Herceptest™ Interpretation Manual - Breast Cancer
Contents
1. 41 9 2 oru anc Eas ee Ud VO Figure 68 Breast carcinoma Score 2 3 XS D UA ee Br 95b 7 7 mz Ae A up ae tn 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 24 These infiltrating tumor cells exhibit complete membrane staining the intensity is moderate ir ME VF te LM a T para uA et ot eee a ver m ka t te Figure 69 Breast carcinoma Score 24 4x magnification Figure 70 Breast carcinoma Score 2 10x magnification Figure 71 Breast carcinoma Score 2 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 24 These infiltrating tumor cells exhibit complete membrane staining the intensity is moderate Figure 74 Breast carcinoma Score 24 Figure 73 Breast carcinoma Score 2 20x magnification Figure 72 Breast carcinoma Score 24 10x magnification 4x magnification t t amp ES i x ip Ad wer eil F As id i 5 has SN PTS tee RIA gt fia amp iA v SUNT x o O O 0 oO eb 03 s 5j E 6 pum OL Y o E D I Q o o 6 Rm i 69 gt O2. E HercepTest Interpretation Manual Breast Cancer ROW Version Crush Artifact m Deposition of the chromogen is characteristic Crush artifact is related closely to edge artifact in areas where the cells are crushed while the This artifact may be encountered more often in central well preserved cells are devoid stereotactic needle biopsies It is presumed that of immunoreactivity the tissue injury occurs during the extraction of the tissue from the needle rather than from the actual Decalcification Artifact biopsy process Regardless the compression of The spinal vertebrae and other areas of the the tissues along the edges of the core can produce human skeleton are sites of metastatic carcinoma a linear staining that has to be interpreted as artifact Interventional radiology has facilitated access to This artifact occurs in less than 1 of cases domains of the body and has provided another source of specimens that can be tested for m Inadvertent crushing of the tissue occasionally analytes such as HER2 However in order to occurs during sectioning resulting in render the tissue soft enough to cut on a histologist s morphologically distorted cellular architecture microtome at 4 5 microns the tissue has to be m When compared to surrounding cells stronger decalcified This traditionally is accomplished by staining may be observed in crushed cells exposing the bony tissue to a variety of available Crushed cells
3. Retraction of the epithelial cells from the stroma with deposition Figure 31 Immunoreactivity in the well preserved area of breast of the chromogen circumferentially around clusters of cells but carcinoma is 1 10x magnification little to no immunoreactivity in the cell to cell interface Score 1 Figure 32 Breast carcinoma with non specific immunoreactivity Figure 30 is apparent as retraction artifact 20x magnification Breast carcinoma 4x magnification Figure 33 Breast carcinoma with non specific immunoreactivity is confirmed 40x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Thermal Artifact This artifact occurs at the preanalytic stage The surgical removal of tissue with an electrocautery instrument is detrimental to the preservation of the tissue This is especially true and inversely proportional to the size of the tissue i e the smaller the biopsy the more damage incurred The frequency is dependent upon the surgeon and his her preferred method of tissue procurement Thermal artifacts may occur in 3 5 of cases Example of Thermal Artifact Figure 34 Breast carcinoma with thermal artifact can best be seen on the H amp E The majority of the injury can be seen at the edge As heat transfers through the tissue less and less can be seen 10x magnification Figure 35 Breast carcinoma with burning around the edges is slightly apparent Central part of the lesion is the b
4. DCIS Score 0 40x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score O No staining is seen in this invasive ductal carcinoma ee te tate ge ee x T T nti Sa B onera i less P i Mi i 5 e 2 el Pag aA a n ae 2 E a wd ANT SL Qe 4 Sb We es E A lt Figure 49 X Uu i P F Wade 2 je Ze pea s 5 il e Y Figure 48 Breast carcinoma Score 0 4x magnification Figure 49 Breast carcinoma Score 0 10x magnification Figure 50 Breast carcinoma Score 0 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 1 The infiltrating tumor cells are weakly stained and do not demonstrate complete membrane staining Figure 51 Breast carcinoma Score 1 4x magnification Figure 52 Breast carcinoma Score 1 10x magnification Figure 53 Breast carcinoma Score 1 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 1 The infiltrating tumor cells are weakly stained and do not demonstrate complete membrane staining 7 te A nn y Ae D J I e Rn C esi CU DE NA m Ua g 2s A Y us Di S Lo a we s E Deon N A A D pe P
5. Score 3 20x magnification 0 o 62 ab P 0 i ae gt 2 07 Q ke D KE Q 0 O RS ale 2 69 gt O a Herceplest Score 3 The majority of infiltrating tumor cells exhibit intense complete membrane staining P407 JU a a Figure 81 Breast carcinoma Score 3 4x magnification Figure 82 Breast carcinoma Score 3 10x magnification Figure 83 Breast carcinoma Score 3 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 3 The majority of infiltrating tumor cells exhibit intense complete membrane staining ROM E XQ EO HercepTest Interpretation Manual Breast Cancer ROW Version Figure 84 Breast carcinoma Score 3 4x magnification Figure 85 Breast carcinoma Score 34 10x magnification Figure 86 Breast carcinoma Score 34 20x magnification Herceplest Score 3 The majority of infiltrating tumor cells exhibit intense complete membrane staining _ L n AP _ Figure 88 g E m it i M Figure 87 Breast carcinoma Score 3 4x magnification Figure 88 Breast carcinoma Score 3 10x magnification
6. cellular staining runs e g low Fig 9 and moderate Fig 10 Figure 8 The 1 control cell line MDA 175 20x may display different categories of HER2 specific cellular stainings Only the HER2 specific staining displayed as a partial brown membrane rimming is used to validate the staining run Note The image only represents approximately 5096 of a 20x microscope visual field Figure 9 1 control cell line MDA 175 20x acceptable staining run with punctate and discontinuous membrane staining in a small number of cells The low limit appearance may reflect the difference in quality between images and true microscopy Note The image only represents approximately 5096 of a 20x microscope visual field Figure 10 1 control cell line MDA 175 20x acceptable staining run with punctate and discontinuous membrane staining in a moderate number of cells Note The image only represents approximately 5096 of a 20x microscope visual field HercepTest Interpretation Manual Breast Cancer ROW Version Guidelines for Scoring Use of the attached scoring system has proved reproducible both within and among laboratories Dako recommends that scoring always be performed within the context of the pathologist s past experience and best judgment in interpreting IHC stains Only patients with invasive breast carcinoma should be scored In cases with carcinoma in situ and invasive carcinoma in the same specimen only the inv
7. performed in a calibrated oven with uniform heat distribution 2 Weak staining 2a Inadequate epitope retrieval Verify that Epitope Retrieval Solution HercepTest Interpretation of slides reaches 95 99 C for full 40 minutes and is Manual Effects of allowed to cool for an additional 20 minutes Insufficient Target Retrieval page 29 2b Inadequate reagent Heview Staining Procedure instructions See incubation times Instructions for Use 2c Inappropriate fixation Ensure that patient tissue is not over fixed HercepTest Interpretation method used or that an alternative fixative was not used Manual Effects of Fixation page 28 2d Excessive heating for more Air dry the tissue sections at room HercepTest Interpretation than one hour at gt 60 C temperature for a minimum of 12 hours or Manual Effects of may cause a significant until dry Alternatively dry at 37 C overnight Excessive Tissue Drying decrease or loss of the specific or at 60 C for a maximum of one hour Loss of specific staining membrane associated Drying of tissue sections at elevated page 30 HER2 immunoreactivity temperatures must only be performed in a calibrated oven with uniform heat distribution HercepTest Interpretation Manual Breast Cancer ROW Version Problem 3 Excessive background staining of slides Probable Cause 3a Paraffin incompletely removed Suggested Action Use fresh clearing solutions and follow procedu
8. 2 positive advanced gastric cancer J Clin Oncol 2009 27 18s suppl abstr LBA2409 http media asco org silver Dako is a registered trademark of Dako Denmark A S HercepTest and Herceptin are trademarks owned by Genentech Inc and or F Hoffman La Roche Ltd HercepTest is subject to an exclusive trademark license to Dako Denmark A S HercepTest Interpretation Manual Breast Cancer ROW Version HER Overview HER2 Protein and HER2 Family The gene encoding HER 2 is located on chromosome 17 and is a member of the EGF erbB growth factor receptor gene family which also includes epidermal growth factor receptor EGFR or HER1 HERS3 erbB3 and HER4 erbB4 All of these genes encode transmembrane growth factor receptors which are tyrosine kinase type 1 receptors with growth stimulating potential Activation of HER family members generally occurs when the ligand and a dimer of the same monomer or other member of the HER family are bound together HER2 has no known ligand Once activation has occurred tyrosine autophoshorylation of cytoplasmic signal proteins transmit signals to the nucleus thus regulating aspects of cell growth division differentiation and migration Overexpression of HER2 receptors results in receptors transmitting excessive signals for cell proliferation to the nucleus This may lead to more aggressive growth of the transformed cell Data supports the hypothesis that the HER2 overexpression
9. 23 Figure 21 Breast carcinoma brown staining is apparent 4x magnification Figure 22 In this breast carcinoma homogeneous non specific cytoplasmic staining can be seen 20x magnification Figure 23 Breast carcinoma with no membranous staining seen 40x magnification Figure 24 Breast carcinoma with dot artifact 4x magnification Figure 25 Breast carcinoma exhibits brown dot artifact staining 10x magnification Figure 26 Breast carcinoma with brown dots representing cytoplasmic staining not membrane staining 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Artifacts Interpreting Artifacts gue eae oe J Ly Edge Artifact CES E Commonly edge artifacts are linked to the ipu war Ue E Ms preanalytic handling of the tissue Often the E er SERE Se ER PORA LEE MEN C method of surgical extraction is the cause LORD ON ahaa CEDE Ce CONES see Crushing and Thermal artifact sections This phenomenon is more frequently observed with the advent of stereotactic needle biopsies This artifact occurs in 3 526 of cases m Inadequate processing of thick tissue samples may mimic edge artifact by rendering the central portion of the tissue sub optimally fixed relative to the peripheral areas In these circumstances the immunoreactivity based on the sub optimal central portion may be mistakenly interpreted as false negative as optimal fixation is onl
10. 4 HercepTest procedure HercepTest Interpretation Manual ROW Version HER2 protein e HRP enzyme Peroxidase Block gt DAB Breast Cancer HER QFISH pharmDxr Kit HER2 IQFISH pharmDXx M kit is a direct fluorescence in situ hybridization FISH assay designed to quantitatively determine HER2 gene amplification in formalin fixed paraffin embedded FFPE breast cancer tissue specimens and FFPE specimens from patients with adenocarcinoma of the stomach including gastroesophageal junction HER2 IQFISH pharmDx kit is indicated as an aid in the assessment of breast and gastric patients for whom Herceptin trastuzumab treatment is being considered see Herceptin package insert For breast cancer patient results from the HER2 IQFISH pharmDx Kit are intended for use as an adjunct to the clinicopathologic information currently used for estimating prognosis in stage ll node positive breast cancer patients The assays includes a chromosone 17 reference probe to correct for HER2 signal number in the event of chromosone 17 aneusomy B CEN 17 PNA probes directly labeled with fluorescein FITO targets the centromeric region of the chromosome green signals B HER2 DNA probe directly labeled with Texas Red fluorochrome targets the HER2 amplicon red signals B Results are expressed as a ratio of HER2 gene copies red signals per number of chromosome 17 copies green signals HER2 IQFISH pharmDx is a
11. a ie iB ORRIN Sore ae my dal es Jf i elis Miti s ie yo v rm 4 oe 3a B pw Feu Ea E 4 1 as A ERES 4 f tie a i P xd 17 A f i J m uel a A w a J n y 96 Figure 73 Herceplest Score 24 These infiltrating tumor cells exhibit complete membrane staining the intensity is moderate p re ot ras ig i AT im lt b s La E ua aee TS m i Fata Yor uu Teu re vM ap RET dis y E71 AC 1 j At T bs m wh E ape L T ust r 3 a Dra US E i ale a m 6 i y ot EC p 4 6 M Y E A j d ni 2 x ES _ FX m om x j EE r A L Eu cmm F m f E i Ja E B P ae xw Pos cn f A x al _ Pm PED i Figure 75 Breast carcinoma Score 24 4x magnification Figure 76 Breast carcinoma Score 2 10x magnification Figure 77 Breast carcinoma Score 24 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 3 The majority of infiltrating tumor cells exhibit intense complete membrane staining AMUCECE Figure 78 Breast carcinoma Score 3 4x magnification Figure 79 Mos Figure 79 Breast carcinoma Score 34 10x magnification Figure 80 Breast carcinoma
12. i ttl fal 04 TA ee E eae WU t ee LAU e LM a A ee E rra Eae phare d dar as ee 2 Fite re i am dua d Ba ee A ges Vis SANTA ten ty Arr i E pe 5 a d mos cen ei ELE TI AT Erga arre ole L Pen Va FIRE s Es a ih ey be nd a at 4 air aa as Y Fe AP pay h 1 amp Y a J L al a ATE T Effects of Insufficient Target Retrieval It is important to adhere to the target retrieval procedure described in the Instructions for Use for HercepTest The stains displayed to the left are sections from the same tissue but exposed to appropriate epitope retrieval and insufficient epitope retrieval respectively Se s zy Er Ze du io we Fa d 7 amp J gt MM 4 Fi y i mu EI y it ns da e i D AT Tr 4 0 LM Sd 21 oS i _ E l AMNEM Figure 40A Breast carcinoma displaying a 24 staining when appropriate epitope retrieval is used 40 min at 95 99 C 20x magnification Figure 40B Breast carcinoma displaying a 1 staining when insufficient epitope retrieval is used 20 min at 90 C 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Effects of Excessive lissue Drying Loss of Specific Staining Excessive heating for more than 1 hour at gt 60 C may cause a significant decrease or loss of the specific membrane associa
13. in HER2 Positive Breast Cancer New England Journal of Medicine October 20 2005 HercepTest Interpretation Manual Breast Cancer ROW Version HER2 Testing Algorithm Tumor Sample HER2 IHC 0 1 2 3 Negative Negative Weakly Positive Positive Negative Positive Non Amplified Amplified Report to Oncologist for Herceptin Consideration Figure 3 Current clinical practices for selection of patients for Herceptin treatment For Herceptin Weakly positive cases 2 may be considered equivocal and reflexed to ISH testing NCON Practice Guidelines in Oncology CAP Conference Summary Laboratories performing HER2 testing should meet quality assurance standards HercepTest Interpretation Manual Breast Cancer ROW Version The Herceplest HercepTest is a semi quantitative immunohistochemical kit system for determination of HER2 protein overexpression in breast cancer tissues routinely processed for histological evaluation and in formalin fixed paraffin embedded cancer tissue from patients with adenocarcinoma of the stomach including gastro esophageal junction Following incubation with the primary antibody to human HER2 protein this kit employs a ready to use Visualization Reagent based on dextran technology This reagent consists of both secondary goat anti rabbit molecules and horseradish peroxidase molecules linked to a common dextran polymer backbone thus eliminating the need for seq
14. minutes after epitope retrieval and prior to staining Pre treatment Using PT Link Preheat the diluted epitope retrieval solution 1 10 in the Dako PT Link tank to 85 C Place the room temperature deparaffinized sections in Autostainer racks and immerse the slides into the preheated epitope retrieval solution Let the PT Link warm up to 97 C and incubate for 40 1 minutes at 97 C Leave the sections to cool in the PT Link until the temperature reaches 85 C Remove the PT Link tanks with the sections from the PT Link and leave the tanks on the table for 10 minutes with the lid off for further cooling Prepare a jar tank eg the PT Link Rinse Station with diluted Dako Wash Buffer and soak sections for 5 20 minutes after epitope retrieval and prior to staining Dedicated PT Link equipment must be used for HercepTest M Proper Incubations All incubation times should be performed according to the package insert Stay within x1 minute of all incubation times If staining must be interrupted slides may be kept in wash buffer following incubation of the primary antibody for up to one hour at room temperature 20 25 C Automated Staining Dako recommends the use of HercepTest on an Autostainer Link or a Dako Autostainer Use of HerceplTest on alternative automated platforms has not been validated and may give erroneous results HercepTest Interpretation Manual Breast Cancer ROW Version Wash Buffer Dilute t
15. of heterogeneous staining due to incomplete spreading of reagent Characteristic feature 3 score on the lower part and 0 score on the upper 10x magnification Figure 15 Breast carcinoma with example of heterogeneous staining due to incomplete spreading of hematoxylin Characteristic feature Weak counterstain to the left appropriate counterstain to the right 70x magnification Artificial Heterogeneous Staining Heterogeneous staining may occur as a consequence of suboptimal performance of the immunohistochemical test m Incomplete spreading of reagent HercepTest Interpretation Manual Breast Cancer ROW Version Background Staining Background staining is defined as diffuse non specific staining of a specimen It is caused by several factors These factors include but are not limited to pre analytic fixation and processing of the specimen incomplete removal of paraffin from sections and incomplete rinsing of slides The use of fixatives other than Neutral Buffered Formalin or Bouin s solution may be a source of background staining Background staining with Herceplest is rare This artifact may occur in 2 396 of cases Background has been reported in breast tissues with abundant hyalinized stroma Possible Cause of Background m Improper drying of slides use a humid chamber for primary antibody negative control and labeled polymer HRP reagent incubations when the assay is performed manually Improper de
16. stained and do not demonstrate complete membrane staining Figure 60 Breast carcinoma Score 1 4x magnification Figure 61 Breast carcinoma Score 1 10x magnification Figure 62 Breast carcinoma Score 1 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 24 These infiltrating tumor cells exhibit complete membrane staining the intensity is moderate pF A X rp dax 3 rp ge per se sie wr a p A Br gt iad CS eee ae A i aL d n Jl I E Ts j wp a 3 or foo r i ER amp P Nm Ty i r 4 bey Es IS O Ek Lo n us 1 Mia E t M4 A a A 2 F Figure 63 Breast carcinoma Score 2 4x magnification Figure 64 Breast carcinoma Score 2 10x magnification Figure 65 Breast carcinoma Score 2 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 24 These infiltrating tumor cells exhibit complete membrane staining the intensity is moderate L d 4 T rea d UM E pk ac J we Ns a T s in 1 A 3 A dr LORI OY T Figure 66 Breast carcinoma Score 24 4x magnification Figure 67 Breast carcinoma Score 2 10x magnification
17. 0 Characteristic feature Diffuse smudgy brown stain in background stroma and cells Figure 16 Breast carcinoma brown staining is apparent 4x magnification Figure 17 Breast carcinoma diffuse non specific background staining can be seen 10x magnification Figure 18 Breast carcinoma minimal membrane staining is seen O score is apparent 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Homogeneous Staining Properly fixed breast cancer tissue with HER2 protein overexpression should reveal relative uniformity of immunostaining in individual tumor cells In cases where variability in fixation of the tissue is present the tissue may not appear homogeneous m Inthe majority of cases breast tumor specimens stain homogenously for HER2 m Evaluation of homogeneous staining should be based on an overall average of all the infiltrative tumor cells Review average staining of the whole section m Carefully evaluate m The percent of infiltrative tumor cells showing complete membrane staining m The intensity of staining e f 21076 of the infiltrative tumor cells exhibit complete membrane staining and there is a moderate intensity of staining the score would be at least 24 e f itis difficult to determine whether gt 10 of the infiltrative tumor cells show complete membrane staining the score should be no greater than 1 Staining of Normal Epithelium Overexpression of HE
18. 1 minute buffer baths O between Peroxidase Block Primary Antibody ie pie Control Reagent Visualization Reagent and Substrate Chromogen Solution DAB steps Peroxidase Blocking Reagent applied for five minutes and specimen fully covered HercepTest Interpretation Manual Breast Cancer ROW Version Recommendations Recommended Data Tracking for HercepTest Immunostaining If the average percent positive cases falls within 15 Use HercepTest data to determine an 20 report results Continue to use HercepTest by average number of percent positive cases following the protocol Continue to monitor results High numbei ot recurrent If patient demographics are normal _ P review HercepTest procedures NI I Lt L LILA 3 matinnt Normal patient AMo0aqran Review Technical Procedures Page Review Interpretation Procedures Page 14 12 14 12 16 13 13 17 19 24 31 Table 1 HercepTest Interpretation Manual Breast Cancer ROW Version Technical Considerations Technical Considerations for Optimal Hercep Test Performance While accurate and consistent interpretation can be achieved technical issues relating to the performance of Herceplest are not always easy to identify If cumulative laboratory test results fall outside the expected range of 15 2096 positive evaluate the patient demographics and then adaress any technical problems Tec
19. 2 854 Parton M Dowsett M Ashley S Hills M Lowe F Smith IE High incidence of HER 2 positivity in inflammatory breast cancer Breast 2004 13 2 97 103 Perez EA Roche PC Jenkins RB Reynolds CA Halling KC Ingle JN Wold LE HER testing in patients with breast cancer Poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization Mayo Clin Proc 2002 77 148 154 Persons DL Bui MM Lowery MC et al Fluorescence in situ hybridization FISH for detection of HER 2 neu amplification in breast cancer a multicenter portability study Ann Clin Lab Sci 2000 30 41 Regitnig P Schippinger W Lindbauer M Samonigg H Lax SF Change of HER 2 neu status in a subset of distant metastases from breast carcinomas J Pathol 2004 Aug 203 4 918 26 Roche PC Suman VJ Jenkins RB Davidson NE Martino S Kaufman PA Addo FK Murphy B Ingle JN Perez EA Concordance between local and central laboratory HER 2 testing in the breast intergroup trial N9831 JNCI 2002 94 855 857 Roche PC Ingle JN Increased HER2 with U S Food and Drug Administration approved antibody J Clin Oncol 17 434 1999 letter B Ropke M Askaa J Key M HER2 neu CAP Today 1999 13 7 11 12 B Ross J Fletcher JA The HER 2 neu oncogene prognostic factor predictive factor and target for therapy Sem Canc Biol 1999 9 125 B Rudlowski C Friedrichs N Faridi A Fuzesi L Moll R Bastert G Rath W Buttner
20. ARKS aa Pe no ad TE Se at E eu QA IQ MN s Po qu UAE i A v M E Y 4 qu E Me Au P 3M A AE p A a 2 7 E Tig e Rik et ns Ra Ee s a We Ve S Ted 2 21644 E Pag Ctt ARCU dics PNE Von f ut T A lt lt PAT PON lt 0 DTE 2e e he M MC e ane 3 LT xe s LE NA 2 4 rac e zy Yi dei p EA ur Figure 54 Breast carcinoma Score 1 4x magnification Figure 55 Breast carcinoma Score 1 10x magnification Figure 56 Breast carcinoma Score 1 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 1 The infiltrating tumor cells are weakly stained and do not demonstrate complete membrane staining WEF PES a 4 oe e A i Ax 4 E A Ye gt SN T i d gt FRE RR p VPE Ag Seu A Fig i ure 57 MEE Ms Ae P E TEESE T NA ES Figure 57 Breast carcinoma Score 1 4x magnification Figure 58 Breast carcinoma Score 1 10x magnification Figure 59 Breast carcinoma Score 1 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Herceplest Score 1 The infiltrating tumor cells are weakly
21. EDUCATION Interpretation Manual ndi Dako Breast Cancer A Better Path HercepTest Table of Contents Gela a Introduction HER2 Overview HER2 Protein and HER2 Family HER2 Testing IHC and FISH HER2 Testing Algorithm The HercepTest Kit HER2 IQFISH pharmDx Kit Hybridizer Instrument for In Situ Hybridization FISH Recommendations Recommended Data Tracking for HercepTest Immunostaining Technical Considerations Technical Considerations for Optimal HercepTest Performance Protocol Recommendations Tissue Processing Considerations Tissue Processing Recommendations Review of HercepTest Scoring Guidelines Validation of the Assay Interpretation Guide for 1 Cell Line Guidelines for Scoring Interpretation Recommendations for Interpretation of HercepTest Breast Cancer Steps for HercepTest Interpretation 19 Staining Patterns 24 COE Interpreting Artifacts Effects of Fixation 29 Effects of Insufficient Target Retrieval 30 Effects of Excessive Tissue Drying Staining Images HER2 Expression in Various Diagnostic Entities 46 MEC T oi Re gt 46 Troubleshooting Guideline for HercepTest 49 MESI r0 0 oO iS O qj ab faa c gt Cc gt is o o ab Q 0 Efe LI Y 62 o Q 0 Herc introduction HercepTest Interpretation Manual Herceplest is a semi quantitative immuno
22. Engl J Med 2012 366 109 19 E Birner P Oberhuber G Stani J et al Evaluation of the United States Food and Drug Administration approved scoring and test system of HER2 protein expression in breast cancer Clin Cancer Res 2001 7 1669 B Blackwell K Mills D Gianni L et al EMILIA Primary results from EMILIA a phase IIl study of trastuzumab emtansine T DM1 and lapatinib L in HER2 positive locally advanced or metastatic breast cancer MBC previously treated with trastuzumab T and a taxane J Clin Oncol 2012 30 suppl abstr LBA1 B Bloom Kenneth Harrington Douglas Enhanced Accuracy of HER 2 nue Immunohistochemical Scoring Using Digital Microscopy Am J Clin Pathol 2004 121 5 619 630 B Brown RE Bernath AM Lewis GO HER 2 neu protein receptor positive breast carcinoma An immunologic perspective Ann Clin Lab Sec 2000 30 249 B Burris HA Docetaxel Taxotere plus trastuzumab Herceptin in breast cancer Sem Oncol 2001 28 38 B Burstein HJ Kuter Campos SM Gelman HS Tribou L Parker LM Manola J Younger J Matulonis U Bunnell CA Partridge AH Richardson PG Clarke K Shulman LN Winer EP Clinical activity of trastuzumab and vinorelbine in women with HER2 overexpressing metastatic breast cancer J Clin Oncol 2001 19 10 2722 2730 B Burstein HJ Recent Readings in the Oncology Literature HER2 Testing Defining the Gold Standard July 21 1999 Medscape Oncology Journal Scan Special Feature B Carlsson J Nor
23. Figure 89 Breast carcinoma Score 3 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Troubleshooting Guide Troubleshooting Guideline for HercepTest Problem Probable Cause ouggested Action Reference 1 No staining 1a Programming error Reagents Check programming grid to verify that the of slides not used in proper order staining run was programmed correctly 1b Reagent vials were not loaded Check the Reagent Map to verify the proper in the correct locations in the location of reagent vials reagent racks 1c Insufficient reagent on Ensure that enough reagent is loaded into HercepTest Interpretation tissue section the reagent vials prior to commencing the Manual Artificial run Refer to the Reagent Map for volumes Heterogeneous Staining required Ensure that spreading of reagent page 20 is optimal 1d Sodium azide in Use fresh preparation of Wash Buffer Wash Solution provided in the kit 1e Excessive heating for more Air dry the tissue sections at room HercepTest Interpretation than one hour at gt 60 C temperature for a minimum of 12 hours Manual Excessive Tissue may cause a significant or until dry Alternatively dry at 37 C Drying Loss of specific decrease or loss of the overnight or dry at 60 C for a maximum staining page 30 specific membrane associatec of one hour Drying of tissue sections at HER2 immunoreactivity elevated temperatures must only be
24. R Her 2 neu gene amplification and protein expression in primary male breast cancer Breast Cancer Res Treat 2004 84 3 215 23 B Sahin AA Biologic and clinical significance of HER 2 neu cerbB 2 in breast cancer Adv Anat Pathol 2000 7 3 158 B Simon R Nocito A Hubscher T Bucher C Torhorst J Schraml p Bubendorf L Mihatsch MM Moch H Wilber K Schotzau A Kononen J Sauter G Patterns of HER 2 neu amplification and overexpression in primary and metastatic breast cancer J Natl Cancer Inst 2001 93 1141 B Tripathy D Plante M HER 2 as a Predictive Marker Data from CALGB NSABP and SWOG San Antonio Breast Cancer oymposium December 12 1998 Medscape Daily Summary B Van Poznak C Tan L Panageas KS Arroyo CD Hudis C Norton L Seidman AD Assessment of molecular markers of clinical sensitivity to single agent taxane therapy for metastatic breast cancer J Clin Oncol 2002 20 9 2319 B Vera Roman JM Rubio Martinez LA Comparative assays for the HER 2 neu oncogene status in breast cancer Arch Pathol Lab Med 2004 128 6 627 33 B Wang S Saboorian MH Frenkel EP Haley BB Siddiqui MT Gokaslan S Wians FH Hynan L Ashfaq R Assessment of HER 2 neu status in breast cancer Automated cellular imaging system assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard Am J Clin Pathol 2001 116 495 B Wisecarver JL HER 2 ne
25. R2 on tumor cells is relative to a baseline level of expression on normal breast epithelium m Normal breast tissue rarely overexpresses HER2 Staining of normal ducts may be observed occasionally The sensitivity of Herceplest V has been established under controlled conditions to stain normal breast epithelium between 0 14 m f normal epithelium is staining 1 the test should be repeated and the protocol should be observed closely This phenomenon may be caused by 1 Fixatives other than Neutral Buffered Formalin or Bouin s solution 2 Use of a steamer or microwave rather than water bath for epitope retrieval Figure 19 Breast carcinoma with no staining of normal ducts on the left and 3 homogeneous staining on the right 10x magnification Figure 20 Breast carcinoma with no staining of normal ducts on the left and 3 homogeneous staining on the right 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Cytoplasmic Staining Homogeneous Cytoplasmic Staining Dot Artifact Diffuse homogeneous staining is specifically The dot artifact is specific to the cytoplasm confined to the cytoplasm Score O This artifact is associated with tumors having neuroendocrine differentiation Score O P aero D E OU at T f 4 L Figure 24 as Figure
26. asive component should be scored Figure 9 shows examples of staining patterns Score to HER2 Protein Report Overexpression Assessment Staining Pattern 0 Negative No staining is observed or membrane staining is observed in 1096 of the tumor cells 1 Negative A faint barely perceptible membrane staining is detected in gt 10 of tumor cells The cells exhibit incomplete membrane staining 2 Weakly Positive A weak to moderate complete membrane staining is observed in gt 10 of tumor cells 3 Positive A strong complete membrane staining is observed in 1096 of tumor cells Figure 11 Examples of staining patterns for tissue scored O 1 2 and 3 at 40x magnification i Weakly positive cases 2 may be considered equivocal and reflexed to ISH testing HercepTest Interpretation Manual Breast Cancer ROW Version interpretation Recommendations for Interpretation of HercepTest Breast Cancer Dako emphasizes that scoring of Hercep Test must be performed in accordance with the guidelines established in the package insert and within the context of best practices and the pathologists experience and best medical judgment This manual will highlight areas of interpretation potentially problematic for Hercep lest users The original Immunohistochemical assay CTA used by Genentech for the Herceptin clinical trials utilized a scoring system lat
27. ation Reagent step 95 99 C substrate Chromogen DAB Solution prepared properly 7 l iner f Epitope Retrieval Solution brought to 95 C after slides 1 erie DAB ma Lucie immersed before 40 minutes incubation started l f Dako Automated Link Platform Mix an appropriate amount Slides allowed to cool for 20 minutes in Epitope Ll of DAB Buffered Substrate with 25 uL DAB Chromogen Retrieval Solution per mL DAB Buffered Substrate Either alcohol or water based hematoxylin O Substrate Chromogen Solution DAB applied for O C counterstains used 10 minutes Manual Procedure instrumentation Equipment Distilled or deionized water not tap water used L L E regular preventative maintenance pe rtermed O for water bath after Substrate Chromogen on the Dako Autostainer Automated Link Platform Solution DAB step Do you have all the necessary equipment to perform O and baths after Peroxidase Block Primary If not specify what is missing in comments below Antibody Negative Control Reagent Visualization Reagent If you answered No to any of the above you have O deviated from protocol and should consult with your Buffer bath s changed between each step 1 local Dako Technical Support Representative Humid chamber used for Primary Antibody for assistance Negative Control Reagent Visualization Reagent incubations Additional observations or comments Slides placed in 5
28. cells directly contribute to the HER2 DNA Target for ISH pathogenesis and clinical aggressiveness of tumors This overexpression is associated with poor prognosis including reduced relapse free and overall survival HER2 Homodimer EE d Fi d Figure 1 Representation of HER family HER2 Testing IHC and FISH Immunohistochemistry IHC measures the level of HER2 receptor overexpression while fluorescence in situ hybridization FISH quantifies the level of HER2 gene amplification Together they are the most commonly used methods of determining HER2 status in routine diagnostic settings HER2 Protein Target for IHC Ze ARIT Seo Breast Cancer Cell Amplified Result Score gt 2 Breast cancer specimen stained with HER2 IQFISH pharmDx Figure 2 IHC and ISH targets for HER2 testing x Positive Result Score 34 Breast cancer specimen stained with HercepTest M Robert W Carlson MD Susan J Moench et al HER2 Testing in Breast Cancer NCCN Task Force Heport and Recommendations Journal of the National Comprehensive Cancer Network July 2005 Edith A Perez Vera J Suman et al HER2 Testing by Local Central and Reference Laboratories in Specimens from the North Central Cancer Treatment Group N9831 Intergroup Adjuvant Trial Journal of Clinical Oncology July 1 2006 Martine J Piccart Gebhart MD Ph D Marion Proctor M Sci et al Trastuzumab after Adjuvant Chemotherapy
29. complete kit and includes B Pre Treatment Solution 20x Pepsin Ready to Use Pepsin Diluent 10x HER2 CEN 17 IQISH Probe Mix otringent Wash Buffer 20x Fluorescence Mounting Medium containing DAPI Wash Buffer 20x B Coverslio Sealant 20 Tests K5731 HERZ IQFISH pharmDx Kit 22 x 22 mm target area Hybridizer Instrument for In Situ Hybridization FISH Hybridizer is a hands free denaturation and hybridization instrument The system allows for semi automation of FISH by eliminating manual steps in the hands on intensive manual procedure 120 volt 240 volt 2450 2451 Hybridizer Hybridizer Figure 5 Dako Hybridizer instrument HercepTest Interpretation Manual Breast Cancer ROW Version Checklist HercepTest Training Checklist Customer Mamer Institution Person Trained Title Manual Staining Run Yes If no complete the infarmation below _ No Dako Autostainer Software Version Dako Autostainer Serial Number Dako Automated Link Platform Software Version Dako Automated Link Platform Serial Number __ Manual Dako Autostainer or Automated Link Platform Procedure Yes No Yes No Control slides and kit stored at 2 8 C L a Specimens fully covered for 30 minutes with three drops 100 pl of Primary Antibody or Cell Line control slides and all reagents warmed to L LI Negative Control Reagent room temperature 20 25 C prior to starting assay Visualization Reagent applie
30. d for 30 minutes O _ Tissues fixed in 10 neutral buffered formalin or O a and specimen fully covered Bouin s fixative only Substrate Chromogen DAB Solution prepared properly E Specimens air dried at room temperature for a C Mix 1 drop of DAB Chromogen with 1 mL DAB minimum of 12 hours or until dry ar at 37 C Buffered Substrate overnight or at 60 C for one hour Substrate Chromogen solution applied for 1 L Specimens stained within 4 6 weeks of tissue O 10 minules and specimen fully covered mounted on slides when stored at room temperature Dako Autostainer or Automated Link Platform Procedure Clearing solutions changed after 40 slides 7 B Slides placed in buffer 5 1 minutes before E Deparaffinization and rehydration protocol followed 3 L loading anto the Dako Autastainer Wash Buffer prepared properly C 7 Appropriate protocol template used O E Prepare sufficient quantity of Wash Buffer mM l by diluting Wash Buffer 10X 1 10 in Reagent For each slide is 200 ul of Primary Antibody _ Quality Water deionized or distilled water or Negative Control Reagent applied m uu Was the Dako Autostainer Automated Link Platform Distilled or deionized water not tap water E awadi used for water washes after last alcohol bath ai da iit i in deparaffinization Slides rinsed with buffer between steps and 1 7 Water bath used and set to proper temperature o C double rinsed after the Visualiz
31. dgren H Sjostrom J Wester K Villman K Bengtsson NO Ostenstad B Lundqvist H Blomqvist C HER2 expression in breast cancer primary tumours and corresponding metastases Original data and literature review Br J Cancer 2004 90 12 2344 8 B Check W More than one way to look for HER2 CAP Today 1999 13 3 1 40 54 B Couturier J Vincent Salomon A Nicolas A et al Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 HER 2 neu gene status in breast carcinoma Mod Pathol 2000 13 1238 Dowsett M Cooke T Ellis Gullick WJ Gusterson B Mallon E Walker R Assessment of HER status in breast cancer why when and how Eur J Cancer 2000 36 170 Espinoza F A Anguiano The HercepTest assay Another perspective Letter Reply J Clin Oncol 1999 17 7 2293 2294 Esteva FJ Valero V Booser D Guerra LT Murray JL Pasztai L Cristofanilli M Arun B Esmaeli B Fritsche HA Sneige N Smith TL Hortobagyi GN Phase Il study of weekly docetaxel and trastuzumab for patients with HER 2 overexpressing metastatic breast cancer J Clin Oncol 2002 20 1800 1808 Field AS Charberlain NL Tran D Morey AL Suggestions for HER2 neu testing in breast carcinoma based on a comparison of immunohistochemistry and fluorescence in situ hybridization Pathology 2001 33 278 Fitzgibbons PL Page DL Weaver D Thor AD Allred DC Clark GM Ruby SG O Malley F Simpson JF Conno
32. er adopted by Dako as an integrated part of HercepTest Steps for HercepTest Interpretation Manual or Automated Interpretation 1 Evaluate the control cell lines to validate the assay run 2 lo verify the percentage of stained tumor cells and completeness of membrane staining use 10x magnification 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 2 Next evaluate the positive and negative If there is complete membrane staining at a control slides weak to moderate intensity in greater than 10 of the tumor cells the score of the specimens is 3 An H amp E stained section of the tissue sample is 2 his is usually accompanied by incomplete recommended for the first evaluation The tumor may not be obvious when looking at the sample stained with HercepTest An H amp E stain allows the pathologist to verify the presence of the invasive tumor Manual Interpretation with Conventional Microscopy 1 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 Invasive infiltrating breast cancer tumor cells are the only component that should be sco
33. ercepTest package insert guidelines will be reviewed and technical tips for ensuring high quality staining in your laboratory will be given Reviewing this HercepTest Interpretation Manual will provide a solid foundation for evaluating slides stained with HercepTest Most metastatic breast cancer tissue specimens tested for HER2 overexpression are scored with either O or 3 staining intensity While the majority of cases are clear cut a small percentage of the remaining 1 and 2 scored samples may be more difficult to interpret In this manual we will focus on these equivocal samples In addition we will review images of sample artifacts and discuss how to best interpret such cases HER2 IQFISH pharmDx Despite the high quality of HercepTest clinical response of weakly positive specimens has remained an area of uncertainty within HER2 assessment HER2 IQFISH pharmDx complements HercepTest by quantitatively determining HER2 gene amplification and clarifying equivocal cases HercepTest and HER2 IQFISH pharmDx M enhance patient care by aiding in proper determination of the appropriate course of treatment Photomicrographs The included photomicrographs are breast carcinoma unless otherwise noted Van Cutsem E Kang Y Chung H Shen L Sawaki A Loraick F et al Efficacy results from the ToGA trial A phase lll study of trastuzumab added to standard chemotherapy in first line human epidermal growth factor receptor
34. est Interpretation Manual Background Staining page 21 4 Tissue detaches from slides 5 Excessively strong specific staining 3h Excessive heating of tissue Refer to 2d 4a Use of incorrect slides 5a Inappropriate fixation method used Use corrective procedure for drying tissue sections Use silanized slides such as Dako silanized Slides Code 53003 SuperFrost Plus or poly L lysine coated slides Ensure that only approved fixatives and fixation methods are used 5b Use of improper heat source for epitope retrieval e g steamer microwave oven or autoclave Ensure that only an approved procedure for target retrieval is applied Refer to the Instructions for Use 5c Reagent incubation times too long Review Staining Procedure instructions 5d Inappropriate wash solution used Use only the Wash Buffer that is recommended for the kit HercepTest Interpretation Manual Breast Cancer ROW Version Troubleshooting Guideline for HercepTest Problem Probable Cause ouggested Action Reference 6 Weak staining of the _ 6a Incorrect epitope retrieval Immerse the slides in the pre heated 1 Control Slide protocol followed Epitope Retrieval Solution Bring temperature Cell Line of the Epitope Retrieval Solution back to 95 99 C and pre treat for a full 40 minutes 6b Lack of reaction with Ensure that the full 10 minute incubation oubstrate Chromogen time is used Ensure that
35. est preserved area 1 score is apparent 4x magnification Figure 36 Breast carcinoma with thermal artifact Score 1 Non specific deposition of the chromogen in a pattern consistent with specific HER2 is localized in areas with typical morphologic features of the thermal injury The centrally located tumor is HER2 negative 10x magnification t 1 Y Tx E hia 2 hm JA mS Ti v ns we lt s 1 4 T _ pes cn t P L a s Lj il d Mean NE reris L E P M i 7 z T Ta Lm amy le f ts eT uid d a ae e k e i Ay mt x 2 i L uo w a 2 e i i ES 1 r Q om d u A t polt p Mr 2 oe D 2 m dut 5 a om oo Pd n e i F z D d 6 u USE E i F gt so zl i IAM P i 1 E Figure 35 ff x y D LA Jar gt zt X t 5 a EE 2 NL RS ur i ia F E a 2 4 er a x P re pt 2 2 d E AP g J r E 4 E he rn gt TA ray J qt e j d s H f E ee te TEN Ae i el n zr 1 P inia Len i bop ks hala Pye 1 2n h rn S Bosch j 5 7 pL 6 Pala M psa LM SaL n So fee GaN EO pay WES u e 5 jJ N E H 4 a LE aor aia e P p a on j
36. he recommended wash buffer 1 10 using distilled or deionized water Store unused diluted solution at 2 8 C up to one month Discard diluted solution if cloudy in appearance Storage of Reagents Reagents should be stored at 2 8 C Do not use after the expiration date stamped on the outside package Tissue Processing Considerations Procedural deviations related to sample handling and processing can affect HercepTest results Some of the variables that affect outcome are as follows m Specimens drying prior to fixation m Type of fixative only neutral buffered formalin Is recommended Temperature age storage pH of fixative Length of fixation specimen size ratio of size to fixative volume Length of time in alcohol after primary fixation Processing time temperature pressure and chemicals used otorage of paraffin blocks otorage of cut sections Section thickness Tissue Processing Recommendations Validated Fixatives m Neutral Buffered Formalin m Bouin s Solution Fixation Times Neutral Buffered Formalin B 18 24 hours Time to fixation and duration of fixation if available should be recorded for each sample Bouin s m 1 12 hours depending on tissue thickness Tissues fixed in Bouin s solution must be washed in 70 ethanol to remove picrates prior to aqueous washes Bouin s solution may not be optimal if FISH testing is needed Specimen Thickness Tissue samples submitted for processing and embedding sh
37. histochemical assay to determine HER2 protein overexpression in breast cancer tissues routinely processed for histological evaluation and formalin fixed paraffin embedded cancer tissue from patients with adenocarcinoma of the stomach including the gastroesophageal junction Herceplest M is indicated as an aid in the assessment of breast and gastric cancer patients for whom Herceptin trastuzumab treatment is being considered see Herceptin package insert HercepTest Interpretation Guidelines Prior to HercepTest immunohistochemistry was practiced largely as a subjective method ideally suited for qualitative analysis HercepTest however changed this paradigm as the determination of positivity was no longer a simple yes or no answer Patients are now evaluated using immunohistochemistry technology applied as a semi quantitative tool with a scoring system reflective of intensity of staining in conjunction with percentage of stained tumor cells This shift in application introduced a change in the way immunohistochemistry was viewed This HercepTest Interpretation Manual for breast cancer is provided as a tool to help guide pathologists and laboratorians to achieve correct and reproducible results The goal of this manual is to familiarize you with the requirements for scoring breast carcinomas stained with HercepTest Example cases of various staining intensities of HER2 expression are provided for reference The H
38. hnical problems may arise in two areas those involving sample collection and preparation prior to performing the test and those involving the actual performance of the test itself Technical problems relating to the performance of the test generally are related to procedural deviations and can be controlled and eliminated through training and where necessary clarification of the product instructions Protocol Recommendations Pre treatment Using Water Bath Water Bath Heat HercepTest Epitope Retrieval Solution in a calibrated water bath capable of maintaining the required temperature of 95 99 C For best results fill a container suitable for holding slides with diluted epitope retrieval 1 10 solution Place container with epitope retrieval solution in a water bath and bring the temperature of the water bath and the epitope retrieval solution to 95 99 C Add the tissue sections mounted on slides to the container and bring the temperature of the epitope retrieval solution back to 95 C before starting the timer Incubation Time Incubate the slides for 40 x1 minutes in the preheated epitope retrieval solution Remove the container with the slides from the water bath but keep them in the epitope retrieval solution while allowing them to cool for 20 x1 minutes at room temperature After cooling decant the epitope retrieval solution and rinse in wash buffer For optimal performance soak sections in wash buffer for 5 20
39. land 48 58 661 1879 Spain 34 93 499 05 06 Sweden 46 8 556 20 600 Switzerland 41 41 760 11 66 United Kingdom 44 0 1 353 66 99 11 United States of America 1 805 566 6655 28630 17SEP14 ROW Version
40. lly JL Hayes DF Edge SB Lichter A Schnitt SJ Prognostic factors in breast cancer College of American Pathologists Consensus Statement 1999 Arch Pathol Lab Med 2000 124 966 Fornier M Esteva FJ Seidman AD Trastuzumab in combination with chemotherapy for the treatment of metastatic breast cancer Sem Oncol 2000 27 38 Hanna W Kahn HJ Trudeau M Evaluation of HER 2 Erb B2 on breast cancer cell lines From bench to bedside Mod Pathol 1999 12 8 827 Hoang MP Sahin AA Ordonez NG et al HER 2 neu gene amplification compared with HER 2 neu protein overexpression and interobserver reproducibility in invasive breast carcinoma Am J Clin Pathol 2000 113 852 Jacobs TW Barnes M Yaziji H et al HER2 neu protein expression in breast cancer determined by immunohistochemistry IHC A study of inter laboratory agreement Mod Pathol 1999 12 23A abstr Jacobs TW Gown AM Yaziji H Barnes MJ Schnitt SU Comparison of fluorescence in situ hybridization and immunohistochemistry for the evaluation of HER 2 neu in breast cancer J Clin Oncol 1999 17 7 1974 1982 Jacobs TW Gown AM Yaziji H Barnes MJ Schnitt SJ Specificity of Herceplest in determining HER 2 neu status of breast cancers using the United States Food and Drug Administration approved scoring system J Clin Oncol 1999 17 7 1983 1987 Koeppen HKW Wright BD Burt AD Quirke P McNicol AM Dybdal NO Sliwkowski MX Hillan KJ Overexpression of HER2 neu in solid tumo
41. on of correctly prepared tissues and proper staining technique The ideal positive tissue control is weakly positive staining tissue The presence of a brown reaction product at the cell membrane is indicative of positive reactivity Figure7 3 control cell line SK BR 3 stained with HercepTest A strong staining of the entire membrane is observed 20x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Interpretation Guide for 1 Cell Line The 1 control cell line can display different categories of HER2 specific cellular staining Cells displaying a partial brown membrane rimming where the immunostaining is punctate and discontinuous Fig 8 1a are the true indicators of a valid staining run In some cells the partial brown membrane rimming is more borderline but still considered positive consisting of a punctate and discontinuous immunostaining of both membrane and cytoplasm Fig 8 1b The borderline cells depicted here may reflect the difference in quality between images and true microscopy In a normal IHC staining run of the 1 control cell line few cells will display a circumferential brown cell membrane staining Fig 8 2 In addition in some cells dot like immunostaining can be observed in the Golgi region of the cytoplasm Fig 8 3 The different categories of HER2 specific cellular stainings may be reflected in the different appearances of acceptable 1
42. only one drop of Solution DAB DAB Chromogen was added to 1 mL of DAB Buffered Substrate 6c Degradation of Control Slide Check kit expiration date and kit storage conditions on outside of package 7 Other artifacts 7a Heterogeneous Staining Hercep Test Interpretation miscellaneous Manual Heterogeneous Staining page 19 7b Cytoplasmic Staining Hercep Test Interpretation Manual Cytoplasmic Staining page 23 7c Edge Artifacts HercepTest Interpretation Manual Edge Artifacts page 24 7d Retraction Artifacts Hercep Test Interpretation Manual Retraction Artifacts page 25 7e Thermal Artifacts Hercep Test Interpretation Manual Thermal Artifacts page 26 7f Crush Artifacts Hercep Test Interpretation Manual Crush Artifacts page 27 7g Decalcification Artifacts Hercep Test Interpretation Manual Decalcification Artifacts page 27 HercepTest Interpretation Manual Breast Cancer ROW Version Bibliography B Alpert LI Chao D Detection of HER 2 overexpression a new laboratory challenge Med Lab Observer June 1999 29 37 B Bartlett JMS Going JJ Mallon EA Watters AD Reeves JR Stanton P Richmond J Donald B Ferrier R Cooke TG Evaluating HER2 amplification and overexpression in breast cancer J Pathol 2001 195 422 428 B Baselga J Cort s J Kim SB Im SA Hegg R Im YH et al Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer N
43. or cells and the score is therefore no greater than 1 By definition focal staining implies that most of the tumor cells are not stained or are stained only partially on their membranes However it is important to verify that fewer than 10 of the tumor cells demonstrate complete membrane staining Staining not Associated with Tumor Cells Occasionally HER2 staining can be observed as luminal secretions of normal breast epithelium or may be seen as extracellular accumulations within the tissue This staining pattern should be disregarded DCIS Cases HercepTest has no indication for ductal carcinoma in situ DCIS at this time Staining of DCIS should be disregarded ACE n EE TE iy San i Figure os ee a gt UR A S Ya WI e Rec m WEZ AS Figure Figure 12 Breast carcinoma with example of heterogeneous staining Characteristic feature 3 score on the left and O score on the lower right with an intermingling of tumor cell subsets in between gt 10 of the infiltrative tumor cells demonstrate complete membrane staining 4x magnification Figure 13 Breast carcinoma with example of heterogeneous staining Characteristic feature 2 score on lower left 1 score on upper middle and negative on normal tissue on lower right 4x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Figure 14 Breast carcinoma with example
44. ould not exceed 3 4 mm in thickness Processing and Embedding After fixation tissues are dehydrated in a series of alcohols and xylene followed by infiltration by melted paraffin held at no more than 60 C Properly fixed and embedded tissues expressing the HER2 protein will keep indefinitely prior to sectioning and slide mounting if stored in a cool place 15 25 C Overheating of tissues during embedding or overheating of sections during drying can induce detrimental effects on immunostaining and therefore should be avoided The slides required for HER2 protein evaluation and tumor presence should be prepared at the same time Io preserve antigenicity tissue sections mounted on slides should be stained within four to six weeks of sectioning when held at room temperature 20 25 C Tissue specimens should be cut into sections of 4 5 um thickness To achieve reproducible results each laboratory performing HercepTest M should monitor its rate of positivity If the positive rate exceeds 20 a complete review of interpretation and technical procedures should be done HercepTest Interpretation Manual Breast Cancer ROW Version Guidelines Review of HercepTest Scoring Guidelines for Breast Tissue Herceplest is a semi quantitative immunohistochemical assay to determine HERZ protein overexpression in breast cancer tissues routinely processed for histological evaluation For the determination of HER2 protein Verify
45. paraffinization procedure Use of a different wash buffer than recommended Code S3006 is recommended m Incomplete rinsing of reagents from slides The non specific background staining of the negative test specimen is useful in ascertaining the level of background staining in the positive test specimen If background staining is significant the specific staining must be interpreted with caution ee M 3 d n a ub b in BP p E i 2 lt lt lt we EN eas n LCD a i vy J A 1 a E f A lt Re x iudi T f i e gt 4 i d ot i NEL a p A LS me Y y ert id MN T r LA Ax un m j i EE IF FE d m 1 gt h LL ib ue ac E W AA 1 TEL no ean CLE at E ea am dod v a NC ei erate Ia x a e L i is ti Fe i T i a E C LL E Ninos Ty Mm g P A T v i i wA y ae T M i ai re MET p uA ET i F j C T EB TS ANGEN es ie i Aig 2 E d i Is L2 i Ee J c AME CES je SA LT Fd M oes i qua al t i F d a A a is f Y k ai E 4 1 x r SP di i E pP NE Al I uod gt C E 5 A t a bd E av a Ve Figure 18 TS XT J t 5 Example of high non specific background Score
46. re as outlined in i Instructions for Use section B 1 Heference HercepTest Interpretation Manual Background Staining page 21 3b Starch additives used in mounting sections to slides Avoid using starch additives for adhering sections to glass sides Many additives are immunoreactive 3c Slides not thoroughly rinsed Ensure that the Autostainer is properly primed prior to running Check to make sure that adequate buffer is provided for entire run Use fresh solutions of buffers and washes HercepTest Interpretation Manual Background Staining page 21 3d Sections dried during staining procedure Verify that the appropriate volume of reagent is applied to slides Make sure the Autostainer is run with the hood in the closed position and is not exposed to excessive heat or drafts HercepTest Interpretation Manual Background Staining page 21 3e Sections dried while loading the Autostainer Ensure sections remain wet with buffer while loading and prior to initiating run HercepTest Interpretation Manual Background Staining page 21 3f Inappropriate fixation method used Ensure that approved fixative was used Alternative fixative may cause excessive background staining HercepTest Interpretation Manual Background Staining page 21 3g Non specific binding of reagents to tissue Check fixation method of the specimen and presence of necrosis HercepT
47. red In situ breast cancer cells should not be scored 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 membrane staining HercepTest Interpretation Manual Breast Cancer ROW Version HercepTest Interpretation Manual Breast Cancer ROW Version Staining Patterns Heterogeneous Staining Heterogeneous staining patterns occur less frequently as true biological entities Consequently when present this staining pattern may represent artifacts of tissue preparation m he pathologist s experience and judgment is important in the evaluation of heterogeneous staining m Review these cases at a low power on the microscope m fthe staining pattern is an artifact the best representative area s should be graded There must be gt 10 of the infiltrative tumor cells demonstrating complete membrane staining for the score to be at least 2 or greater m Inthe absence of clear evidence for biological heterogeneity the best representative area s should be scored as long as gt 10 of the infiltrative tumor cells in these areas demonstrate complete membrane staining at a moderate to strong intensity Focus on the most well preserved and well stained areas to make the determination Focal Staining Focal staining is usually 1 Focal staining usually occurs in lt 10 of tum
48. ted HER2 immunoreactivity The decreased HER2 immunostaining is likely caused by the destruction of the epitope s recognized by the HER2 antibodies Use proper procedure for tissue drying The drying temperature should be 60 C for a maximum of 1 hour 37 C overnight or room temperature for 12 hours or longer Use validated equipment oven thermometer when conducting the tissue drying Figure 41A Breast carcinoma displaying a 2 staining after appropriate tissue drying Figure 41B Breast carcinoma displaying a noticeably weaker 1 staining after excessive tissue drying Figure 42A Breast carcinoma displaying a strong 3 staining after appropriate tissue drying Figure 42B Breast carcinoma displaying a negative staining after excessive tissue drying HercepTest Interpretation Manual Breast Cancer ROW Version otaining Images HER2 Expression in Various Diagnostic Entities T Figure 45 Fa Figure 43 Example of poorly differentiated ductal carcinoma Score 0 40x magnification Figure 44 Example of well differentiated ductal carcinoma Score 1 40x magnification Figure 45 Example of moderately differentiated ductal carcinoma Score 2 40x magnification Figure 46 Example of poorly differentiated ductal carcinoma Score 34 40x magnification Figure 47 Example of intraductal carcinoma
49. that the negative tissue control slide from overexpression only the membrane staining the same staining run demonstrates no reactivity intensity and pattern should be evaluated using the scale presented on page 16 Slide evaluation should be performed using a light microscope Validation of the Assay Included in each HercepTest kit are control slides representing different levels of HER2 protein expression MDA 231 0 MDA 175 1 and SK BR 3 3 The first step of interpretation is to evaluate the control cell lines The control cell lines have been provided for qualifying the procedure and reagents not as an interpretation reference No staining of the O control cell line MDA 231 partial brown membrane rimming in the 1 control cell line MDA 175 refer to the Interpretation Guide for 1 Cell Line on next page and presence of complete intense brown membrane staining rimming in the 3 control cell line SK BR 3 indicates a valid assay If any of the control cell lines perform outside of these criteria all results with the patient specimens should be considered invalid l Figure 6 0 control cell line MDA 231 stained with HercepTest Next the positive tissue control slide known No staining of the membrane is observed 20x magnification to contain the HER2 antigen stained with HercepTest and fixed and processed similarly to the patient slides should be evaluated for indicati
50. typically demonstrate condensed decalcification solutions This renders the tissue nuclei Crushed cells should be avoided soft enough to obtain good histologic section in grading but also renders the tissue less than optimal for immunostains The use of HercepTest on decalcified tissues has not been validated and is not recommended Carcinoma has darker staining on crushed areas Score 1 Figure 37 Breast carcinoma showing crush artifact 40x magnification HercepTest Interpretation Manual Breast Cancer ROW Version 1 5 of Fixation standardization of fixation is very important when using HercepTest These stains have been fixed for 18 24 hours and for one week respectively Figure 38A Breast carcinoma shows a strong 3 staining with the appropriate fixation time Figure 38B Breast carcinoma shows a noticeably weaker staining but still 3 after the extended fixation Figure 39A Breast carcinoma shows 2 staining with the appropriate fixation time Figure 39B Breast carcinoma shows negative staining after the extended fixation HercepTest Interpretation Manual Breast Cancer ROW Version z Tra dns 3 724 LE 3 k D T T fd a Rig mif NT TT E VI d LIES i Tar de X Hu 85 E og po ea ah ye S a Ul s OCT Vio F e L
51. u testing comes of age Am J Clin Pathol 1999 111 3 299 301 B 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 Acknowledgements Dako would like to thank Dr Froilan Espinoza at Quest Diagnostics and Dr Jim Thompson at Impath Laboratories for their generosity in contributing to this project Impath Laboratories and Quest Diagnostics process a large volume of HercepTest slides every month Dr Espinoza and Dr Thompson contributed by offering their expertise and providing many of the images throughout this manual HercepTest Interpretation Manual Breast Cancer Relentless In our commitment to fighting cancer E Dako An Agilent Technologies Company Corporate Headquarters Australia Canada France Japan Denmark 61 2 9922 0700 1 905 335 3256 33 1 64 53 61 44 81 3 5802 7211 M EE Austria China Germany Korea 82 2 4026775 The Netherlands 31 20 42 11 100 43 1 408 43 340 86 21 3612 7091 49 40 69 69 470 Ireland 4353 1 479 0568 Denmark 45 44 85 97 56 Belgium www dako com 32 0 16 38 72 20 Finland Italy 358 9 348 73 950 39 02 58 078 1 Norway 47 23 14 05 40 Brazil 55 11 50708300 Represented in more than 100 countries Po
52. uential application of link antibody and peroxidase conjugate The enzymatic conversion of the subsequently added chromogen results in formation of a visible reaction product at the antigen site The specimen may then be counterstained and coverslipped Control cell line slides are provided HercepTest is a complete kit and includes m Peroxidase Blocking Reagent m Rabbit Anti Human HER2 Protein m Visualization Reagent Water bath 40 minutes 95 99 C Application of peroxidase block Incubate for 5 minutes Application of primary antibody Incubate for 30 minutes Ait Negative Control Reagent DAB Buffered Substrate DAB Chromogen Epitope Retrieval Solution 10x Wash Buffer 10x not included in 001 User Fillable Bottles only included in SKOO1 Hecommended hematoxylin counterstain not provided m Mayer s Hematoxylin for Dako Autostainer Autostainer Plus Code 3301 m Mayer s Hematoxylin for Automated Link Platforms Code SK308 Three HercepTest kit configurations are available K5204 HercepTest for manual use K5207 35 Tests 50 Tests HercepTest for the Dako Autostainer SK001 HercepTest for Automated Link Platforms 50 Tests e tom Application of Application of HRP labeled polymer chromogenic substrate Incubate for 30 minutes Incubate for 10 minutes A HER2 antibody AN J Tissue proteins Dextran backbone PN Secondary antibody Figure
53. urs an immunohistochemical survey Histopathol 2001 38 96 HercepTest Interpretation Manual Breast Cancer ROW Version Lal P Tan LK Chen B Correlation of HER 2 status with estrogen and progesterone receptors and histologic features in 3 655 invasive breast carcinomas Am J Clin Pathol 2005 123 4 541 Lebeau A Deimling D Kaltz C et al Her 2 neu analysis in archival tissue samples of human breast cancer comparison of immunohistochemistry and fluorescence in situ hybridization J Clin Oncol 2001 19 354 Lohrisch C Piccart M An overview of HER2 Sem Oncol 2001 28 No 6 Suppl 18 3 11 Lottner C Schwarz S Diermeier S Hartmann A Knuechel R Hofstaedter F Brockhoff G Simultaneous detection of HER2 neu gene amplification and protein overexpression in paraffin embedded breast cancer J Pathol 2005 205 5 577 84 Maia DM Immunohistochemical assays for HER2 overexpression Letter J Clin Oncol 17 5 1650 1999 Masood S Bui MM Assessment of HER 2 neu overexpression in primary breast cancers and their metastatic lesions An immunohistochemical study Ann Clin Laboratory Sci 2000 30 3 259 McNeil C How should HER2 status be determined J Natl Cancer Inst 91 2 111 1999 Paik S Bryant J Tan Chiu E Romond E Hiller W Park K Brown A Yothers G Anderson 5 Smith Wickerham DL Wolmark N Real world performance of HER2 testing National Surgical Adjuvant Breast and Bowel Project Experience JNCI 2002 94 85
54. y present at the periphery m Frequently increased staining is observed around the periphery of the tissue specimen known as the edge effect m The edge effect represents artifact due to tissue drying prior to fixation m fthe positive reaction is only at the edge of the tissue section i e a few layers of staining at the periphery and ending abruptly with penetration into the centrally located tumor grading at the edge of the tissue specimen should be avoided Figure 27 Breast carcinoma edge artifact is obvious 10x magnification Figure 28 Breast carcinoma edge artifact 20x magnification Figure 29 Breast carcinoma edge artifact 40x magnification HercepTest Interpretation Manual Breast Cancer ROW Version Retraction Artifact Hetraction artifact is edge artifact on a cellular level and can be observed in diagnostic entities such as basal cell carcinoma Unfortunately in many infiltrating breast carcinomas the desmoplastic status may cause retraction of the epithelial cells from the stroma This creates small spaces where antibody and chromogen can pool around the epithelial cells forming circumferential deposition of the brown stain This artifact requires thorough examination of the intercellular areas i e cell to cell interfaces not the cell to stroma interface Retraction artifacts occur in 2 5 of cases SS
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