intramural grant (project 441)., Conflict of Interest: None

DOI: 10.4103/0377-4929.97855

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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