AmoyDx HER-2 Gene Amplification Analysis Kit
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1 AmoyDx HER-2 Gene Amplification Analysis Kit Detection of HER-2 gene amplification Instruction for Use Instruction Version: B1.0 Revision Date: August 2016 Store at -20±5
2 Background For: ADx-FHE03 The proto-oncogene HER-2 gene (also called ERBB2 or HER2/neu) is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family, which is located at the long arm of human chromosome 17 (17q12-q21). HER-2 gene encodes a 185-kDa HER-2 protein, which is a membrane receptor tyrosine kinase with homology to EGFR (HER-1). Amplification of HER-2 gene has been shown to play an important role in the development and progression of several aggressive types of human cancers, which occurs in 20-30% of breast cancer, approx. 20% of gastric cancer, also prostate, ovarian cancer and uterine serous endometrial carcinoma. In recent years the protein has become an important biomarker and target of therapy for breast and gastric cancer patients. HER-2 gene amplification in cancer carries prognostic and predictive significance for HER-2 targeted therapies, like monoclonal antibody trastuzumab (Herceptin ). Clinical studies have shown that breast and gastric cancer patients whose tumors have HER-2 gene amplification benefit most from Herceptin. The AmoyDx HER-2 Gene Amplification Analysis Kit is a fluorescence in situ hybridization (FISH) test designed to qualitatively detect HER-2 gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimen. The purpose of the kit is to aid physicians and clinical researchers in identifying breast and gastric cancer patients who are eligible for HER-2 targeted monoclonal antibody treatment. Intended Use The AmoyDx HER-2 Gene Amplification Analysis Kit is a qualitative test to accurately identify HER-2 gene amplification and via fluorescence in situ hybridization (FISH) technology. The used specimen is formalin-fixed paraffin-embedded (FFPE) tissue of breast and gastric cancers. Kit Contents This kit contains sufficient reagents to carry out 20 tests (Table 1). Table 1 Kit Contents Tube No. Reagents Supplied Volume (μl) Signal 1 LSP HER-2/CSP17 Probe Mix 210 Cy3 and FITC 2 DAPI Counterstain 1000 / Equipment and Reagents Not Supplied With Kit 1. Fluorescence microscope with Cy3 (orange signal with excitation wavelength 550nm and emission wavelength 575nm) and FITC (green signal with excitation wavelength 494nm and emission wavelength 519nm) filters. 2. Glass microscope slides and coverslips. 3. Thermostatic water bath with lid (37~99 adjustable). 4. Pre-treatment solution SSC (saline-sodium citrate) solution. 6. NP Xylene. 8. Proteinase K (recommend use of Proteinase K from Merck or Amresco). 9. Absolute ethanol (96~100%) % formaldehyde solution. 11. Sterile, deionized water. 12. Rubber cement. 1/9
3 Shipping and Storage The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at -20±5 in the dark in a constant temperature freezer. Once opened, this reagent is stable at -20±5 until the expiry. Stability The shelf-life of the kit is twelve months when stored under the recommended conditions and in the original packaging. Do not use the kit after the stated expiry date. Specimen Material The kit uses breast and gastric cancer formalin-fixed paraffin-embedded (FFPE) tissue specimen. The tissue specimens are fixed in formalin (10% neutral buffered formalin) and those are well processed and produce good tissue sections. The fresh tissue samples should be immediately fixed with 4~20 times volume of formalin within 0.5~1 hour. Specimen should be blocked into a thickness of 3~5 μm and fixed for 6~72 hours. The ready-made FFPE tissue sections should be used for FISH analysis, if not, the sections should be stored at -20±5 for no more than six weeks. Avoid using FFPE tissue blocks have been stored for more than 2 years. Technological Principles The kit uses fluorescence in situ hybridization (FISH) technology, which employs a dual-color HER-2/CSP17 FISH probe mix with two fluorescent-labeled DNA probes to detect HER-2 gene amplification. The tissue specimens were handled with a series of pre-treatment procedures, and the targeted DNA is denatured to single-stranded form and hybridized with the FISH probes. Following a series of wash and DAPI counterstaining steps, the hybridization of the probes is viewed using a fluorescence microscope equipped with appropriate excitation and emission filters, and then the HER-2/CSP17 ratio is calculated to identify the HER-2 gene amplification status. Protocol 1. The LSP HER-2/CSP17 Probe Mix includes two fluorescent-labeled DNA probes. It contains Cy3-labeled DNA probe with orange fluorescent signal specific for HER-2 gene on chromosome 17, and FITC-labeled DNA probe with green fluorescent signal specific for the centromeric region of chromosome 17 (CSP17). 2. The DAPI Counterstain is a blue fluorescent stain with DAPI (diamidino-2-phenylindole) specific for cell nucleus to make the hybridized probes more easily visible. Working Reagents and Equipment Preparation A. Reagents Preparation 1. Pre-treatment Solution (ph7.0): Dissolve 22.6 g of Na 2 EDTA 2H 2 O and 6.34 g Tris in 800 ml of deionized water, and adjust the PH value to 7.0, then dilute the solution to 1 L volume with deionized water. Store it at 2~8 under seal for no more than 12 months. Please discard it if any turbidity or contamination SSC Solution (ph7.0): Dissolve 44 g sodium citrate and 88 g NaCl in 800 ml of deionized water, and adjust the PH value to 7.0, then dilute the solution to 1 L volume with deionized water. Store it at 2~8 under seal for no more than 12 months. Please discard it if any turbidity or contamination SSC Solution (ph7.0): 10 times dilute 20 SSC Solution (ph7.0) with deionized water, and adjust the ph value to 7.0. Store it at 2~8 under seal for no more than 12 months. Please discard it if any turbidity or contamination mg/ml Proteinase K Concentrate: Dissolve 100 mg dry Proteinase K in 5 ml 2 SSC Solution (ph7.0), shake it gently without vortexing and Subpackage into 100 μl/vial. The Proteinase K concentrate should be stored at -20 and avoid frequently freeze-thaw. 2/9
4 5. Slow Wash Buffer: Dissolve 40 μl NP-40 in 40 ml 2 SSC Solution (ph7.0), mix them thoroughly. The solution should be formulated just prior to use. 6. Fast Wash Buffer: Dissolve 120 μl NP-40 in 40 ml 2 SSC Solution (ph7.0), mix them thoroughly. The solution should be formulated just prior to use %, 85% Ethanol: Respectively dilute 700 ml, 850 ml absolute ethanol to 1000 ml with deionized water, and store them at room temperature under seal. B. Equipment Preparation 1. Before the experiment: Take 40 ml 2 SSC Solution (ph7.0) into a Coplin jar, and preheat it at 37 for 20 min. 2. Before pre-treatment procedure: Heat Pre-treatment Solution (ph7.0) till boiled with magnetic heated stirrer or other heater. 3. Before wash steps: Adjust the thermostatic water bath to 46 and preheat Slow Wash Buffer in it for 20 min if choose slow wash mode. Or adjust the thermostatic water bath to 72 and preheat Fast Wash Buffer in it for 20 min if choose fast wash mode. Experimental Procedure 1. Specimen Pre-treatment 1) Bake the unstained specimens slides at 56 for 15~30 min on a heater. 2) Immerse the slides in a xylene jar for 10 min in the fume cupboard. Change clean jars and repeat twice. 3) Tap off excess xylene, place and shake the slides in an absolute ethanol (96~100%) jar for 5 seconds. Change clean jars and repeat twice. 4) Successively place the slides in the following substances: in absolute ethanol (96~100%) for 1 min, in 85% ethanol for 1 min, in 70% ethanol for 1 min and in deionized water for 1 min. 5) Immerse and boil the slides (keep the specimen upwards) in the boiled Pre-treatment Solution (ph7.0) for 20 min. 6) Wash the slides in deionized water for 1 min. 7) Wash the slides in 2 SSC Solution (ph7.0) for 1 min. 8) Take out a vial of 100 μl 20 mg/ml Proteinase K Concentrate, thaw it completely at 37 and centrifuge briefly to collect the contents at the bottom of the tube. 9) Add the above 100 μl 20 mg/ml Proteinase K Concentrate into 40 ml 37 preheated 2 SSC Solution (ph7.0) to generate 0.05 mg/ml Proteinase K working solution. 10) Incubate the above slides in 0.05 mg/ml Proteinase K working solution at 37 for 5~15 min. Do not place more than 8 slides in a Coplin jar. Note: The digestion duration will vary by different cancer types or thickness of FFPE tissue slides. For breast cancer specimen, it should be digested for a short time, and for gastric cancer specimen, the digestion duration should be appropriately extended. The digestion duration should be reduced if there are thin FFPE tissue slides. The degree of digestion has a high impact on the following hybridization performance. 11) Wash the slides in 2 SSC Solution (ph7.0) for 1 min. 12) Fix the slides in 10% formaldehyde solution for 7 min. (optional) 13) Successively place the slides in the following substances: in 70% ethanol for 3 min, in 85% ethanol for 3 min and in absolute ethanol (96~100%) for 3 min. 14) Take out the slides and air-dry them in the fume cupboard or clean bench. 3/9
5 2. Denaturation and Hybridization (in dark condition) 1) Mark the specimen area at the back of the slides. 2) Moisten the humidity cards with water and place in the card slots, to ensure hybridization in certain humidity. 3) Take out LSP HER-2/CSP17 Probe Mix, mix the reagent by vortexing and then briefly centrifuge for 2~5 seconds. 4) Apply 10 μl LSP HER-2/CSP17 Probe Mix to the center of the specimen area for each slide, immediately apply a coverslip and allow the probe mix to spread evenly under the coverslip. Avoid air bubbles in the probe mix. 5) Seal the coverslip with rubber cement. 6) Open the hybridizer, setup the program for denaturation and hybridization: denature at 85 for 5 min, hybridize at 37 overnight for 12~16 hours. 7) Place the slides on the hybridizer and start the denaturation and hybridization program. 3. Wash Procedure (in dark condition, slow or fast wash optional) a) Slow Wash: 1) When hybridization finished, take the hybridized slides out of the hybridizer, remove the rubber cement from each slide using forceps, and avoid disturbing the coverslip. 2) Immerse the slides in 2 SSC Solution (ph7.0) for 5 min at room temperature, and then remove the coverslips. 3) Immerse the slides in the 46 preheated Slow Wash Buffer for 7 min. 4) Dehydrate the slides through a graded series of ethanol: in 70% ethanol for 1 min, in 85% ethanol for 1 min, and in absolute ethanol (96~100%) for 3 min. 5) Take out the slides and air-dry them completely in the fume cupboard or clean bench. b) Fast Wash: 1) When hybridization finished, take the hybridized slides out of the hybridizer, remove the rubber cement from each slide using forceps, and avoid disturbing the coverslip. 2) Immerse the slides in 2 SSC Solution (ph7.0) for 5 min at room temperature, and then remove the coverslips. 3) Immerse the slides in the 72 preheated Fast Wash Buffer for 2 min. 4) Dehydrate the slides through a graded series of ethanol: in 70% ethanol for 1 min, in 85% ethanol for 1 min, and in absolute ethanol (96~100%) for 3 min. 5) Take out the slides and air-dry them completely in the fume cupboard or clean bench. 4. Counterstaining Procedure (in dark condition) 1) Mark the specimen area at the back of the slides. 2) Apply 10 μl DAPI counterstain to the center of the specimen area for each slide, immediately apply a coverslip and allow the DAPI counterstain to spread evenly under the coverslip. Avoid air bubbles are in the probe mix. 3) Incubate at room temperature for 15 min. 5. Slide Examination (in dark condition) 1) View the hybridized slides using a fluorescence microscope equipped suitable filters. 2) When slide examination finished, store the hybridized slides at -20 in dark condition. Quality Control 1. Sample During the sample preparation process, abnormal slides should be distinguished according to the following criteria: 1) Tissue should be firmly adhered to the slide without air bubbles. 2) The cut surface of the tissue should be integrated without crack and fold. 3) The thickness of the section should be between 3-5μm. If the slide with any one or more the above indications, the slide should be discarded and repeat the sample preparation. 4/9
6 2. Experiment Procedure 1) Prepare the solutions correctly according to the instruction. 2) Using thermometer to adjust the solution temperature accurately. 3) Keep the tissue side of the slide upwards during the boiling procedure. 4) Check the digestion degree of the sample carefully. 5) Remove the rubber cement and coverslip carefully. 6) The hybridization, washing, counterstaining and slide examination steps should be operated in dark condition. 3. Result analysis If any of the following situations occurs, the result should be discarded. 1) There is no result on the control slide. 2) The tumor area is too small to find two fields for cell counting. 3) The cell nucleus is not distinguishable. 4) There is too high autofluorescence. 5) Tissue is excessive proteinase digested, and the cell nucleus has been damaged. 6) Tissue is inadequate proteinase digested and no fluorescent signals. Result Interpretation A. Slide Assessment Rules 1. Only specimen areas where tumor cells are centralized (identified by pathologist) should be analyzed. 2. Count the cells with both orange signal indicating hybridized HER-2 gene and green signal indicating hybridized CSP17. The signals should be bright, distinct and easy to evaluate. 3. The specimen areas hybridized unevenly should be considered invalid. 4. The background should not be too dark or contain particles that interfere with enumeration. 5. Avoid areas of necrosis and where the nuclear borders are ambiguous, overlapped. 6. Avoid areas where more than 25% of nuclei have weak signals or more than 10% of cytoplasm have signals. 7. Slide evaluation should be conducted by two skin technicians and results should be concordant. B. Slide Evaluation Procedure 1. Using a 10 or 20 objective to scan the entire marked specimen area and locate the area where tumor cells are centralized. 2. Using a 40 objective to observe the tumor cells and signals, examine the quality of tissue morphology and select the representative area where more than 75% of nuclei have hybridization signals for enumeration. 3. Using a 100 oil objective and prescribed filters to analyze the cells selected for enumeration and record signals in each cell. Move to the next eligible area for enumeration. 4. Stop enumeration until 30 cells have been enumerated, and calculate the Orange/Green Ratio and Average HER-2 Signals Number. Orange/Green Ratio = total number of orange signals in 30 cell nuclei / total number of green signals in 30 cell nuclei. Average HER-2 Signals Number = total number of orange signals in 30 cell nuclei / Interpret the results according to the guideline provided below. C. Result Interpretation Guide 1. For breast cancer: result interpretation could refer to 2013 ASCO/CAP Guideline on HER-2 testing. 1) If the Orange/Green Ratio 2.0 or Orange/Green Ratio < 2.0 but the Average HER-2 Signal Number 6.0, the specimen is classified as positive, which indicates HER-2 gene amplified. 2) If the Orange/Green Ratio < 2.0 and the Average HER-2 Signals Number < 4.0, the specimen is classified as 5/9
7 negative, which indicates no HER-2 gene amplified. 3) If the Orange/Green Ratio < 2.0 and the Average HER-2 Signals Number between 4.0 ~ 6.0, the specimen is undetermined, that indicates it required to count additional 20 cells and recalculate to interpret the result or redo the experiment. 2. For gastric cancer: result interpretation could refer to Gastric cancer guideline on HER-2 testing. [10] 1) If the Orange/Green Ratio 2.2 or HER-2 signals are connected as cluster, the specimen is classified as positive, which indicates HER-2 gene amplified. 2) If the Orange/Green Ratio < 1.8, the specimen is classified as negative, which indicates no HER-2 gene amplified. 3) If the Orange/Green Ratio is between 1.8 ~2.2, the specimen is undetermined, that indicates it required to count additional 20 cells and recalculate the Orange/Green Ratio, if the Orange/Green Ratio 2.0, the specimen is classified as positive, otherwise the specimen will be classified as negative. Redo the experiment if necessary. Typical Signal Patterns Sample Typical signal Result analysis and interpretation Positive: Orange/Green Ratio 2.0. Positive: HER-2 Signals are connected as cluster, (HER-2 Signals Number 6.0). Breast Cancer Negative: Orange/Green Ratio < 2.0 and the Average HER-2 Signals Number < 4.0 Undetermined: Orange/Green Ratio < 2.0 and 4.0 HER-2 signal number < 6.0. The specimen is undetermined, extend the counting scope and recalculate the ratio. Gastric cancer Positive: Orange/Green Ratio /9
8 Positive: HER-2 signals are connected as cluster. Negative: Ratio < 1.8. Undetermined: Orange/Green Ratio is between 1.8~2.2. The specimen is undetermined, extend the counting scope and recalculate the ratio. Limitations 1. The kits only analyze the formalin-fixed paraffin-embedded (FFPE) tissue specimen from invasive breast and gastric cancer patients. 2. The kits only detect HER-2 gene amplification, not HER-2 base mutation. 3. The performance of the kits requires to strictly following the procedure provided in this instruction. Modifications to these procedures may alter the performance of the assay. Troubleshooting Problem Probable Cause Suggested Solution Specimen damaged or lost Unsuitable slides. FFPE section with excessive air bubbles. Tissue not tightly fixed. Slides baked inadequately or too high baking temperature. Specimen with excessive pre-treatment. Coverslip improperly removed before wash. Use poly-lysine handle slides with strong positive specimen. Re-prepare the FFPE section. Improve the fixation condition and duration time. The slides should be baked at 56 for 15~30 min or more time. During specimen pre-treatment, keep the specimen upwards on the slides. Use 0.05 mg/ml Proteinase K working solution for digestion and reduce digestion time. Gently remove the coverslip in 2 SSC Solution. No signals or weak signals Microscope not functioning properly or aged. Inappropriate filter set used to view slides Probe mix loses efficiency. Insufficient or lack of probe mix. Inadequate denaturation. Inappropriate hybridization. Call microscope manufacturer s technical support to examine and replace the questionable accessories. Use appropriate filters. Store probe mix in recommended condition. Do not use probe mix after the stated expiry date. Handle probe mix in dark condition to protect from light. Apply 10 μl probe mix for each sample. Ensure denaturation at 85 for 5 min. Recommend use of hybridizer, properly setup the hybridization program. Keep certain humidity during the hybridization. 7/9
9 Excessive background Improper DAPI counterstain Inappropriate wash. Inadequate or excessive specimen pre-treatment. Hybridization with air bubbles under the coverslip. DAPI counterstain loses efficiency. Inappropriate filter set used to view slides Unclean slides used for FFPE section preparation Unclean reagents Inadequate wash Excessive cell debris or impurities DAPI counterstain loses efficiency Excessive counterstain Insufficient counterstain Prepare the wash buffer according to formula provided in this instruction. Ensure the correct wash temperature. Ensure the pre-treatment solution correctly prepared and appropriate pre-treatment procedure. Apply coverslip by first touching the surface of the probe mixture. Store DAPI counterstain in recommended condition. Do not use it after the stated expiry date. Use appropriate filters. Use absolute ethanol to immerse slides and air-dry them. Recommend use of fresh reagents and avoid using the same reagents over and over again. Prepare the wash buffer according to formula provided in this instruction. Ensure the correct wash temperature. Prolong the wash time. Avoid analyzing the kind of area or repeat testing with new slide. Store DAPI counterstain in recommended condition. Do not use it after the stated expiry date. Reduce the usage of DAPI counterstain. Repeat counterstain. Warnings and Precautions 1. The kit is for in vitro diagnostic use only. 2. Please read the instruction carefully and become familiar with all components of the kit prior to use. 3. The performance of the kits would suffer impact of sample source, specimen handling, preparation and storage and other conditions during the experiment. Otherwise, false positive or false negative may be produced. 4. Fluorescent probes area readily photo bleached by exposure to light. To minimize this degradation, the whole experiment procedures except for specimen pre-treatment should be performed in dark condition to protect from light. 5. If any working reagents precipitate or become cloudy, they should be discarded and fresh solutions prepared. 6. All the chemicals are potential hazard, only trained professionals could use this kit. Please wear suitable lab coat and disposable gloves. Handle xylene in the fume cupboard. 7. The used kits or any reagents should be disposed of as hazardous waste. 8. Wipe down the work area, spray down the pipettes and equipment with 75% ethanol or 10% hypochlorous acid. 8/9
10 Notes 1. Symbol for "AUTHORISED REPRESENTATIVE IN THE EUROPEAN COMMUNITY" 2. Symbol for "IN VITRO DIAGNOSTIC MEDICAL DEVICE" 3. Symbol for "KEEP DRY" 4. Symbol for "THIS WAY UP" 5. Symbol for "FRAGILE,HANDLE WITH CARE" Information of European Authorised Representative Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom References 1. Fox J. L et al., Clin. Chem. 41(11): Ross J. S et al., The Oncologist. 8: Tsuda H et al., Cancer. 92 (12): Lago L. D et al., Mol. Cancer. Ther. 5 (10): Wolff A. C et al., J. Clin. Oncol. 31 (31): Salmon D. J et al., N. Engl. J. Med. 344 (11): Vogel C. L et al., J. Clin. Oncol. 20 (3): Tanner M et al., Annals. Oncol. 16: Bang Y. J et al., Lancet. 376: Chin J Pathol, August 2011, Vol. 40, No. 8 9/9
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