RealLine EGFR Detect-2M

Transcription

1 Instructions for use REALLINE KIT FOR THE DETECTION OF THE L858R MUTATION AND DELETION IN THE EGF-RECEPTOR GENE BY REAL-TIME PCR For research use only (RUO) 12 reactions REF MED reactions REF MED21810 valid from: April 2016 Rev _EN page 1 of 21

2 Explanation of symbols used in labelling: RUO LOT REF For research use only Batch code Catalogue number Expiry Date Temperature limitation Consult instructions for use Keep out of sunlight Manufacturer BIORON Diagnostics GmbH Rheinhorststr Ludwigshafen (Germany) Phone Fax: This kit is validated for the real-time PCR cyclers: IQ 5 icycler (Bio-Rad) CFX96 (Bio-Rad) DTprime (DT-96) (DNA-Technology) LightCycler Nano (Roche) Legals: Limited Product Warranty: This warranty limits our liability for the replacement of this product. warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. BIORON Diagnostics GmbH shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. Trademarks: iq and CFX96 are trademarks of Bio-Rad Laboratories, Inc. (USA). LightCycler is a trademark of Roche Diagnostics GmbH (Germany). FAM and ROX are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries. MED21801_21810 page 2 of 21

3 Content 1. Introduction and Clinical Information Product Description Content of the Kit Suitable Sample Material... 5 DNA extracted from fresh, fresh-frozen or formalin fixed paraffin embedded (FFPE) tumor tissue section can be tested Principle of the Kit... 6 Step1: Evaluation of unknown DNA Samples by the Control PCR (Co-PCR)... 6 Step 2: Allele-specific real time PCR for EGFR mutations (AS-PCR) Materials and Devices required but not supplied Shipping and Storage Product Use Limitations Precautions Things to Remember Before Procedure Test of Unknown DNA Samples in Control PCR (Step 1) Preparation of dilutions of DNA samples Preparation of PCR mix Programming of the Real-time PCR cycler Data analysis Test of EGFR mutations by allele-specific real time PCR (AS-PCR) Preparation of PCR mix Programming of the Real-Time PCR Cycler Data Analysis References Recommendations for DNA Isolation from FFPE Tissue Blocks List with known mutation in EGFR Technical Support MED21801_21810 page 3 of 21

4 1. Introduction and Clinical Information n-small cell lung cancer (NSCLC) is the most common type of the lung cancer. About % of NSCLC cases have activating mutations in the epidermal growth factor receptor (EGFR) gene [1-4]. Most of these somatic mutations (~90 %) are within the tyrosine kinase (TK) domain of the EGFR and occur as either an in-frame deletion of 3-6 amino acids that corresponds to codons (del ) (see list in the Table 7, p.18), or a missense mutation T2573G that leads to change of Leucine (L) to Arginine (R) at position 858 (L858R). Tumors with this mutant form of the EGFR are sensitive to the selective inhibitors of EGFR-TK - Gefitinib (IRESSA ) or Erlotinib (TARCEVA ). The European Society for Medical Oncology (ESMO) and American Society of Clinical Oncology (ASCO) recommend an EGFR mutation testing for selection of NSCLC patients who could benefit from anti-egfr therapy [5,6]. 2. Product Description The kit is designed to detect the missense mutation CTG CGG at codon 858 (mutated nucleotide is underlined) and deletions in exon 19 of the EGFR gene (see list in the Table 7, p.18) that are associated with sensitivity to therapy with EGFR-TK inhibitors. The mutations are detected by allele specific real-time PCR. post-pcr processing is needed and the risk of contamination is minimized, as detection is performed in the same sealed tube without any further handling steps. The kit is adapted for use on real-time PCR instrument IQ 5 Cycler, CFX96 (Bio-Rad Laboratories, Inc., USA), DT96 (DNA-Technology, Russia), LightCycler Nano (Roche Diagnostics GmbH, Germany). The use of other instruments has to be tested and validated by the lab. te: The use of other instruments, other extraction kits or other volumes has to be validated by the user in the lab. MED21801_21810 page 4 of 21

5 3. Content of the Kit The Kit contains sufficient reagents for 12 (REF MED21801) or 36 (REF MED21810) reactions with unknown samples and controls. Table 2. Content of the Kit. Tube Color cap Content MED21801 MED Green Control PCR mix* (PCR Mix 1) 740 μl 2200 μl 2 Yellow PCR mix L858R* (PCR Mix 2) 280 μl 840 μl 3 Yellow PCR mix ex19del* (PCR Mix 3) 280 μl 840 μl 4 Blue Taq DNA polymerase 25 μl 55 μl 5 Red Positive DNA Control** 45 μl 135 μl 6 White PCR grade water 1.4 ml 4 ml * PCR mixes does not contain Taq DNA-Polymerase; ** The mix has 1 % of mutant DNA-copies of each of 2 mutations of EGFR and normal human genomic DNA; The label at the outer box does have the information of Quality control chart (QC) with the relevant numbers for the evaluation of the data. 4. Suitable Sample Material DNA extracted from fresh, fresh-frozen or formalin fixed paraffin embedded (FFPE) tumor tissue section can be tested. Tissue for DNA extraction should be verified by pathologist and should contain not less than 20 % of tumor cells. About copies of the EGFR gene (3-6 ng of genomic DNA) are sufficient to perform PCR. Under such conditions, DNA containing more than 1 % of mutant copies of the EGFR gene yield positive result in the test. The kit does not provide reagents for DNA isolation. For the extraction of the DNA the use of the RealLine FFPE DNA Extraction Kit (BIORON Diagnostics GmbH, REF MED20301) or RealLine FFPE DNA Extraction Mag Kit (BIORON Diagnostics GmbH, REF MED32501) is recommended. MED21801_21810 page 5 of 21

6 5. Principle of the Kit The kit includes 3 PCR mixes: 1 Control PCR mix (Mix 1) 1 mix specific for mutations EGFR-L858R (Mix 2) 1 mix specific for deletions ex19del (Mix 3) RealLine Cancer Mutation Analysis Kits The products of the EGFR PCR are detected by 5'-nuclease assay with FAM labeled probes. Enclosed controls in the kit: Internal Control: the PCR mixes contain an Internal Control IC probe labelled with ROX and with corresponding primers. The IC is for the testing of the presence of inhibitors of the PCR which may lead to false negative results. The target gene is not from human source. Positive DNA Control: this control is added as an extra tube and contains copies of the EGFR gene with mutations. The mix has 1 % of mutant DNA-copies of both mutations and normal human genomic DNA; Negative Control: as negative control PCR grade water is added. The PCR mastermixes contain all reagents except Taq DNA-polymerase that is supplied in a separate tube and have to be added before the assay. The EGFR mutation assay is composed from two parts: Step1: Evaluation of unknown DNA Samples by the Control PCR (Co-PCR) First the Control PCR assay has to be performed to test if the unknown DNA - extracted from clinical samples - is suitable for the EGFR test. te: A determination of DNA concentration by measurement of the optical density at 260 nm may not provide the correct concentration of DNA and whether the DNA is suitable for the test. The reasons can be a partial degradation and chemical modifications of DNA during the tissue fixation that may inhibit or decrease efficiency of PCR. This assay is specific for a constant region of the EGFR gene. To test and compare different dilutions of unknown samples the kit is additionally supplied with the Positive DNA Control provided with the kit. For each unknown DNA sample a dilution should be identified that has the Ct value most close to the Ct of the Positive DNA control. The selected dilutions of unknown DNA samples are tested in the Part II of the assay. MED21801_21810 page 6 of 21

7 Step 2: Allele-specific real time PCR for EGFR mutations (AS-PCR) After dilutions of unknown DNA samples are optimized, the DNA is tested in an allele-specific real time PCR using 2 different PCR mixes with primers specific to the mutations EGFR-L858R and EGFR-del19ex. If the DNA sample has no mutation (wild type), the Ct in allele-specific real time PCR increases more than 5 cycles in comparison to DNA with mutation. The Control PCR reaction should be repeated for all samples. A Positive DNA Control PC as well as a no template control NTC with PCR-grade water should be included in each PCR run. Result of the allele-specific PCR (AS-PCR) for each mutation is analyzed by comparison of dct value of unknown DNA sample (dct sample) with dct value of Positive DNA control (dct m) (Fig.1). Values for dct sample and dct m are determined by the equation: = where (Ct AS) is Ct value in allele-specific reaction, (Ct C) is Ct value in control reaction; If the dct sample dct m, the DNA sample is positive and has the mutation. If the dct sample > dct m, the DNA sample is negative (no mutation or percentage of mutant EGFR alleles is less than 1 %) (See Table 1 for example). Table 1. Analysis of mutation EGFR-L858R in tumor DNA samples. Sample Ct AS Ct c dct** Result NSCLC1* rmal NSCLC Mutant NSCLC rmal NSCLC Mutant Positive DNA Control * NSCLC - non-small-cell lung cancer; ** dct = Ct AS Ct C. MED21801_21810 page 7 of 21

8 Figure 1. Schematic fluorescence curves for control PCR (dashed lines) and allele-specific PCR (solid lines) of the positive DNA control (red line) and unknown DNA samples (blue and green lines). 1 - Ct C for Positive DNA Control and Unknown DNA samples, 2 - Ct AS for Unknown DNA sample #1, 3 - Ct AS for Positive DNA Control, 4 - Ct AS for Unknown DNA sample #2. DNA sample #1 is positive (dct#1<dct m). DNA sample #2 is negative (dct#2>dct m). DNA samples without mutation could also show Ct AS values more than 35. MED21801_21810 page 8 of 21

9 6. Materials and Devices required but not supplied RealLine Cancer Mutation Analysis Kits Disposable powder-free gloves and coat; Real-Time PCR Device e.g iq 5 icycler, CFX96 (Bio-Rad Laboratories, Inc., USA), DTprime (DT-96) (DNA-Technology, Russia), LightCycler Nano (Roche Diagnostics GmbH, Germany); 8-Well Flat Cap PCR Strips or 96-Well PCR Plates; Pipetting tools ( μl, 5-50 μl; μl; μl ) with sterile filter tips; Sterile 1.5 ml microcentrifuge tubes; Microcentrifuge ( rpm); Vortexer; Refrigerator +2 C +8 С and freezer -15 C - 25 С; Heating block for +37 C +95 C; Reagents or kit for extracting DNA from FFPE tissue, e.g. RealLine FFPE DNA Extraction Kit (REF MED20301) or RealLine FFPE DNA Extraction Mag Kit (REF MED32501). 7. Shipping and Storage RealLine EGFR Detect -2M Kit is shipped with cooling blocks. All components of the kit should be stored at -15 C -25 С. Avoid more than 4-5 freeze/thaw cycles. Do not expose to light. Attention! If kit reagents are not frozen on arrival or the package has been damaged during transportation, please contact BIORON Diagnostics GmbH 8. Product Use Limitations Kit is for research use only; The kits must be used by skilled personnel only; Strictly follow the manufacturer s instructions to obtain optimal results; Use only the Taq DNA Polymerase that is provided in the kit; Do not pool reagents from different lots; Do not use kit after expiration date. MED21801_21810 page 9 of 21

10 9. Precautions All kit components in the working concentrations are non-toxic; Follow good laboratory practice when conducting DNA amplification; Use separate rooms, eventually PCR hoods, for each step of analysis: pre-pcr procedure (sample preparation); PCR setup; Wear a laboratory coat; Wear powder-free disposable gloves; Use a separate set of pipettes for each step of analysis; Use only disposable consumables (tips with filters, tubes, etc.); Store control and tissue DNAs separately from all other PCR reagents; Used kit components should be utilized as clinical waste and the disposal have to be carried out in accordance with the regulations of the respective country. 10. Things to Remember Before Procedure Always run tests with at no template control (supplied); Thaw PCR mix and DNA control completely at room temperature; Mix on vortex and spin briefly to collect solution at the bottom before opening; Do not vortex Taq DNA polymerase or any mix containing it; Work quickly and store unused reagents at -15 C C immediately after use. MED21801_21810 page 10 of 21

11 11. Test of Unknown DNA Samples in Control PCR (Step 1) RealLine Cancer Mutation Analysis Kits te: It is strongly recommended to perform duplicates from all samples and controls. PC (Positive DNA Control) and NC ( template or water control) should be added to each experiment Preparation of dilutions of DNA samples For the most clinical DNA samples 5 to 20-fold dilutions are sufficient. Prepare two 1.5 ml tubes for each DNA sample. Dilute DNA in PCR-grade water according to Table 3. Mix on vortex and spin the tubes briefly to collect solution at the bottom. Store diluted DNA for 1 day at +4 C or up to 12 months at -15 C C. Table 3. Preparation of DNA dilutions. Dilution ratio DNA PCR-grade water 1:5 30 µl 120 µl 1:20 10 µl 190 µl Preparation of PCR mix Calculate the amount of control PCR mix and Taq DNA polymerase that are required to test in duplicates sample DNA dilutions, Positive DNA Control PC and PCR-grade water NTC. Use amount of reagents per reaction as given in Table 4 and scale-up volumes by factor (4+4) 1.1, where N -= number of analyzed clinical DNA samples. Table 4. Reagents per reaction: Reagent Amount, μl Control PCR mix 19.8 Taq DNA polymerase 0.2 Example of calculation of amount of reagents for analysis of 4 DNA samples: Total amount of Control PCR mix = 19.8 μl x (4 x 4 + 4) x 1.1 = μl Total amount of Taq DNA polymerase = 0.2 μl x (4 x 4 + 4) x 1.1 = 4.4 μl Thaw control PCR mix (tube #1, green cap) at room temperature. Mix on vortex and spin briefly to collect solution at the bottom. Use immediately. Exposure to room temperature should be minimal. Prepare ready-to-use PCR mix according to calculation as described. First, add PCR mix into clean 1.5 ml microcentrifuge tube, then add Taq DNA polymerase (tube #4, blue cap) and mix gently by pipetting 8-10 times (Do not vortex!). Place the rest of PCR mix #1 and Taq DNA polymerase at -15 C...25 C immediately. Add 20 μl of Control PCR Mix with Taq DNA-polymerase into each well. Thaw Positive DNA control (tube #5, red cap) at room temperature or 37 ºС. Vortex 3 to 5 sec and spin briefly to collect all drops at the bottom of the tube. Add 5 μl of each DNA sample (dilutions 1:5 and 1:20), Positive DNA Control and PCR-grade water to appropriate wells in duplicate. Seal plate/strips with caps or sealing film. Mix gently by tapping with fingers on the side of the plate/strips. MED21801_21810 page 11 of 21

12 Centrifuge for 3 min at 2000 rpm to remove bubbles and collect solution at the bottom of the wells. Place the plate/strips into the real-time PCR cycler Programming of the Real-time PCR cycler For basic information about programming of the real-time PCR instrument please read the appropriate Instruction manual. For detailed information about using of the kit with specific real-time PCR cycler please contact Technical Support. Reaction volume: 25 μl Program the cycler with the temperature profile and fluorescence acquisition according to the Table 5. Use the Ramp Rate 3 C/sec Table 5. Protocol for EGFR control and EGFR allele-specific PCR. Cycle Optical Data Step Temperature Time number collection 1 1 Initial activation 95 C 5 min 1 Denaturation 95 C 15 sec 10 2 Annealing 56 C 30 sec 3 Extension 72 C 20 sec 30 1 Denaturation 95 C 15 sec 2 Optical data collection 56 C 1 min Select channels: FAM (green channel) for detection the EGFR DNA and ROX (red channel) for detection of the Internal Control DNA. Specify the samples and controls location according to prepared plate/strips Data analysis For basic information about data analysis on the specific real-time PCR instrument please read the appropriate Instruction manual. For detailed information about data analysis of the kit results from specific real-time PCR cycler please contact BIORON Technical Support. Check Ct values of signals from ROX and FAM channels for NTC, Positive DNA Control and DNA samples dilutions and compare the Ct value for FAM of the Positive DNA Control and dilutions of unknown DNA samples along the flow chart on Figure 2. For the Allel-specific PCR test, select DNA dilutions of clinical DNA samples which have Ct of FAM fluorescence most similar to the Positive DNA control. A further dilution of clinical DNA sample is suitable for the Allel-specific test if in the FAM channel Ct of the DNA is different from Ct of the Positive DNA Control not more than 2 cycles. MED21801_21810 page 12 of 21

13 Start Is ROX Ct value in NTC wells QC ROX *? Is FAM Ct value in the same well > 25? PCR contamination. Repeat reaction.** Is ROX Ct value in Positive DNA Control wells QC ROX *? Is FAM Ct value in the same well QC FAM *? Bad PCR mix. Repeat reaction.** Is ROX Ct value in DNA dilution wells QC ROX *? Is FAM Ct value in the same well Ct m #? PCR inhibitors in the DNA sample. Repeat DNA extraction. Is FAM Ct value in NTC wells > 25? PCR contamination. Repeat reaction.** Is FAM Ct value in Positive DNA control with Control PCR mix QC FAM contrpcr *? Is FAM Ct value in DNA sample dilution Ct m +2 #? Is FAM Ct value in DNA sample dilution Ct m -2 #? DNA dilution can be used for allele-specific real time PCR. Bad PCR mix or DNA control. Repeat reaction.** Concentration of the DNA sample dilution is too low. Repeat control PCR using more concentrated DNA. Concentration of the DNA sample dilution is too high. Repeat control PCR using more diluted DNA. Figure 2. Flow chart for analysis of the control PCR data for the selection of optimal DNA sample dilution. * Please see QC ROX and QC FAM contrpcr lot-specific values in quality control chart supplied with the kit at the label. ** The data must be discarded as data may lead to false results. Repeat PCR for all samples with corresponding and control PCR mix. If Ct value is in the same range again, this PCR mix should be changed for a new one. # Ct m Ct of Positive DNA control. MED21801_21810 page 13 of 21

14 12. Test of EGFR mutations by allel-specific real time PCR (AS-PCR) RealLine Cancer Mutation Analysis Kits Preparation of PCR mix Thaw tubes containing Control PCR mix (tube #1, green cap) and Allel-specific PCR mixes (tubes #2 and #3, yellow caps) at room temperature. Mix by turning over the tube 6-8 times or vortex, centrifuge briefly to collect all drops at the tube bottoms. Use immediately. Exposing to room temperature should be minimal. Prepare three 1.5 ml microcentrifuge tubes. Label tubes #1 to #3. Calculate amount of PCR mixes and Taq DNA-polymerase. Use amount of reagents per reaction as given in Table 4 and scale-up volumes by factor (2+4)., where N - number of analyzed clinical DNA samples. We recommend testing each DNA sample in duplicates. te: Positive DNA control and template (water) controls should be added in each experiment. Add control, L858R and ex19del PCR mixes to tubes #1, #2 and #3, accordingly. Then add Taq DNA-polymerase to each tube and gently mix by pipetting 8-10 times (Do not vortex!). Ready-to-use PCR mixes are prepared. Place the rest of PCR mixes at -15 C...25 C immediately. Add 20 µl of ready-to use PCR mixes to appropriate wells of 96-well PCR plate or PCR strips. Example of plate setup to test 4 DNA samples is shown (Fig. 3). Use optimal dilutions of unknown DNA samples as determined in step Add 5 μl of DNA sample #1 to all wells of column 1 and 2, DNA Sample #2 to all wells of column 3 and 4, etc. (Fig. 3). Add 5 μl of PCR-grade water to wells of columns 9 and 10. Add 5 μl of Positive DNA control to wells of columns 11 and 12. (Fig. 3). Seal plate/strips with caps or sealing film. Mix gently by tapping with fingers along the side of the plate/strips. Centrifuge for 2 min at 2000 rpm to eliminate bubbles and collect solution at the bottom of the wells. Place the plate into amplification block of real-time PCR instrument. MED21801_21810 page 14 of 21

15 Figure 3. Plate setup for Allel-specific PCR of 4 unknown DNA Samples, template control (H 2O) NTC and Positive DNA control (DNA Control). Control, L858R and ex19del PCR mixes are added to rows D, E and F accordingly. All samples are tested in duplicates. Unknown DNA samples 1-4 are added to wells in columns 1-8; Positive DNA control (DNA C) is added to wells in columns 11 and 12; PCR-grade water is added to wells in columns 9 and Programming of the Real-Time PCR Cycler Create and run the protocol as described in step 11.3 and Table Data Analysis For basic information about data analysis on the specific real-time PCR instrument please read the appropriate Instruction manual. For detailed information about data analysis of the kit results from specific real-time PCR cycler please contact BIORON Technical Support. Check Ct values of ROX and FAM signal for NTC, Positive DNA control and unknown DNA samples along the flow chart on Figure 4. MED21801_21810 page 15 of 21

16 Start Is ROX Ct value in NTC wells QC ROX *? Is FAM Ct value in the same well > 28? PCR contamination. Repeat reaction.** Is ROX Ct value in Positive DNA Control wells QC ROX *? Is FAM Ct value in the same well QC FAM *? Bad PCR mix. Repeat reaction.** Is ROX Ct value in Clinical DNA samples wells QC ROX *? Is FAM Ct value in the same well Ct m #? PCR inhibitors in the DNA sample. Repeat DNA extraction. Is FAM Ct value in NTC wells with Control PCR mix > 25? Is FAM Ct value in NTC wells with allele-specific PCR mix >28? Is FAM Ct value in Positive DNA control with Control PCR mix QC FAM contrpcr *? Is FAM Ct value in Positive DNA control with allele-specific PCR mix QC FAM aspcr *? Is FAM Ct value in Clinical DNA sample with Control PCR mix Ct m -2 and Ct m +2 #? Go further on with protocol PCR contamination. Repeat reaction.** PCR contamination. Repeat reaction.** Bad PCR mix or DNA control. Repeat reaction.** Bad PCR mix or DNA control. Repeat reaction.** The DNA sample is not suitable for test. Determine optimal dilution of DNA sample. Figure 4. Flow chart for check of the PCR data suitability for the detection of mutation. * Please see QC ROX, QC FAM contrpcr and QC FAM aspcr lot-specific values in quality control chart supplied with the kit (s. label). ** The data must be discarded as data may lead to false results. Repeat PCR for all samples with corresponding and control PCR mix. If Ct value is in the same range again, this PCR mix should be changed for a new one. # Ct m Ct of Positive DNA control in reaction with corresponding PCR mix. MED21801_21810 page 16 of 21

17 If Ct value in FAM channel for DNA sample cannot be determined (is 0 or N/A) replace it with 30. Check standard deviation values for duplicate DNA samples (data in Ct Std. Dev column). If Ct sample Ct m +3 and Ct Std. Dev > 0.4 data must be discarded as data may lead to false results. Repeat PCR. For Positive DNA control calculate dc m values for each EGFR mutations as following: dct m = Ct ASm Ct Cm where dct m is dct value for Positive DNA control in reaction for a particular mutation, Ct ASm is mean Ct value of a Positive DNA control in allele-specific reaction for a particular mutation, Ct Cm is mean Ct value of a Positive DNA control in control reaction. Analog, for unknown DNA sample calculate dct sample values for each EGFR mutations by subtracting mean Ct value for sample in control PCR (Ct C samplen) from mean Ct value for the same sample in allel-specific reaction for a particular EGFR mutation (Ct AS samplen): = Compare dct sample value with corresponding dct m for each mutation. (Table 6). Table 6. Analysis of allel-specific real-time PCR data for EGFR test. Mutation/PCR mix # Positive Control* Sample N** L858R/PCR mix 2 dct 2m=Ct AS2m-Ct Cm Compare dct 2 samplen=ct AS2 samplen-ct C samplen del /pcr mix 3 dct 3m=Ct AS3m-Ct Cm Compare dct 3 samplen=ct AS3 samplen-ct C samplen * dct Xm - dct value of Positive DNA control for mutation that corresponds to PCR mix #X Ct ASXm mean Ct value of Positive DNA control in reaction with PCR mix #X. Ct Cm mean Ct value of Positive DNA control in reaction with control PCR mix ** N number of sample dct X samplen - dct value of Sample N for mutation that corresponds to PCR mix #X Ct ASX samplen mean Ct value of Sample N in reaction with PCR mix #X Ct C samplen mean Ct value of Sample N in reaction with control PCR mix The DNA sample is positive (has mutation) if the dct sample dct m. The DNA sample is negative (has no mutation or percentage of mutant EGFR allel is less than 1 %) if the dct sample > dct m. MED21801_21810 page 17 of 21

18 13. References 1. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350: Eberhard DA, Johnson BE, Amler LC, et al: Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 23: , Riely GJ. (2008). The use of first-generation tyrosine kinase inhibitors in patients with NSCLC and somatic EGFR mutations. Lung Cancer. Jun;60 Suppl 2:S Sequist LV, Bell DW, Lynch TJ, Haber DA. (2007). Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer. J Clin Oncol. 25(5): D'Addario G, Felip E; ESMO Guidelines Working Group. (2009). n-small-cell lung cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol. May;20 Suppl 4: Keedy VL, Temin S, Somerfield MR, Beasley MB, Johnson DH, McShane LM, Milton DT, Strawn JR, Wakelee HA, Giaccone G. (2011). American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) Mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol. May 20;29(15): Recommendations for DNA Isolation from FFPE Tissue Blocks To extract DNA from FFPE tissue: Cut off any excess paraffin with scalpel and cut serial 5 μm sections with a surface area of up to mm 2 per section. If tissue block has been exposed to air for a long time, discard the first 2-3 sections and use the next 6-8 sections for test. The first and the last sections are reserved for pathologist to estimate percentage of tumor cells. Place remaining 4-6 sections in a 1.5 ml microcentrifuge tubes for DNA extraction. We recommend using RealLine FFPE DNA Extraction Kit (BIORON Diagnostics, REF MED20301) or RealLine FFPE DNA Extraction Mag Kit (BIORON Diagnostics, REF MED32501) for DNA isolation from FFPE tissue blocks. DNA isolated from paraffin blocks may be partially degraded or contain PCR inhibitors. Therefore, efficiency and sensitivity of PCR may vary dramatically for DNA extracted from different paraffin blocks. It is strongly recommended to test DNA performance in control PCR before testing for mutations. For that reason additional amount of control PCR mix is provided in the kit. MED21801_21810 page 18 of 21

19 15. List with known mutation in EGFR Table 7. List of mutations in EGFR exon 19 that can be detected by RealLine EGFR-2M Detect AA Mutation Mutation ID Type Detectable* Count** (COSM) 1 p.k745_e746insipvaik insertion p.k745_e746insipvaik insertion p.k745_a750delkelrea deletion p.i744_k745inskipvai insertion p.k745_e749delkelre deletion p.k745_e746instpvaik insertion p.k745_e746insipvaik insertion p.e746_t751>a complex p.e746_s752>v complex p.e746_t751delelreat deletion p.e746_t751>i complex p.e746_s752>i complex p.e746_t751>ip complex p.e746_a750>ip complex p.e746_e749delelre deletion p.e746_a750delelrea 6223 deletion p.e746_p753>is complex p.e746_t751>ip complex p.e746_t751>i complex p.e746_s752>i complex p.e746_t751>s complex p.e746_t751>q complex p.e746_t751>l complex p.e746_s752>i complex p.e746_p753>ls complex p.e746_s752delelreats deletion p.e746_a750>rp complex p.e746_a750>qp complex p.e746_t751delelreat deletion p.k745_e746insvpvaik insertion p.e746_a750delelrea 6225 deletion p.e746_t751>vp complex p.e746_a750>vp complex p.e746_p753>vq complex p.e746_s752>v complex p.e746_t751>v complex p.e746_t751>va complex p.e746_p753>vs complex p.e746_t751>v complex p.e746_s752>v complex p.e746_t751>vp complex p.e746_t751>v complex p.e746_s752>v complex + 5 MED21801_21810 page 19 of 21

20 AA Mutation Mutation ID Type Detectable* Count** (COSM) 44 p.e746_t751>va complex p.e746_s752>a deletion p.e746_t751>a deletion p.e746_s752>v complex p.e746_a750>dp complex p.l747_p753dellreatsp deletion p.l747_t751dellreat deletion p.l747_t751>p complex p.l747_s752>q complex p.l747_s752>qh complex p.l747_t751>q complex p.e746_s752>d 6220 deletion p.l747_a750>p complex p.l747_t751dellreat deletion p.l747_a750>p complex p.l747_t751>a complex p.l747_a755>an complex p.l747_t751>q complex p.l747_s752>qh complex p.l747_t751>q complex p.l747_s752>q complex p.l747_k754dellreatspk deletion p.l747_p753>s complex p.l747_p753>q complex p.l747_e749dellre 6218 deletion p.l747_t751>p complex p.l747_t751dellreat 6254 deletion p.l747_s752dellreats 6255 deletion p.l747_a750>p complex p.l747_k754>st complex p.l747_t751>s 6210 deletion p.l747_t751dellreat deletion p.l747_p753>s deletion p.l747_r748>fp complex p.t751_a755deltspka deletion p.a750_e758delatspkanke deletion p.a750_e758>p complex p.t751_e758deltspkanke deletion p.t751_i759>s complex p.t751_i759>s complex p.t751fs* complex p.t751_i759>rea complex p.t751_e758deltspkanke deletion p.t751_i759>n complex p.t751_i759>n complex p.s752_i759delspkankei deletion p.s752_i759delspkankei 6256 deletion - 8 MED21801_21810 page 20 of 21

21 AA Mutation Mutation ID Type Detectable* Count** (COSM) 91 p.p753_i759delpkankei deletion - 1 Total * mutations that can be detected by RealLine EGFR Detect 2M Kit are shown by the sign + ; ** number of tumors with such mutation in the COSMIC database: COSMIC database ( as of February 2013 had 1605 tumors with 91 different types of actionable mutations in EGFR exon 19. In silico PCR indicated that 70 of these mutation types present in 1552 tumors could be detected by the RealLine EGFR Detect 2M Kit; that accounts for 96,7% of the actionable cases. 16. Technical Support For troubleshooting or technical assistance, please contact the technical support department of BIORON Diagnostics GmbH: techsupport@bioron.de MED21801_21810 page 21 of 21