SALSA MLPA probemix P315-B1 EGFR

SALSA MLPA probemix P315-B1 EGFR Lot B1-0215 and B1-0112. As compared to the previous A1 version (lot 0208), two mutation-specific probes for the EGFR mutations L858R and T709M as well as one additional probe for exon 1 were added. The total number of reference probes was increased to 12 and all old reference probes replaced. In addition, the 88 and 96 nt control fragments have been replaced (QDX2). The epidermal growth factor receptor (EGFR) is cell surface tyrosine kinase (TK) enzyme involved in controlling cell growth. Following binding of a ligand, EGFR stimulation leads to activation of downstream events including cell proliferation, differentiation, survival and DNA synthesis. EGFR is involved in the development of many cancers and undergoes various types of alterations to gain oncogenic properties. Moreover, patients with tumours which have alterations in EGFR tend to have a more aggressive form of the disease; and expression levels of EGFR are highly predictive of clinical outcome for cancer patients. The mechanisms for oncogenic conversion of EGFR in cancer include amplified copy number, structural rearrangements of the gene, and activating mutations that have all been detected in various malignancies. One of the most common deletions detected in tumours is the EGFR deletion variant III (EGFRvIII), which contains an in-frame deletion of exons 2-7 from the extracellular domain of EGFR. In addition numerous other (exon) deletions and duplications are found in biopsies. EGFR mutations cluster in the kinase domain of EGFR (exons 18-21), and cause ligand-independent activation of the receptor, representing possible targets for therapeutic intervention. In this regard, somatic EGFR mutations as well as gene amplification in patients with non-small cell lung cancer (NSCLC) highly correlate with the clinical response to TK inhibitors. Two frequent mutations, c.2573t>g=p.l858r and c.2369c>t=p.t790m, are shown to be an important source of resistance to drugs acting on the TK domain of EGFR. The EGFR gene comprises 28 exons, spans about 188 kb of genomic DNA and is located at chromosome 7p11.2 (55 Mb from p-telomere). This P315-B1 EGFR probemix contains one probe for each EGFR exon, except of exon 11; exon 1 is covered by two probes. Furthermore, the probemix contains two mutationspecific probes for the L858R and one for the T790M mutation, which are involved in drug resistance. Note that other mutations in EGFR are described as well, which will not be detected by this MLPA probemix. In addition, 12 reference probes have been included in this probemix, detecting different autosomal chromosomal locations which are relatively quiet in in most cancer types. However, it should be noticed that cancer karyotypes typically harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes SD006 Sample DNA Please note that the mutation-specific probes have only been tested on control plasmids and not on positive human DNA samples with the L858R and the T790M mutation! This SD006 sample DNA is provided with each probemix vial and can be used in data binning in the fragment analysis and as a positive control for the mutation-specific probes (see next page). This SALSA probemix is designed to detect deletions/duplications and mutation of one or more sequences in the aforementioned gene. Heterozygous deletions of probe recognition sequences will be apparent by a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA probemix. SALSA probemixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA MLPA test probemixes and reagents includes a limited license to use these products for research purposes. SALSA probemix P315 EGFR Page 1 of 8

The use of this SALSA probemix requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). References for SALSA probemix P315 EGFR probemix Fiala O et al (2016) Epidermal Growth Factor Receptor Gene Amplification in Patients with Advancedstage NSCLC. Anticancer Res. 2:455-60. Cimino PJ et al (2015) A wide spectrum of EGFR mutations in glioblastoma is detected by a single clinical oncology targeted next-generation sequencing panel. Exp Mol Pathol. 98:568-73. Molenaar RJ et al (2014) The combination of IDH1 mutations and MGMT methylation status predicts survival in glioblastoma better than either IDH1 or MGMT alone. Neuro Oncol. 16:1263-73. Kim Y et al (2013) Spectrum of EGFR gene copy number changes and KRAS gene mutation status in Korean triple negative breast cancer patients. PloS One. 8:e79014. Jeuken J et al. (2011) The nature and timing of specific copy number changes in the course of molecular progression in diffuse gliomas: further elucidation of their genetic "life story". Brain Pathol. 21:308-20. Minarik M et al. (2010) A novel high-resolution chipce assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA. Electrophoresis. 31:3518-24. Jeuken J et al. (2009) Robust detection of EGFR copy number changes and EGFR variant III: technical aspects and relevance for glioma diagnostics. Brain Pathol. 19:661-71. More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Data analysis The P315-B1 EGFR probemix contains 42 MLPA probes with amplification products between 124 and 439 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA denaturation control fragments (Dfragments) at 88-92-96 nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. SD006 Sample DNA The SD006 Sample DNA provided with this probemix can be used as Binning DNA sample for binning of the c.2573t>g=p.l858r and the c.2369c>t=p.t790m mutation-specific probes (246 nt probe 17162- SP0448-L21565 and 281 nt probe 17163-SP0449-L21566). Inclusion of one reaction with SD006 Sample DNA in MLPA experiments is recommended as it can be used to aid in data binning of the peak pattern using Coffalyser.NET software and as an artificial positive control for the specific point mutations. Please note that SD006 Sample DNA consists of female DNA mixed with a plasmid that contains the target sequences detected by the above mentioned probes + the sequence of the 105 nt chromosome Y specific control fragment. The amount of plasmid used (relative to the genomic DNA) results in a relative probe signal for the 105 nt probe on this female DNA which is identical to the relative probe signal obtained on male DNA samples. As a result, the 100 nt and 105 nt control fragments indicate the presence of two copies chromosome X and one copy chromosome Y and one copy of the mutationspecific probes (heterozygous mutation). The product description of the SD006 can be found on www.mlpa.com. This product is for research use only. Data generated by this probemix should be normalised with a more robust method, as the target sites of the reference probes maybe gained or lost. (1) Intra-sample normalisation should be performed by dividing the signal of each target-specific probe by the signal of every single reference probe in that sample, thus creating as many ratios per target-specific probe as there are reference probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. (2) Secondly, inter-sample comparison should be performed by dividing the Normalisation Constant of each probe in a given sample by the average Normalisation Constant of that probe in all the reference samples. SALSA probemix P315 EGFR Page 2 of 8

Data normalisation should be performed within one experiment. Always use sample and reference DNA extracted with the same method and derived from the same source of tissue. Confirmation of deletions, duplications and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website www.mlpa.com. Warning: MLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. Gains or losses of genomic regions or genes may not be detected if the percentage of tumour cells is low. Furthermore, although reference probes are located in silent regions that are not frequently altered in copy number in multiple cancers, there is always a possibility that one or more reference probes do show a copy number alteration in a sample. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. Users should always verify the latest updates of the database and scientific literature when interpreting their findings. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P315 EGFR Page 3 of 8

Table 1. SALSA MLPA P315-B1 EGFR probemix Length Chromosomal position SALSA MLPA probe (nt) References EGFR 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 124 * Reference probe S0156-L21341 7q21.2 130 *~ Reference probe 13720-L15201 11p15.5 137 EGFR probe 06121-L20393 Exon 6 142 EGFR probe 05955-20394 Exon 9 148 * EGFR probe 12961-L20662 Exon 1 157 EGFR probe 05438-L20424 Exon 5 160 EGFR probe 06405-L11395 Exon 1 166 EGFR probe 05956-L05373 Exon 10 172 * Reference probe 06046-L05501 4q31.3 179 EGFR probe 05965-L20425 Exon 19 184 EGFR probe 05440-L20433 Exon 7 190 *~ Reference probe 13382-L14839 6q12 196 * Reference probe 13232-L14565 1p21.1 205 EGFR probe 17511-L21356 Exon 21 211 * Reference probe 07019-L20663 14q11.2 218 * EGFR probe 17208-L21000 Exon 4 224 EGFR probe 06408-L20396 Exon 20 231 EGFR probe 06407-L20428 Exon 18 238 EGFR probe 05968-L05385 Exon 22 246 * Ж «EGFR probe 17162-SP0448-L21565 c.2369c>t=p.t790m 254 EGFR probe 05959-L20429 Exon 13 260 * Reference probe 07592-L14699 21q21.3 267 EGFR probe 05969-L21001 Exon 23 274 EGFR probe 05960-L05377 Exon 14 281 * Ж «EGFR probe 17163-SP0449-L21566 c.2573t>g=p.l858r 288 * Reference probe 15289-L21567 12q13.1 295 EGFR probe 05970-L20431 Exon 24 303 EGFR probe 05961-L20432 Exon 15 310 * EGFR probe 17209-L20443 Exon 12 322 * Reference probe 07130-L20666 2p22.3 328 EGFR probe 05971-L20436 Exon 25 337 * EGFR probe 17210-L20444 Exon 16 348 EGFR probe 05972-L20437 Exon 26 356 EGFR probe 05963-L20438 Exon 17 369 *~ Reference probe 05770-L05208 16q11.2 380 * EGFR probe 12901-L20439 Exon 28 391 EGFR probe 05436-L04852 Exon 3 404 * EGFR probe 12900-L20440 Exon 27 414 * Reference probe 12787-L20671 2q12.3 423 EGFR probe 05435-L21568 Exon 2 430 EGFR probe 02063-L21569 Exon 8 439 * Reference probe 16538-L21570 15q21.1 * New in version B1 (from lot B1-0112 onwards). Changed in version B1 (from lot B1-0112 onwards). Small change in length, no change in sequence detected. Mutation-specific probe. This probe will only generate a signal when the respective EGFR mutation is present. It has been tested on artificial test DNA but not on positive human samples! Ж This probe consists of three parts and has two ligation sites. «This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. ~ This probe has been reported to be deleted / duplicated in a healthy individuals. SALSA probemix P315 EGFR Page 4 of 8

Note. The EGFR exon numbering has changed. From description version v08 onwards, we have adopted the NCBI exon numbering that is present in the NM_ 055228.3 sequence for EGFR gene, which is similar to the LRG exon numbering (LRG_304). This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2a. Table 2. P315 probes arranged according to chromosomal location Table 2a. EGFR probes. Length SALSA MLPA Ligation site Partial sequence Distance to EGFR (nt) probe NM_005228.3 (24 nt adjacent to ligation site) next probe EGFR, at 7p11.2. Indicated ligation sites are in NM_005228.3 which is a reference standard in the RefSeqGene project. start codon 247-249 (exon 1) 160 06405-L11395 Exon 1 208-209 CCGTCCAGTATT-GATCGGGAGAGC 0.1 kb 148 12961-L20662 Exon 1 310-311 TCTGCCCGGCGA-GTCGGGCTCTGG 123.0 kb 423 05435-L21568 Exon 2 425-426 TAACTGTGAGGT-GGTCCTTGGGAA 1.0 kb 391 05436-L04852 Exon 3 518-519 TTATGTCCTCAT-TGCCCTCAACAC 3.4 kb 218 17208-L21000 Exon 4 802-803 ACCACCTGGGCA-GCTGTAAGTGTC 4.6 kb 157 05438-L20424 Exon 5 837-838 TGTCCCAATGGG-AGCTGCTGGGGT 1.2 kb 137 06121-L20393 Exon 6 899-900 CTGTGCCCAGCA-GTGCTCCGGGCG 1.6 kb 184 05440-L20433 Exon 7 1102-1103 AGGGCAAATACA-GCTTTGGTGCCA 1.8 kb 430 02063-L21569 Exon 8 1200-1201 AGCTATGAGATG-GAGGAAGACGGC 0.7 kb 142 05955-L20394 Exon 9 1345-1346 CCTCCATCAGTG-GCGATCTCCACA 0.2 kb 166 05956-L05373 Exon 10 1419-1420 GATCCACAGGAA-CTGGATATTCTG 3.4 kb 310 17209-L20443 Exon 12 1594-1595 CATCCTTGGGAT-TACGCTCCCTCA 1.4 kb 254 05959-L20429 Exon 13 1847-1848 CCGAGGCAGGGA-ATGCGTGGACAA 2.2 kb 274 05960-L05377 Exon 14 1913-1914 CTCTGAGTGCAT-ACAGTGCCACCC 1.6 kb 303 05961-L20432 Exon 15 2053-2054 TCATGGGAGAAA-ACAACACCCTGG 5.8 kb 337 17210-L20444 Exon 16 (18) 2154-2155 CTTGAAGGCTGT-CCAACGAATGGG 1.9 kb 356 05963-L20438 Exon 17 (19) 2253-2254 CTCTTCATGCGA-AGGCGCCACATC 0.9 kb 231 06407-L20428 Exon 18 (20) 2331-2332 CTTACACCCAGT-GGAGAAGCTCCC 0.8 kb 179 05965-L20425 Exon 19 (21) 2459-2460 AGGTGAGAAAGT-TAAAATTCCCGT 6.6 kb 246 Ж «17162-SP0448- c.2369c>t=p.t790m 2615-2614; GCATGAGCTGCA-45nt spanning L21565 (Exon 20) 2570-3569 reverse oligo-acacgtgggggt 0.1 kb 224 06408-L20396 Exon 20 (22) 2648-2649 CCTCCTGGACTA-TGTCCGGGAACA 10.3 kb 205 17511-L21356 Exon 21 (23) 2728-2727 reverse ACGGTCCTCCAA-GTAGTTCATGCC 0.1 kb 281 Ж «17163-SP0449- c.2573t>g=p.l858r 2819-2820; AGATTTTGGGCG-31nt spanning L21566 (Exon 21) 2850-2851 oligo-taccatgcagaa 1.0 kb 238 05968-L05385 Exon 22 (24) 9 nt after exon 24 CGGTGAGTCATA-ATCCTGATGCTA 5.9 kb 267 05969-L21001 Exon 23 (25) 3022-3023 AGATCTCCTCCA-TCCTGGAGAAAG 1.5 kb 295 05970-L20431 Exon 24 (26) 3124-3125 ATAGTCGCCCAA-AGTTCCGTGAGT 0.9 kb 328 05971-L20436 Exon 25 (27) 3250-3251 ACCGTGCCCTGA-TGGATGAAGAAG 0.5 kb 348 05972-L20437 Exon 26 (28) 3382-3383 GCAACAATTCCA-CCGTGGCTTGCA 0.8 kb 404 12900-L20440 Exon 27 (29) 3451-3452 TCTTGCAGCGAT-ACAGCTCAGACC 2.8 kb 380 12901-L20439 Exon 28 (30) 3623-3624 CCCACACTACCA-GGACCCCCACAG stop codon 3877-3879 (exon 28) Ж This probe consists of three parts and has two ligation sites. Mutation-specific probe. This probe will only generate a signal when the respective EGFR mutation is present. It has been tested on artificial test DNA but not on positive human samples! «This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Note. The EGFR exon numbering has changed. From description version v08 onwards, we have adopted the NCBI exon numbering that is present in the NM_ 055228.3 sequence for EGFR gene, which is similar to the LRG exon numbering (LRG_304). This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2a. SALSA probemix P315 EGFR Page 5 of 8

Table 2b. Reference probes. Length SALSA MLPA Mapview Location Gene Location (nt) probe (HG18) 196 13232-L14565 COL11A1 1p21.1 01-103,151765 322 07130-L20666 DPY30 2p22.3 02-032,102615 414 12787-L20671 EDAR 2q12.3 02-108,913892 172 06046-L05501 FBXW7 4q31.3 04-153,471353 190 ~ 13382-L14839 EYS 6q12 06-065,653303 124 S0156-L21341 KRIT1 7q21.2 07-091,703661 130 ~ 13720-L15201 AP2A2 11p15.5 11-000,974640 288 15289-L21567 COL2A1 12q13.1 12-046,662085 211 07019-L20663 RPGRIP1 14q11.2 14-020,838936 439 16538-L21570 SPG11 15q21.1 15-042,671892 369 ~ 05770-L05208 VPS35 16q11.2 16-045,260344 260 07592-L14699 ADAMTS5 21q21.3 21-027,228886 ~ This probe has been reported to be deleted / duplicated in a healthy individuals. SALSA probemix P315 EGFR Page 6 of 8

SALSA MLPA probemix P315-B1 EGFR sample pictures Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P315-B1 EGFR (lot B1-0215). Figure 2. Capillary electrophoresis pattern from a SD006 sample (approximately 50 ng human control DNA), containing the sequences of the 105 nt chromosome Y-specific probe and the L858R and T709M mutations, and analysed with SALSA probemix P315-B1 EGFR (lot B1-0215). The locations of the L858R and T709M mutation-specific probes are indicated. SALSA probemix P315 EGFR Page 7 of 8

Implemented Changes compared to the previous product description version(s). Version 08 22 June 2016 (T15) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Exon numbering of the EGFR gene has been changed on page 4 and 5 (Table 1 and 2a) and notes added about the change on page 5. - Various minor textual changes on page 1 and 2. - New references added on page 2. Version 07 - New references added on page 2. Version 06 (52) - Adjusted SD information on page 1 and 2. Version 05 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 04 (48) - Product description adapted to a new product version (version number changed, lot number added, new picture included). - Various minor textual changes on page 1. - Additional information about the use of SD006 sample DNA. - Tables have been adapted. Version 03 (44) - Various minor textual changes on page 1. - Data analysis method has been changed. - Tables have been numbered. - Data analysis paragraph When only a small has been changed. SALSA probemix P315 EGFR Page 8 of 8