INFORMTM HER2 - HER
Contents
1. If a condition persists across multiple cases and repeated attempts to correct the condition contact your local Ventana office 20 Ventana Medical Systems References Slamon DJ Godolphin W Jones LA et al Studies of the HER 2 neu proto ongene in human breast and ovarian cancer Science 1989 244 707 712 Gschwind A Fischer OM Ullrich A The discovery of receptor tyrosine kinases target for cancer therapy Nat Rev Cancer 2004 4 361 370 Muleris M et al Assignment of v erb b2 avian erythroblastic leukemic viral oncogene homolog 2 ERBB2 to human chromosome band 17121 1 by in situ hybridization Cytogenet Cell Genet 1997 76 34 5 Coussens L Yang Feng TL Liao Y C Chen E Gray A McGrath et al Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene Science 1985 230 1132 1139 Slamon DJ Clark GM Wong SG et al Human breast cancer correlation of relapse and survival with amplification of the HER 2 neu oncogene Science 1987 235 177 182 Narita M Nakao K Ogino N et al Independent prognostic factors in breast cancer patients Am J Surg 1998 175 73 75 O Reilly SM Barnes DM Camplejohn RS et al The relationship between c erbB 2 expression S phase fraction and prognosis in breast cancer Br J Cancer 1991 63 444 446 Pegram MD Finn RS Arzoo K et al The effect of HER 2 neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian c2. For the minority of cases in which the HER2 Chr17 ratio falls within the range of 1 4 to 4 0 the reader must proceed 8 Ventana Medical Systems Figure 7 Frequency Distribution of HER2 Chr17 Ratios by FISH 400 15 3 350 300 gt 250 o c S 200 c We 150 100 50 0 honor un Go GZ O O Q 6 4 O O QN co X O O6 SS ei OO e e N o cO x TCU OOOO N nr OD DF rz rz FISH Ratio Method 2 Quantitatively determine HER2 Chr17 Ratio lt 1 8 S gt 2 2 Ratio Negative Equivocal Positive to Method 2 a quantitative method This is recommended to ensure accuracy in determining HER2 gene status As cited above approximately 15 of breast carcinoma cases will fall within the range of 1 4 and 4 0 Alternately a reader may choose to skip Method 1 and proceed directly to Method 2 the quantitative method for enumeration of the HER2 Chr17 ratio Method 1 Semi Quantitative Method Using Method 1 the reader semi quantitatively estimates the average number of HER2 and Chr17 signals in the target area and records the HER2 Chr17 ratio Utilizing stained serial slides that meet the criteria for Slide Adequacy locate the same target area on each slide of HER2 and Chr17 and determine semi quantitatively the average number of signals in the Target Area according to Table 2 Signal Enumeration This is HER2 and Chr17 within the target area Calculate the HER2 Chr17 ratio by dividing the average number of HER2
3. 131 18 43 Press MF et al Diagnostic Evaluation of HER 2 as a Molecular Target An Assessment of Accuracy and Reproducibility of Laboratory Testing in Large Prospective Randomized Clinical Trials Clin Cancer Res 2005 11 18 September 15 2005 www ventanamed com 21 Appendix A Score Sheets Interpretation of Results Score Sheet for Method 1 Method 1 HER2 Estimated average number of HER2 Signals in Target Area 1 a Chr17 Estimated average number of Chr17 Signals in Target Area 1 b HER2 Chr17 Ratio c a b O Negative HER2 Chr17 lt 1 4 O Positive HER2 Chr17 gt 4 0 O 1 4 lt HER2 Chr17 lt 4 0 Use Method 2 Interpretation of Results Score Sheet for Method 2 and Method 2A Method 2 Target Area 1 Target Area 2 Cell HER2 Count Cell Chr17 Count Cell HER2 Count Cell Chr17 Count 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8 9 9 9 9 ll D O ll D o0 ll D O ch O 12 sel sc sesch O A OO 0 0 IL e Oo 0 4 oO 0 4 CO IL el e O A OO C 4 0 sesch O A ODO C 4 20 20 20 20 Total number of HER2 Total number of Chr17 Total number of HER2 Total number of Chr17 signals in Target Area 1 signals in Target Area 1 signals in Target Area 2 signals in Target Area 2 d e g h Target Area 1 HER2 Chr17 Ratio Target Areas 1 and 2 HER2 Ch
4. Interpretation Guide INFORM HER2 DNA Probe Staining of Breast Carcinoma CA mm gem A 4 D We Ki e R 1 A a an D A Ps gt v P 7 e ch WW e on oe A A A a e KI 8 i A q 117 wa a L m e i gt 4 y a D Thomas M Grogan M D we Lidija Pestic Dragovich Ph D E Abigail McElhinny Ph D ts Frank Vladich M S Hiro Nitta Ph D Eric Walk M D us Christina Reita NY e Christopher Roberts e i Patrick Roche Ph D e Se a INTRODUCTION General Description of INFORM HER2 DNA Probe Purpose of Interpretive Guide IDENTIFICATION OF APPROPRIATE STAINING PATTERN HER2 Gene Status 2 2 Signal Visualization and Enumeration of SISH Staining 4 Target Area Slide Adequacy Selecting Carcinoma Nuclei for Enumeration Additional Observations DETERMINING HER2 GENE STATUS Method 1 Semi Quantitiative Method Method 2 Quantitative Method Method 2A Additional Quantitation EXAMPLES OF HERZ AND CHR17 STAINING PATTERNS IN CLINICAL CASES 4 5 6 6 11 12 QUALITY CONTROL FOR SISH STAINING Positive and Negative Controls for SISH Staining HER2 3 in 1 Xenograft Control Slides PRE ANALYTICAL CONSIDERATIONS AND OPTIMIZATION OF THE ASSAY Fixation Sample Thickness Other Considerations TROUBLESHOOTING REFERENCES APPENDIX A Score Sheets 18 21 22 22 Introduction General De
5. mutated p53 and high nuclear grade HER2 gene status is reported as a function of the ratio of the average number of copies of the HER2 gene to the average number of copies of Chromosome 17 Chr17 per cell in an invasive breast carcinoma HER2 gene status is classified using the following guidelines A sample is negative for HER2 gene amplification if the HER2 Chr17 ratio is less than 1 8 Figure 1 Case 1 A sample is equivocal for HER2 gene amplification if the HER2 Chr17 Figure 1 HER2 Gene Status in Representative Cases Case 1 is negative for HER2 gene amplification ratio is either equal to or falls between 1 8 and 2 2 Figure 1 Case 2 Finally if the HER2 Chr17 ratio is greater than 2 2 a sample is positive for HER2 gene amplification Figure 1 Case 3 These classifications are summarized in Table 1 Ventana has designed this assay so that the INFORM HER2 DNA Probe is detectable on one slide and the INFORM Chromosome 17 Probe is detectable on a matched slide The probes are visualized using silver n situ hybridization SISH and appear as discrete black dots in the nuclei of normal cells serving as internal positive controls for staining and in invasive carcinoma This strategy allows HER2 gene status to be determined in the context of its chromosomal state using standard light microscopy with 20X 40X and or 60x objectives Ventana has developed two methods for enumerating HER2 gene status a semi q
6. Slides Cell Line INFORM HER2 DNA Probe INFORM Chromosome 17 Probe Calu 3 or BT474 ZR 75 1 MCF 7 800 227 2155 www ventanamed com 15 Pre Analytical Considerations and Optimization of the Assay Pre analytical sample preparation is an essential factor to consider before using any in situ hybridization assay For the INFORM HER2 DNA Probe Ventana recommends that the tissue be fixed in 10 neutral buffered formalin NBF for 6 24 hours paraffin embedded and sectioned at approximately four microns Ventana has developed the INFORM HER2 DNA Probe assay with protease treatment options that will enable optimization of the assay in different laboratories and for subsequent troubleshooting of individual slides that may exhibit sub optimal staining It is recommended that each laboratory perform staining runs on representative biopsy samples that have been prepared under the identical conditions as the clinical samples to be tested This will aid in optimizing the specific SISH staining conditions for individual laboratories that may vary in their exact specimen preparation procedures Fixation For samples fixed in 10 NBF and sectioned at approximately four microns Ventana ISH Protease 3 is recommended for eight minutes for both HER2 and Chr17 probes Tissue fixed in either 10 NBF recommended or Zinc formalin are suitable specimen types for the INFORM HER2 DNA Probe Assay Tissue fixed in alcohol
7. area must satisfy two criteria to be deemed adequate for enumeration If the slide does not meet these criteria it cannot be enumerated The user should refer to the troubleshooting section and evaluate the appropriate conditions for re staining the slide Internal Positive Control Staining must be present Normal HER2 or Chr17 signals one to two copies per cell that are visible as distinct Single Copy staining act as internal positive controls and should be visible using 20X 40X or 60X objectives This distinct nuclear staining may be located in various non neoplastic cells including stromal fibroblasts endothelial cells lymphocytes and benign breast epithelial cells Figure 3 Due to truncation artifacts in the plane of sectioning it usually is not possible to visualize single HER2 gene or Chr17 copy number in all cells on the slide nor in all regions of the tissue However accurate enumeration requires Figure 3 Normal Staining in Internal Positive Control Nuclei Case 2 is equivocal for HER2 gene amplification that single copy SISH signals are visible in normal cells within and or adjacent to the target area See Figures 1 3 Figure 4 shows an example of a slide that would be considered inadequate due to a lack of internal positive control staining in or adjacent to the target area SISH staining within the invasive breast carcinoma cells in the target area must be enumerable Using 20X 40X and or 60X o
8. of nuclei in which multiple discrete copies are visible Clusters In some nuclei clusters of dots representing many copies of HER2 gene are apparent A small cluster of multiple signals is counted as six signals and a large cluster as 12 signals It is possible for a single nucleus to have multiple small clusters multiple large clusters or a combination of large and small clusters Figure 2 Case 6 shows a number of nuclei in which clusters are visible both small and large Target Area It is important that the reader identify acceptable target areas for signal visualization and enumeration within the sample An acceptable target area must be located within the invasive breast carcinoma carcinoma n situ should not be scored Once located the target area must satisfy two criteria to be deemed adequate for enumeration This is discussed thoroughly in Slide Adequacy D ae Se KR P 3 Case 4 H amp E 60X HERZ 60X Chr17 60X pm v X N amp i Ka g al D y b Y E 5 5 NF e CN a i P D a a A i M Neg u S gt t t 4 Ze A p H 1 d gt Case 5 H amp E 60X Multiple copies and small clusters 4 Ventana Medical Systems Case 6 HER2 60X Small A and large B clusters Slide Adequacy Before enumerating a HER2 or Chr17 stained slide it is critical that the reader determine whether the target area is adequately stained The target
9. positive controls stained on the same staining run should be checked to determine whether the failure is due to the control slide or the reagents used Ventana s HER2 3 in 1 Xenograft Control Slides serve as run controls to ensure that the reagents and instrument are functioning properly Oxidizing or Fading of SISH Signal Oxidation fading and or disappearance of the SISH signal may be due to certain brands of mounting media The following mounting media are known to cause oxidation EUKITT EMS Entellan Merck and Entellan NEW Merck Please refer to Table 5 for mounting media compatibility If the mounting media you are using is not included in this list we recommend that you validate that the media is compatible with the test system prior to use with patient samples Internal studies have shown that fading usually occurs within a few hours to a week As further guidance Table 6 has been created to help troubleshoot some of the issues described above For additional information refer to the Step By Step Procedure section in the automated slide stainer Operator s Manual or contact your local Ventana office Table 5 Mounting Media Compatibility Compatible Mounting Media Manufacturer with SISH Eukitt EMS No Entellan New Merck No Entellan Merck No Cytoseal XYL Richard Allan Scientific Yes Paramount Protaqs Quartett Dako Yes DPX BDH Raymond Lam
10. ER e d AA La b e e e a amp i q KP ep a7 P i UM e d a i Ki e F a a Pd d e A E LI Ld a A a Case 14 HER2 60X Case 15 HER260X 12 Ventana Medical Systems Photoset B cont Cases Positive for HER2 Gene Amplification Note normal lymphocytes and fibroblasts denoted with an arrow adjacent to tumor gt A dt A mm w adi c e 3 HER2 60X Chr17 60X ai P oe Case 17 HER2 60X Case 18 HER2 60X Photoset C A Case Initially Equivocal for HER2 Gene Amplification Corrected to Negative by Polysomy 17 Case 19 is an initially borderline HER2 case corrected by polysomy 17 chromosome ratio to negative for HER2 gene amplification Case 19 HER2 60X s m Li e f amp RB F D 9 e 7 T E V D e a e 7 LA a 8 V e a e e e i a ke 8 Sa P e k e d e e nO 7 K a YA i P e e Be ki i a i H x LI e is Li a Ey a e E Chr 17 60X 800 227 2155 www ventanamed com 13 Quality Control For SISH Staining Positive and Negative Controls for SISH Staining Normal HER2 signals 1 to 2 copies per cell act as internal positive controls for each case and must be visible in the sample Specific SISH nuclear staining may be located in various cells including stromal fibroblasts endoth
11. Results are and have a HER2 Chr17 ratio either equal to or between reported as 1 8 and 2 2 Method 2A must be utilized The reader a Negative for HER2 gene amplification Defined as a selects a second target area for each of HER2 and Chr17 HER2 Chr17 ratio less than 1 8 and counts the number of signals in 20 additional nuclei b Equivocal for HER2 gene amplification Defined as a The HER2 Chr17 ratio using counts from both target HER2 Chr17 ratio equal to or between 1 8 and 2 2 areas is then calculated A total of 40 nuclei will have c Positive for HER2 gene amplification Defined as a been read between both target areas Calculate the HER2 HER2 Chr17 ratio greater than 2 2 Chr17 ratio by dividing the total number of HER2 signals Figure 11 Method 2A Scoring Algorithm HER2 Chr17 Ratio Equivocal Positive for HER2 Gene Amplification Method 2A HERZ stained slide Identify and select target area 2 Count HER2 signals in 20 nuceli Chr17 stained slide Locate target area 2 Count Chr17 signals in 20 nuceli Calculate HER2 Chr17 ratio by dividing the total number of HERZ signals from target area 1 and 2 by the total number of Chr17 signals fron target area 1 and 2 Report Results Negative HER2 Chr17 1 8 Equivocal 1 8 lt HER2 Chr17 s 2 2 Positive HER2 Chr17 gt 2 2 www ventanamed com 11 Examples of HER2 and Chr17 Staining Patterns in Clinical Cases The following images are provided to illustrate
12. a variety of Photoset A provides an example of a case negative for staining patterns that may be present in invasive breast HER2 gene amplification with matched Chr17 staining carcinoma cases when stained with INFORM HER2 DNA Photoset B provides examples of cases positive for HER2 Probe positive negative and equivocal for HER2 gene gene amplification cases 14 and 15 Case 16 shows amplification The intended use of these photographs is to a HER2 amplified case with H amp E and matched Chr17 allow the new user of this test to become familiar with the Cases 17 and 18 provide examples of HER2 amplified spectrum of staining patterns they may encounter cases with emphasis on the presence of normal gene copy number in lymphocytes and fibroblasts adjacent Any staining performed in the end user s laboratory to tumor should be interpreted within the context of the controls Photoset C provides an example of a case that was run with the clinical cases at the time of evaluation See initially equivocal for HER2 gene amplification corrected Quality Control for SISH Staining section to negative by polysomy Chr17 staining This case illustrates the importance of a dual HER2 Chr17 result Photoset A Case Negative for HER2 Gene Amplification sf a PS Case 13 H amp E 60X HER2 60X Chr17 60X Photoset B Cases Positive for HER2 Gene Amplification t 2 r d e a ep a a Ve CN e A e
13. ancer cells Oncogene 1997 15 537 547 Press MF Pike MC Chazin VR et al HER 2 neu expression in node negative breast cancer Direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease Cancer Res 1993 53 4960 4970 Press MF Bernstein L Thomas PA Meisner LF et al HER 2 neu gene amplification characterized by fluorescence n situ hybridization Poor prognosis in node negative breast carcinomas J Clin Oncol 1997 15 2894 2904 Gusterson BA Gelber RD Goldhirsch A et al Prognostic importance of c erbB 2 expression in breast cancer J Clin Oncol 1992 10 1049 1056 Bianchi S Paglierani M Zampi G et al Prognostic significance of c erbB 2 expression in node negative breast cancer Br J Cancer 1993 67 625 629 Kallioniemi O P Holli K Visakorpi T et al Association of c erB 2 protein expression with high rate of cell proliferation increased risk of visceral metastasis and poor long term survival in breast cancer Int J Cancer 1991 49 650 655 14 15 16 17 18 19 20 21 22 23 Vogel CL Cobleigh MA Tripathy D et al Efficacy and safety of trastuzumab as a single agent in first line treatment of HER2 overexpressing metastatic breast cancer J Clin Oncol 2002 20 719 726 Baselga J Carbonell X Castaneda Soto NJ et al Phase Il study of efficacy safety and pharmacokinetics of trastuzumab monot
14. b Yes Cytoseal 60 Richard Allan Scientific Yes Permount Fisher Yes Histomount Raymond Lamb Yes Ultramount Dako Yes Thermo EZ Mount Thermo Scientific Yes SureMount Triangle Biomedical Sciences Yes Flo Texx Lerner Labs Yes Mountex Histolab Yes Shandon Consul mount Thermo Scientific Yes MM24 SurgiPath Yes Pertex Cell Path Yes MicroMount SurgiPath Yes Diamount Diapath Yes Alcolmount Diapath Yes BioMount 2 BB International Yes Acrytol SurgiPath Yes Gel Mount Biomeda Yes Mount Quick Daido Sangyo Co Yes www ventanamed com 19 Table 6 Troubleshooting Lack of staining on xenograft control slides Weak or absent staining Weak or absent counterstain Non specific staining slide drying artifact Non specific staining nuclear dust or haze Weak or absent staining of positive control Oxidizing or fading of SISH signal am Lj e If there is a lack of expected staining in the HERZ 3 in 1 Xenograft Control Slides e an absence of single copy staining of HER2 or Chromosome 17 the run is invalid Check to ensure that all dispensers are functioning properly Determine if weak staining interferes with slide enumeration If staining is weak or absent use a stronger protease treatment and or increase the hybridization time to greater than 6 hours Determine if weak counterstaining interferes with slide enumeration If weak counterstaining interferes with slide enumeration then repeat the staining of
15. bjectives the invasive breast carcinoma in the target area must exhibit an enumerable field of HER2 or Chr17 SISH signals Due to truncation in the plane of sectioning it is likely that not every carcinoma nucleus will contain SISH signals However it is important that the target area contains an acceptable region that is enumerable If a particular target area is deemed too weak to enumerate it is often possible to enumerate a different target area on the same slide If acceptable SISH staining within any of the target areas is not present then the slide is considered inadequate and cannot be enumerated Figure 5 is an example of a target area that would be considered inadequate ed F L4 j de adi F d m CH E Fe m E rd p 4 r i Ap e A db i bh vi ha e i 7 A a Case 7 H amp E 60X HER2 60X Chr17 60X Figure 4 Inadequate Due to a Lack of Figure 5 Inadequate Due to Weak Internal Positive Control Staining in or SISH Staining Within the Target Area Adjacent to the Target Area Case 8 HER2 60X Case 9 HER2 60X www ventanamed com 5 Selecting Carcinoma Nuclei for Enumeration Once a target area that exhibits adequate SISH staining is located only representative carcinoma nuclei should be enumerated Signal enumeration should not be performed in areas that contain weak SISH signals e no internal control cell staining present c
16. counter Additionally the images allow users to become familiar with determination of slide adequacy enumeration methods and troubleshooting of the assay The intent is to provide pathologists with a tool to facilitate interpretation of INFORM HER2 DNA and Chromosome 17 Probes staining patterns Any staining performed in the end user s lab should be interpreted within the context of the controls run with the clinical cases at the time of evaluation See the package insert provided with these products for further information and the Quality Control section of this guide The images contained in this interpretive guide were obtained using the INFORM HER2 DNA and Chromosome 17 Probes in the assay developed and validated on Ventana automated slide stainers under the direction of Ventana Medical Systems Inc www ventanamed com 1 Identification of Appropriate Staining Pattern HER2 Gene Status The HER2 gene is located on human Chromosome 17 and encodes the HER2 protein Amplification of the HER2 gene occurs in approximately 15 to 25 percent of breast cancers and is associated with aggressive tumor behavior In many clinical studies amplification and or overexpression of HER2 has been shown to be associated with a poor clinical outcome for women with invasive breast cancer and correlated with several negative prognostic variables including estrogen receptor ER negative status high S phase fraction positive nodal status
17. elial cells lymphocytes and non neoplastic breast epithelial cells However not all cells will exhibit single gene copy due to biological heterogeneity and plane of sectioning If normal HER2 signals are not evident the slide is inadequate for interpretation A positive control may be included with every staining procedure performed The controls are used to confirm that the reagents and the instrument have functioned properly It is important that the optimal staining conditions be established on biopsy control samples prior to running patient samples Positive tissue controls should include biopsy specimens prepared in a manner identical to the patient specimens to be tested These specimens should be used because they are known to have normal copies of HER2 in both the control stromal cells and the invasive carcinoma cells These serve as quality controls for all steps of the procedure from the pre analytical specimen preparation through the staining process Use of a specimen prepared differently from the test specimens will provide control for all reagents and procedures except fixation and specimen processing Figure 12 HERZ 3 in 1 Xenograft Control Slides 14 Ventana Medical Systems Separate positive controls are not required Since the HER2 gene and Chromosome 17 are present in every human cell there is no true negative tissue control For the purpose of a negative reagent control Ventana produces an ISH negat
18. f signals in 20 cells located in the Target Area according to Table 2 Signal Enumeration Calculate the HER2 Chr17 ratio by dividing the total number of HER2 signals by the total number of Chr17 signals Cases with a HER2 Chr17 ratio less than 1 8 are negative for HER2 gene amplification while cases with a HER2 Chr17 ratio greater than 2 2 are positive for HER2 gene amplification and can be reported as such Cases with a HER2 Chr17 ratio that is either equal to or falls between 1 8 and 2 2 must be enumerated using Method 2A to ensure accuracy Figure 10 Figure 10 Method 2 Scoring Algorithm HER2 Chr17 Ratio Positive for HER2 Gene Amplification HER2 stained slide Method 2 Slide adequate Unsatisfactory Yes Identify and select target area 1 Count HER2 signals in 20 nuceli Chr17 stained slide Slide adequate Unsatisfactory No Yes Locate target area 1 Count Chr17 signals in 20 nuceli Calculate HER2 Chr17 ratio by dividing the total number of HERZ signals from target area 1 by the total number of Chr17 signals fron target area 1 Report Results Negative 1 8 lt HER2 Chr17 s 2 2 HER2 Chr17 1 8 Positive HER2 Chr17 gt 2 2 Yes Method 2A 10 Ventana Medical Systems Method 2A Additional Quantitative in both target areas 40 cells by the total number of For cases that have been enumerated using Method 2 Chr17 signals in both target areas 40 cells
19. herapy administered on a 3 weekly schedule J Clin Oncol 2005 23 2162 2171 Slamon DJ Leyland Jones B Shak S et al Concurrent administration of anti HER2 monoclonal antibody and first line chemotherapy for HER2 overexpressing metastatic breast cancer A phase lll multinational randomized controlled trial N Engl J Med 2001 344 783 792 Marty M Cognetti F Maraninichi D et al Randomized phase ll trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2 positive metastatic breast cancer administered as first line treatments The M77001 Study Group J Clin Oncol 2005 23 4265 4274 Piccart Gebhart MJ Procter M Leyland Jones B et al Trastuzumab after adjuvant chemotherapy in HER2 positive breast cancer N Engl J Med 2005 353 1659 1672 Romond EH Perez EA Bryant J et al Trastuzumab plus adjuvant chemotherapy for operable HER2 positive breast cancer N Engl J Med 2005 353 1673 1684 Bilous M et al Current perspectives on HER2 Testing A review of national testing guidelines Mod Pathol 2003 173 182 Sheehan DC Hrapchak BB Theory and practice of histotechnology 2nd Edition The C V Mosby Company St Louis 1980 Wolff AC Hammond MEH Schwartz JN et al American Society of Clinical Oncology College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer Arch Pathol Lab Med 2007
20. ic formalin may be a suitable specimen type but may not stain as robustly as tissue fixed in either 10 NBF or Zinc formalin Prefer and Bouin s fixatives were demonstrated to be incompatible with this assay as samples fixed using these two fixatives failed to yield single copy gene detection Tissues that have been fixed inappropriately sometimes can be identified by the presence of peripheral SISH staining with weak or absent staining in the center of the tissue Troubleshooting for weak or absent staining as discussed in the Troubleshooting Section is recommended This 16 Ventana Medical Systems involves treatment of the tissue with a stronger protease e g ISH Protease 2 for four minutes or ISH Protease 3 for 12 24 minutes For additional information on fixation for ISH refer to the CAP ASCO Guideline Recommendations for HER2 Testing Sample Thickness samples with a thickness of greater than four microns likely will require the use of a stronger protease treatment such as Ventana s ISH Protease 2 for a treatment time of four minutes or longer This protease treatment is effective at unmasking DNA in thicker samples Thinner sections may require gentler protease treatments i e ISH Protease 3 for four minutes if excessive nuclear background staining is observed See Troubleshooting Other Considerations Type of specimen It also is important to consider the type of specimen to be tested in the assay for example t
21. in that is weak or absent making it difficult to discern nuclear boundaries increase the counterstaining conditions from 4 minutes to 8 minutes Weak counterstaining can indicate that the specimen has not been prepared as recommended Also see the Pre Analytical Considerations section for more information on the balance between nuclear morphology and SISH signal intensity Non Specific Staining Slide Drying Artifact If a case or xenograft control slide exhibits uninterpretable SISH staining with brown or black background throughout the tissue the slide has dried and must be re run If excess silver is deposited on the slide making it difficult to enumerate the nuclear SISH signal the slide 18 Ventana Medical Systems must be re run Check that the instrument and reagent dispensers are functioning properly and that the bulk reagents are adequately filled Also check to make sure that the dispensers have not been stored without their Caps on as silver acetate is known to oxidize over time Non Specific Staining Nuclear Dust or Haze If the slides exhibit excessive background staining that interferes with enumeration of the specific SISH signals i e nuclear dust or haze the use of a gentler protease treatment e g ISH Protease 3 for four minutes instead of 8 minutes is recommended Weak or Absent Staining of Positive Control If the positive control is negative or exhibits weaker staining than expected other
22. issue obtained from a needle biopsy vs an excisional biopsy Optimizing the ISH Protease treatment conditions on each specimen type may be necessary if differences in staining quality are observed Overall sample quality SISH staining may be sub optimal in tissue containing areas of crush artifact necrosis or in tissue of overall poor morphology It is recommended that H amp E staining be evaluated from each case to evaluate overall specimen morphology The balance between nuclear morphology and SISH signal intensity In specimens prepared differently from the recommended procedures it often is possible to visualize adequate SISH staining by optimizing the ISH Protease treatment However the use of ISH Protease 2 or 3 for extended amounts of time may affect the nuclear morphology and nuclear counterstain Each reader should be aware of this and balance SISH signal intensity with nuclear morphology and counterstain Furthermore HER2 or Chr17 SISH staining may decrease in intensity if too harsh a Protease treatment is used For samples that exhibit a weak counterstain increasing the Hematoxylin Il staining time and the Bluing Reagent staining time from the recommended four minutes to eight minutes may enhance the intensity of the counterstain Tissue that has been pre analytically processed in a suboptimal manner may exhibit nuclear bubbling In many cases the nuclear bubbling does not interfere with the SISH staining
23. ive control reagent that can be used in place of the DNA probes HER2 3 in 1 Xenograft Control Slides Xenograft controls are useful for a preliminary validation of the processing methods used for staining clinical slides and for use as controls on individual runs Ventana offers the HER2 3 in 1 Xenograft Control Slides p n 783 4332 containing three distinct xenograft sections that are formalin fixed and embedded in a single paraffin block Figure 12 The cell lines Calu 3 or BT474 ZR 75 1 and MCF 7 generated as xenograft sections have been selected based upon their molecular characterization of HER2 gene copy number and protein levels from published literature and represent the dynamic range of HER2 copies observed in clinical samples Tables 3 and 4 Note that the xenografts contain host mouse cells interspersed with the human carcinoma cells Human HER2 and Chr17 are not detectable in these mouse cells II o mg C O 2 ct o Table 3 Characterization of HER2 3 in 1 Xenograft Control Slides Cell HERZ Protein Approximate HER2 Approximate Chr17 HER2 Gene Line Expression Level Gene Copy by FISH Copy by FISH Amplification Calu 3 or Equal to or greater than 20 BTA74 3 copies nudlei 3 copies nuclei Positive ZR 75 1 1 3 copies nuclei 2 copies nuclei Negative MCF 7 0 1 2 copies nuclei 2 copies nuclei Negative Table 4 Characteristic Staining Pattern of HER2 3 in 1 Xenograft Control
24. ly 2 but note that Chromosome 17 is polysomic Thus Chromosome 17 was duplicated but there was also a genetic loss of the HER2 allele The case report should note Monoallelic Deletion for Case 12 i gt pa 2 e m e BR wf CS i Se e D D ta i i d vi F m D Mi i e en n P S D Ss de r s K A u ne t m 2 a ie A D be D Case 12 H amp E 60X HERZ 60X Chr17 60X 800 227 2155 www ventanamed com 7 Determining HER2 Gene Status In a study that analyzed fluorescence n situ hybridization FISH testing in clinical laboratories the frequency of HER2 Chr17 ratios in invasive breast carcinoma was determined to have the distribution shown in Figure 7 To develop a scoring algorithm that fits within the workflow of a pathology laboratory and generates results that are reproducible between readers Ventana has developed a two part approach Figure 8 Figure 8 Overview of HER2 SISH Scoring Algorithm Semi Quantitatively determine HER2 Chr17 Ratio 1 4 lt Ratio lt 4 0 Negative Positive Method 1 allows the reader to rapidly and semi quantitatively determine HER2 gene status for approximately 85 of all cases which are either clearly negative or positive for HER2 gene amplification i e cases with a HER2 Chr17 ratio less than 1 4 or greater than 4 0 This is done by analyzing the overall staining patterns of HER2 and Chromosome 17 within a target area
25. ompressed or overlapping nuclei or necrosis Additionally signal enumeration should not be performed in nuclei that are not representative of the general population of invasive carcinoma nuclei in the target area Abnormal giant nuclei 2 fold or greater the size of other carcinoma nuclei and small nuclei about half the size of the other carcinoma nuclei should not be enumerated Finally in target areas that are genetically heterogeneous for HER2 copy number count only nuclei that are representative of the population of invasive carcinoma nuclei with the highest average number of signals Heterogeneity is discussed in greater detail in Additional Observations Additional Observations Other observations regarding the HER2 or Chr17 SISH staining may be clinically significant and should be noted as comments on the pathologist s report Heterogeneity Occasionally tissue samples may contain breast carcinoma nuclei that are genetically heterogeneous 6 Ventana Medical Systems for HER2 copy number In these cases there can be a mixture of nuclei that are amplified and not amplified and or a mixture of nuclei containing various numbers of copies of HERZ This may be observed among carcinoma cells within the same target area or between two different target areas within the tissue It is recommended that the nuclei with the higher numbers of HER2 copy number be chosen for enumeration but that the heterogeneity in relative copy numbe
26. r be noted on the patient s report Polysomy Polysomy is a condition in which the carcinoma nuclei contain at least one more chromosome than normal i e the average number of Chromosome 17 copies is not diploid In these nuclei there may be three or more copies of the chromosome Figure 6 Case 11 and Case 12 Note This is not to be confused with diploid dividing nuclei present throughout the tissue that contain 3 4 copies of Chromosome 17 Monosomy Monosomy is a condition in which the carcinoma nuclei contain only one copy of Chromosome 17 Monoallelic Deletion Monoallelic Deletion is the deletion of the HER2 gene from Chromosome 17 Figure 6 Case 12 Figure 6 Additional Observations Regarding HER2 or Chr17 Staining The HER2 image for Case 10 contains both invasive carcinoma A and atypical ductal hyperplasia B If the hyperplasia were enumerated the case would be negative It is important to only enumerate invasive carcinoma and use the H amp E slide as a guide wu E p ls Sech sw 2 bk Ba E am D x a E amp E a 1 a D a A E j k 4 ln H g ke a d e 5 e En Chr17 60X 7m Ty 5 j D M hol Y a f e A A Se Pa LE 1 E ee Ka D amp De amp SP A F L e x e Adi g et A Un E Ce e E ia D x 7 4 Case 11 HER2 60X Case 9 HER2 60X Chr17 60X HER2 copy number for Case 12 is approximate
27. r17 Ratio f d e k d g e h O Negative HER2 Chr17 lt 1 8 O Negative HER2 Chr17 lt 1 8 O Positive HER2 Chr17 gt 2 2 O Positive HER2 Chr17 gt 2 2 O 1 8 lt HER2 Chr17 lt 2 2 Use Method 2A O Equivocal 1 8 x HER2 Chr17 x 2 2 22 Ventana Medical Systems Patient Centric Pathology WORKFLOW DIAGNOSTICS Ventana Medical Systems Inc 1910 E Innovation Park Drive Tucson Arizona 85755 USA 1 520 887 2155 800 227 2155 USA only Roche Diagnostics GmbH Sandhofer Strasse 116 68305 Mannheim Germany www ventanamed com 2008 Ventana Medical Systems Inc VENTANA and BenchMark are registered trademarks of Ventana Medical Systems Inc INFORM is a trademark of Ventana Medical Systems Inc HERCEPTIN is a registered trademark of Genentech Inc E616 1008B
28. rete dots are present Large Cluster Count as 16 One large cluster 12 and 4 discrete dots are present 800 227 2155 www ventanamed com 3 Signal Visualization and Enumeration of SISH Staining SISH signals are visualized as single copies copies and clusters Table 2 Single Copy multiple A discrete dot is counted as a single copy of HER2 or Chr17 Discrete single dots visualized in the internal positive control nuclei represent the size of a single copy in invasive carcinoma cells See Figure 2 Case 4 It is important to note that the discrete dots representing a single copy of either HER2 or Chr17 may appear larger in some patient samples than in others Because of this it is important to use the single dots visualized in the internal positive control nuclei adjacent to the target area as a reference The internal positive control nuclei occur within normal adjacent stromal cells e g fibroblasts fibrocytes and endothelials and leukocytes e g lymphocytes and macrophages Figure 3 Multiple Copies As described above discrete single dots visualized in the internal positive control nuclei represent the size of a single copy in invasive carcinoma cells The size of these single dots is used as a reference to determine Figure 2 Signal Visualization single Copy the relative number of amplified copies in the cancer nuclei Figure 2 Case 5 shows a number
29. scription of INFORM HER2 DNA Probe Ventana Medical Systems INFORM HER2 DNA Probe is designed to quantitatively detect amplification of the HERZ gene via chromogenic silver in situ hybridization SISH in formalin fixed paraffin embedded human breast cancer tissue specimens following staining on Ventana automated slide stainers using light microscopy The INFORM HER2 DNA Probe is indicated as an aid in the assessment of patients for whom Herceptin trastuzumab treatment is being considered Results from the INFORM HER2 DNA Probe are intended for use as an adjunct to existing clinical and pathologic information currently used for estimating prognosis in patients with invasive breast cancer A qualified reader experienced in the microscopic interpretation of breast carcinoma specimens ISH procedures and the recognition of single and amplified HER2 copies which may require microscopic examination using objectives as high as 40X to 60X must evaluate controls before interpreting results Purpose of Interpretive Guide The following cases illustrate the variety of staining patterns that may be present in breast biopsies when stained with INFORM HER2 DNA Probe and Chromosome 17 Probe The photomicrographs allow new users to become familiar with the spectrum of staining patterns including single copy staining of HER2 and Chromosome 17 amplified gene copies and clusters of HER2 staining as well as artifact staining that they may en
30. signals by the average number of Chr17 signals Cases with a HER2 Chr17 ratio less than 1 4 are negative for HER2 gene amplification while cases with a HER2 Chr17 ratio more than 4 0 are positive for HER2 gene amplification and can be reported as such Cases with a HER2 Chr17 ratio that is either equal to or falls between 1 4 and 4 0 must be enumerated using Method 2 to ensure accuracy Figure 9 performed by analyzing the overall staining pattern for Figure 9 Method 1 Scoring Algorithm 1 4 4 0 HER2 Chr17 Ratio Go to Method 2 Positive for HER2 Gene Amplification HER2 stained slide Slide adequate Method 1 Unsatisfactory Yes Identify and select target area 1 Semi quantitatively determine the average number of HER2 signals Chr17 stained slide Slide adequate Unsatisfactory Locate target area 1 Semi quantitatively determine the average number of Chr17 signals Calculate HER2 Chr17 ratio Report Results Negative HER2 Chr17 1 4 NO Positive HER2 Chr17 gt 4 0 Use Method 2 www ventanamed com 9 Method 2 Quantitative Method Using Method 2 the reader records the quantitative enumeration of HER2 and Chr17 signals in 20 nuclei within a target area and calculates the HER2 Chr17 ratio Utilizing stained serial slides that meet the criteria for Slide Adequacy locate the same target area on each slide of HER2 and Chr17 and count the number o
31. such as in Case 20 and cells may be enumerated if SISH signal is adequate However if bubbling precludes the SISH signal affected cells should not be scored Case 21 Case 20 a e Case 21 www ventanamed com 17 Troubleshooting This section addresses issues that may be encountered with the assay Lack of Staining on Xenograft Control Slides If there is a lack of expected staining of the HER2 3 in 1 Xenograft Control Slides i e an absence of single copy staining of HER2 or Chromosome 17 check to ensure that all dispensers are functioning properly Weak or Absent Staining When cases both HER2 and Chr17 exhibit weak or absent staining and the weak staining is found to interfere with cell signal enumeration this often indicates a problem with specimen preparation Repeat staining on the case using a stronger protease treatment e g ISH Protease 2 for four minutes or a hybridization time longer than six hours If a case has stained appropriately for one probe but not for the other e g HER2 stains appropriately but Chr17 exhibits absent staining repeat the staining of the affected slide utilizing the same protocol If the staining is still inadequate this often indicates a problem with specimen preparation and staining should be repeated utilizing a stronger protease treatment or a longer hybridization time Weak or Absent Counterstain For cases that exhibit a nuclear countersta
32. the affected slide s and either increase the counterstain time or utilize a gentler protease treatment i e ISH Protease 3 for 4 min For more information regarding the balance between nuclear morphology and SISH signal intensity review the section on Pre Analytical Considerations and Optimization of the assay Check to make sure all bulk fluids are filled and reagent dispensers are working properly Repeat the staining of the affected slide s using the same protocol If staining is still unacceptable follow instrument user s manual troubleshooting Determine whether non specific staining interferes with slide eneumeration If non specific staining interferes with slide enumeration then repeat the staining of the affected slide with a gentler protease treatment i e ISH Protease 3 for 4 min and or reduce hybridization time to less than 6 hours If the positive control is negative or exhibits weaker staining than expected other positive controls stained on the same staining run should be checked to determine whether the failure is due to the control slide or the reagents used Ventana s HER2 3 in 1 Xenograft Control Slides serve as run controls to ensure that the reagents and instrument are functioning properly This may be due to certain brands of mounting media If the SISH signal of the sample begins to oxidize turning orange or completely disappears restain the case and coverslip using a different mounting media
33. uantitative method Method 1 and a quantitative method Method 2 These methods are discussed in greater detail in the Determining HER2 Gene Status Section Case 1 H amp E 20X HER2 60X 2 Ventana Medical Systems Chr17 60X Figure 1 cont HER2 Gene Status in Representative Cases Case 2 is equivocal for HER2 gen e amplification Ce Me an K i y an s e Zb Dy te 3 A wf E d d H a DH i E di AS D d ai E E 4 e a E s LI L ge E s Gi EI D B s a 3 a V E P a i a HER2 60X Chr17 60X Case 3 is positive for HER2 gene amplification i RE ai m i P m D LE chi e s y a i e LA gt D L E E D or Ka e e D Z gt y Fr a e d H a gt Y Pa E u e e x a D ha a 5 i d gt EL C d d e vi i t4 8 D a D HER2 60X Chr17 60X Case 3 H amp E 20 Table 1 HER2 Testing Reported Results Average HER2 Reported Result Chr17 ratio HER2 Chr17 lt 1 8 Negative for HER2 gene amplification 1 8 x HER2 Chr17 x 2 2 Equivocal for HER2 gene amplification HER2 Chr17 gt 2 2 Positive for HER2 gene amplification Table 2 Signal Enumeration Single Copy Count as 1 Multiple Copies Count as 2 Small Clusters Count as 8 One small cluster 6 and 2 disc
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