Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2012  |  Volume : 55  |  Issue : 4  |  Page : 481--484

Correlation between HER2 gene amplification and protein overexpression through fluorescence in situ hybridization and immunohistochemistry in breast carcinoma patients


RN Makroo1, Mohit Chowdhry1, Manoj Kumar1, Priyanka Srivastava1, Richa Tyagi1, Preeti Bhadauria1, Sumaid Kaul2, Ramesh Sarin3, PK Das4, Harsh Dua4,  
1 Transplant Immunology, Molecular Biology and Transfusion Medicine, Apollo Hospitals, Sarita Vihar, Delhi Mathura Road, New Delhi, India
2 Department of Histopathology, Apollo Hospitals, Sarita Vihar, Delhi Mathura Road, New Delhi, India
3 Department of Surgical Oncology, Apollo Hospitals, Sarita Vihar, Delhi Mathura Road, New Delhi, India
4 Department of Medical Oncology, Apollo Hospitals, Sarita Vihar, Delhi Mathura Road, New Delhi, India

Correspondence Address:
Mohit Chowdhry
Transplant Immunology, Molecular Biology and Transfusion Medicine, Apollo Hospitals, Sarita Vihar, Delhi Mathura Road, New Delhi
India

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 this study was to compare the efficacy of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for determining HER2/neu alteration in breast carcinoma. Materials and Methods : A total of 188 histologically proven breast carcinoma cases between the years 2007 and 2011 were retrospectively analyzed on the paraffin tissue sections by both IHC and 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 the remaining 124 (65.9%) cases were found to be unamplified through FISH. Patients observed with 3+ reading on IHC were later confirmed as unamplified in 29.5% cases through FISH. 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.



How to cite this article:
Makroo R N, Chowdhry M, Kumar M, Srivastava P, Tyagi R, Bhadauria P, Kaul S, Sarin R, Das P K, Dua H. Correlation between HER2 gene amplification and protein overexpression through fluorescence in situ hybridization and immunohistochemistry in breast carcinoma patients.Indian J Pathol Microbiol 2012;55:481-484


How to cite this URL:
Makroo R N, Chowdhry M, Kumar M, Srivastava P, Tyagi R, Bhadauria P, Kaul S, Sarin R, Das P K, Dua H. Correlation between HER2 gene amplification and protein overexpression through fluorescence in situ hybridization and immunohistochemistry in breast carcinoma patients. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Sep 18 ];55:481-484
Available from: http://www.ijpmonline.org/text.asp?2012/55/4/481/107785


Full Text

 Introduction



Breast cancer is one of the most common malignancies according to the global cancer statistics, with high incidence and mortality all over the world. [1] India accounts for nearly 6% of deaths due to breast cancer globally. In breast carcinoma, HER2/neu gene amplification is correlated with prognostification and therapeutic implications with overexpression in 20-25% of invasive cancers. [2],[3] HER2 (erbB2/neu) proto-oncogene is mapped to chromosome 17q12-21.32 region and is tyrosine kinase of 185kDa transmembrane receptor protein of epidermal growth factor receptor (EGFR) family. [4] HER2/neu overexpression has been reported to be associated with positive lymph nodes, high histologic grade, high proliferation rate, and lack of expression of estrogen and progesterone receptors associated with a more aggressive phenotype with decreased survival rate. [2],[3],[4],[5],[6],[7] The incidence of overexpression 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 overexpression in HER2/neu gene by attaching itself 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 overexpression of HER2/neu gene, which include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and reverse transcriptase polymerase chain reaction (RT-PCR). [7] IHC and FISH are the most commonly used diagnostic procedures for determining the HER2 status in breast carcinoma and are 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],[8],[9],[10],[11],[12],[13],[14] Researchers have documented the discordance rate between HER2 by FISH and IHC and is high in all four IHC scores (0, 1+, 2+, 3+), and a FISH-alone screening strategy has been alternatively suggested. [12] The aim of our study was to retrospectively analyze the 188 cases of invasive ductal carcinoma of breast cancer for HER2/neu FISH and compare between IHC and FISH findings.

 Materials and Methods



Patients and Samples

A total of 188 histologically proven breast cancer cases from 2007 to 2011, aged between 28 and 84 years were received at our department.

Immunohistochemical Assessment

The representative sections were stained immunohistochemically for estrogen receptors (ERs) (Neo Markers; Clone SP1; dilution 1:200), progesterone receptors (PRs) (Neo Markers; Clone SP2; dilution 1:400), human EGFR, i.e. HER2/neu (DAKO Cytomation, Polyclonal Rabbit Antihuman C-erbB-2 oncoprotein; dilution 1:250), and Ki-67 (DAKO Cytomation; Clone D5/16 B4; dilution 1:75) using standard immunoperoxidase method, i.e., avidin-biotin complex peroxidase method.

Maximum invasive tumor components were selected and IHC was performed for ER, PR, Ki-67, and HER2/neu on the tissue paraffin sections (3-4 μm 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, +1, +2, and +3. 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, IL, USA). Probes used were PathVysion HER2/neu DNA probe kit, a dual colored probe kit comprising locus specific identifiers (LSI), HER2/neu spectrum orange and 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 apochromatic 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 tumor nuclei were scored to calculate the ratio of total number of HER2/neu spectrum orange signals to CEP 17 spectrum green signals. If the ratio of HER2 probe and CEP 17 probe is <1.8, it is considered as non-amplified, a ratio of 1.8-2.2 is considered as low amplification or equivocal, and a ratio >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 tumor grades and clinical stages) were evaluated by IHC and FISH for HER2/neu gene amplification.

Immunohistochemical Evaluation

IHC evaluation of 188 breast carcinoma patients shows the relationship between the ER and PR as given in [Table 1]. On the basis of their ER/PR values, patients were divided into four categories: ER+/PR+ (27.1%), ER+/PR- (14.89%), ER-/PR+ (8.51%), and ER+/PR- (49.46%) [Table 1]. {Table 1}

FISH Evaluation

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 (1+), 2 (15.4%) were amplified and 11 (84.6%) cases were unamplified by FISH method. Out of 127 IHC determined equivocal cases (2+), 31 (24.4%) were amplified and 96 (75.5%) cases were unamplified by FISH method. Out of 44 IHC determined positive cases (3+), 31 (70.4%) were amplified and 13 (29.5%) cases were unamplified by FISH method. Out of four IHC determined cases with positivity of 2+/focally 3+, all were unamplified by FISH method [Figure 1]a-d [Table 1].{Figure 1}

 Discussion



In the present study, of the 188 cases tested for HER2 gene amplification, 64 (34.0%) cases were amplified and 124 (66.0%) cases were unamplified by FISH method. HER2 amplification is reported in various studies to be in the range of 18-20 to 25-30% of breast cancers. [3],[15],[16],[17],[18],[19],[20],[] 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, 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 toward the detection of protein, the technical considerations such as pre-analytical 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 criteria. [22],[23],[24] FISH assay being quantitative, has a very high specificity and high inter-observer correlation as shown in our results. Published reports have documented that the 1+ and 2+ categories are the most difficult to assess and FISH should be considered in these cases. [9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29]

HER2/neu gene amplification by FISH was noted in 15.4% IHC negative cases. Press et al. [30] have reported an incidence of 7.4% 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 2+ group is characterized by high heterogeneity as only 24.4% of the cases showed HER2/neu gene amplification through FISH and 75.5% cases were negative for HER2/neu gene amplification. Previous studies reported 6-25% incidence of IHC 2+/FISH amplified cases.[32],[33],[34],[35] In the IHC 2+/3+ (2.1%) group of the present study, all samples were found negative for amplification by FISH. Out of the 23.9% patients with 3+ IHC grade, 70.4% cases were amplified on FISH. Panjwani et al.[36] showed positive correlation of grade III with HER2/neu amplification which was concordant with our study. Thirteen (29.5%) cases with the score of 3+ by IHC were FISH negative for gene amplification (>2.2). Although there is good correlation between HER2 gene amplification and protein overexpression, many studies also have shown that 3-15% of breast carcinomas overexpress the HER-2 protein without gene amplification and a small subset of breast carcinomas amplify the HER2 gene without overexpression. Various theories have been proposed to account for these discrepancies. Increased receptor expression in breast cancer without genetic alterations has been reported in approximately 10% of cases, probably caused by transcriptional or post-translational activation. Other proposed explanations for the phenomenon of overexpression without amplification include artifactual high sensitivity of immunohistochemical assays, single copy overexpression of the HER2 gene at the messenger RNA (mRNA) transcription level, or gene amplification below the level of detection of FISH assays. [9]

 Conclusion



We conclude that FISH is a very accurate and highly specific method for the determination of HER2/neu gene amplification in breast cancer. Equivocal (2+) and contradictory histological grades (2+/3+) 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.

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