Journal of Cytology
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 Table of Contents    
ORIGINAL ARTICLE  
Year : 2011  |  Volume : 28  |  Issue : 2  |  Page : 54-56
Her-2 neu (Cerb-B2) expression in fine needle aspiration samples of breast carcinoma: A pilot study comparing FISH, CISH and immunocytochemistry


1 Department of Pathology, Kuwait University; Department of Cytology Laboratory, Mubarak Al-Kabeer Hospital, Jabriya, Kuwait
2 Department of Medicine, Kuwait University, Jabriya, Kuwait
3 Department of Pathology, Kuwait University, Jabriya, Kuwait

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Date of Web Publication12-May-2011
 

   Abstract 

Background: Breast cancers with Her-2 neu gene amplification are recognized as important markers for aggressive disease and targets which respond to therapy with trastuzumab. Her-2 neu testing on histological sections is routinely performed to select patients who may benefit from anti- Her-2 neu therapy. Few reports are available which document Her-2 neu status on fine needle aspirates (FNA).
Aim: This pilot study is to document expression of Her-2 neu (Cerb-B2) on cytospin smears from FNA of patients with breast carcinoma.
Materials and Methods: Twenty samples of FNA already collected for diagnostic purposes from patients with primary breast carcinoma were studied for demonstration of Her-2 neu expression by immunohistochemistry (IHC), Fluorescent in-situ hybridization (FISH) and chromogenic in-situ hybridization (CISH) on cytospin smears from FNA. Their expression was compared with tissue sections where possible.
Results: Good correlation was observed between Her-2 neu protein expression and gene amplification in cytospin smears. Three of five (60%) breast carcinomas cases with 2+ and 3+ staining on IHC showed gene amplification by FISH and CISH. Three of 7 (43%) and 5 of 7 (71%) cases negative/1+ staining on IHC did not show gene amplification by FISH and CISH respectively. Tissue sections from 10 cases with 2+ and 3+ staining for Her2neu by IHC showed gene amplification in 8 cases.
Conclusion: Demonstration of Her-2 neu by IHC, FISH or CISH in FNA is possible and may play a role in the management of patients with advanced breast cancer or those cases where surgical resection is not advisable.

Keywords: Fine needle breast aspirates; FISH; Her-2 neu; Immunohistochemistry

How to cite this article:
Kapila K, Al-Awadhi S, Francis I M. Her-2 neu (Cerb-B2) expression in fine needle aspiration samples of breast carcinoma: A pilot study comparing FISH, CISH and immunocytochemistry. J Cytol 2011;28:54-6

How to cite this URL:
Kapila K, Al-Awadhi S, Francis I M. Her-2 neu (Cerb-B2) expression in fine needle aspiration samples of breast carcinoma: A pilot study comparing FISH, CISH and immunocytochemistry. J Cytol [serial online] 2011 [cited 2019 Oct 18];28:54-6. Available from: http://www.jcytol.org/text.asp?2011/28/2/54/80731



   Introduction Top


Her-2 neu (or Cerb B-2) protooncogene is amplified in nearly 10%-30% of breast carcinomas and is an indicator of clinical tumor aggressiveness and poor prognosis. [1] Her-2 neu has become an essential part of the clinical evaluation of breast cancer patients to select those patients suitable for a treatment regimen with a humanized monoclonal antibody trastuzumab (Herceptin; Genentech, San Francisco, CA, USA). [2] Fluorescent in-situ hybridization (FISH) and immunohistochemistry (IHC) are two major detection methods in routine clinical practice. In most centres, IHC is used as a screening technique for assessing Her-2 neu status and then FISH is used for those cases that are weakly positive or show indeterminate staining pattern. [1] Her-2 neu expression has been extensively studied on surgically excised breast tissue sections but few studies demonstrate it by FISH technique on cytological specimens namely imprints and aspirate smears. [3] Occasional studies have applied chromogenic in-situ hybridization (CISH) technique on cytologic samples [4] and found monolayer preparations to give excellent results. Evaluation of Her-2 neu status in cytology samples has a lot of clinical relevance as the sample can be obtained prior to surgery and help plan management of patients with inoperable breast carcinoma. This pilot study was undertaken to evaluate Her-2 neu on cytospin smears from fine needle aspirates (FNA) of breast carcinoma patients and determine if IHC, FISH or CISH is better suited.


   Materials and Methods Top


Twenty samples of FNA collected for diagnostic purposes from histologically documented primary ductal breast carcinoma were studied. In 18 patients Her-2 neu expression had also been determined on tissue sections for patient care.

Cytospin smears prepared from FNA were fixed in 95% ethanol. The prefixed unstained cytospin smear was used to demonstrate Her-2 neu by IHC, FISH and CISH using standard protocols. [5] The source of the antibody for IHC was DAKO (Cat No. A0485), dilution of antibody (1:50) and detection system LSAB2; System-HRP, DAKO (Cat No. K 0675); FISH VYSIS PathvisionR HER-2 DNA Probe kit and CISH. CISH - ZYMED SPoT-Light; HER-2 CISH TM Kit (Cat 84-0146)

Scoring System for Her-2 neu by IHC

0 - No immunostaining

1+ - Weak immunostaining, less than 30% of tumor cells

2+ - Complete membranous staining, either uniform or weak in at least 10% of cells

3+ - Uniform intense membranous staining in at least 30% of cells [Figure 1].
Figure 1: Her-2 neu expression by immunocytochemical studies on fine needle aspriates from breast carcinoma. a, b (Score 3); c (Score 1); d (Negative). (a,b,c,d ×400)

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Enumeration of Her-2 neu Amplification by FISH

The total number of Her-2 neu and CEP 17 signals in 20-60 interphase tumor cells were counted. The Her-2/CEP 17 ratio was calculated by dividing the total counts of the Her-2 neu signals by the total counts of CEP 17 signals. The ratio of >2.0 was defined as Her-2 neu DNA amplification. Ratio at or near the cut-off (1.8-<2.0) was considered as borderline amplification [Figure 2]. Ratios below 1.8 were considered as not amplified.
Figure 2: Fluorescence in-situ hybridization (FISH) on fine needle aspriates from breast carcinoma. a, b (Case 1); c, d (Case 5): Cases 1 and 5 show amplified Her-2 neu (red) and normal CEP-17 (green). c, f (Case 12) Normal Her-2 neu (red) and CEP-17 (green). (a, c, e ×400; b, d, f ×1000)

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Evaluation of CISH Results

Amplification was considered to be high when more than 10 copies, or large clusters of the Her-2 neu gene were present in >50% of cancer cell nuclei, whereas 6-10 copies of the Her-2 neu gene or a small Her-2 neu gene cluster, in the same percentage of cells were considered to be of low amplification [Figure 3] . Tumors were not considered to be amplified when 1-5 copies of the Her-2 neu gene were identified per nucleus.
Figure 3: CISH detection of Her-2 neu on fine needle aspirates from breast carcinoma. a,b (Case 3) low amplification (5 or more Her-2 neu gene signals); c,d (Case 5) High Amplification (brown clusters seen in the nuclei); e (Case 12): No amplification of Her-2 neu (2-3 Her-2 neu gene signals in each nucleus). (a, c, e ×400; b, d ×1000)

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   Results Top


In 12 of the 20 cytospin smears examined Her-2 neu detection was available by all the three methods. In 10 of these corresponding Her-2 neu results were available by IHC and FISH in tissue sections.

[Table 1] correlates the expression of Her-2 neu by IHC, FISH and CISH in cytospin smears and tissue sections. Of the 7 cases negative/1+ for Her-2 neu by IHC - 3 and 5 were also negative by FISH and CISH, respectively. Three of the 5 cases with 2+ and 3+ staining by IHC also showed gene amplification by FISH and CISH.
Table 1: Correlation of expression of Her-2 neu by IHC, FISH and CISH in cytospin smears from fine needle aspirates of breast carcinoma

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   Discussion Top


The accuracy of diagnostic assays for Her-2 neu in breast cancer is extremely important as its over expression is clinically useful for predicting prognosis and identifying patients who may benefit from herceptin treatment. FNA is an established reliable method for the diagnosis of breast carcinoma. Its application has been extended to immunocytochemical analysis of prognostic indicators. [6] Limited studies are available which document Her-2 neu status of breast carcinoma in FNA and most of the studies have used conventional smears. [3] We have tried to document the reliability of Her-2 neu expression on cytospin smears from FNA by IHC, FISH and CISH to see which is better suited. Unfortunately, we were unable to demonstrate any consistency. Others have also shown that Her-2 neu protein expression on cytologic preparations by IHC is insufficiently reliable while gene amplification determined by FISH on FNA has been found to have consistent correlation with tissue sample. [7] Within the FNA specimens, the agreement between protein overexpression (IHC) and gene amplification (FISH and CISH) was variable. [3],[4],[7] This could be attributed to the method of fixation. It has been recently reported that poorest agreement was observed with immunocytochemical analysis performed on slides fixed in ethanol whereas there was little difference between formalin fixation and Thin-Prep slides. [5] To conclude, it is possible to study Her-2 neu by IHC, FISH and CISH in cytologic material. However, it is not very reliable. Further studies are needed to standardize fixation techniques. Determining Her-2 neu status on aspirates may still be useful in patients with advanced cancer where surgical resection of the tumor may pose a problem and documenting Her-2 neu status is imperative prior to starting therapy.


   Acknowledgments Top


The support of the Research Core Facility Project No. GM01/01, GM01/05 and Kuwait University Research Grant No MGO 1/07 is gratefully acknowledged. We wish to thank Mr. James Luke for the secretarial assistance.

 
   References Top

1.Gown AM. Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol 2008; 21 Suppl 2:S8-S15.  Back to cited text no. 1
    
2.Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783-92.  Back to cited text no. 2
    
3.Bofin AM, Ytterhus B, Hagmar BM. TOP2A and HER-2 gene amplification in fine needle aspirates from breast carcinomas. Cytopathlogy 2003;14:314-9.  Back to cited text no. 3
    
4.Kim GY, Oh YL. Chromogenic in situ hybridization analysis of HER-2/neu status in cytological samples of breast carcinoma. Cytopathlogy 2004;15:315-20.  Back to cited text no. 4
    
5.Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol 2009;27:1323-33.  Back to cited text no. 5
    
6.Kapila K, Anim JT, Francis IM, Al-Mulla F, George SS, Behbehani AI. Expression of estrogen receptor alpha and estrogen receptor beta in fine needle aspirates from breast carcinoma. Acta Cytol 2010;54:25-30.  Back to cited text no. 6
    
7.Beatty BG, Bryant R, Wang W, Ashikaga T, Gibson PC, Leiman G, et al. HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in-situ hybridization and immunocytochemical analysis. Am J Clin Pathol 2004;122:246-55.  Back to cited text no. 7
    

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Correspondence Address:
Kusum Kapila
Department of Pathology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110
Kuwait
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Source of Support: Research Core Facility Project No. GM01/01 and GM01/05, Conflict of Interest: None


DOI: 10.4103/0970-9371.80731

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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]

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