- Email: [email protected]
neu gene amplification in breast carcinoma patients: Our experience with fluorescence in situ hybridization (FISH) technique
Apollo Medicine 2012 June Volume 9, Number 2; pp. 146e150
Review Article
Her2/neu gene amplification in breast carcinoma patients: Our experience with fluorescence in situ hybridization (FISH) technique R.N. Makrooa,d,*, M. Chowdhrya,d, A. Fauzdara,d, M. Mishraa,d, P. Srivastavaa,d, P. Bhaudauriaa,d, S. Kaulb,d, R. Sarinc,d, P.K. Dasc,d, H. Duac,d
ABSTRACT Background: In India the incidence of breast cancer has increased in the urban population with 1 in every 22 women diagnosed with breast cancer. It is important to know the Her2/neu gene status for a better prognostication of these patients. Aim: The aim of study was to compare the efficacy of fluorescent in situ hybridisation (FISH) and immunohistochemistry (IHC) for determining Her2/neu alteration in breast carcinoma. Material & methods: A total of 188 histologically proven breast carcinoma cases between years 2007e2011 and were retrospectively analysed on the paraffin tissue sections by both IHC & FISH techniques. FISH for Her2/neu gene amplification was performed on cases where the IHC status was already known and the results were compared. Results: A total of 64 (30%) patients were found to be amplified and remaining 124 (65.9%) cases were found to be unamplified through FISH. Patients observed with 3þ reading on IHC were later confirmed as unamplified in 29.5% cases through FISH. Interpretation & conclusion: It has been confirmed with the present study that IHC is a prudent first-step technique to screen tissue samples for Her2/neu gene status but should be supplemented with the FISH technique especially in equivocal cases. Copyright © 2012, Indraprastha Medical Corporation Ltd. All rights reserved. Keywords: HEr2/NEu, Breast carcinoma, IHC, FISH, Cytogenetics
INTRODUCTION Breast cancer is one of the most common malignancies according to the global cancer statistics with high incidence and mortality of breast cancer all over the world.1 India accounts for nearly six percent of deaths due to breast cancer globally. In breast carcinoma, Her2/neu gene amplification is correlated with prognostification and therapeutic implications with over expression in 20e25% of invasive cancers.2,3 HER2 (erbB2/neu) proto-oncogene is mapped to chromosome 17q12-21.324 region and is tyrosine kinase of 185 kDa transmembrane receptor protein of epidermal
growth factor receptor family (EGFR). Her2/neu over expression has been reported to be associated with positive lymph nodes, high histologic grade, high proliferation rate and lack of expression of oestrogen and progesterone receptors associated with a more aggressive phenotype with decreased survival rate.2e7 The incidence of over expression of Her2/neu is highest among all human malignancies, ranging from 9 to 34% in breast cancer patients tested. Her2/neu is the only oncogene with a specific molecular targeted therapy available through monoclonal antibody drug named Herceptin. This therapy functions to treat patients with an over expression in Her2/neu gene by attaching itself
a Department of Transfusion Medicine, Transplant Immunology & Molecular Biology, bDepartment of Histopathology, Oncology, dDepartment of Medical Oncology, Indraprastha Apollo Hospitals, New Delhi, India. * Corresponding author. email: [email protected] Available online: 9.5.2012 Copyright Ó 2012, Indraprastha Medical Corporation Ltd. All rights reserved.
doi:10.1016/j.apme.2012.05.002
c
Department of Surgical
Her2/neu gene amplification in breast Ca
to the HER2 protein, which restricts the epidermal growth factor ligand from reaching the breast cancer cells.5,6 Ultimately, this blocks the ability of cells to divide and grow. There are several methods to determine the amplification or over expression of Her2/neu gene, which includes Immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and reverse transcriptase polymerase chain reaction (RT-PCR).7 IHC and FISH are most commonly used diagnostic procedures for determining the HER2 status in breast carcinoma and widely used in clinical laboratories. Various studies have been done to compare and correlate the results of IHC with FISH, but the results are ambiguous at best and are quite contrasting with some studies showing IHC being superior while others showing FISH being the best assay for determining the Her2/neu status.3,8e14 Researchers have documented the discordance rate between HER2 by FISH and IHC and is high in all four IHC scores (0, 1þ, 2þ, 3þ), and a FISH-alone screening strategy has been alternatively suggested.12 The aim of our study was to retrospective analysize the 188 cases of invasive ductal carcinoma of breast cancer for Her2/neu FISH and compare between IHC and FISH findings.
Review Article
147
appochromatic objective and single band pass filter for DAPI, FITC and TRITC and a dual band pass filter for TRITC and FITC (Olympus Japan). Image acquisition was performed with an epifluorescence microscope (Olympus BX60) with a cooled charge-coupled device (CCD) camera with karyotype software package (Cytovision, Applied Imaging, Sunderland, UK). FISH signal enumeration: In each case at least 20 tumour nuclei were scored to calculated ratio of total number Her2/ neu spectrum orange signals to CEP17 spectrum green signals was calculated. If ratio of HER2 probe and CEP17 probe is <1.8, it is considered as non-amplified, if 1.8e2.2 it is considered as low amplification or equivocal and >2.2 is considered as Her2/neu amplification of the Her2/neu gene.
RESULTS In our study, 188 histopathologically confirmed breast cancer patients (invasive ductal carcinoma with varying tumour grades and clinical stages) were evaluated by IHC and FISH for Her2/neu gene amplification.
Immunohistochemical evaluation
MATERIALS & METHODS Patients & samples A total of 188 histologically proven breast cancer cases from 2007 to 2011, aged between 28 yrs and 84 yrs were received at our Molecular Biology & Transplant Immunology laboratory at Indraprastha Apollo Hospitals, New Delhi. Immunohistochemical assessment: Maximum invasive tumour component were selected and IHC was performed for ER, PR, Ki67 and HER2 neu on the tissue paraffin sections (3e4 mm thickness). IHC for Her2/neu gene was already done for the initial diagnosis of the patients. Patients were categorized on the basis of IHC values as 0, þ1, þ2, þ3. Fluorescence in situ hybridization (FISH) was performed on sections from the same paraffin blocks on which IHC was already done. Fluorescence in situ hybridization: FISH was carried out by following standard protocols and hybridization procedure was modified according to the probe manufacturer (Vysis-Abbott Molecular Abbott Park, Illinois, U.S.A). Probes used were PathVysion Her2/neu DNA probe kit, a dual coloured probe kit comprising locus specific identifier (LSI) Her2/neu spectrum orange & centromere enumeration probe (CEP) 17 spectrum green. The signal analysis was done using an Olympus BX 60 fluorescent microscope equipped with a 100-W mercury bulb, 100 plane
IHC evaluation of 188 breast carcinoma patients shows relationship between the oestrogen receptor (ER) and progesterone receptor (PR) in Table 1. On the basis of their ER/PR values, patients are divided into four categories which are: ERþ/PRþ (27.1%), ERþ/PR (14.89%), ER/ PRþ (8.51%) and ER/PR (49.46%).
FISH evaluation (Figs. 1 and 2) Out of 188 cases tested for HER2 gene amplification, 64 (34.0%) cases were amplified and 124(66.0%) cases were unamplified by FISH method. (Table 1). Out of 13 IHC determined negative cases (1þ), 2 (15.3%) were amplified and 11 (84.6%) cases were unamplified by FISH method. Out of 127 IHC determined equivocal cases (2þ), 31 (24.5%) were amplified and 96 (75.5%) cases were unamplified by FISH method. Out of 44 IHC determined positive cases (3þ), 31 (70.5%) were amplified and 13 (29.5%) cases were unamplified by FISH method. Out of 4 IHC determined cases with positivity of 2þ/focally 3þ, all were unamplified by FISH method. (Table 1).
DISCUSSION In the present study, 188 cases tested for HER2 gene amplification, 64 (34.0%) cases were amplified and 124(66.0%)
148
Apollo Medicine 2012 June; Vol. 9, No. 2
Makroo et al.
Table 1 Comparison of IHC results with FISH (n ¼ 188). HER2 protein expression by IHC
HER2 testing by FISH No.(%) Amplified
Negative (0/1þ) (n ¼ 13) (6.9%) Equivocal (2þ) (n ¼ 127) (67.02%) Positive (3þ) (n ¼ 44) (23.9%) þ2/þ3 (n ¼ 4) (2.1%) n ¼ 188
Non-amplified
2 (15.4) 31 (24.6) 31 (68.9) 0 34
11 96 13 04 124
(84.6) (76.2) (28.9) (1.0)
Equivocal 0 0 0 0 0
criteria.22e24 FISH assay being quantitative, has a very high specificity & has high inter-observer correlation as shown in our results. Published reports have documented that the 1þ and 2þ categories are the most difficult to assess and FISH should be considered in these cases.
cases were unamplified by FISH method. HER2 amplification is reported in various studies in the range of 18e20 to 25e30 percent of breast cancers.3,15e20 Accurate detection of the Her2/neu gene alteration in human breast cancer specimens has become increasingly important in determining patient prognosis as well as response to standard chemotherapeutic agents (Herceptin). Generally, IHC is currently extensively used as a diagnostic tool for determining the presence or absence of particular proteins and certain carbohydrates in routinely fixed and embedded tissue specimens,21 while FISH is one of the most important techniques for visualization of gene expression at the cellular level in tissues.21 IHC, however, has significant shortcomings, the most important of which is the loss of sensitivity.3 Since the assay is directed towards the detection of protein, the technical considerations such as preanalytical tissue processing, reagent variability, antigen retrieval and very subjective scoring might adversely affect the result. A FISH evaluation directed at the gene or molecular level itself might overcome all these drawbacks. FISH has several advantages over IHC such as ease to use, reproducibility and very objective and accurate scoring
Her2/neu Gene amplification by FISH was noted in 15.4% IHC negative cases. Press et al30 have reported an incidence of 7.4 percent of gene amplification in IHC negative cases. Possible reasons for this discrepancy may be insufficient tissue preservation leading to low levels of protein detection and cases with low level gene amplification.31 Our results indicate that the IHC 2þ group is characterized by high heterogeneity as only 24.6% of the cases showed Her2/neu gene amplification through FISH and the 75.4% cases were negative for Her2/neu gene amplification. Previous studies reported 6e25 percent incidence of IHC 2þ/FISH amplified cases.32e35 In the IHC 2þ/3þ (2.3%) group of the present study, all samples were found negative for amplification by FISH. Out of the 23.9% patients with 3þ IHC grade, 68.9% cases were amplified on FISH.
Fig. 1 Tumour nuclei with amplified with 2 spectrum green signals for CEP17 with amplified spectrum orange signals for Her2/neu gene.
Fig. 2 Tumour nuclei with 2 spectrum green signals for CEP17 and two spectrum orange signals for Her2/neu gene.
8,13,25e29
Her2/neu gene amplification in breast Ca
CONCLUSION We conclude that FISH is very accurate and highly specific method for the determination of Her2/neu gene amplification in breast cancer. Equivocal (2þ) and Contradictory histological grades (2þ/3þ) on IHC can be accurately assessed by FISH. Falsely categorizing the patients as being Her2/neu positive or negative will have a negative impact on the therapeutic usefulness of Herceptin in any given patient.
REFERENCES 1. Schwab M. Amplification of oncogenes in human cancer cells. Bioessays. 1998;20:473e479. 2. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the Her2/neu oncogene. Science. 1987;235:177e182. 3. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the Her2/neu proto-oncogene in human breast and ovarian cancer. Science (Wash. DC). 1989;244:707e712. 4. Popescu NC, King CR, Kraus MH. Localization of the human erbB-2 gene on normal and rearranged chromosomes17 to bands q12-21.32. Genomics. 1989;4:362e366. 5. DiGiovanna MP. Clinical significance of Her2/neu overexpression: part I and II. PPO Updates. 1999;13(9)13(10)(1e10): 1e14. 6. Bell R. What can we learn from herceptin trials in metastatic breast cancer? Oncology. 2002;1(63 Suppl):39e46. 7. Marks A, Seth A, Blondal J, Pienkowska M, Kahn HJ, Hanna WM. Defining a test for Her2/neu evaluation in breast cancer in the diagnostic setting. Mod Pathol. 2001;14: 677e685. 8. Pauletti G, Dandekar S, Rong H, et al. Assessment of methods for tissue-based detection of the Her2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol. 2000;18:3651e3664. 9. Penault-Llorca F, Adelaide J, Houvenaeghel G, et al. Optimization of immunohistochemical detection of erbB2 in human breast cancer: impact of fixation. J Pathol. 1994;173:65e75. 10. Press MF, Hung G, Godolphi W, Slamon DJ. Sensitivity of Her2/neu antibodies in archival tissue samples: potential source of error in the immunohistochemical studies of oncogene expression. Cancer Res. 1994;54:2771e2777. 11. Jiminez RE, Wallis T, Tabasczka BS, Visscher DW. Determination of Her2/neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescence in situ hybridization. Mod Pathol. 2000;13:37e45.
Review Article
149
12. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Comparison of fluorescence in situ hybridization and immunohistochemistry for the evaluation of Her2/neu in breast cancer. J Clin Oncol. 1999;17:1974e1982. 13. Lebeau A, Deimling D, Kaltz C, et al. Her2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization. J Clin Oncol. 2001;19:354e363. 14. Onody P, Bertrand F, Muzeau F, Bieche I, Lidereau R. Fluorescence in situ hybridization and immunohistochemical assays for Her2/neu status determination. Arch Pathol Lab Med. 2001;125:746e750. 15. Wolff AC, Hammond ME, Schwartz JN, et al. American society of clinical oncology/college of American pathologists guideline recommendations for human epidermal growth factor receptor 2 resting in breast cancer. J Clin Oncol. 2007;25:118e145. 16. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metatstatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783e792. 17. Baselga J, Perez EA, Pienkowski T, Bell R. Adjuvant trastuzumab: a milestone in the treatment of HER-2 positive early breast cancer. Oncologist. 2006;11(Suppl 1):4e12. 18. Bilancia D, Rosati G, Dinota A, Germano D, Romano R, Manzione L. Lapatinib in breast cancer. Ann Oncol. 2007;18(Suppl 6):vi26evi30. 19. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing-National Surgical adjuvant breast and Bowel Project experience. J Natl Cancer Inst. 2002;94:852e854. 20. Payne SJ, Bowen RL, Jones JL, Wells CA. Predictive markers in breast cancer e the present. Histopathology. 2008;52: 82e90. 21. Hayat MA. Comparison of immunohistochemistry, in situ hybridization, fluorescence in situ hybridization and chromogenic in situ hybridization. In: Hayat MA, ed. Handbook of immunohistochemistry and in situ hybridization of human carcinomas. Molecular genetics; lung and breast carcinomas. Elsevier Inc; 2004:3e11. 22. Bartlett JM, Going JJ, Mallon EA, et al. Evaluating HER2 amplification and overexpression in breast cancer. J Pathol. 2001;195:422e428. 23. Gupta D, Middleton LP, Whitaker MJ, Abrams J. Comparison of fluorescence and chromogenic in situ hybridization for detection of Her2/neu oncogenes in breast cancer. Am J Clin Pathol. 2003;119:381e387. 24. Gancberg D, Jarvinen T, di Leo A, et al. Evaluation of Her2/ neu protein expression in breast cancer by immunohistochemistry: an interlaboratory study assessing the reproducibility of Her2/neu testing. Breast Cancer Res Treat. 2002;74: 113e120.
150
Apollo Medicine 2012 June; Vol. 9, No. 2
25. Hoang MP, Sahin AA, Ordonez NG, Sneige N. Her2/neu gene amplification compared with Her2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma. Am J Clin Pathol. 2000;113:852e859. 26. Mass R, Sanders C, Kasian C. The concordance between the clinical trials assays (CTA) and fluorescence in situ hybridization in the herceptin pivotal trails. Proc Am Soc Clin Oncol. 2000;19:75. 27. Perez EA, Roche PC, Jenkins RB, et al. HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clinic Proc. 2002;77:148e154. 28. Kobayashi M, Ooi A, Oda Y, Nakanishi I. Protein overexpression and gen amplification of c-erbB-2 in breast carcinomas: a comparative study of immunohistochemistry and fluorescence in situ hybridization of formalin-fixed, paraffinembedded tissues. Hum Pathol. 2002;33:21e28. 29. Tsuda H, Akiyama F, Terasaki H, et al. Detection of Her2/neu (c-erb B-2) DNA amplification in primary breast carcinoma. Cancer. 2001;92:2965e2974. 30. Press MF, Sauter G, Bernstein L, et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy
Makroo et al.
31.
32.
33.
34.
35.
and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res. 2005;11: 6598e6607. Birner P, Oberhuber G, Stani J, et al. Evaluation of the united states food and drug administration-approved scoring and test system of HER-2 protein expression in breast cancer. Clin Cancer Res. 2001;7:1669e1675. Yaziji H, Goldstein LC, Barry TS, et al. HER-2 testing in breast cancer using parallel tissue-based methods. J Am Med Assoc. 2004;291:1972e1977. Hammock L, Lewis M, Phillips C, Cohen C. Strong Her2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization. Hum Pathol. 2003;34:1043e1047. Perez EA, Suman VJ, Davidson NE, et al. HER2 testing by local, central, and reference laboratories in specimens from the north central cancer treatment group N9831 intergroup adjuvant trial. J Clin Oncol. 2006;24:3032e3038. Tsuda H, Akiyama F, Terasaki H, et al. Detection of Her2/neu (c-erbB-2) DNA amplification in primary breast carcinoma: interobserver reproducibility and correlation with immunohistochemical HER-2 overexpression. Cancer. 2001;92: 2965e2974.
Review Article
Her2/neu gene amplification in breast carcinoma patients: Our experience with fluorescence in situ hybridization (FISH) technique R.N. Makrooa,d,*, M. Chowdhrya,d, A. Fauzdara,d, M. Mishraa,d, P. Srivastavaa,d, P. Bhaudauriaa,d, S. Kaulb,d, R. Sarinc,d, P.K. Dasc,d, H. Duac,d
ABSTRACT Background: In India the incidence of breast cancer has increased in the urban population with 1 in every 22 women diagnosed with breast cancer. It is important to know the Her2/neu gene status for a better prognostication of these patients. Aim: The aim of study was to compare the efficacy of fluorescent in situ hybridisation (FISH) and immunohistochemistry (IHC) for determining Her2/neu alteration in breast carcinoma. Material & methods: A total of 188 histologically proven breast carcinoma cases between years 2007e2011 and were retrospectively analysed on the paraffin tissue sections by both IHC & FISH techniques. FISH for Her2/neu gene amplification was performed on cases where the IHC status was already known and the results were compared. Results: A total of 64 (30%) patients were found to be amplified and remaining 124 (65.9%) cases were found to be unamplified through FISH. Patients observed with 3þ reading on IHC were later confirmed as unamplified in 29.5% cases through FISH. Interpretation & conclusion: It has been confirmed with the present study that IHC is a prudent first-step technique to screen tissue samples for Her2/neu gene status but should be supplemented with the FISH technique especially in equivocal cases. Copyright © 2012, Indraprastha Medical Corporation Ltd. All rights reserved. Keywords: HEr2/NEu, Breast carcinoma, IHC, FISH, Cytogenetics
INTRODUCTION Breast cancer is one of the most common malignancies according to the global cancer statistics with high incidence and mortality of breast cancer all over the world.1 India accounts for nearly six percent of deaths due to breast cancer globally. In breast carcinoma, Her2/neu gene amplification is correlated with prognostification and therapeutic implications with over expression in 20e25% of invasive cancers.2,3 HER2 (erbB2/neu) proto-oncogene is mapped to chromosome 17q12-21.324 region and is tyrosine kinase of 185 kDa transmembrane receptor protein of epidermal
growth factor receptor family (EGFR). Her2/neu over expression has been reported to be associated with positive lymph nodes, high histologic grade, high proliferation rate and lack of expression of oestrogen and progesterone receptors associated with a more aggressive phenotype with decreased survival rate.2e7 The incidence of over expression of Her2/neu is highest among all human malignancies, ranging from 9 to 34% in breast cancer patients tested. Her2/neu is the only oncogene with a specific molecular targeted therapy available through monoclonal antibody drug named Herceptin. This therapy functions to treat patients with an over expression in Her2/neu gene by attaching itself
a Department of Transfusion Medicine, Transplant Immunology & Molecular Biology, bDepartment of Histopathology, Oncology, dDepartment of Medical Oncology, Indraprastha Apollo Hospitals, New Delhi, India. * Corresponding author. email: [email protected] Available online: 9.5.2012 Copyright Ó 2012, Indraprastha Medical Corporation Ltd. All rights reserved.
doi:10.1016/j.apme.2012.05.002
c
Department of Surgical
Her2/neu gene amplification in breast Ca
to the HER2 protein, which restricts the epidermal growth factor ligand from reaching the breast cancer cells.5,6 Ultimately, this blocks the ability of cells to divide and grow. There are several methods to determine the amplification or over expression of Her2/neu gene, which includes Immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and reverse transcriptase polymerase chain reaction (RT-PCR).7 IHC and FISH are most commonly used diagnostic procedures for determining the HER2 status in breast carcinoma and widely used in clinical laboratories. Various studies have been done to compare and correlate the results of IHC with FISH, but the results are ambiguous at best and are quite contrasting with some studies showing IHC being superior while others showing FISH being the best assay for determining the Her2/neu status.3,8e14 Researchers have documented the discordance rate between HER2 by FISH and IHC and is high in all four IHC scores (0, 1þ, 2þ, 3þ), and a FISH-alone screening strategy has been alternatively suggested.12 The aim of our study was to retrospective analysize the 188 cases of invasive ductal carcinoma of breast cancer for Her2/neu FISH and compare between IHC and FISH findings.
Review Article
147
appochromatic objective and single band pass filter for DAPI, FITC and TRITC and a dual band pass filter for TRITC and FITC (Olympus Japan). Image acquisition was performed with an epifluorescence microscope (Olympus BX60) with a cooled charge-coupled device (CCD) camera with karyotype software package (Cytovision, Applied Imaging, Sunderland, UK). FISH signal enumeration: In each case at least 20 tumour nuclei were scored to calculated ratio of total number Her2/ neu spectrum orange signals to CEP17 spectrum green signals was calculated. If ratio of HER2 probe and CEP17 probe is <1.8, it is considered as non-amplified, if 1.8e2.2 it is considered as low amplification or equivocal and >2.2 is considered as Her2/neu amplification of the Her2/neu gene.
RESULTS In our study, 188 histopathologically confirmed breast cancer patients (invasive ductal carcinoma with varying tumour grades and clinical stages) were evaluated by IHC and FISH for Her2/neu gene amplification.
Immunohistochemical evaluation
MATERIALS & METHODS Patients & samples A total of 188 histologically proven breast cancer cases from 2007 to 2011, aged between 28 yrs and 84 yrs were received at our Molecular Biology & Transplant Immunology laboratory at Indraprastha Apollo Hospitals, New Delhi. Immunohistochemical assessment: Maximum invasive tumour component were selected and IHC was performed for ER, PR, Ki67 and HER2 neu on the tissue paraffin sections (3e4 mm thickness). IHC for Her2/neu gene was already done for the initial diagnosis of the patients. Patients were categorized on the basis of IHC values as 0, þ1, þ2, þ3. Fluorescence in situ hybridization (FISH) was performed on sections from the same paraffin blocks on which IHC was already done. Fluorescence in situ hybridization: FISH was carried out by following standard protocols and hybridization procedure was modified according to the probe manufacturer (Vysis-Abbott Molecular Abbott Park, Illinois, U.S.A). Probes used were PathVysion Her2/neu DNA probe kit, a dual coloured probe kit comprising locus specific identifier (LSI) Her2/neu spectrum orange & centromere enumeration probe (CEP) 17 spectrum green. The signal analysis was done using an Olympus BX 60 fluorescent microscope equipped with a 100-W mercury bulb, 100 plane
IHC evaluation of 188 breast carcinoma patients shows relationship between the oestrogen receptor (ER) and progesterone receptor (PR) in Table 1. On the basis of their ER/PR values, patients are divided into four categories which are: ERþ/PRþ (27.1%), ERþ/PR (14.89%), ER/ PRþ (8.51%) and ER/PR (49.46%).
FISH evaluation (Figs. 1 and 2) Out of 188 cases tested for HER2 gene amplification, 64 (34.0%) cases were amplified and 124(66.0%) cases were unamplified by FISH method. (Table 1). Out of 13 IHC determined negative cases (1þ), 2 (15.3%) were amplified and 11 (84.6%) cases were unamplified by FISH method. Out of 127 IHC determined equivocal cases (2þ), 31 (24.5%) were amplified and 96 (75.5%) cases were unamplified by FISH method. Out of 44 IHC determined positive cases (3þ), 31 (70.5%) were amplified and 13 (29.5%) cases were unamplified by FISH method. Out of 4 IHC determined cases with positivity of 2þ/focally 3þ, all were unamplified by FISH method. (Table 1).
DISCUSSION In the present study, 188 cases tested for HER2 gene amplification, 64 (34.0%) cases were amplified and 124(66.0%)
148
Apollo Medicine 2012 June; Vol. 9, No. 2
Makroo et al.
Table 1 Comparison of IHC results with FISH (n ¼ 188). HER2 protein expression by IHC
HER2 testing by FISH No.(%) Amplified
Negative (0/1þ) (n ¼ 13) (6.9%) Equivocal (2þ) (n ¼ 127) (67.02%) Positive (3þ) (n ¼ 44) (23.9%) þ2/þ3 (n ¼ 4) (2.1%) n ¼ 188
Non-amplified
2 (15.4) 31 (24.6) 31 (68.9) 0 34
11 96 13 04 124
(84.6) (76.2) (28.9) (1.0)
Equivocal 0 0 0 0 0
criteria.22e24 FISH assay being quantitative, has a very high specificity & has high inter-observer correlation as shown in our results. Published reports have documented that the 1þ and 2þ categories are the most difficult to assess and FISH should be considered in these cases.
cases were unamplified by FISH method. HER2 amplification is reported in various studies in the range of 18e20 to 25e30 percent of breast cancers.3,15e20 Accurate detection of the Her2/neu gene alteration in human breast cancer specimens has become increasingly important in determining patient prognosis as well as response to standard chemotherapeutic agents (Herceptin). Generally, IHC is currently extensively used as a diagnostic tool for determining the presence or absence of particular proteins and certain carbohydrates in routinely fixed and embedded tissue specimens,21 while FISH is one of the most important techniques for visualization of gene expression at the cellular level in tissues.21 IHC, however, has significant shortcomings, the most important of which is the loss of sensitivity.3 Since the assay is directed towards the detection of protein, the technical considerations such as preanalytical tissue processing, reagent variability, antigen retrieval and very subjective scoring might adversely affect the result. A FISH evaluation directed at the gene or molecular level itself might overcome all these drawbacks. FISH has several advantages over IHC such as ease to use, reproducibility and very objective and accurate scoring
Her2/neu Gene amplification by FISH was noted in 15.4% IHC negative cases. Press et al30 have reported an incidence of 7.4 percent of gene amplification in IHC negative cases. Possible reasons for this discrepancy may be insufficient tissue preservation leading to low levels of protein detection and cases with low level gene amplification.31 Our results indicate that the IHC 2þ group is characterized by high heterogeneity as only 24.6% of the cases showed Her2/neu gene amplification through FISH and the 75.4% cases were negative for Her2/neu gene amplification. Previous studies reported 6e25 percent incidence of IHC 2þ/FISH amplified cases.32e35 In the IHC 2þ/3þ (2.3%) group of the present study, all samples were found negative for amplification by FISH. Out of the 23.9% patients with 3þ IHC grade, 68.9% cases were amplified on FISH.
Fig. 1 Tumour nuclei with amplified with 2 spectrum green signals for CEP17 with amplified spectrum orange signals for Her2/neu gene.
Fig. 2 Tumour nuclei with 2 spectrum green signals for CEP17 and two spectrum orange signals for Her2/neu gene.
8,13,25e29
Her2/neu gene amplification in breast Ca
CONCLUSION We conclude that FISH is very accurate and highly specific method for the determination of Her2/neu gene amplification in breast cancer. Equivocal (2þ) and Contradictory histological grades (2þ/3þ) on IHC can be accurately assessed by FISH. Falsely categorizing the patients as being Her2/neu positive or negative will have a negative impact on the therapeutic usefulness of Herceptin in any given patient.
REFERENCES 1. Schwab M. Amplification of oncogenes in human cancer cells. Bioessays. 1998;20:473e479. 2. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the Her2/neu oncogene. Science. 1987;235:177e182. 3. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the Her2/neu proto-oncogene in human breast and ovarian cancer. Science (Wash. DC). 1989;244:707e712. 4. Popescu NC, King CR, Kraus MH. Localization of the human erbB-2 gene on normal and rearranged chromosomes17 to bands q12-21.32. Genomics. 1989;4:362e366. 5. DiGiovanna MP. Clinical significance of Her2/neu overexpression: part I and II. PPO Updates. 1999;13(9)13(10)(1e10): 1e14. 6. Bell R. What can we learn from herceptin trials in metastatic breast cancer? Oncology. 2002;1(63 Suppl):39e46. 7. Marks A, Seth A, Blondal J, Pienkowska M, Kahn HJ, Hanna WM. Defining a test for Her2/neu evaluation in breast cancer in the diagnostic setting. Mod Pathol. 2001;14: 677e685. 8. Pauletti G, Dandekar S, Rong H, et al. Assessment of methods for tissue-based detection of the Her2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol. 2000;18:3651e3664. 9. Penault-Llorca F, Adelaide J, Houvenaeghel G, et al. Optimization of immunohistochemical detection of erbB2 in human breast cancer: impact of fixation. J Pathol. 1994;173:65e75. 10. Press MF, Hung G, Godolphi W, Slamon DJ. Sensitivity of Her2/neu antibodies in archival tissue samples: potential source of error in the immunohistochemical studies of oncogene expression. Cancer Res. 1994;54:2771e2777. 11. Jiminez RE, Wallis T, Tabasczka BS, Visscher DW. Determination of Her2/neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescence in situ hybridization. Mod Pathol. 2000;13:37e45.
Review Article
149
12. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Comparison of fluorescence in situ hybridization and immunohistochemistry for the evaluation of Her2/neu in breast cancer. J Clin Oncol. 1999;17:1974e1982. 13. Lebeau A, Deimling D, Kaltz C, et al. Her2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization. J Clin Oncol. 2001;19:354e363. 14. Onody P, Bertrand F, Muzeau F, Bieche I, Lidereau R. Fluorescence in situ hybridization and immunohistochemical assays for Her2/neu status determination. Arch Pathol Lab Med. 2001;125:746e750. 15. Wolff AC, Hammond ME, Schwartz JN, et al. American society of clinical oncology/college of American pathologists guideline recommendations for human epidermal growth factor receptor 2 resting in breast cancer. J Clin Oncol. 2007;25:118e145. 16. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metatstatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783e792. 17. Baselga J, Perez EA, Pienkowski T, Bell R. Adjuvant trastuzumab: a milestone in the treatment of HER-2 positive early breast cancer. Oncologist. 2006;11(Suppl 1):4e12. 18. Bilancia D, Rosati G, Dinota A, Germano D, Romano R, Manzione L. Lapatinib in breast cancer. Ann Oncol. 2007;18(Suppl 6):vi26evi30. 19. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing-National Surgical adjuvant breast and Bowel Project experience. J Natl Cancer Inst. 2002;94:852e854. 20. Payne SJ, Bowen RL, Jones JL, Wells CA. Predictive markers in breast cancer e the present. Histopathology. 2008;52: 82e90. 21. Hayat MA. Comparison of immunohistochemistry, in situ hybridization, fluorescence in situ hybridization and chromogenic in situ hybridization. In: Hayat MA, ed. Handbook of immunohistochemistry and in situ hybridization of human carcinomas. Molecular genetics; lung and breast carcinomas. Elsevier Inc; 2004:3e11. 22. Bartlett JM, Going JJ, Mallon EA, et al. Evaluating HER2 amplification and overexpression in breast cancer. J Pathol. 2001;195:422e428. 23. Gupta D, Middleton LP, Whitaker MJ, Abrams J. Comparison of fluorescence and chromogenic in situ hybridization for detection of Her2/neu oncogenes in breast cancer. Am J Clin Pathol. 2003;119:381e387. 24. Gancberg D, Jarvinen T, di Leo A, et al. Evaluation of Her2/ neu protein expression in breast cancer by immunohistochemistry: an interlaboratory study assessing the reproducibility of Her2/neu testing. Breast Cancer Res Treat. 2002;74: 113e120.
150
Apollo Medicine 2012 June; Vol. 9, No. 2
25. Hoang MP, Sahin AA, Ordonez NG, Sneige N. Her2/neu gene amplification compared with Her2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma. Am J Clin Pathol. 2000;113:852e859. 26. Mass R, Sanders C, Kasian C. The concordance between the clinical trials assays (CTA) and fluorescence in situ hybridization in the herceptin pivotal trails. Proc Am Soc Clin Oncol. 2000;19:75. 27. Perez EA, Roche PC, Jenkins RB, et al. HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clinic Proc. 2002;77:148e154. 28. Kobayashi M, Ooi A, Oda Y, Nakanishi I. Protein overexpression and gen amplification of c-erbB-2 in breast carcinomas: a comparative study of immunohistochemistry and fluorescence in situ hybridization of formalin-fixed, paraffinembedded tissues. Hum Pathol. 2002;33:21e28. 29. Tsuda H, Akiyama F, Terasaki H, et al. Detection of Her2/neu (c-erb B-2) DNA amplification in primary breast carcinoma. Cancer. 2001;92:2965e2974. 30. Press MF, Sauter G, Bernstein L, et al. Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy
Makroo et al.
31.
32.
33.
34.
35.
and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res. 2005;11: 6598e6607. Birner P, Oberhuber G, Stani J, et al. Evaluation of the united states food and drug administration-approved scoring and test system of HER-2 protein expression in breast cancer. Clin Cancer Res. 2001;7:1669e1675. Yaziji H, Goldstein LC, Barry TS, et al. HER-2 testing in breast cancer using parallel tissue-based methods. J Am Med Assoc. 2004;291:1972e1977. Hammock L, Lewis M, Phillips C, Cohen C. Strong Her2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization. Hum Pathol. 2003;34:1043e1047. Perez EA, Suman VJ, Davidson NE, et al. HER2 testing by local, central, and reference laboratories in specimens from the north central cancer treatment group N9831 intergroup adjuvant trial. J Clin Oncol. 2006;24:3032e3038. Tsuda H, Akiyama F, Terasaki H, et al. Detection of Her2/neu (c-erbB-2) DNA amplification in primary breast carcinoma: interobserver reproducibility and correlation with immunohistochemical HER-2 overexpression. Cancer. 2001;92: 2965e2974.