SALSA MLPA KIT P078-B1 Breast Tumour Lot 0210, 0109

SALSA MLPA KIT P078-B1 Breast Tumour Lot 0210, 0109 This P078-B1 Breast Tumour probemix contains probes for several genes (including ERBB2, BIRC5, MYC, TOP2A, ESR1, MTDH, CCND1, CCNE1, EGFR and C11orf30) that frequently show copy number changes and that have been shown to play a significant role in the development, progression and response to therapy in invasive breast tumours. This probe mix contains 39 probes for 10 different genes / chromosomal regions, which are suggested to be prognostically and therapeutically important in breast cancers. In addition, 11 reference probes are included in this probemix detecting 11 different autosomal chromosomal locations, which are relatively quiet in breast tumours. However, it should be noticed that breast cancer karyotypes may harbour complex numerical and structural aberrations, which can complicate interpretation of these reference probes. This SALSA MLPA kit is designed to detect amplifications, gains and losses of one or more genes / chromosomal regions in breast tumours. Heterozygote deletions of probe recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. However, a mutation or polymorphism (e.g. SNP) in the sequence detected by a probe can also cause a reduction in relative peak area, even when not located exactly on the ligation site! In addition, some probe signals may be more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions / duplications and amplifications detected by MLPA should always be confirmed by other methods or by MLPA probemixes with higher resolution in the gene or chromosomal area of interest. Users should always verify the latest scientific literature when interpreting their findings. SALSA kits are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. This kit is not CE/FDA certified for use in diagnostic procedures. SALSA MLPA kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA MLPA test kits includes a limited license to use these products for research purposes. The use of this SALSA MLPA kit 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). Related SALSA kits P004 ERBB2: contains probes for ERBB2, TOP2A and several flanking genes in 17p and 17q. Suitable also for detecting CEP17. P002/P045 BRCA1/BRCA2: contain probes for every exon of BRCA1 and BRCA2 genes. References for SALSA kit P078 Breast tumour Moelans CB. et al (2010) Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes. Mod Pathol. Epub ahead of print. See also detailed information and references on included chromosomal areas and genes in Table 2. More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA kit P078 Breast tumour Page 1 of 6

Data analysis The P078-B1 Breast tumour probemix contains 50 MLPA probes with amplification products between 113 and 500 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 110 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA denaturation control fragments (D-fragments) 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. Data generated by this probemix should be normalised with a more robust method, as the target sites of the reference probes may be 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. 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. FISH, Southern blotting, long range PCR, qpcr. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website www.mlpa.com. 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. Reference probes are located in silent regions that are not frequently altered in copy number in breast cancer, but there is always a possibility that one or more reference probes have a copy number alteration in a sample. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://projects.tcag.ca/variation. Reference probes are carefully selected not to have normal copy number variation, but when in doubt, users should always verify the latest updates of the database and scientific literature when interpreting their findings. This probemix was developed by C. Moelans and R. Vijzelaar at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the first probemix designer could be made a coauthor. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA kit P078 Breast tumour Page 2 of 6

SALSA MLPA P078-B1 Breast Tumour probemix Length (nt) SALSA MLPA probe Chromosomal position reference target gene 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 113 ERBB2 probe S0393-L12911 17q12 118 MYC probe S0247-L08464 8q24 124 TRAF4 probe 09176-L09350 17q11 132 C11orf30 (EMSY) probe 09173-L09347 11q13 136 ADAM9 probe 11992-L12820 8p11 142 ERBB2 probe 00675-L00146 17q12 148 IKBKB probe 11993-L12821 8p11 157 MYC probe 00580-L00625 8q24 166 CCNE1 probe 02881-L02348 19q12 172 TOP2A probe 11994-L12822 17q21 178 CDH1 probe 02410-L02237 16q22 184 Reference probe 11350-L12075 12p13 190 CCNE1 probe 09170-L09344 19q12 196 CDC6 probe 08611-L13204 17q21 202 Reference probe 05730-L06767 13q11 208 Reference probe 10223-L10704 3q26 215 ESR1 probe 11996-L12824 6q25 220 Reference probe 09570-L10024 22q13 226 CPD probe 09628-L09913 17q11 232 ESR1 probe 11998-L12826 6q25 238 MYC probe 00672-L00169 8q24 244 ERBB2 probe 12048-L12913 17q12 256 C11orf30 (EMSY) probe 09175-L09349 11q13 264 EGFR probe 05969-L05386 7p11 274 Reference probe 00978-L00565 15q25 281 MTDH probe 04151-L03506 8q22 292 CCND1 probe 00583-L00148 11q13 301 Reference probe 00971-L09490 10q22 310 ERBB2 probe 00986-L00406 17q12 316 BIRC5 probe 03717-L02410 17q25 328 TOP2A probe 11999-L13177 17q21 337 MTDH probe 04152-L03507 8q22 346 MED1 probe 09963-L13205 17q12 355 CDH1 probe 02860-L01849 16q22 364 TOP2A probe 12000-L12828 17q21 373 FGFR1 probe 01046-L00624 8p11 384 BIRC5 probe 03025-L14708 17q25 391 Reference probe 08964-L09059 5q33 400 FGFR1 probe 04440-L03826 8p11 409 Reference probe 00963-L00550 2p16 416 MAPT probe 08358-L08211 17q21 427 EGFR probe 02063-L03283 7p11 436 BIRC5 probe 03189-L02540 17q25 445 PRDM14 probe 12002-L12830 8p21 453 IKBKB probe 12003-L12831 8p11 463 CCND1 probe 05402-L04808 11q13 472 Reference probe 09205-L09581 18p11 481 AURKA probe 10236-L10717 20q13 490 Reference probe 10224-L10705 9q21 500 Reference probe 10218-L14675 7q22 SALSA kit P078 Breast tumour Page 3 of 6

P078 probes arranged according to chromosomal location Length SALSA MLPA Mapview 36 Partial sequence Gene / exon (nt) probe position (hg 18) (24 nt adjacent to ligation site) Reference probes on 2p, 3q, 5q 409 00963-L00550 RTN4 02-055.068269 CTGGAGAGACAT-TAAGAAGACTGG 208 10223-L10704 SLITRK3 03-166.390414 GTCTGACTCTGA-GGTAGAGGCTAG 391 08964-L09059 GLRA1 05-151.214886 ATCCCAGTTGGA-TATACGATGAAT Distance to next probe ESR1 gene, at 6q25.1 Amplification of estrogen receptor alpha (ESR1) gene seems to a rare event in breast cancer. Amplification of ESR1 has been detected in 0-20% of the breast cancer tumours and both the frequency and the prognostic value is still controversial and under debate (Holst F et al. (2007) Nature Genet. 39:655-660; Albertson DG (2008) Nature Genet. 40:821-822). In addition, new independent analyses of series of breast cancer are warranted to clarify this issue. 215 11996-L12824 ESR1, ex 10 06-152.423838 TTCGACATGCTG-CTGGCTACATCA 33.4 kb 232 11998-L12826 ESR1, ex 7 06-152.457215 GTCCAGCACCCT-GAAGTCTCTGGA EGFR gene, at 7p11.2 EGFR is a putative therapeutic target in breast cancer, since EGFR amplification has been detected in 8% of breast cancers and high expression level of EGFR gene has been shown to be an adverse prognostic factor for the disease-free and overall survival in breast cancer patients (Park K. (2007) Eur J Surg Oncol. 33:956-60). 427 02063-L03283 EGFR, ex 2 07-055.191055 AGCTATGAGATG-GAGGAAGACGGC 42.9 kb 264 05969-L05386 EGFR, ex 25 07-055.233957 AGATCTCCTCCA-TCCTGGAGAAAG 47630.5 kb Reference probe on 7q 500 10218-L14675 RELN 07-102.864424 GGGCTATTGATG-AGATTATCATGA 8p11-12 amplifications Amplification of 8p11-12 is detected in ~15% of breast cancer patients and it is associated with poor prognosis. Interestingly, evidence suggests that 8p11-12 amplifications are associated with 11q13 amplifications in ER+ breast cancers (Kwek SS. et al (2009) Oncogene 28:1892-1903). FGFR1 amplification is suggested to be the best marker of poor prognosis in this chromosomal area. Moreover, FGFR1 is a putative therapeutic target, since it is major contributor in endocrine therapy resistance (Turner N. et al (2010) Cancer Res 70:2085-94). ADAM9 is matrix metalloproteinase, which is amplified and highly expressed in breast cancer samples and making it also an attractive molecular therapeutic target (O Shea C et al (2003) Int J Cancer 105:754-61). IKBKB is a kinase associated with IKK/NF-kB activation pathway, which makes it a potential therapeutic target within 8p11-12 amplicon (Chin K. et al (2006) Cancer Cell 10:529-41). 400 04440-L03826 FGFR1, ex 14 08-038.391533 TGCATACACCGA-GACCTGGCAGCC 42.6 kb 373 01046-L00624 FGFR1, ex 2 08-038.434092 CAACCTCTAACT-GCAGAACTGGGA 564.2 kb 136 11992-L12820 ADAM9, ex 8 08-038.998319 AATCAGACTGCT-GTGAGAGAAGAG 3294.6 kb 148 11993-L12821 IKBKB, ex 10 08-042.292902 CAACTGATGCTG-ATGTGGCACCCC 9.8 kb 453 12003-L12831 IKBKB, ex 20 08-042.302676 GCCTCTCGACTT-AGCCAGCCTGGG 28827.4 kb 8q amplifications (PRDM14 gene at 8q13.3, MTDH gene at 8q22.1 and MYC gene at 8q24.12) PRDM14 is frequently amplified and subsequently overexpressed in breast cancer samples (Nishikawa N. et al (2007) Cancer Res. 67:9649-57). MTDH activation by 8q22 genomic gain promotes chemoresistance and metastasis of poor-prognosis breast cancer (Hu et al (2009): Cancer Cell 15:9-20). MYC amplification is detected in ~15% of breast cancer patients and is a marker of poor survival (Deming SL. et al (2000) Bri J of Cancer. 83:1688-98). 445 12002-L12830 PRDM14, ex 7 08-071.130073 CACTCTGGAGAC-AGACCATACCAG 27612.4 kb 281 04151-L03506 MTDH, ex 2 08-098.742504 ACCTCAAAGTGT-AACAGCAAAGCA 45.6 kb 337 04152-L03507 MTDH, ex 8 08-098.788082 GAAGAAAGAGCT-TCACTTCTAAAG 30033.7 kb 118 S0247-L08464 MYC, ex 3 08-128.821796 TCTTAAAGAGGA-GGAACAAGAAGA 0.2 kb 238 00672-L00169 MYC, ex 3 08-128.822001 AGGACTATCCTG-CTGCCAAGAGGG 0.2 kb 157 00580-L00625 MYC, ex 3 08-128.822151 GAACGAGCTAAA-ACGGAGCTTTTT Reference probes on 9q and 10q 490 10224-L10705 TRPM3 09-072.566364 TACAGAAGACTT-TCACATACACTC 301 00971-L09490 ANXA7 10-074.828036 CCTCCAATGGGA-GGAGGTGCCTAC SALSA kit P078 Breast tumour Page 4 of 6

Length SALSA MLPA Mapview 36 Partial sequence Distance to Gene / exon (nt) probe position (hg 18) (24 nt adjacent to ligation site) next probe 11q13 amplifications (CCND1 gene, at 11q13.2 and C11orf30 (EMSY) gene, at 11q13.5) CCND1 amplifications are detected in ~15% of breast cancer patients and are associated with poor overall survival in ER+ patients. C11orf30 (EMSY) amplifications are detected in 7-13% of BC patients and they are also associated with poor clinical outcome (Kirkegaard T. et al (2007) Histopathology 52:698-705). 463 05402-L04808 CCND1, ex 10 11-069.167779 CCTGGTGAACAA-GCTCAAGTGGAA 7.3 kb 292 00583-L00148 CCND1, ex 5 11-069.175089 CCCTGCTGGAGT-CAAGCCTGCGCC 6727.0 kb 132 09173-L09347 C11orf30, ex 10 11-075.902087 AACCAAGTAAAA-TCTTACCCAAAC 24.5 kb 256 09175-L09349 C11orf30, ex 16 11-075.926543 ATGACCCAGGAA-AAGAGACATTCT Reference probes on 12p, 13q, 15q 184 11350-L12075 VWF 12-006.015847 TGGCCCTGGAAA-GGTGTCCCTGCT 202 05730-L06767 ZNF198 13-019.465729 AGACTCTGAAAT-TCAGATTGCTAA 274 00978-L00565 IDH3A 15-076.239491 GAGGAGCGGAAC-GTCACTGCCATT 16q deletions (CDH1 gene, at 16q22.1) Loss of heterozygosity and deletions at 16q arm are one of the most common genetic alterations in breast cancer, occurring in ~50% of all ductal carcinomas and even more frequently in lobular breast cancer. Loss of E-cadherin (CDH1) is thought to contribute to progression in breast cancer, especially in ductal and lobular breast carcinomas. 355 02860-L01849 CDH1 16-067.328716 TTGCGGAAGTCA-GTTCAGACTCCA 76.1 kb 178 02410-L02237 CDH1 16-067.404826 GACAATTTGTCG-TCACCACAAATC 17q amplifications ERBB2 gene, at 17q12. Amplification of ERBB2 (HER2-Neu) is detected in 15-30% of breast cancers. This ERBB2-amplification defines an aggressive subtype of breast cancer that can be treated with targeted therapy (tratuzumab). Moreover, amplification of ERBB2 has been shown to correlate with poor prognosis and resistance to conventional adjuvant chemotherapy and tamoxifen. TFAF4, CPD and MED1 centromeric and CDC6 telomeric to ERBB2 gene are frequently co-amplified with ERBB2 and show increased expression. TOP2A gene, at 17q21.2. TOP2A, which is a direct molecular target of anthracycline drug action, is amplified in 25-40% of ERBB2 amplified breast cancers. Several studies have shown that TOP2 amplification is a marker of sensitivity for anthracyclines (Nielsen KV et al (2008) Acta Oncol 47:725-34). Moreover, a recent study has reported that TOP2 deletion is a significant prognostic factor for poor survival in breast cancer (Bartlett JMS et al (2010) Lancet 11:266-274). MAPT gene, at 17q21.31. Low expression of MAPT (Tau) is a marker for paclitaxel sensitivity in breast cancer (Rouzier R et al (2005) PNAS 102:8315-20). BIRC5 gene, at 17q25.3. BIRC5 amplification has been shown to predict distant recurrence in breast carcinoma (Davis LM. et al (2007) J Mol Diagn. 9:327-36). 124 09176-L09350 TRAF4 17-024.098403 ACACTGCCAGGA-AGAAGCCCAAGC 1696.6 kb 226 09628-L09913 CPD 17-025.795018 CCAGTGACTACT-TACAAAACTGGA 9045.8 kb 346 09963-L13205 MED1 17-034.840858 TATCTCACACCA-AGGAGTGGGGGT 277.2 kb 142 00675-L00146 ERBB2, ex 7 17-035.118101 GGTGCAGGGCTA-CGTGCTCATCGC 9.1 kb 310 00986-L00406 ERBB2, ex 19 17-035.127183 CCATCTGGAAGT-TTCCAGATGAGG 6.0 kb 113 S0393-L12911 ERBB2, ex 21 17-035.133169 CGACGGCAGCAG-AAGATCCGGAAG 3.2 kb 244 12048-L12913 ERBB2, ex 28 17-035.136344 TGTCGGCCAAGA-TTCCGGGAGTTG 562.9 kb 196 08611-L13204 CDC6 17-035.699283 GAACCAACAAAT-GTCCAAACCGTA 113.4 kb 328 11999-L13177 TOP2A, ex 20 17-035.812698 AGTTTGGTACCA-GGCTACATGGTG 4.0 kb 364 12000-L12828 TOP2A, ex 14 17-035.816651 AAAGGCTTGCTG-ATTAATTTTATC 1.6 kb 172 11994-L12822 TOP2A, ex 11 17-035.818297 CAAGTCCAGTTA-AACAAGAAGTGT 5604.8 kb 416 08358-L08211 MAPT, ex 8 17-041.423085 TAAAACCTTGAA-AAATAGGCCTTG 32299.0 kb 316 03717-L02410 BIRC5, ex 1 17-073.722036 CTCTACATTCAA-GAACTGGCCCTT 0.4 kb 436 03189-L02540 BIRC5, ex 2 17-073.722396 AGTGTTTCTTCT-GCTTCAAGGAGC 1.9 kb 384 03025-L14708 BIRC5, ex 4 17-073.724340 GCATTCGTCCGG-TTGCGCTTTCCT Reference probe on 18p 472 09205-L09581 LPIN2 18-002.950707 CTCTGGGTGCAT-TGATGTCATCGT CCNE1 gene, at 19q12 CCNE1 is often amplified in breast cancer cells and CCNE1 overexpression has been associated with an increased risk of breast cancer relapse and death (Keyomarsi K. et al (2002) N Engl J Med. 347:1566 75). 190 09170-L09344 CCNE1, ex 6 19-035.000150 GGAAGTCTGGAA-AATCATGTTAAA 5.1 kb 166 02881-L02348 CCNE1, ex 11 19-035.005214 GATGGTTCCATT-TGCCATGGTTAT SALSA kit P078 Breast tumour Page 5 of 6

Length SALSA MLPA Mapview 36 Partial sequence Distance to Gene / exon (nt) probe position (hg 18) (24 nt adjacent to ligation site) next probe AURKA gene, at 20q13.31 20q13 is amplified in 5-15% of breast carcinoma samples and the putative target gene of the amplicon is AURKA gene. A recent study has suggested that AURKA amplification is especially frequent in breast carcinomas of basal-like phenotype (Staff S. et al (2010) Oncol Rep. 23:307-12). Moreover, high-level 20q13 amplifications, including AURKA, have been suggested to be an indicator of poor clinical outcome in breast cancer (Tanner MM. et al (1995) Clin Cancer Res. 1:1455-61). 481 10236-L10717 AURKA, ex 8 20-054.389980 AGGCATCCTAAT-ATTCTTAGACTG Reference probe on 22q 220 09570-L10024 PLA2G6 22-036.865968 CTGCTGTGCAAT-GCTCGGTGCAAC Note: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA kit P078-B1 Breast Tumour sample picture 50000 45000 40000 35000 30000 25000 20000 1 8 3,3 2 1 7 7,0 4 2 0 7,6 3 3 0 1,3 9 3 6 3,5 1 2 3 1,3 3 3 5 4,0 3 2 8 0,0 1 3 2 7,1 6 2 2 4,8 0 3 8 4,0 2 1 5 5,0 4 2 6 2,8 9 1 3 9,8 2 4 2 6,6 2 8 5,4 3 2 9 1,1 9 1 2 9,6 2 4 1 5,1 3 4 5 3,8 0 2 2 0,2 4 2 5 4,6 1 1 1 5,5 7 2 3 7,2 9 1 0 9,6 9 1 9 6,0 5 1 0 5,4 0 1 8 9,2 2 1 2 1,2 9 4 0 0,6 0 3 3 6,2 1 9 1,0 0 1 3 4,0 3 3 4 5,6 8 3 9 1,7 6 1 4 5,8 3 2 4 3,4 6 2 1 4,3 2 9 5,7 7 2 7 3,4 4 1 6 4,5 1 3 0 9,0 8 4 4 5,7 4 2 0 2,0 5 1 7 0,5 8 3 7 4,2 4 4 6 3,0 0 3 1 5,8 6 4 8 2,6 9 5 0 1,4 6 4 7 2,4 8 4 8 9,5 9 4 0 7,7 7 1 0 0,5 5 4 3 5,6 3 15000 10000 Dye Signal 5000 0 100 150 200 250 300 350 400 450 500 Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P078-B1 Breast tumour (lot 0210). Please note a non-specific peak is present at 310 nt in the no DNA control reaction. Implemented Changes the following has been altered compared to the previous product description version(s). Version 03 (45) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Sentence it should be noticed that breast cancer karyotypes may harbour has been added to page 1. - Data analysis paragraph, on page 2, has been updated. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Exon numbers have been included in Table 2 for target specific probes. - New information and references on genes/chromosomal areas included in the mix are added in Table 2. Version 02 (44) - Not applicable, new document. SALSA kit P078 Breast tumour Page 6 of 6