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

Transcription

1 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 : 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

2 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 nt, three DNA denaturation control fragments (D-fragments) at 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 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: 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

3 SALSA MLPA P078-B1 Breast Tumour probemix Length (nt) SALSA MLPA probe Chromosomal position reference target gene Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 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-L q MYC probe S0247-L q TRAF4 probe L q C11orf30 (EMSY) probe L q ADAM9 probe L p ERBB2 probe L q IKBKB probe L p MYC probe L q CCNE1 probe L q TOP2A probe L q CDH1 probe L q Reference probe L p CCNE1 probe L q CDC6 probe L q Reference probe L q Reference probe L q ESR1 probe L q Reference probe L q CPD probe L q ESR1 probe L q MYC probe L q ERBB2 probe L q C11orf30 (EMSY) probe L q EGFR probe L p Reference probe L q MTDH probe L q CCND1 probe L q Reference probe L q ERBB2 probe L q BIRC5 probe L q TOP2A probe L q MTDH probe L q MED1 probe L q CDH1 probe L q TOP2A probe L q FGFR1 probe L p BIRC5 probe L q Reference probe L q FGFR1 probe L p Reference probe L p MAPT probe L q EGFR probe L p BIRC5 probe L q PRDM14 probe L p IKBKB probe L p CCND1 probe L q Reference probe L p AURKA probe L q Reference probe L q Reference probe L q22 SALSA kit P078 Breast tumour Page 3 of 6

4 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 L00550 RTN CTGGAGAGACAT-TAAGAAGACTGG L10704 SLITRK GTCTGACTCTGA-GGTAGAGGCTAG L09059 GLRA 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: ; Albertson DG (2008) Nature Genet. 40: ). In addition, new independent analyses of series of breast cancer are warranted to clarify this issue L12824 ESR1, ex TTCGACATGCTG-CTGGCTACATCA 33.4 kb L12826 ESR1, ex 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) L03283 EGFR, ex AGCTATGAGATG-GAGGAAGACGGC 42.9 kb L05386 EGFR, ex AGATCTCCTCCA-TCCTGGAGAAAG kb Reference probe on 7q L14675 RELN 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: ). 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: ). 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) L03826 FGFR1, ex TGCATACACCGA-GACCTGGCAGCC 42.6 kb L00624 FGFR1, ex CAACCTCTAACT-GCAGAACTGGGA kb L12820 ADAM9, ex AATCAGACTGCT-GTGAGAGAAGAG kb L12821 IKBKB, ex CAACTGATGCTG-ATGTGGCACCCC 9.8 kb L12831 IKBKB, ex GCCTCTCGACTT-AGCCAGCCTGGG 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: ). 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: ) L12830 PRDM14, ex CACTCTGGAGAC-AGACCATACCAG kb L03506 MTDH, ex ACCTCAAAGTGT-AACAGCAAAGCA 45.6 kb L03507 MTDH, ex GAAGAAAGAGCT-TCACTTCTAAAG kb 118 S0247-L08464 MYC, ex TCTTAAAGAGGA-GGAACAAGAAGA 0.2 kb L00169 MYC, ex AGGACTATCCTG-CTGCCAAGAGGG 0.2 kb L00625 MYC, ex GAACGAGCTAAA-ACGGAGCTTTTT Reference probes on 9q and 10q L10705 TRPM TACAGAAGACTT-TCACATACACTC L09490 ANXA CCTCCAATGGGA-GGAGGTGCCTAC SALSA kit P078 Breast tumour Page 4 of 6

5 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: ) L04808 CCND1, ex CCTGGTGAACAA-GCTCAAGTGGAA 7.3 kb L00148 CCND1, ex CCCTGCTGGAGT-CAAGCCTGCGCC kb L09347 C11orf30, ex AACCAAGTAAAA-TCTTACCCAAAC 24.5 kb L09349 C11orf30, ex ATGACCCAGGAA-AAGAGACATTCT Reference probes on 12p, 13q, 15q L12075 VWF TGGCCCTGGAAA-GGTGTCCCTGCT L06767 ZNF AGACTCTGAAAT-TCAGATTGCTAA L00565 IDH3A 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 L01849 CDH TTGCGGAAGTCA-GTTCAGACTCCA 76.1 kb L02237 CDH 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: ). MAPT gene, at 17q Low expression of MAPT (Tau) is a marker for paclitaxel sensitivity in breast cancer (Rouzier R et al (2005) PNAS 102: ). 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) L09350 TRAF ACACTGCCAGGA-AGAAGCCCAAGC kb L09913 CPD CCAGTGACTACT-TACAAAACTGGA kb L13205 MED TATCTCACACCA-AGGAGTGGGGGT kb L00146 ERBB2, ex GGTGCAGGGCTA-CGTGCTCATCGC 9.1 kb L00406 ERBB2, ex CCATCTGGAAGT-TTCCAGATGAGG 6.0 kb 113 S0393-L12911 ERBB2, ex CGACGGCAGCAG-AAGATCCGGAAG 3.2 kb L12913 ERBB2, ex TGTCGGCCAAGA-TTCCGGGAGTTG kb L13204 CDC GAACCAACAAAT-GTCCAAACCGTA kb L13177 TOP2A, ex AGTTTGGTACCA-GGCTACATGGTG 4.0 kb L12828 TOP2A, ex AAAGGCTTGCTG-ATTAATTTTATC 1.6 kb L12822 TOP2A, ex CAAGTCCAGTTA-AACAAGAAGTGT kb L08211 MAPT, ex TAAAACCTTGAA-AAATAGGCCTTG kb L02410 BIRC5, ex CTCTACATTCAA-GAACTGGCCCTT 0.4 kb L02540 BIRC5, ex AGTGTTTCTTCT-GCTTCAAGGAGC 1.9 kb L14708 BIRC5, ex GCATTCGTCCGG-TTGCGCTTTCCT Reference probe on 18p L09581 LPIN 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: ) L09344 CCNE1, ex GGAAGTCTGGAA-AATCATGTTAAA 5.1 kb L02348 CCNE1, ex GATGGTTCCATT-TGCCATGGTTAT SALSA kit P078 Breast tumour Page 5 of 6

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 20q q13 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: ) L10717 AURKA, ex AGGCATCCTAAT-ATTCTTAGACTG Reference probe on 22q L10024 PLA2G 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 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Dye Signal 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