HER2/CEP17

Product Name

Human HER2 gene amplification detection kit

Package Specifications

10 Tests/box

Intended Usage

This kit uses fluorescence in situ hybridization to detect the HER2 gene amplification status. The test sample is a paraffin-embedded specimen of breast cancer tissue. The product has not been clinically validated in conjunction with HER2 targeted therapies, and its clinical testing capabilities have been confirmed by comparative trial studies with companion diagnostics that have targeted drug validation. The test results of the product should not be used as the sole basis for individualized treatment. The clinician should make comprehensive judgment on the test resultsin combination with the patient’s condition, drug indications, treatment response, and other laboratory testing indicators.
Human epidermal growth factor receptor 2 (HER2, also known as Neu, ErbB-2, CD340, or p185) is a proto-oncogene HER2/neu located at the 17q12 of the human chromosome 17 long arm. Encoded, it is one of epidermal growth factor receptor (EGFR/ErbB) family members, has tyrosine kinase activity and is involved in signal transduction of cell growth and differentiation. The carcinogenic mechanism of HER2 oncogene includes inhibition of cell apoptosis, promotion of cell proliferation, increase of tumor cell invasiveness, and promotion of tumor angiogenesis and lymphangiogenesis.
Breast cancer is one of the most serious malignant tumors that threaten women’s health. Its incidence has been increasing year by year. HER2 gene amplification is found in 20% to 30% of diagnosed breast cancer patients. Patients with HER2-positive breast cancer often show high levels of tumor malignancy, poor treatment outcomes, and poor prognosis, but can benefit from specific HER2-targeted drug therapies. Therefore, the detection of HER2 gene amplification status in breast cancer patientsis the precondition for the screening of HER2-targeted breast cancer patients and the curative effect prediction.

Detection Principle

The kit is based on fluorescence in situ hybridization technology. A nucleic acid probe is labeled with fluorescein. The target gene is detected with homologous complementary to the nucleic acid probe used. Both after denaturation, annealing and renaturation, the hybrid of the target gene and the nucleic acid probe can be formed, and the qualitative, quantitative or relative positioning analysis of the gene to be measured under the microscope can be performed by the fluorescence detection system.
This kit uses the rhodamine fluorescein (RHO)-labeled orange probe (HER2 probe) to detect the HER2 gene, and the fluorescein isothiocyanate (FITC)-labeled green probe (CEP17 probe) to detect chromosome 17 centromere sequence can be used to bind two probes to the target detection area by in situ hybridization. The number of signals corresponding to the CEP17 probe reflects the number of chromosomes at the target area, and the number of signals from the HER2 probe reflects the copy number of the HER2 gene at the target site. By the ratio of the number of HER2 probes and the number of CEP17 probe signals, the amplified state of the HER2 gene in the tissue to be detected can be determined.

Product Main Components

The kit consists of HER2 orange probe and CEP17 green probe, as shown in Table 1.
The reagents not provided in the kit are shown in Table 2.

Storage conditions & Validity

Keep sealed away from light at -20^oC±5^oC. The product is valid for 12 months. Avoid unnecessary repeated freezing and thawing that should not exceed 10 times. After opening, within 24 hours for short-term preservation, keep sealed at 2~8^oC in dark. For long-term preservation after opening, keep the lid sealed at -20^oC±5^oC away from light. See the label of the kit for the production date and expiration date.

Applicable Instruments

1. Fluorescence microscopy imaging system includesfluorescence microscope and filtersets. The kit islabeled with orange fluorescein, and the filter set compatible with the fluorescent labeled dye should be selected.
Orange fluorescence: The maximum excitation wavelength is 547nm and the maximum emission wavelength is 565nm.
Green fluorescence: The maximum excitation wavelength is 495nm and the maximum emission wavelength is 517nm.
Fluorescence microscopy imaging system should use a microscope with orange and green channels.
2. Automatic hybridization instrument: Strict temperature uniformity isrequired, and the temperature difference should be ≤1^oC.

Sample Requirements

• Applicable specimen types: Paraffin-embedded specimens from surgical resection of breast cancer.
• The tissue should be fixed with 4% neutral formaldehyde solution within 1 hour after isolation. After tissue fixation, it is routinely dehydrated and embedded in paraffin.
• Paraffin section thickness could affect the experimental results. The recommended slice thickness is 4~5μm.
• Paraffin-embedded tissue samples from breast cancer should be selected from representative tumor tissue wax blocks and confirmed by HE staining.
• It is recommended to select paraffin-embedded tissue specimens within a 5-year preservation period.

Testing Method

• Related reagents
  The following reagents are required for the experiment but not provided in this kit
  1. 20×SSC (sodium citrate buffer), pH 5.3±0.2

  Sodium chloride

  176g

  Sodium citrate

  88g

  Weigh 176g of sodium chloride and 88g of sodium citrate, dissolve in 800mL of deionized water, adjust the pH to 5.3±0.2 at room temperature, and complete to 1 L with deionized water. High-pressure steam sterilization, stored at 2~8°C, the solution shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.

• 2×SSC, pH 7.0±0.2
  Take 100mL of the above 20xSSC, dilute with 800mL deionized water, mix, adjust the pH to 7.0±0.2 at room temperature, complete to 1L with deionized water, stored at 2~8^oC, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• Ethanol Solution: 70% ethanol, 85% ethanol
  Dilute 700ml, 850ml of ethanol with deionized water to 1L. The shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.
• Protease K
  Protease K stock solution (20mg/mL): Weigh 0.1g of proteinase K dry powder, dissolve in 5mL 2xSSC (pH value 7.0). Gently mix the solution till completely dissolved, and store at -20°C. Shelf life is 6 months. Protease K working solution (200μg/mL): Dissolve 0.8mL Proteinase K stock solution in 80mL 2×SSC (pH value 7.0), mix well and the solution is ready for immediate use.
• 0.3% NP-40/0.4xSSC solution, pH 7.0 ~ 7.5

  NP-40

  0.6mL

  20xSSC

  4mL

  Take 0.6mL NP-40 and 4mL 20×SSC, add 150mL deionized water, mix, adjust the pH to 7.0 ~ 7.5 at room temperature, with deionized water complete to a volume of 200mL. Stored at 2~8oC, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.

• 0.1% NP-40/2×SSC solution, pH 7.0 ± 0.2

  NP-40

  0.2mL

  20xSSC

  20mL

  Take 0.2mL NP-40 and 20mL 20×SSC, add 150mL deionized water, mix, adjust the pH 7.0±0.2 at room temperature, with deionized water complete to a volume of 200mL. Stored at 2~8oC, the shelf life is of 6 months. Discard if the reagent appears cloudy (turbid) or contaminated.

• Diamidinyl phenylindole (DAPI) counterstain Use commercially available anti-quenching DAPI counterstain
• Xylene.

Pretreatment

It is recommended to select specimens with known positive and negative HER2 gene amplification as controls.

• Sectioning: Place on clean slides tissue section fixed in neutral formalin and paraffin-embedded.
• Heating slices: Place the slices in the heating machine and heat overnight at 65°C.
• Dewaxing: Immerse the tissue sections in xylene for 10 minutes, dewax, repeat once, and then immediately immerse in 100% ethanol for 5 minutes.
• Rehydration: At room temperature, place the tissue sections in 100% ethanol, 85% ethanol, and 70% ethanol for 2 minutes each, then immediately immerse in deionized water for 3 minutes. Take out the slices and use a lint-free tissue to absorb excess water around the tissue.
• Water treatment: Soak tissue sections in deionized water at 95°C for 30 to 40 minutes (deionized water is applied in a water bath to preheat).
• Washing: At room temperature, soak the tissue sections in 2xSSC solution and rinse twice for 5 minutes each.
• Proteinase K treatment: Soak the tissue sections in proteinase K working solution and treat at 37°C for 5-30 minutes.
• Washing: At room temperature, soak the tissue sections in 2xSSC solution and rinse twice for 5 minutes each.
• Dehydration: Place the tissue sections in 70% ethanol, 85% ethanol, and 100% ethanol for 2 minutes each, and then dry naturally.

Denaturation and Hybridization

The following operations should be performed in a darkroom.

• Take the dual-color probe. Mix and centrifuge briefly. Take 10μL droplet in the cell and drop in the hybridization zone, immediately cover 22mmx22mm glass slide area;spread evenly without bubbles the probe under the glass slide covered area and seal edges with rubber (edge sealing must be thorough to prevent dry film from affecting the test results during hybridization).
• Place the glass slides in the hybridization instrument, denature at 83°C for 5 minutes (the hybridizer should be preheated to 83°C) and hybridize at 42°C for 2 to 16 hours.

Washing

The following operations should be performed in a darkroom.

• Take out the hybridized glass slides, remove the rubber on the coverslip and immediately place the slides in a 0.3% NP-40/0.4x SSC solution at 67°C. Shake for 1-3 seconds, remove the coverslip and continue to soak the glass slides for 1-2 minutes.
• At room temperature, place the slices in 0.1%NP-40/2xSSC solution, oscillate for 1-3 seconds and soak for 1-2 minutes.
• At room temperature, place the slices in 70% ethanol, soak for 1-3 minutes, and naturally dry in the dark.

Counterstaining

The following operations should be performed in a darkroom.
Dip 10~15μL of DAPI counterstain into the hybridization area of the glass slide, immediately cover and place in dark for 10~20 minutes, and then use the suitable filter to observe the sections under the fluorescence microscope.

FISH results observation

Place the stained sections under a fluorescence microscope and the area of the breast cancer cells is first confirmed under a low magnification objective (10x); under magnification objective (40x) a uniform cells distribution is observed; then the nucleus size uniformity, nuclear boundary integrity, DAPI staining uniformity, no nuclei overlapping, cells clear signal are observed in the high magnification objective (60x, 100x). Select randomly 20 tumor cells at least and count the orange and green signals in the nuclei.

Table 3: Dual-color for signal counting guide

The number of HER2 signals and the number of signals of CEP17 in 20 tumor cells were observed and recorded (Table 3). The total number of HER2 signals indicates the total HER2 copy number, and the total number of CEP17 signals indicates the total number of chromosomes 17 in the target area. Calculate the ratio value (Ratio = Total number of HER2 signals / Total number of CEP17 signals).

Breast cancer FISH result determination

• When the ratio is ≥2.0, the tissue is considered positive for HER2 gene amplification.
• The ratio is <2.0, but HER2 gene amplification is also positive when HER2 gene copy number/cell number ≥6.0.
• If the HER2 orange signal is clustered or the ratio is greater than 20, the HER2 gene is directly considered to be amplified.

Negative HER2 gene amplification

Ratio <2.0, and HER2 gene copy number/cell number<4.0 is negative for HER2 gene amplification.
The HER2 gene amplification status is uncertain When the ratio<2.0, 4.0 ≤HER2 gene copy number/cell number <6.0, the amplification result of HER2 gene is uncertain. For cases with uncertain results, it is necessary to count the signalsin 20 nuclei,sum up the two count results or count again the results by another analyst. If results still in critical value range, immunohistochemistry detection should performed (if FISH is not done anteriorly). Different tissue blocks can be selected for re-testing.

Results Interpretation

• The CEP17 probe is used as an internal control in this kit. The hybridization is regarded as successful when more than 75% of the nuclei in the tissue show a dual-color signal. The dual-color signals are used as mutual controls to compare the cancerous and non-cancerous cells.
• The CEP17 probe is added to the fluorescence in situ hybridization dual-probe assay to detect chromosome 17 at the same time as detecting the HER2 gene, thereby differentiating between chromosome 17 aneuploidy and HER2 gene amplification, especially at low levels of amplification. The mean signal number of the centromere of chromosome 17 in a tumor cell is ≥3, and it is a chromosome 17 polymorphism. Studies have shown that whole chromosome 17 polymorphism is rare, and the polymorphism of the chromosome 17 centromere does not represent the entire chromosome 17 polymorphism. There is a co-amplification of the HER2 gene and the centromere on chromosome 17 in some breast cancers. More and more scholars believe that HER2 copy number is more important for the judgment of HER2 gene amplification than the HER2/CEP17 ratio. Therefore, in addition to the HER2/CEP17 ratio reported in the HER2 gene in situ hybridization assay, the HER2 gene copy number and the chromosome 17 centromere should also be reported separately.
• FISH test failure should be considered when the following conditions occur, including:
  • No positive or negative results were expected.
  • Tumor lesions are too small, making it difficult to observe two tumor regions and count.
  • Countable signal cells < 75%.
  • Over 10% of the fluorescence signal is located outside the nucleus.
  • Difficulty to distinguish the nuclei structure
  • Very strong auto-fluorescence.

For details and support, please contact Gene Bio Solution. Technical Department at info@genebiosolution.com

Product Performance Index

• The kit packaging should be intact and not damaged. The labels should be complete and clear; each liquid reagent should be marked clearly and without leakage.
• After the probe effective hybridization with peripheral blood lymphocytes, it should emit a fluorescent signal that can be identified by the naked eye under a fluorescence microscope.
• Paraffin-embedded tissue sections from 4 breast cancer patients with negative HER2 gene amplification were analyzed for fluorescent signals and the results were negative.
• Paraffin sections from four cases of breast cancer patients positive for HER2 gene amplification were examined and analyzed for fluorescent signals. Results were all positive
• Sensitive detection reference (product) analyzed 100 sets of chromosome 17 of 50 cells in the metaphase division. At least 98 sets of chromosome 17 showed a green fluorescent signal and 1 orange-red fluorescent signal.
• Specific detection reference (product) analyzed 100 chromosomes 17 of 50 cells in the metaphase division, and at least 98 chromosomes 17 display 1 specific green fluorescence signal in the centromere region, 1 specific orange fluorescent signal in the target region.
• The clinical study of this kit was designed using a synchronous blind test method, and a companion diagnostic reagent that has been targeted for drug validation (Abbott Molecular’s “HER-2 Gene Detection Kit for Breast Cancer (FISH Method)” (CFDA Registration (2013) No. 3402619) was used as a comparative reagent and a total of 1,053 effective samples were tested. The kit tested positive, negative, and overall compliance rates were 100% and Kappa values were 1.000 (p=0.00), the consistency of the two reagents for the detection was good.

Precautions

• Please read this manual carefully before testing. Operators should undergo professional technical training. Signal counting personnel must be able to observe orange and green signals.
• When testing clinical samples, the test will not provide any test results when the hybridization signal is difficult to count and the sample is not sufficient for repeated retests. If the amount of cells is not sufficient for analysis, the test will not provide test results.
• The xylene, formamide, and DAPI dye used in this experiment are potentially toxic or carcinogenic and should be handled in a fume hood. Wear masks and gloves to avoid direct contact.
• The results of this kit will be affected by various factors of the sample itself, as well as restrictions such as enzyme digestion time, hybridization temperature and time, operating environment, and limitations of current molecular biology techniques, which may result in the wrong amplification of the HER2 gene.