|Year : 2019 | Volume
| Issue : 2 | Page : 119-123
FISH and HER2/neu equivocal immunohistochemistry in breast carcinoma
Geeta Vikram Patil Okaly1, Dipti Panwar1, Kavitha Bidadli Lingappa1, Prasanna Kumari2, Abhishek Anand3, Prashantha Kumar1, Manju Hosur Chikkalingaiah1, Rekha Vijay Kumar1
1 Department of Pathology, Kidwai Cancer Institute, Bengaluru, Karnataka, India
2 Department of Pathology, Cytogenetics Unit, Kidwai Cancer Institute, Bengaluru, Karnataka, India
3 Department of Medical Oncology, Kidwai Cancer Institute, Bengaluru, Karnataka, India
|Date of Web Publication||2-May-2019|
Kavitha Bidadli Lingappa
Department of Pathology, Kidwai Cancer Institute, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
AIM: The aim of this study was to validate the role of fluorescence in situ hybridization (FISH) in investigating HER2/neu gene amplification (human epidermal growth factor receptor 2) in patients with HER2/neu equivocal breast cancer diagnosed on immunohistochemistry (IHC).
MATERIALS AND METHODS: This was a retrospective study conducted from January 2013 to October 2017. A total of 134 patients diagnosed with invasive breast carcinoma and HER2/neu equivocal status on IHC were analyzed. Also, the cases for the years 2016 and 2017 formed a subgroup that was analyzed further to study the impact of pre-analytical factors on IHC and FISH results.
RESULTS: A total of 134 women with HER2/neu IHC equivocal breast cancer were included in the study with a median age of 50 years (range 25–81). HER2/neu amplification by FISH was noted in 72 (54%) cases, whereas it was non-amplified in 52 (39%) cases. Ten cases were reported as equivocal even on FISH (ASCO/CAP 2013 guidelines). Polysomy 17 was noted in 55 cases (41%), of which 26 patients were≤50 years and 29 patients were >50 years of age. Twenty (36%) of these 55 cases showed HER2/neu amplification, whereas 26 (48%) cases were non-amplified and 9 (16%) cases were reported as equivocal on FISH. Also, more than half of the polysomy cases were hormone receptor negative.
CONCLUSION: IHC is a good screening tool for negative and positive results. Any patient targeted for trastuzumab therapy should undergo confirmation of HER2/neu equivocal status by FISH analysis. We also suggest that if a non-classical FISH pattern is seen, the test should be repeated with a non-centromeric chromosome 17 reference locus probe for better treatment planning.
Keywords: Breast carcinoma, FISH, HER2/neu, immunohistochemistry
|How to cite this article:|
Patil Okaly GV, Panwar D, Lingappa KB, Kumari P, Anand A, Kumar P, Chikkalingaiah MH, Kumar RV. FISH and HER2/neu equivocal immunohistochemistry in breast carcinoma. Indian J Cancer 2019;56:119-23
|How to cite this URL:|
Patil Okaly GV, Panwar D, Lingappa KB, Kumari P, Anand A, Kumar P, Chikkalingaiah MH, Kumar RV. FISH and HER2/neu equivocal immunohistochemistry in breast carcinoma. Indian J Cancer [serial online] 2019 [cited 2020 Jun 5];56:119-23. Available from: http://www.indianjcancer.com/text.asp?2019/56/2/119/257551
| » Introduction|| |
Breast cancer is the second most common cancer in Indian women, usually seen in females between 40 and 50 years of age, with a mean age of 47 years. HER2/neu (human epidermal growth factor receptor 2) was first reported as a poor prognostic factor for breast cancer in 1987. It also has an adverse prognostic effect in cancers of the stomach, ovary, lung, and pancreas.,,, The long arm of chromosome 17 bears this gene and encodes a 185-kDa glycoprotein which belongs to the family of type 1 growth factor receptor. As the ligand binds to HER2/neu protein, it undergoes dimerization followed by transphosphorylation of the intracellular domains. These phosphorylated tyrosine residues lead to the activation of various second messengers and transmembrane signaling pathways that cumulate in various biological effects. HER2/neu gene overexpression has been observed in up to 25% of primary breast carcinomas. These patients have poor prognosis due to an aggressive tumor phenotype, increased risk of metastasis, and poor survival. The development of trastuzumab, a HER2/neu targeted humanized monoclonal antibody, was a boon to patients and their oncologists. In 1998, the United States, Food and Drug Administration (US FDA) approved trastuzumab for use in metastatic breast cancer, and it was approved as an adjuvant therapy for patients with HER2/neu positive and lymph node positive breast cancer in 2006.,, In addition, HER2/neu status is also an important predictive marker for better and worse response respectively to other anti-cancer agents such as anthracyclines and cyclophosphamides. Accurate identification of the expression pattern of HER2/neu in patients withinvasive breast carcinoma is, therefore, an important pre-requisite for appropriate management.
HER2/neu assessment can be carried out by various methods like immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) (protein level), quantitative real-time polymerase chain reaction and microarray (RNA level), and fluorescence in situ hybridization (FISH)/chromogenic in situ hybridization (DNA level). IHC and FISH are the most commonly used FDA-approved tests. IHC has some advantages over FISH in that it is quick, easy, and economical, whereas FISH is more expensive and takes a longer period of time. However, due to the quantitative nature of FISH, it is more reliable than IHC which is a semi-quantitative technique with the disadvantage of discrepancies in results due to variations in laboratory parameters., There is good concordance reported between these two methods for IHC scores of 3+ and 1+., IHC 2+ is an equivocal score with variable concordance with FISH results. This study is an attempt to validate the role of FISH in investigating HER2/neu gene amplification in patients with HER2/neu equivocal breast cancer diagnosed on IHC.
| » Materials and Methods|| |
This is a retrospective study from the archives of the Department of Pathology, Kidwai Cancer Institute, from January 2013 to October 2017. A total of 134 patients diagnosed with invasive breast carcinoma and HER2/neu equivocal status on IHC were analyzed. The details of the antibodies used on the Ventana BenchMark XT are given in [Table 1].
The PathVysion HER2/neu DNA probe kit II (Abbott) was used, which is designed to detect amplification of the HER2/neu gene via FISH in formalin-fixed paraffin-embedded human breast cancer tissue specimens. It uses a 226kb spectrum orange (SO) directly labelled fluorescent DNA probe specific for the HER2/neu gene locus 17q11.2-q12. The chromosome enumeration probe (CEP) 17 DNA probe is approximately 5.4 kb spectrum green for alpha satellite DNA sequence at the centromeric region of chromosome 17 (17p11.1-q11.1). The FFPE tissue block of breast cancer was selected and the area of interest marked on hematoxylin and eosin (H and E) slides. The matching area on the subsequent section cut from the block was taken for FISH analysis. The tissue was subjected to a series of deparaffinization, dehydration, and prehybridization treatments. After this, the probe was added and the sections were kept for overnight incubation. After the post hybridization wash, the sections were mounted and checked for signals. The whole slide was screened and every single discrete nucleus was examined for red and green signals. In all the cases, 100 nuclei were scored as positive, negative, and equivocal based on the American Society of Clinical Oncology (ASCO)/college of American pathologists (CAP) 2013 guidelines. According to these guidelines, a positive HER2/neu test is defined as positive if dual-probe HER2/CEP17 ratio is ≥2.0 with an average HER2 copy number ≥4.0 signals/cell or dual-probe HER2/CEP17 ratio ≥2.0 with an average HER2 copy number <4.0 signals/cell or dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥6.0 signals/cell. It is considered equivocal if dual-probe HER2/CEP17 ratio is <2.0 with an average HER2 copy number ≥4.0 and <6.0 signals/cell and negative when dual-probe HER2/CEP17 ratio is <2.0 with an average HER2 copy number <4.0 signals/cell. The readings were taken by two pathologists with discordant cases being reviewed and resolved in consultation.
The cases for the years 2016 and 2017 formed a subgroup that was analyzed further to study the impact of pre-analytical factors on IHC and FISH results.
Chi-square test was used to find the statistical significance of results in the two age groups (≤50 and >50 years).
| » Results|| |
A total of 134 women with HER2/neu IHC equivocal breast cancer were included in the study, with a median age of 50 years and mode of 40 years (range 25–81). HER2/neu amplification by FISH was noted in 72 (54%) cases [Figure 1]a, whereas it was non amplified in 52 (39%) cases [Figure 1]b. Ten cases were reported as equivocal even on FISH [Figure 1]c. More than half of our cases were ≤50 years (70, 52%), while the rest were >50 years (64, 48%). However, there was no statistically significant difference in HER2/neu amplification patterns between the two age groups (amplified 56% vs. 52%, equivocal 8% vs. 6%, and non-amplified 36% vs. 42%). Polysomy 17 was noted in 55 cases (41%), of which 26 cases were ≤50 years and 29 cases were >50 years of age. Twenty (36%) of these 55 cases showed HER2/neu amplification, whereas 26 (48%) cases were non-amplified and 9 (16%) cases were reported as equivocal on FISH. Also, more than half of the polysomy cases were hormone receptor negative.
|Figure 1: (a) Fluorescence in situ hybridization image showing positive results for HER2/neu gene amplification: red signals in clusters of 8–10 and 2 green signals per nucleus. (b) Fluorescence in situ hybridization image showing negative results for HER2/neu gene amplification: 2–3 red signals and 2 green signals per nucleus. (c) Fluorescence in situ hybridization image showing equivocal results for HER2/neu gene amplification: 2–5 red signals and 2 green signals per nucleus|
Click here to view
The hormone receptor status for estrogen receptor (ER) and progesterone receptor (PR) was also analyzed for all the cases. Most of our cases were hormone receptor negative. When the expression patterns of hormone receptors correlated with that of HER2/neu status [Table 2], 74% and 69% of ER negative and PR negative cases, respectively, showed HER2/neu amplification, whereas an inverse correlation was noted between ER expression and HER2/neu status. Several PR positive cases, however, showed HER2/neu amplification.
|Table 2: Relation between hormone receptor expression and human epidermal growth factor receptor 2 status|
Click here to view
A total of 78 cases of invasive breast cancer with an age range of 30–81 years had undergone FISH assay following HER2/neu equivocal on IHC in the years 2016 and 2017. Of these cases, 29 were needle core biopsies (NCBs), 48 were excised specimens, and there was 1 cell block. All cases were evaluated for HER2/neu expression. The incidence of HER2/neu amplified, HER2/neu non-amplified, and HER2/neu equivocal in NCBs and excised specimens were 9 (31%) and 24 (50%), 12 (41%) and 23 (48%), and 8 (28%) and 1 (2%), respectively [Table 3].
|Table 3: Human epidermal growth factor receptor 2 expression on fluorescence in situ hybridization in biopsies and excised specimens in 2016 and 2017|
Click here to view
| » Discussion|| |
IHC and FISH are the most commonly used FDA-approved tests and also the most viable tests for determining HER2/neu status in breast cancer for both clinical practice and research purposes. IHC scores of 3+ and 1+ have shown a good concordance rate with FISH results,, but when it comes to IHC 2+ there is more variability. This study is an attempt to validate the role of FISH in investigating HER2/neu gene amplification in patients with HER2/neu equivocal breast cancer diagnosed on IHC.
In our study, more than half (54%) of the patients with IHC 2+ showed HER2/neu amplification on FISH, which is higher than that observed in some studies.,, However, the studies carried out at other tertiary centers like ours show similar results., This has been attributed to a possible referral bias. HER2/neu was non-amplified in 39% of cases, whereas equivocal FISH was observed in 7% of cases which is higher than the lower limit (<3%) recommended by the 2013 ASCO/CAP guidelines. It is hypothesized that IHC equivocal results which do not show gene amplification (39%) are possibly due to variations in pre-analytical factors like tissue fixation and processing (which can affect the epitope retrieval process) and protein expression due to polysomy.,,,,, The equivocal HER2 FISH category has been done away with in the 2018 ASCO/CAP Focused Update, with the recommendation that a definitive diagnosis will be rendered on additional workup with an additional observer, blinded to previous FISH results, doing a recount of at least 20 cells. If the count remains as before, the report is HER2/neu negative with a comment.
Furthermore, HER2/neu status was compared between patients ≤50 years and >50 years, but there was no statistically significant difference between these two arbitrary groups. However, women ≤50 years showed comparatively more HER2/neu amplification. Incidence of polysomy 17 ranges from 10% to 50% and its identification by FISH assay is challenging., The presence of polysomy is associated with more unfavorable pathological features than with disomic tumors., We observed 55 cases of polysomy 17 in IHC equivocal women. Twenty-six women were ≤50 years and 29 women were >50 years of age. On FISH assay, 20 of these 55 cases showed HER2/neu amplification (36%). Similar findings have been observed in other studies,,,, and it has been concluded that polysomy is an important cause of equivocal results.
Review of the literature shows that the term “polysomy 17” has been used when FISH shows an increase in the CEP17 signals. However, FISH analysis cannot assess the copy number of the entire chromosome because it is a targeted assay. Therefore, an increased number of CEP17 signals may represent a focal gain in the centromeric region of chromosome 17 and not a true polysomy 17. By using single nucleotide polymorphism array karyotype, one can differentiate between true polysomy 17 and focal gain of CEP17. True polysomy of chromosome 17 is rare and noted only in 1% of all analyzed cases. Increase in CEP17 copy number by FISH attributed to focal gains rather than true polysomy 17 have been reported by other authors as well.,
When the expression patterns of hormone receptors and HER2/neu status were studied, as observed in other studies, our study also showed that ER and PR negative women had higher HER2/neu amplification: 74% and 69%, respectively. Interestingly, despite this inverse relation, a number of cases showed ER+ and HER2/neu amplification (43%) and PR+ and HER2/neu amplification (50%). Such dual positivity (ER/PR and HER2/neu) reduces the efficiency of tamoxifen (Selective estrogen receptor modulator (SERM)-selective ER modulator) because it acts as an estrogen agonist and enhances the growth of tumor cells expressing HER2/neu. Therefore, such tumors are more aggressive. We also noted that more than half of the cases with “polysomy 17” were hormone receptor negative.
Recently, Pai et al. conducted a study to assess HER2/neu gene status in cases showing non classical FISH patterns with the CEP17 probe by using a non centromeric chromosome 17 reference locus (D17S122) probe and concluded that denominator-stable alternate probe can be used to analyze HER2/neu status in cases with FISH equivocal and complex patterns. This will help to stratify patients as HER2/neu positive or negative and aid their management.
| » Conclusion|| |
Although IHC is a good screening tool for negative and positive results, any patient targeted for trastuzumab therapy should undergo HER2/neu IHC equivocal status confirmation by FISH analysis. As our country is a resource deprived one, it is recommended to confirm HER2/neu IHC equivocal cases by FISH analysis. If a non-classical FISH pattern is seen, then the test should be repeated with a non-centromeric chromosome 17 reference locus probe for better treatment planning.
All faculty members from the Department of Histopathology are gratefully acknowledged.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| » References|| |
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL, et al.
Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177-82.
Dagrada GP, Mezzelani A, Alasio L, Ruggeri M, Romanò R, Pierotti MA, et al.
HER-2/neu assessment in primary chemotherapy treated breast carcinoma: No evidence of gene profile changing. Breast Cancer Res Treat 2003;80:207-14.
Lear-Kaul KC, Yoon HR, Kleinschmidt-DeMasters BK, McGavran L, Singh M. Her-2/neu status in breast cancer metastases to the central nervous system. Arch Pathol Lab Med 2003;127:1451-7.
Durbecq V, Di Leo A, Cardoso F, Rouas G, Leroy JY, Piccart M, et al.
Comparison of topoisomerase-II alpha gene status between primary breast cancer and corresponding distant metastatic sites. Breast Cancer Res Treat 2003;77:199-204.
Moasser MM. The oncogene HER2: Its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene 2007;26:6469-87.
Emde A, Köstler WJ, Yarden Y, Association of Radiotherapy and Oncology of the Mediterranean arEa (AROME). Therapeutic strategies and mechanisms of tumorigenesis of HER2-overexpressing breast cancer. Crit Rev Oncol Hematol 2012;84 Suppl 1:e49-57.
Gown AM, Goldstein LC, Barry TS, Kussick SJ, Kandalaft PL, Kim PM, et al.
High concordance between immunohistochemistry and fluorescence in situ
hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system. Mod Pathol 2008;21:1271-7.
Joensuu H, Kellokumpu-Lehtinen PL, Bono P, Alanko T, Kataja V, Asola R, et al.
Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer. N Engl J Med 2006;354:809-20.
Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr., Davidson NE, et al.
Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005;353:1673-84.
Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, et al.
Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005;353:1659-72.
Villman K, Sjöström J, Heikkilä R, Hultborn R, Malmström P, Bengtsson NO, et al.
TOP2A and HER2 gene amplification as predictors of response to anthracycline treatment in breast cancer. Acta Oncol 2006;45:590-6.
Ménard S, Valagussa P, Pilotti S, Gianni L, Biganzoli E, Boracchi P, et al.
Response to cyclophosphamide, methotrexate, and fluorouracil in lymph node-positive breast cancer according to HER2 overexpression and other tumor biologic variables. J Clin Oncol 2001;19:329-35.
Moelans CB, de Weger RA, Van der Wall E, van Diest PJ. Current technologies for HER2 testing in breast cancer. Crit Rev Oncol Hematol 2011;80:380-92.
Olsson H, Jansson A, Holmlund B, Gunnarsson C. Methods for evaluating HER2 status in breast cancer: Comparison of IHC, FISH, and real-time PCR analysis of formalin fixed paraffin-embedded tissue. Pathol Lab Med Int 2013;5:31-7.
Rosa FE, Santos RM, Rogatto SR, Domingues MA. Chromogenic in situ
hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas. Braz J Med Biol Res 2013;46:207-16.
Mrozkowiak A, Olszewski WP, Piaścik A, Olszewski WT. HER2 status in breast cancer determined by IHC and FISH: Comparison of the results. Pol J Pathol 2004;55:165-71.
Kuo SJ, Wang BB, Chang CS, Chen TH, Yeh KT, Lee DJ, et al.
Comparison of immunohistochemical and fluorescence in situ
hybridization assessment for HER-2/neu status in Taiwanese breast cancer patients. Taiwan J Obstet Gynecol 2007;46:146-51.
Yosepovich A, Avivi C, Bar J, Polak-Charcon S, Mardoukh C, Barshack I, et al.
Breast cancer HER2 equivocal cases: Is there an alternative to FISH testing? A pilot study using two different antibodies sequentially. Isr Med Assoc J 2010;12:353-6.
Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Comparison of fluorescence in situ
hybridization and immunohistochemistry for the evaluation of HER-2/neu in breast cancer. Journal of Clinical Oncology 1999;17:1974
Henry NL, Hayes DF. Uses and abuses of tumor markers in the diagnosis, monitoring, and treatment of primary and metastatic breast cancer. Oncologist 2006;11:541-52.
Walker RA. Use and assessment of diagnostic and predictive markers in breast pathology. Curr Diagn Pathol 2007;13:126-34.
Eswarachary V, Mohammed IG, Jayanna PK, Patilokaly GV, Nargund AR, Dhondalay GK, et al.
HER2/neu testing in 432 consecutive breast cancer cases using FISH and IHC – A comparative study. J Clin Diagn Res 2017;11:EC01-5.
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.
] [Full text]
Keyhani E, Muhammadnejad A, Karimlou M. Prevalence of HER-2-positive invasive breast cancer: A systematic review from Iran. Asian Pac J Cancer Prev 2012;13:5477-82.
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. Arch Pathol Lab Med 2007;131:18-43.
Hanna WM, Rüschoff J, Bilous M, Coudry RA, Dowsett M, Osamura RY, et al.
HER2 in situ
hybridization in breast cancer: Clinical implications of polysomy 17 and genetic heterogeneity. Mod Pathol 2014;27:4-18.
Vanden Bempt I, Van Loo P, Drijkoningen M, Neven P, Smeets A, Christiaens MR, et al.
Polysomy 17 in breast cancer: Clinicopathologic significance and impact on HER-2 testing. J Clin Oncol 2008;26:4869-74.
Hyun CL, Lee HE, Kim KS, Kim SW, Kim JH, Choe G, et al.
The effect of chromosome 17 polysomy on HER-2/neu status in breast cancer. J Clin Pathol 2008;61:317-21.
Vranic S, Teruya B, Repertinger S, Ulmer P, Hagenkord J, Gatalica Z, et al.
Assessment of HER2 gene status in breast carcinomas with polysomy of chromosome 17. Cancer 2011;117:48-53.
Goud KI, Dayakar S, Vijayalaxmi K, Babu SJ, Reddy PV. Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) and fluorescence in-situ
hybridization (FISH) assay. Indian J Med Res 2012;135:312-7. [Full text]
Makroo RN, Chowdhry M, Kumar M, Srivastava P, Tyagi R, Bhadauria P, et al.
Correlation between HER2 gene amplification and protein overexpression through fluorescence in situ
hybridization and immunohistochemistry in breast carcinoma patients. Indian J Pathol Microbiol 2012;55:481-4.
] [Full text]
Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, Bartlett JMS, et al.
Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. Arch Pathol Lab Med 2018;142:1364-82.
Downs-Kelly E, Yoder BJ, Stoler M, Tubbs RR, Skacel M, Grogan T, et al.
The influence of polysomy 17 on HER2 gene and protein expression in adenocarcinoma of the breast: A fluorescent in situ
hybridization, immunohistochemical, and isotopic mRNA in situ
hybridization study. Am J Surg Pathol 2005;29:1221-7.
Zhang Y, Martens JW, Yu JX, Jiang J, Sieuwerts AM, Smid M, et al.
Copy number alterations that predict metastatic capability of human breast cancer. Cancer Res 2009;69:3795-801.
Marchiò C, Lambros MB, Gugliotta P, Di Cantogno LV, Botta C, Pasini B, et al.
Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ
hybridization and microarray-based CGH analysis. J Pathol 2009;219:16-24.
Alqaisi A, Chen L, Romond E, Chambers M, Stevens M, Pasley G, et al.
Impact of estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) co-expression on breast cancer disease characteristics: Implications for tumor biology and research. Breast Cancer Res Treat 2014;148:437-44.
Pai T, Shet T, Patil A, Shetty O, Singh A, Desai SB. HER2 FISH with alternate probe in breast cancer. Arch Pathol Lab Med 2018;142:626-33.
[Table 1], [Table 2], [Table 3]