ALK

Product Name

Human ALK gene fusion detection kit.

Package Specifications

10 Tests/box.

Intended Usage

This kit uses fluorescence in situ hybridization to detect the fusion status of ALK gene in non-small cell lung cancer. The detection samples are surgical resection or biopsy samples of non-small cell lung cancer embedded in 4% neutral formaldehyde fixed paraffin. The product has not been clinically verified in combination with ALK targeted therapeutic drugs, and its clinical detection performance has been confirmed by comparative test with accompanying diagnostic reagents that have been verified by targeted drugs. The test results of the product should not be used as the only basis for the individualized treatment of patients. Clinicians should comprehensively judge the test results in combination with the patient’s condition, drug indications, treatment response and other laboratory test indicators.
Anaplastic lymphoma kinase (ALK) wasfirst found as a subtype of anaplastic large cell lymphoma (ALCL). ALK gene islocated at chromosome 2p23. Normally, human ALK can be transcribed to produce 6222 BP mRNA, which is composed of 29 exons, encoding 1620 amino acid sequences and 200kda type I transmembrane protein. This protein is a receptor tyrosine kinase (RTK) and a member of RTK insulin superfamily. Subsequently, it was found that many types of ALK gene fusion were found in non-small cell lung cancer, diffuse large B-cell lymphoma and inflammatory myofibroblast tumor, which proved that ALK gene fusion was a strong carcinogenic driver gene.
At present, the incidence rate and mortality rate of lung cancer are first of the malignant tumors in China, of which 80%~85% is non-small- cell lung cancer (NSCLC). Individualized molecular targeted therapy based on epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) has become a research hotspot of NSCLC. Many clinical studies have proved that ALK inhibitors can benefit NSCLC patients with ALK fusion gene positive. Therefore, it is of great clinical significance to detect ALK gene status in patients with NSCLC.

Detection Principle

Based on the fluorescence in situ hybridization technology, a nucleotide of the nucleic acid probe is labeled with fluorescein. The detected target gene is homologous and complementary with the used nucleic acid probe. After denaturation, annealing and renaturation, they can form a hybrid between the target gene and the nucleic acid probe. The detected gene is analyzed qualitatively, quantitatively or relatively under the microscope by the fluorescence detection system. The kit adopts rhodamine fluorescein (rho) labeled orange probe and fluorescein isothiocyanate (FITC) labeled green probe. The two probes can be combined with the target detection site by in situ hybridization. Under normal conditions (ALK gene does not fuse), it is displayed as green and orange signals close to each other or yellow signals overlapping each other under fluorescence microscope. When there is gene fusion, the green and red signals are “broken” due to the replacement of recombinant fusion partner genes, and appear as signals far apart. The fusion of ALK gene in the tissues of patients with non-small cell lung cancer was detected by this broken fluorescence in situ hybridization method, so asto provide a reference basis for the treatment prognosis and medication of patients with non-small cell lung cancer.

Product Main Components

This kit consists of ALK dual color probe, asshown 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: Surgical resection or biopsy tissue paraffin-embedded specimens.
• Tissue should be fixed in 4% neutral formaldehyde solution within 1 hour after ex vivo. After the tissue isfixed, it isroutinely dehydrated and embedded in paraffin.
• Paraffin section thickness affectsthe experimental results and slice thickness of 4~5μm is appropriate.
• It is recommended to choose paraffin-embedded tissue specimens for 5 years preservation time.

Testing Method

1. Related reagents
The following reagents are required for the experiment but not provided in this kit

• 120×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^oC, 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 atroom temperature, with deionized water complete to a volume of 200mL. Stored at 2~8^oC, 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 2~8^oC, 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.

2. Pretreatment

• Sectioning: Neutral formalin fixed paraffin-embedded tissue sections are placed on clean glass slides.
• Baking: The tissue slices are placed on the baking machine at 65°C overnight (30 min at 80°C baking for old slices).
• Dewaxing: Tissue sections are soaked for 10 minutes in a xylene dye tank for dewaxing, repeated once, and then immediately immersed in 100% ethanol for 5 minutes.
• Rehydration: At room temperature, the tissue slices are placed in 100% ethanol, 85% ethanol, and 70% ethanol for 2 minutes, and then immersed in deionized water for 3 minutes. After taking out the slices, remove excess moisture with sterile clean tissue paper.
• Water treatment: Under a 95°C water bath, the tissue slices are soaked in deionized water for 30 to 40 minutes (deionized water is preheated by the water bath).
• Washing: At room temperature, the tissue sections are soaked in 2xSSC solution and rinsed twice for 5 minutes each.
• Protease treatment: The tissue slices are immersed in the protease working solution at 37°C for 5 to 30 minutes.
• Washing: At room temperature, the tissue sections are soaked in 2xSSC solution and rinsed twice for 5 minutes each.
• Dehydration: The tissue slices are placed in 70% ethanol, 85% ethanol, and 100% ethanol for 2 minutes each, then taken out and air-dried.

3.Denaturation and Hybridization
The following operations should be performed in a darkroom.

• Take ALK dual-color probe at static room temperature for 5 minutes. Briefly centrifuge manually (do not use vortex or shaker instrument). 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 slide in the hybridization instrument, denature at 83°C for 5 minutes (the hybridizer should be preheated to 83^oC) and hybridize at 42°C for 2 to 16 hours.

4.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.

5.Counterstaining
The following operations should be performed in a darkroom 10-15μl DAPI compound dye is dropped in the hybridization area of the glass slide and immediately covered. The suitable filter is selected for glass slide observation under the fluorescence microscope.

6.FISH results observation
Place the slides under the fluorescence microscope after counterstaining, then under the natural light, use a low-power objective (10x) to locate the NSCLC cell area, and then switch to 40× objective to locate an area where cells are well-distributed. Use high-power objective (60x, 100x) to select the cells which have same size of nuclei, complete nuclei boundary, well DAPI staining, no overlapping of nuclei and show clear signal, randomly choose at least 50 tumor cells, enumerate the orange and green signal in these nuclei.

Table 3: Dual Color Signal Counting Guide

Enumerate 50 tumor cells, record the number of fused or adjacent signals, single orange signal and single green signal in each cell. Enumerate once for each tumor cell, and record the cells with hybridization signal (both orange and green signal), but these cells with no signal, only single color signal, weak signal or extremely diffuse signal should not be counted.

Results determination

Enumerate the positive cells and negative cells based on the determination methods described in Table 4 and 5, calculate Ratio value, Ratio = number of positive cells/ total number of enumerated cells × 100%.

• Positive results
  The tissue specimen is considered positive for ALK Gene Break Apart if Ratio > 50%.
• Negative results
  The tissue specimen is considered negative for ALK Gene Break Apart if Ratio < 10%.
• Indeterminate results
  If Ratio is between 10%-50%, choose another cell area to enumerate 50 cells once more, add the first and second cell count readings together and calculate the final Ratio. If Ratio < 15%, the tissue specimen is considered negative, if Ratio ≥ 15%, the tissue specimen is considered positive.

Interpretation of test results

• • In this kit, a two-color probe is used to detect the fusion status of ALK gene. When ≥ 75% of the nuclei in the tissue show two-colorsignals, it is regarded as successful hybridization, and the two-color signals are compared with each other, and cancer cells and non-cancer cells are compared with each other.
  • Fish testshall be deemed to have failed in the following cases and shall be retested, including:
    1. The positive or negative controlsample does notshow the expected results;
    2. The tumorfocusistoo smallto observe and count two tumor areas;
    3. Cells with countable signal < 75%;
    4. More than 10% of the fluorescence signals were located outside the nucleus;
    5. The nuclearstructure is difficult to distinguish;
    6. It hasstrong spontaneous fluorescence.

• The common factors affecting the test results and treatment methodsin the experiment are shown in Table 6:

Table 6: Frequent problems and handling methods

Problem	Probable cause	Recommended solution
Strong background of slides	Inadequate wash of glass slide before preparation of specimens	Wash the glass slide using the absolute ethyl alcohol.
	Inadequate wash after hybridization	Assure that the wash buffer is prepared in line with Instruction For Use, assure the correct pH value and temperature of wash buffer, remove the coverslip and repeat the washing steps.
	Improper use of filter sets	Replace with suitable filter sets to reduce the background light.
	Improper hybridization condition	Assure the temperature of hybridization instrument is set as 42ºC.
	The temperature is too low when washing / The washing intensity of wash buffer is too low	Assure that the wash buffer reaches the required temperature when washing the slides. Assure the wash buffer is prepared in line with Instruction For Use. (low SSC concentration or high NP-40 concentration would help improving the washing intensity of wash buffer).
	The washing intensity of wash buffer is too low	Assure the wash buffer is prepared in line with Instruction For Use. (low SSC concentration or high NP-40 concentration would help improving the washing intensity of wash buffer).
Weak counterstaining	Weak counterstaining	Remove coverslip, at room temperature, immerse the slides in the wash buffer containing 2 × SSC/0.1%NP-40 for 5 minutes. Then sequentially immerse the slides in 70%, 85% and 100% ethanol solution for 1 minute respectively, and then perform the counterstaining.
	The counter stain has been kept under long-term storage or excessive light	Assure the counter stain is stored at -20ºC and protected away from light, assure its effect.
No signal or weak signals	Inadequate denaturation of specimens	Assure the temperature of hybridization instrument is set as 83ºC, at least 10 minutes in advance is needed to preheat hybridization instrument.
	The probe mixture and hybridization buffer were not mixed sufficiently before use	Blow the probe mixture and mix the probe sufficiently, centrifuge for a short time.
	The probe mixture on tissue slides dries too fast	After dropping probe mixture the target area should be covered by coverslip immediately, when washing the slides you can only remove one coverslip at a time, and dip it into wash buffer immediately before removing next coverslip.
	Air bubbles formed under coverslip during hybridization	The coverslip should cover the probe mixture in order to gently squeeze out air bubbles.
	Inappropriate hybridization condition	Ensure to comply with the time and temperature required by hybridization and do not leave gaps when sealing the slides with rubber cement. The hybridization time should be adjusted according to the situation.
	Improper wash buffer or incorrect washing conditions	Be sure to follow the requirements of Instruction for Use to formulate the wash buffer. Ensure that the temperature of wash buffer reaches the temperature predetermined in washing step. The thermometer and pH meter should be accurately calibrated. Remove coverslip before immersing the slide into wash buffer.
	Inappropriate storage of probe or specimens slides	Make sure that the probe mixture is stored at -20ºC and protected from light. Place the slides without hybridization at -20ºC for long-term storage or at room temperature for short-term storage. Place the hybridized slides at -20ºC, away from light, and store for less than 6 months.
	Incorrect use of DAPI counter stain, excessively high brightness of counter stain	Remove the coverslip, immerse the slides in 2 × SSC/ 0.1%NP-40 for 5 minutes at room temperature. Sequentially immerse the slides in 70%, 85% and 100% ethanol solution for 1 minute respectively, and then perform the counterstaining after air drying the slides.
	Inappropriate filter sets were selected for observation	Use correct filter sets to observe the probe fluorescence. For the detailed information, please consult the technical service department of Gene Bio Solution.

Product performance index

• • The outer package of the kit shall be complete without damage, and the marks shall be complete and clear; Each liquid reagent shall be clearly marked without leakage.
  • After the probe effectively hybridizes with peripheral blood (or its culture medium) lymphocytes, it should send out fluorescence signals that can be recognized by the naked eye under the fluorescence microscope.
  • The paraffin sections (negative reference) of 5 patients with non-small cell lung cancer with negative ALK gene fusion were detected and the fluorescence signals were analyzed. The results were negative.
  • Paraffin sections (positive reference) of 5 patients with non-small cell lung cancer with ALK gene fusion positive were detected, and the fluorescence signals were analyzed. The results were all positive.
  • Detect the sensitivity reference (karyotype reference), analyze 100 chromosomes 2 of 50 cells in metaphase, and at least 98 chromosomes 2 show a green fluorescence signal and a red fluorescence signal.
  • Detect the specific reference (karyotype reference), analyze 100 chromosomes 2 of 50 cells in metaphase division phase, and at least 98 chromosomes 2 show a specific green fluorescence signal and a specific red fluorescence signal in the target area.

The clinical study of this kit adopts the experimental design of a synchronous blind method and uses the concomitant diagnostic reagent (Abbott Molecular’s “ALK gene recombination detection kit (fluorescence in situ hybridization)” (Registration Certificate No.: GXZZ 20143405183) that has been verified by targeted drugs as the comparison reagent. A total of 1189 effective samples have been tested, and the positive coincidence rate of this kit, the negative coincidence rate, and the overall coincidence rate were 100%, and the kappa value was 1.000 (P = 0.00).

Precautions

• • Please read this manual carefully before testing. The testing personnel shall receive professional technical training, and the signal counting personnel must be able to observe and distinguish orange and green signals.
  • When testing clinical samples, when it is difficult to count the hybridization signal and the samples are not enough to repeat the retest, the test will not provide any test results. If the amount of cells is insufficient for analysis, the test will not provide test results.
  • Xylene, formamide and DAPI re dye used in this experiment have potential toxicity or carcinogenicity. It is necessary to operate in the fume hood and wear masks and gloves to avoid direct contact.

The results of this kit will be affected by various factors of the sample itself, but also limited by enzyme digestion time, hybridization
temperature and time, operating environment and the limitations of current molecular biology technology, which may lead to wrong ALK gene fusion results. Users must understand the potential errors and accuracy limitations that may exist in the detection process.