SMGr up Immunohistochemistry in Breast Pathology- Brief Overview of the Technique and Applications in Breast Pathology Bhanumathi K Rao 1 * 1 Department of Biochemistry, JSS Medical College, a constituent of JSS University, India *Corresponding author: Nandini Manoli, Department of Pathology, JSS Medical College, a constituent of JSS University, Mysore, Karnataka, India, Email: nandinimanoli65@gmail.com Published Date: April 30, 3016 INTRODUCTION AND TECHNICAL ASPECTS Immunohistochemistry (IHC) is a powerful technique by which specific antigens are identified in Formalin-Fixed, Paraffin-Embedded (FFPE) tissues. The method is based on antigen-antibody interaction (Taylor and Burns, 1974). The technique is widely used in breast cancer diagnosis and prognosis, and its applications continue to be extended because of its ease of use, comparative low cost, reliability, and versatility. In IHC an antigen-antibody reaction is visualized through light microscopy by means of a color signal which is produced by labeling or tagging the antibody by various methods. The morphology of the tissue around the specific antigen is clearly visualized by counter staining usually with Hematoxylin (blue). Results of stained IHC markers are reported semi quantitatively and have been used widely by pathologists for prognostication of breast tumors and also for diagnosis and differentiation of certain lesions of the breast. 1
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STEP1: FIXATION Fixation is a very crucial factor for optimal immunostaining. The tissues must be promptly and adequately fixed and well processed for crisp, clear and unambiguous results. Problems of interpretation can arise due to under fixation due to elution of the stain or over fixation which causes masking of antigen sites, and hence false negative results. Optimal times can be determined by the individual laboratory. A General Guideline for the Various Types of Samples is Mastectomy specimens and core biopsies: The tissue is fixed immediately in neutral buffered formalin. Tissue must not be allowed to dry. The fixation time is paramount, requiring between 6 and 48 hours. Breast FNAC/Cytology smears: Must be dropped into 10 %neutral buffered formalin and fixed for10 minutes. Alternately centrifuge the fluid to make a pellet and fix similarly to tissue. Ether alcohol or acetone may be poured on to fresh smears on the slide. STEP 2: ANTIGEN RETRIEVAL Formalin fixation is good for retaining morphology of the tissue, but causes masking of several epitopes. However these can be unmasked by antigen retrieval which is the next step. The FFPE tissue should be cut into 3- to 4-micron thin sections and cut on to glass slide coated with an adhesive, the most popular being Poly L Lysine (PLL) or alternately onto APES coated slides. Two methods are available, Heat retrieval Enzyme retrieval Heat Retrieval The most common antigen retrieval technique to restore the tertiary structure is heating tissue sections in water or buffered solutions (e.g., citrate or EDTA buffer). Microwave, pressure cooker, water bath and now automated pressure cooking based technology are available for heat retrieval. Enzyme digestion By trypsin or protease is the alternative method. 3
STEP 3: ANTIGEN-ANTIBODY INTERACTION AND LABELING/ DETECTION Basically two methods are available, the direct and the indirect methods, the basic difference being that the direct method uses labeled monospecific antibody and the indirect uses unlabeled primary antibodies. Direct Method The labeled antibody reacts directly with antigen (Figure 1) (a) Biotin labeled antigen-specific primary antibody (b) Biotin binds to avidin/streptavidin. This amplifies the staining. c) Color visualization is achieved through enzymatic reaction of horseradish peroxidase/ alkaline phosphatase. Advantage Short, quick and easy Disadvantages Very specific labeled antibodies have to be manufactured and tested against each antigen Poor signal amplification, hence not sensitive Indirect Method Here two layers of antibodies are utilized (Figure 1 and Figure 1c). (a) The primary antibody is unlabeled and reacts with the tissue antigen. (b) The secondary antibody is biotin-labeled and binds to primary antibody. (c) Secondary antibody is raised to the antibody of the same species which produced the primary. Advantage Increases versatility because the same primary antibody can be used with different secondary antibodies. Sensitive due to signal amplification, because the primary antibody can react with different antigenic sites. Also this allows higher dilution of the primary-reduces cost and nonspecific background staining Further developments are 4
Unlabeled Method Here the secondary antibody acts as bridge between the primary antibody and the enzyme detection complex. The peroxidase- antiperoxidase technique was the first of these methods to be used. It is a multistep procedure, using unlabeled primary and secondary antibodies and preformed enzyme detection complexes consisting of enzymes-peroxidase antiperoxidase/alkaline phosphatase linked with Avidin/Streptavidin (glycoproteins) and the vitamin Biotin which binds easily to both enzymes and antibodies. Advantage Signal amplification is increased in this multistep process Figure 1: Schematic diagram of Immunohistochemical techniques. (a) Direct method: the antigen-specific primary antibody is biotin labeled. Biotin binds to avidin/streptavidin. Color visualization is achieved through enzymatic reaction of horseradish peroxidase/alkaline phosphatase. (b) Indirect method: the antigen-specific primary antibody is unlabeled. The secondary, biotinlabeled antibody binds to primary antibody. Visualization is achieved accordingly through avidin/ streptavidin and peroxidase/alkaline phosphatase complexes. The indirect method increases versatility because unlabeled primary antibodies can be used. 5
(c) Indirect method with polymerchain detection system. Biotin and avidin/streptavidin are replaced by a polymer backbone. Indirect Method with Polymer Chain Detection System Biotin and avidin/streptavidin are replaced by a labeled polymer chain, allowing for increased sensitivity and specificity. Multiple secondary antibodies and enzymes conjugate with a polymer backbone (Figure 1c). These are highly sensitive as well as specific and hence there is much less nonspecific background staining when compared to enzyme detection techniques. Using two different colors in one tissue section is also possible by combining two immunoenzymatic systems or one immunoenzymatic system with different substrates. STEP 4: VISUALIZATION In the previous step active enzymes such as horseradish peroxidase or calf intestine alkaline phosphatase are added to the tissue sections to tag or label the biotin, avidin/streptavidin labeled antibodies so that they can be visualized. These in turn react with a chromogen such as DAB (3, 3 diaminobenzidine tetrahydrochloride) or AEC (3-amino-9-ethylcarbazole) which gets oxidized by the enzyme, so that a colored product is formed. A brown color is obtained when DAB is used as a chromogen and a red product when AEC is used. 6
QUALITY CONTROL IN IMMUNOHISTOCHEMISTRY Quality Control is imperative to standardize the IHC protocol in order to ensure that IHC staining is sensitive and specific and that the results are reproducible. Positive and negative controls should be used with every run. Tissue samples containing the antigen to be detected may 7
be identified for use as both positive and negative control. Sections are cut and stained along with the sample to be tested. For the negative control, the primary antibody is replaced by nonbinding Immunoglobulin from the same species (Figure 2 & Figure 3). Figure 2: Smooth muscle actin highlighting the myoepithelial layer of benign ducts. Figure 3: Negative SMA immunostain in a case of invasive carcinoma. The smooth muscle around the blood vessels has taken up the stain, acting as an internal control. False-Negative Results These are mainly due to faulty pre analytical procedures: Improper tissue fixation 8
Improper processing Improper pretreatment Troubleshooting Buffered neutral Formalin is mandatory for fixation; ER gives optimal reaction with this fixative. All reagents to be checked for ph before use and maintained at 7.2. If a Formalin alternative is used, it must be validated against NBF, laboratory director assuming responsibility for the validity of these assay results. False-positive results can occur through nonspecific background staining. Ionic binding of antibodies to charged connective tissue elements, e.g., collagen fibers. To avoid this, it is recommended that the tissue be incubated with normal serum of the same species as the secondary antibody (blocking). Endogenous enzyme activity must be blocked-taking into account the fixation and retrieval method-to further avoid false-positive reactions. Undissolved precipitates of chromogen or counter stain can also be mistaken for a positive reaction. Validation of IHC methodologies can be achieved by: 1. Participation in quality assurance (proficiency testing) programs. 2. Staining various tissue and tumor types to determine sensitivity and specificity 3. Comparing staining results of different antibodies that recognize similar proteins [1-4]. Prognostic and predictive ER, PR Her-2- neu ER/ PR: It is routine practice to evaluate all invasive breast cancers for Estrogen Receptor (ER) and Progesterone Receptor (PR) status, in order to predict prognosis and responsiveness to endocrine treatment should cancer recur after initial treatment. Earlier performed by biochemical assay, Immunohistochemistry is now the standard method as IHC has been proven to be equal and in fact, superior in predicting tamoxifen response in a clinically relevant manner. Levels as low as 1% positive staining of the carcinoma cells are associated with significant clinical response to hormonal therapy. 1. Prognostic role ER/PR serves as good biomarkers for treatment decisions 2. Predictive role 3. Role in pre-invasive stage 9
Prognostic role: ER/PR positive tumors have better prognosis than those which are negative, especially if both ER and PR are positive. These tumors are hormone responsive Adjuvant chemotherapy and endocrine treatment prevent or delay recurrence in ER positive cancers ER/PR serves as good biomarkers for treatment decisions Predictive Role St Gallen treatment guidelines partition breast cancer into 3 endocrine groups, which predicts the type of response depending on the hormone receptor status. Standardized, reproducible ER/ PR staining is necessary to classify patients into these groups. 1. Responsive high- 10% positivity 2. Response uncertain 3. Response Low (1%-9%) 4. Non responsive none (0) ER-negative invasive breast cancers do not benefit from endocrine therapy. PR status has an independent predictive role irrespective of the ER status especially among premenopausal women [5-7]. 1% of staining of tumor cell nuclei has a predictive value 10. Greater than 1% of carcinoma cells positive for PR corresponding to an ALL red score of more than 2 is associated with a disease free interval and overall survival for those who receive adjuvant chemotherapy Hormone Receptor IHC in DCIS: Immunohistochemical staining for ER in DCIS, lacking associated invasive carcinoma, has an emerging role in estimating potential tamoxifen benefit. Adjuvant tamoxifen in these patients reduced both ipsilateral recurrence and new contra lateral cancers [5]. Guidelines for ER/PR testing: ER and PR status should be determined on: All newly diagnosed invasive breast cancers. At least one and preferably the largest of multiple synchronous tumors. Breast recurrences (to ensure that prior negative results of ER and/or PR were not false negatives and to evaluate the specimen for biologic changes since the previous testing) Newly diagnosed cases of DCIS also being analyzed for receptor content 10
TECHNICAL ASPECTS Preanalytic Immediate fixation after resection is ideal Fixative: Buffered Neutral Formalin solution only. ph to be maintained at 7.2 Time of fixation more than 6 and less than 72 hours, time to be recorded. Check ph of all reagents before assay If tumor comes from a remote location, it should be bisected neatly through the tumor on removal and sent to the laboratory immersed in a sufficient volume of NBF. Cold ischemia time, fixative type, and time the sample was placed in NBF must be recorded. Time tissue is removed from patient, time tissue is placed in fixative, duration of fixation, and fixative type must be recorded and noted on accession slip or in report. Storage of slides for more than 6 weeks before analysis is not recommended for Her-2 staining Analytic Ideally known negative, intermediate positive and strong positive controls to be run or at least positive and negative controls to be run with each run Endometrial tissue with known receptor content may be used as external control Internal control from normal breast tissue to be run: It should show heterogenous staining of the luminal cells with weak, moderate and strong intensity. If not, i.e if the staining is homogenous and weak, it indicates low sensitivity of the assay and will lead to false negative interpretation of the test. Normal breast tissue acts as a built-in negative control, the myoepithelial cells and the stromal cells being negative for receptor content. Check controls before reading the slides - assay to be rejected if controls do not work and repeated under standard test conditions till desired till acceptable staining is achieved Analyze the invasive component. Choose non necrotic areas. When there is no normal breast epithelium and henceno built in control, the pathologist must exercise judgment as to whether the assay can be interpreted based on the level of ER and/ or PR positivity of the tumor cells, the histologic type of the tumor, the fixation status ofthe tumor, and the status of external controls. If a tubular, mucinous, or lobular morphology or a tumour with Nottingham score of 1 tests 11
ER /PR negative contrary to expectations, retesting on another bit of tissue or sending the specimen to referral laboratory for confirmation may be necessary (Figure 4). Figure 4: IHC in a case of lobular carcinoma breast. ER, Cytokeratin and CK7 positivity with negative E-cadherin confirmed a diagnosis of metastatic lobular in this patient who presented with abdominal metastasis at a later date. Post Analytic Participating in proficiency testing programs; interlaboratory comparison. Guidelines for reporting receptor status: Reporting ER/PR, (Figure 5 & Figure 6) Nuclear positivity Report contains percentage positivity and intensity The percentage of tumor cells is quantified by counting cells manually. Image analysis may also be used. Cytology specimen: At least 100 cells should be counted to estimate the percentage. The intensity of staining is reported as weak, Moderate, or strong (1+,2+,3+) in comparison with the positive controls and should be indicative of the average staining intensity of the tumour nuclei on the entire slide. Receptor positive:1% positive tumor nuclei using internal (normal epithelial elements) and external controls 12
Receptor negative: When less than 1% of tumor cells stain for ER or PR Receptor uninterpretable: Tissues negative for ER/PR that are without intrinsic elements (normal breast epithelium) should be repeated using another tumor block or another tumor specimen and reported as uninterpretable rather than as negative. Figure 5: An example of strong ER positive staining of almost 98% of the nuclei. By the Allred score, this would qualify for a proportion score of 5 and an intensity score of 3+. Figure 6: PR positive staining of almost 99% of the nuclei. This would qualify for a proportion score of 5 and an intensity score of 3+. 13
The reason for the uninterpretable result should be specified. This usually occurs when sample preanalytic guidelines were not met with, for example: Use of alcoholic fixatives for needle biopsies or cytology samples Fixatives other than 10% Neutral Buffered Formalin (NBF) Sub optimal fixation-under or over fixation (less than 6 hours than 72 hours) Delay in initiation of fixation by more than 1 hour Samples treated with acids as part of a decalcification procedure. Tissues with unsatisfactory staining of controls. Staining of adjacent normal breast indicates that the assay is too sensitive-leads to false positive Samples on unstained slides for >6 weeks prior to staining Needle/core biopsies with crush artifact (eg, under fixed sample -fixation for less than 6 hours), and an alternative potential sample for retesting should be suggested, if appropriate. ER/ PR negativity in histological types expected to be ER/PR-positive: Tubular, lobular, and mucinous histologic types or tumors with a Nottingham score of 1 being ER/PR negative. The statement should convey that such histologic subtypes or Nottingham score in most cases test ER/PR positive. HER-2/neu: (Human epidermal growth factor) (Figure 7). Oncogene which gives membranous staining of the carcinoma cells. Its presence signifies bad prognosis. Biomarker for Herceptin sensitivity (trastuzumab) monoclonal antibody against the HER-2 receptor) Its presence denotes resistance to tamoxifen Identification is by IHC. Should be performed on all newly diagnosed cases of primary breast cancer On subsequent metastatic site if any and if tissue is available. An FDA-approved IHC kit should be used preferentially. Manual Interpretation with Conventional Microscopy: Interpretation of Her 2 neu: Staining: membrane 14
Normal breast tissue should not take up stain. Can be done on core biopsies. (Concordance with resection is 85 to 90%) Only invasive carcinoma is scored; scoring is semi quantitative to be clinically relevant. Evaluate the HER2 sections for estimation of the percentage of tumor cells showing membrane staining at low power first, 4x magnification. The majority of strongly positive cases will be obvious at 4x magnification. In situ breast cancer cells should not be scored and has no relevance. To verify the percentage of stained tumor cells and completeness of membrane staining, use 10x magnifications. Well-preserved and well-stained areas of the specimen should be used to make a determination of the percent of positive infiltrating tumor cells. If determination of equivocal 1+/2+ cases is difficult using 10x magnification, confirm score using 20x or 40x magnification. If there is complete membrane staining at a weak to moderate intensity in greater than 10% of the tumor cells, the score of the specimens is 2+. This is usually accompanied by incomplete membrane staining of the majority of the remaining tumor cells. In the majority of 3+ cases, staining is usually homogeneous with approximately 80% of the tumor cells positive with intense uniform staining HER2 test indeterminate may be reported if technical issues prevent one or both tests from being reported as positive, negative, or equivocal such as Inadequate specimen handling Artifacts (crush or edge artifacts) that make interpretation difficult Analytic testing failure Multiple scoring systems have been used by various authors to predict treatment such as the H score, Allred score, or quick score using the percentage and intensity measurements. The Allred score which gives both percentage and intensity is recommended by the food and drug administration Dako Cytomation Hercep Test and the Ventana CB1are approved by Food and Drug Administration [5,8]. 15
Figure 7: HER 2 neu stain. Other Prognostic Indicators Ki-67 (MIB-1) stains the nuclei of cell in the G-phase and hence is a proliferation marker, the percentage of stained nuclei expressed as the proliferation index. It is being increasingly used as part of the prognostic panel as a low proliferation index indicates slower tumor growth and therefore better prognosis, whereas a high Ki-67 indicate indicates bad prognosis [5]. SUMMARY AND CONCLUSION To summarize, Immunohistochemistry is a technique that has wide applications in the field of Breast pathology, ranging from diagnostic, prognostic and therapeutic uses and also as a research tool. The reason for its popularity is that it is a fairly inexpensive, reliable and versatile method requiring relatively basic infrastructure and can be used not only in breast resection specimens but also in core biopsies and cytological specimens. The results are reported semi quantitatively and help to categorize breast carcinomas into therapeutic groups predicting response to therapy. Apart from prognostic assays, other diagnostic difficulties IHC can help resolve are to recognize micro-invasion, differentiation between usual and atypical hyperplasia, benign mimickers of malignancy from malignant conditions, to sub type breast tumors and determine the origin of intra and extra mammary metastasis. The last, though there is no specific marker for mammary tissue, is possible by using a panel of markers. IHC has also become an important research tool and is widely used in microarrays to determine various breast cancer histologic subtypes and phenotypes. As a concluding remark, the use and interpretation of immunohistochemistry should be done in the light of the clinical history, radiologic findings, and histologic characteristics of the tumors. A panel based approach used in conjunction with standard Hematoxylin-eosinstain is probably most successful. 16
References 1. Dabbs DJ. Diagnostic Immunohistochemistry: Theranostic and Genomic Applications, 4th edn. Saunders: Philadelphia, PA. 2013. 2. Schacht V, Kern JS. Basics of immunohistochemistry. J Invest Dermatol. 2015; 135: e30. 3. Taylor CR. Introduction to Immunohistochemistry. 1: 10. 4. Shi SR, Liu C, Taylor CR. Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis. J Histochem Cytochem. 2007; 55: 105-109. 5. Yeh IT, Mies C. Application of immunohistochemistry to breast lesions. Arch Pathol Lab Med. 2008; 132: 349-358. 6. Goldhirsch A, Coates AS, Gelber RD, Glick JH, Thürlimann B, Senn HJ, et al. First-select the target: better choice of adjuvant treatments for breast cancer patients. Ann Oncol. 2006; 17: 1772-1776. 7. Goldhirsch A, Glick JH, Gelber RD, Coates AS, Thürlimann B, Senn HJ; et al. Meeting highlights: international expert consensus on the primary therapy of early breast cancer 2005. Ann Oncol. 2005; 16: 1569-1583. 8. Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013; 31: 3997-4013. 17