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Product description version B4-01; Issued 18 March 2021

SALSA MLPA Probemix P376 BRCA1ness Page 1 of 12

Product Description SALSA® MLPA® Probemix P376-B4 BRCA1ness

To be used with the MLPA General Protocol.

Version B4 As compared to version B3, one target probe was changed, with no change in sequence detected. For complete product history see page 11.

Catalogue numbers: P376-025R: SALSA MLPA Probemix P376 BRCA1ness, 25 reactions. P376-050R: SALSA MLPA Probemix P376 BRCA1ness, 50 reactions. P376-100R: SALSA MLPA Probemix P376 BRCA1ness, 100 reactions.

To be used in combination with a SALSA MLPA reagent kit and Coffalyser.Net data analysis software. MLPA reagent kits are either provided with FAM or Cy5.0 dye-labelled PCR primer, suitable for Applied Biosystems and Beckman/SCIEX capillary sequencers, respectively (see www.mrcholland.com).

Certificate of Analysis Information regarding storage conditions, quality tests, and a sample electropherogram from the current sales lot is available at www.mrcholland.com.

Precautions and warnings For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: www.mrcholland.com. It is the responsibility of the user to be aware of the latest scientific knowledge of the application before drawing any conclusions from findings generated with this product. Note: For the classification of breast cancer patient samples using the P376 BRCA1ness probemix, the PAM algorithm in the statistical programming language R should be used.

Instructions for using the classifier in the statistical programming language R for BRCA1ness classification can be found on our website (www.mrcholland.com).

Users of the classification tool in R should be aware that the R code does not provide any internal quality control nor error reports for performing classification incorrectly. Users should be aware that classification can result in false positive and/or negative calls for BRCA1ness when the instructions are not followed accurately.

General information The SALSA MLPA Probemix P376 is a research use only (RUO) assay for the detection of deletions or duplications in several chromosomal regions associated with BRCA1ness. Additionally, two probes targeting BRCA1 and BRCA2 are included in the probemix.

BRCA1 and BRCA2 are involved in homologous recombination, the double-stranded DNA repair mechanism, which plays an important role in maintaining the stability of the human genome. For individuals carrying germ-line mutations in these genes, this process is impaired. This is known as homologous recombination deficiency (HRD). Cells with HRD are more sensitive to DNA crosslinking agents, such as alkylators and platinum drugs (Kennedy et al. 2004), which are used as chemotherapy regimens.

In patients carrying a BRCA1 mutation, a characteristic pattern of gains and losses of certain chromosomal regions can be detected (Wessels et al. 2002). An array CGH-based classifier recognising this genomic pattern of BRCA1-mutated breast tumours was developed and an accuracy of 91% was shown in the validation set (Joosse et al. 2009).



In addition to hereditary BRCA1-mutated tumors, similar genomic profiles are found in about half of sporadic triple-negative tumours without BRCA1 mutations (Lips et al. 2011; Vollebergh et al. 2011). This phenomenon is referred to as BRCAness (Turner et al. 2004). BRCA1-like profiles were suggested to be predictive for benefit from PARP inhibitors, platinum agents (Severson et al. 2017) and an intensified alkylating regimen (Lips E et al. 2011, Vollebergh et al. 2011).

This P376 BRCA1ness probemix is intended for classification of BRCA1-like profiles in sporadic breast tumours. Regarding the most important classifiers, the following regions have been found gained in previous studies: 3q22-q29, 6p21-p22, 10p14, 12p13, 13q31-q34; and the following regions have been found lost in previous studies: 3p21, 5q12-q23, PTEN region (10q23), 12q21-q23, 14q22-q24 and 15q15-q21 (Joosse et al. 2009; Lips et al. 2011). The P376 BRCA1ness probemix contains probes for each of these regions. Additionally, two probes targeting BRCA1 and two probes targeting BRCA2 are included in the probemix.

BRCA1ness classification To classify a sample as BRCA1-like or non-BRCA1-like, a classifier in the statistical programming language R should be used. This classifier was developed using prediction analysis for microarrays (PAM) (Tibshirani et al. 2002). Before unknown data can be classified, a training data set including reference samples and test samples of which the type is already known has to be made. A set of sample data in ABIF format that can be used to make a training data set, and a description how to run PAM using R in combination with Coffalyser can be downloaded from our website (www.mrcholland.com). The cut-off value to classify a sample as BRCA1-like should be set at 0.5. Below this score, a sample should be classified as non-BRCA1-like. This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.

Gene structure and transcript variants: Entrez Gene shows transcript variants of each gene: http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene For NM_ mRNA reference sequences: http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide Locus Reference Genomic (LRG) database: http://www.lrg-sequence.org/ PhenoGram Plot: http://visualization.ritchielab.org/phenograms/plot

Exon numbering The BRCA1 exon numbering used in this P376-B4 BRCA1ness product description is the exon numbering from the LRG_292 sequence. The BRCA2 exon numbering used in this P376-B4 BRCA1ness product description is the exon numbering from the LRG_293 sequence. The exon numbering of the NM_ sequence that was used for determining a probe’s ligation site does not always correspond to the exon numbering obtained from the LRG sequences. As changes to the databases can occur after release of this product description, the NM_ sequence and exon numbering may not be up-to-date.

Probemix content The SALSA MLPA Probemix P376-B4 BRCA1ness contains 48 MLPA probes with amplification products between 124 and 500 nucleotides (nt). This includes two probes for the BRCA1 gene, two probes for the BRCA2 gene and 34 probes for 11 different chromosomal regions that are suggested to be clinically relevant in prediction of BRCA1-association. In addition, 10 reference probes are included that target relatively copy number stable autosomal regions in breast cancer, which have been validated in a large collaborative study (Lips et al. 2011). However, it should be noticed that breast cancer karyotypes can harbour multiple numerical and structural aberrations, which can complicate data normalisation using these reference probes. Complete probe sequences and the identity of the genes detected by the reference probes are available in table 3 and online (www.mrcholland.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment (see table below). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com.



MLPA technique The principles of the MLPA technique (Schouten et al. 2002) are described in the MLPA General Protocol (www.mrcholland.com). More information on the use of MLPA in tumour applications can be found in Hömig-Hölzel and Savola (2012).

MLPA technique validation Internal validation of the MLPA technique using 16 DNA samples from healthy individuals is required, in particular when using MLPA for the first time, or when changing the sample handling procedure, DNA extraction method or instruments used. This validation experiment should result in a standard deviation ≤0.10 for all probes over the experiment.

Required specimens Extracted DNA, which includes DNA derived from paraffin-embedded tissues, free from impurities known to affect MLPA reactions. For more information please refer to the section on DNA sample treatment found in the MLPA General Protocol. More information on the use of FFPE tissue samples for MLPA can be found in Atanesyan et al. (2017).

Reference samples A sufficient number (≥3) of reference samples should be included in each MLPA experiment for data normalisation. All samples tested, including reference DNA samples, should be derived from the same tissue type, handled using the same procedure, and prepared using the same DNA extraction method when possible. Reference samples should be derived from different healthy individuals without a family history of breast cancer. More information regarding the selection and use of reference samples can be found in the MLPA General Protocol (www.mrcholland.com).

Positive control DNA samples MRC Holland cannot provide positive DNA samples. Inclusion of a positive sample in each experiment is recommended. Coriell Institute (https://catalog.coriell.org) and Leibniz Institute DSMZ (https://www.dsmz.de/) have diverse collections of biological resources which may be used as positive control DNA samples in your MLPA experiments. The samples in the table below have been tested with this P376-B4 probemix at MRC Holland and can be used as a positive control samples to detect duplications and deletions in different chromosomal regions. The quality of cell lines can change; therefore samples should be validated before use.

Length (nt) Name

64-70-76-82 Q-fragments (only visible with <100 ng sample DNA)

88-96 D-fragments (low signal indicates incomplete denaturation)

92 Benchmark fragment

100 X-fragment (X chromosome specific)

105 Y-fragment (Y chromosome specific)

Sample name Source

Chromosomal position of

copy number alteration*

Altered target genes in P376-B4

Expected copy number alteration

NA04127 Coriell Institute 3p21.31 SEMA3B, RASSF1 Heterozygous duplication

NA03563 Coriell Institute 3q21-q26 CASR, ATR, HLTF, PDCD10, PIK3CA

Heterozygous duplication

NA08778 Coriell Institute 3q21.1 CASR Heterozygous deletion

NA20022 Coriell Institute 3q23-q26 ATR, HLTF, PDCD10,

PIK3CA Heterozygous duplication

NA11428 Coriell Institute 3q23-q26 ATR, HLTF, PDCD10,

PIK3CA Heterozygous duplication



a For sample NA07981, homozygous duplications and heterozygous triplications cannot be discrimated. For further information, please refer to Reynolds et al. (2013).

* Indicated chromosomal bands accommodate genes targeted by MLPA probes, however, the whole extent of copy number alteration (CNA) present in this cell line cannot be determined by this P376-B4 BRCA1ness probemix.

Data analysis Coffalyser.Net software should be used for data analysis in combination with the appropriate lot-specific MLPA Coffalyser sheet. For both, the latest version should be used. Coffalyser.Net software is freely downloadable at www.mrcholland.com. Use of other non-proprietary software may lead to inconclusive or false results. For more details on MLPA quality control and data analysis, including normalisation, see the Coffalyser.Net Reference Manual.

For the classification of breast cancer patient samples as BRCA1-like or non-BRCA1-like using the P376-B4 BRCA1ness probemix, a classifier in the statistical programming language R should be used, for which R programming skills are required. Instructions for using the classifier in the statistical programming language R can be found on our website (www.mrcholland.com).

Interpretation of results The standard deviation of each individual probe over all the reference samples should be ≤0.10. When this criterion is fulfilled, the following cut-off values for the final ratio of the probes can be used to interpret MLPA results for autosomal chromosomes or pseudo-autosomal regions:

Note: The term “dosage quotient”, used in older product description versions, has been replaced by “final ratio” to become consistent with the terminology of the Coffalyser.Net software. (Calculations, cut-offs and interpretation remain unchanged.) Please note that the Coffalyser.Net software also shows arbitrary borders

NA10175 Coriell Institute 3q26 PDCD10, PIK3CA Heterozygous duplication NA14234 Coriell Institute 5q22.2 APC Heterozygous deletion NA14230 Coriell Institute 5q23.2 LMNB1 Heterozygous deletion NA03493 Coriell Institute 6p22.3 TPMT Heterozygous duplication NA03047 Coriell Institute 10p14 CELF2 Heterozygous deletion NA06936 Coriell Institute 10p14 GATA3, CELF2 Heterozygous deletion NA20125 Coriell Institute 10q23.31 PTEN Heterozygous duplication NA08035 Coriell Institute 12p13 FGF23, ETV6 Heterozygous duplication

NA07981 Coriell Institute 12p13 FGF23, ETV6 Homozygous

duplication/heterozygous triplication a

NA12606 Coriell Institute 13q13.1 BRCA2 Heterozygous duplication

NA03330 Coriell Institute 13q13-q33 BRCA2, ABCC4, PCCA,

FGF14 Heterozygous duplication

NA03887 Coriell Institute 13q32-q33 ABCC4, PCCA, FGF14 Heterozygous deletion NA05966 Coriell Institute 14q22-q24 GCH1, RDH12 Heterozygous duplication NA03184 Coriell Institute 15q14-q21 THBS1, CAPN3, FBN1 Heterozygous duplication

Copy number status Final ratio (FR) Normal 0.80 < FR < 1.20 Homozygous deletion FR = 0 Heterozygous deletion 0.40 < FR < 0.65 Heterozygous duplication 1.30 < FR < 1.65 Heterozygous triplication/homozygous duplication 1.75 < FR < 2.15 Ambiguous copy number All other values



as part of the statistical analysis of results obtained in an experiment. As such, arbitrary borders are different from the final ratio cut-off values shown here above.

Please note that these above mentioned final ratios are only valid for germline testing. Final ratios are affected both by percentage of tumour cells and by possible subclonality.

- Arranging probes according to chromosomal location facilitates interpretation of the results and may reveal more subtle changes such as those observed in subclonal cases. Analysis of parental samples may be necessary for correct interpretation of complex results.

- False positive results: Please note that abnormalities detected by a single probe (or multiple consecutive probes) still have a considerable chance of being a false positive result. Sequence changes (e.g. SNVs, point mutations) in the target sequence detected by a probe can be one cause. Incomplete DNA denaturation (e.g. due to salt contamination) can also lead to a decreased probe signal, in particular for probes located in or near a GC-rich region. The use of an additional purification step or an alternative DNA extraction method may resolve such cases. Additionally, contamination of DNA samples with cDNA or PCR amplicons of individual exons can lead to an increased probe signal (Varga et al. 2012). Analysis of an independently collected secondary DNA sample can exclude these kinds of contamination artefacts.

- 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 consult the latest update of the database and scientific literature when interpreting their findings.

- Not all abnormalities detected by MLPA are pathogenic. In some genes, intragenic deletions are known that result in very mild or no disease (as described for DMD by Schwartz et al. 2007). For many genes, more than one transcript variant exists. Copy number changes of exons that are not present in all transcript variants may not have clinical significance. Duplications that include the first or last exon of a gene (e.g. exons 1-3) might not result in inactivation of that gene copy.

- Copy number changes detected by reference probes or flanking probes are unlikely to have any relation to the condition tested for.

- False results can be obtained if one or more peaks are off-scale. For example, a duplication of one or more exons can be obscured when peaks are off-scale, resulting in a false negative result. The risk on off-scale peaks is higher when probemixes are used that contain a relatively low number of probes. Coffalyser.Net software warns for off-scale peaks while other software does not. If one or more peaks are off-scale, rerun the PCR products using either: a lower injection voltage or a shorter injection time, or a reduced amount of sample by diluting PCR products.

Limitations of the procedure - In most populations, the major cause of genetic defects in the BRCA1 and BRCA2 genes are small (point)

mutations, most of which will not be detected by using SALSA MLPA Probemix P376 BRCA1ness. - MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect

copy number neutral inversions or translocations. Even when MLPA did not detect any aberrations, the possibility remains that biological changes in that gene or chromosomal region do exist but remain undetected.

- Sequence changes (e.g. SNVs, point mutations) in the target sequence detected by a probe can cause false positive results. Mutations/SNVs (even when >20 nt from the probe ligation site) can reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA.

- 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. In addition, subclonality of the aberration affects the final ratio of the corresponding probe. Furthermore, there is always a possibility that one or more reference probes do show a copy number alteration in a patient sample, especially in solid tumours with more chaotic karyotypes.



Confirmation of results Copy number changes detected by only a single probe always require confirmation by another method. An apparent deletion detected by a single probe can be due to e.g. a mutation/polymorphism that prevents ligation or destabilises the binding of probe oligonucleotides to the DNA sample. Sequence analysis can establish whether mutations or polymorphisms are present in the probe target sequence. The finding of a heterozygous mutation or polymorphism indicates that two different alleles of the sequence are present in the sample DNA and that a false positive MLPA result was obtained.

Copy number changes detected by more than one consecutive probe should be confirmed by another independent technique such as long range PCR, qPCR, array CGH or Southern blotting, whenever possible. Deletions/duplications of more than 50 kb in length can often be confirmed by FISH.

COSMIC mutation database http://cancer.sanger.ac.uk/cosmic. We strongly encourage users to deposit positive results in the COSMIC Database. Recommendations for the nomenclature to describe deletions/duplications of one or more exons can be found on http://varnomen.hgvs.org/.

Please report false positive results due to SNVs and any other unusual results to MRC Holland: [email protected].

Table 1. SALSA MLPA Probemix P376-B4 BRCA1ness

Length (nt)

SALSA MLPA probe

Chromosomal position (hg18)a Location

(hg18) in kb Reference Lost in BRCA1-

like aCGH profile

Gained in BRCA1-like

aCGH profile

BRCA1/ BRCA2

64-105 Control fragments – see table in probemix content section for more information 124 Reference probe 15370-L13762 7q11 07-075,448 130 BRCA2 probe 12289-L13282 Exon 11 13-031,812 136 GATA3 probe 07632-L07317 10p14 10-008,137 141 SEMA3B probe 15373-L02625 3p21.31 03-050,283 148 CAPN3 probe 05795-L05242 15q15.1 15-040,479

156 BTG1 AREA probe 14116-L17758 12q21.33 12-090,905

160 Reference probe 00453-L17937 7q31 07-116,126 167 BRCA1 probe 02810-L02239 Exon 2 17-038,530 172 FBN1 probe 02449-L01893 15q21.1 15-046,680

178 « Reference probe 05506-L04929 10q11 10-042,932 184 ADGRV1 probe 03994-L03261 5q14.3 05-090,495 193 HLTF probe 02758-L02207 3q24 03-150,287 202 PAH probe 02332-L01819 12q23.2 12-101,813 208 Reference probe 05731-L27148 14q11 14-019,995 214 LMNB1 probe 09223-L09541 5q23.2 05-126,185 220 Reference probe 01119-L17938 10q22 10-075,548 226 APC probe 14902-L16646 5q22.2 05-112,071 233 PTEN probe 13685-L17760 10q23.31 10-089,614 241 DEPDC1B probe 09847-L16616 5q12.1 05-060,019 247 ABCC4 probe 15371-L17942 13q32.1 13-094,526 255 Reference probe 06236-L27147 21q11 21-014,510 265 TPMT probe 09451-L09707 6p22.3 06-018,248 274 FANCE probe 02431-L01878 6p21.31 06-035,535 283 GCH1 probe 04404-L02610 14q22.2 14-054,381

290 ¥ PCCA probe 21105-L08700 13q32.3 13-099,719 301 HFE probe 14344-L16013 6p22.1 06-026,199 310 Reference probe 15372-L00892 2p25 02-000,267 319 RDH12 probe 08892-L08854 14q24.1 14-067,263



¥ Changed in version B4. Minor alteration, no change in sequence detected. Δ More variable. This probe may be sensitive to certain experimental variations. Aberrant results should be treated with caution. « Probe located in or near a GC-rich region. A low signal can be caused by salt contamination in the DNA sample leading to incomplete DNA denaturation, especially of GC-rich regions. SNVs located in the target sequence of a probe can influence probe hybridization and/or probe ligation. Single probe aberration(s) must be confirmed by another method.

Table 2. P376-B4 probes arranged according to chromosomal location

Length (nt)

SALSA MLPA probe

Chromosomal position (hg18)a Location

(hg18) in kb Reference Lost in BRCA1-

like aCGH profile

Gained in BRCA1-like

aCGH profile

BRCA1/ BRCA2

328 RASSF1 probe 02248-L01734 3p21.31 03-050,353 338 BRCA2 probe 12313-L13306 Exon 5 13-031,798 346 THBS1 probe 01678-L01258 15q14 15-037,660 355 Reference probe 00965-L00552 2p13 02-071,431

364 CELF2-region probe 01234-L12680 10p14 10-011,017

373 ATR probe 02560-L02023 3q23 03-143,651 382 PDCD10 probe 04397-L03795 3q26.1 03-168,905 390 BRCA1 probe 02827-L02256 Exon 20 17-038,463

400 Δ CEP290 probe 08886-L08848 12q21.32 12-087,019 409 MCCC2 probe 06551-L17944 5q13.2 05-070,984 418 PCCA probe 08682-L08694 13q32.3 13-099,608

427 Δ PIK3CA probe 03827-L21157 3q26.32 03-180,400 436 FGF23 probe 07116-L06696 12p13.32 12-004,359 445 Reference probe 15368-L05185 2p11 02-088,674 454 BMP4 probe 12473-L13488 14q22.2 14-053,489 463 PTEN probe 13695-L15164 10q23.31 10-089,715 474 ETV6 probe 13871-L15389 12p13.2 12-011,914 484 CASR probe 02683-L02148 3q21.1 03-123,485 492 FGF14 probe 15376-L17945 13q33.1 13-101,367 500 Reference probe 06676-L21510 11p15 11-006,369

Length (nt)

SALSA MLPA probe Gene/Exon Location /

Ligation site Partial sequenceb

(24 nt adjacent to ligation site) Distance to next probe

Location (hg18) in kb

3p21 Chromosomal location 3p21 is lost in BRCA1-like aCGH profile (Lips et al. 2011).

141 15373-L02625 SEMA3B 3p21.31 ACCTGGACAACA-TCAGCAAGCGGG 70.7 kb 03-050,283 328 02248-L01734 RASSF1 3p21.31 CCCAGGTTTCCA-TTGCGCGGCTCT 73.1 Mb 03-050,353

3q21-26 Chromosomal location 3q21-q26 is gained in BRCA1-like aCGH profile (Lips et al. 2011).

484 02683-L02148 CASR 3q21.1 GCCCAGATGACT-TCTGGTCCAATG 20.2 Mb 03-123,485 373 02560-L02023 ATR 3q23 GTTCTTGACATT-GAGCAGCGACTA 6.6 Mb 03-143,651 193 02758-L02207 HLTF 3q24 GACTTACCTTTC-AGTCGTGCGCTC 18.6 Mb 03-150,287 382 04397-L03795 PDCD10 3q26.1 AGCCCAGACACT-GAGAGCCGCTTT 11.5 Mb 03-168,905

427 Δ 03827-L21157 PIK3CA 3q26.32 GGCAACCGTGAA-GAAAAGATCCTC 03-180,400 5q12-23 Chromosomal location 5q12-q23 is lost in BRCA1-like aCGH profile (Lips et al. 2011).

241 09847-L16616 DEPDC1B 5q12.1 TTTTTCGTGCTA-AGATGCCGTTAC 11.0 Mb 05-060,019 409 06551-L17944 MCCC2 5q13.2 TGAAGCGGCTTT-AAAAGAGCCCAT 19.5 Mb 05-070,984 184 03994-L03261 ADGRV1 5q14.3 TCAGTGATAATG-AATCTGGTCAAG 21.6 Mb 05-090,495 226 14902-L16646 APC 5q22.2 CCCGAGAACTGA-GGGTGGTACAGA 14.1 Mb 05-112,071 214 09223-L09541 LMNB1 5q23.2 GAGGCGAGTAGT-AGTGTTAGCATC 05-126,185



a See section Exon numbering on page 2 for more information. b Only partial probe sequences are shown. Complete probe sequences are available at www.mrcholland.com. Please notify us of any mistakes: [email protected].

Length (nt)

SALSA MLPA probe Gene/Exon Location /

Ligation site Partial sequenceb

(24 nt adjacent to ligation site) Distance to next probe


6p21-22 Chromosomal location 5q12-q23 is gained in BRCA1-like aCGH profile (Lips et al. 2011).

265 09451-L09707 TPMT 6p22.3 ATCTTCCCAGGT-AGGTTGAATACT 8.0 Mb 06-018,248 301 14344-L16013 HFE 6p22.1 CTACACATGGTT-AAGGCCTGTTGC 9.3 Mb 06-026,199

10p14 Chromosomal location 10p14 is gained in BRCA1-like aCGH profile (Lips et al. 2011).

136 07632-L07317 GATA3 10p14 GAGCAACGCAAT-CTGACCGAGCAG 2.9 Mb 10-008,137 364 01234-L12680 CELF2-

region 10p14 GACATTCACTGT-GGAAATTTGGTG 31.9 Mb 10-011,017

10q23 Chromosomal location 10q23 is lost in BRCA1-like aCGH profile (Lips et al. 2011).

233 13685-L17760 PTEN 10q23.31 CCTGCAGAAGAA-GCCCCGCCACCA 100.9 kb 10-089,614 463 13695-L15164 PTEN 10q23.31 TAAATTTTCTTT-CTCTAGGTGAAG 10-089,715

12p13 Chromosomal location 12p13 is gained in BRCA1-like aCGH profile (Lips et al. 2011).

436 07116-L06696 FGF23 12p13.32 GGGTCTGTGCCT-TGTGCAGCGTCT 7.6 Mb 12-004,359 474 13871-L15389 ETV6 12p13.2 AATGTGCACCAT-AACCCTCCCACC 75.1 Mb 12-011,914

12q21-23 Chromosomal location 12q21-q23 is lost in BRCA1-like aCGH profile (Lips et al. 2011). 400 Δ 08886-L08848 CEP290 12q21.32 TGTAATTGTGAA-TATCTCATACCT 3.9 Mb 12-087,019 156 14116-L17758 BTG1 AREA 12q21.33 GCATTGCTCTCT-GCTTATCTGAGC 10.9 Mb 12-090,905 202 02332-L01819 PAH 12q23.2 AAACATCATCAA-GATCTTGAGGCA 12-101,813

BRCA2 Ligation sites are indicated according to NM_000059.4. SALSA MLPA probemixes P090 BRCA2 (CE), P077 BRCA2 Confirmation (CE) and P045 BRCA2/CHEK2 (CE) can be used for the identification of duplications and deletions in BRCA2.

338 12313-L13306 Exon 5 206 nt before exon 5

GCTGAAATTTGT-GAGTACATATGT 14.0 kb 13-031,798

130 12289-L13282 Exon 11 5735-5736

CTGCATTTAGGA-TAGCCAGTGGTA 62.7 Mb 13-031,812

13q32-33 Chromosomal location 13q32-q34 is gained in BRCA1-like aCGH profile (Lips et al. 2011).

247 15371-L17942 ABCC4 13q32.1 ATCCGGGCATAC-AAAGCAGAAGAG 5.1 Mb 13-094,526 418 08682-L08694 PCCA 13q32.3 TTCAGAAAACAA-AGAATTTGCCAG 111.4 kb 13-099,608 290 21105-L08700 PCCA 13q32.3 CTTAATGAAAGA-GAGTGCTCAATT 1.6 Mb 13-099,719 492 15376-L17945 FGF14 13q33.1 TCGGCCTCAAGA-AGCGCAGGTTGC 13-101,367

14q22-24 Chromosomal location 14q22-q24 is lost in BRCA1-like aCGH profile (Lips et al. 2011).

454 12473-L13488 BMP4 14q22.2 TGGTAACCGAAT-GCTGATGGTCGT 891.9 kb 14-053,489 283 04404-L02610 GCH1 14q22.2 GCGAGGTGTACA-GAAAATGAACAG 12.9 Mb 14-054,381 319 08892-L08854 RDH12 14q24.1 GTCATTCCACTT-TCAATCTTCCCT 14-067,263

15q14-21 Chromosomal location 15q14-q21 is lost in BRCA1-like aCGH profile (Lips et al. 2011).

346 01678-L01258 THBS1 15q14 ATTGGCCGGAGG-AATCCCCAGGAA 2.8 Mb 15-037,660 148 05795-L05242 CAPN3 15q15.1 TGGAGATCTGCA-ACCTCACGGCCG 6.2 Mb 15-040,479 172 02449-L01893 FBN1 15q21.1 ATTCGCTGTATG-AATGGAGGTAGC 15-046,680

BRCA1 Ligation sites are indicated according to NM_007294.4. SALSA MLPA probemixes P002 BRCA1 (CE) and P087 BRCA1 Confirmation (CE) can be used for the identifications of duplications and deletions in BRCA1.

390 02827-L02256 Exon 20 27 nt before exon 20

TTTCTCTTATCC-TGATGGGTTGTG 66.9 kb 17-038,463

167 02810-L02239 Exon 2 180-179 reverse

GATGGGACACTC-TAAGATTTTCTG 17-038,530



Δ More variable. This probe may be sensitive to certain experimental variations. Aberrant results should be treated with caution. SNVs located in the target sequence of a probe can influence probe hybridization and/or probe ligation. Single probe aberration(s) must be confirmed by another method.

Table 3. Reference probes arranged according to chromosomal location.

« Probe located in or near a GC-rich region. A low signal can be caused by salt contamination in the DNA sample leading to incomplete DNA denaturation, especially of GC-rich regions. Complete probe sequences are available at www.mrcholland.com.

Related SALSA MLPA probemixes P002 BRCA1 (CE) Contains probes for each exon of the BRCA1 gene. P045 BRCA2/CHEK2 (CE) Contains probes for each exon of the BRCA2 gene, and 3 probes for

the CHEK2 region. P077 BRCA2 Confirmation (CE) Can be used for confirmation of results obtained with P045 and

P090. Contains probes for the BRCA2 gene with different ligation sites.

P078 Breast tumour Contains probes targeting chromosomal regions 6q25 (ESR1), 7p11 (EGFR), 8p12-p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q13-q24 (PRDM14, MTDH, MYC), 11q13 (CCND1, EMSY), 16q22 (CDH1), 17q11-q25 (ERBB2, TOP2A, MAPT, PPM1D, BIRC5), 19q12 (CCNE1) and 20q13 (AURKA), involved in breast cancer.

P087 BRCA1 Confirmation (CE) Can be used for confirmation of results obtained with the P002 BRCA1 probemix. Includes probes for each exon of the BRCA1 gene, with different ligation sites as compared to probes in P002.

P090 BRCA2 (CE) Contains probes for each exon of the BRCA2 gene. P190 CHEK2 (CE) Contains probes for the CHEK2, ATM and TP53 genes, involved in

breast cancer. P239 BRCA1 region Contains probes for upstream and downstream regions of the

BRCA1 gene. P260 PALB2-RAD50-RAD51C-RAD51D (CE)

Contains probes for PALB2, RAD50, RAD51C, RAD51D, involved in BRCA1 and BRCA2 mutation-negative breast cancer.

P489 BARD1 Contains probes for the BARD1 gene, which is associated with hereditary breast cancer.

P041/P042 ATM (CE) Together contain probes for every exon of the ATM gene, associated with hereditary predisposition to develop cancer, including, but not limited to breast cancer.

Length (nt)

SALSA MLPA probe

Gene Chromosomal band (hg18)

Partial sequence (24 nt adjacent to ligation site)


310 15372-L00892 ACP1 2p25 AGACCCAACACA-TTTGTATCCTGC 02-000,267 355 00965-L00552 ZNF638 2p13 CATTCCCATTCG-GTCTCCCTTTGG 02-071,431 445 15368-L05185 EIF2AK3 2p11 TAGTGACGAAAT-GGAACAAGAGGA 02-088,674 124 15370-L13762 POR 7q11 GATGGGAAGTGA-GTGCCCACCCTG 07-075,448 160 00453-L17937 MET 7q31 GCTCCTGTTTAC-CTTGGTGCAGAG 07-116,126

178 « 05506-L04929 RET 10q11 GGGAATTCCCTC-GGAAGAACTTGG 10-042,932 220 01119-L17938 VCL 10q22 AGAGATGCTGGT-TCACAATGCCCA 10-075,548 500 06676-L21510 SMPD1 11p15 CTGCTGAAGATA-GCACCACCTGCC 11-006,369 208 05731-L27148 APEX1 14q11 ACCAAATGTTCA-GAGAACAAACTA 14-019,995 255 06236-L27147 RBM11 21q11 GTTTGTTGGGAA-TTTAGAGGCCCG 21-014,510



References Atanesyan L et al. (2017). Optimal fixation conditions and DNA extraction methods for MLPA analysis on

FFPE tissue-derived DNA. Am J Clin Pathol. 147:60-8. Hömig-Hölzel C and Savola S. (2012). Multiplex ligation-dependent probe amplification (MLPA) in tumor

diagnostics and prognostics. Diagn Mol Pathol. 21:189-206. Joosse SA et al. (2009). Prediction of BRCA1-association in hereditary non-BRCA1/2 breast carcinomas

with array-CGH. Breast Cancer Res Treat. 116:479-89. Kennedy RD et al. (2004). The role of BRCA1 in the cellular response to chemotherapy. J Natl Cancer Inst.

96:1659-68. Lips EH et al. (2011). Quantitative copy number analysis by Multiplex Ligation-dependent Probe

Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness. Breast Cancer Res. 13:R107.

Reynolds JF et al. (1987). Isochromosome 12p mosaicism (Pallister mosaic aneuploidy or Pallister-Killian syndrome): report of 11 cases. Am J Med Genet. 27:257-74.

Schouten JP et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 30:e57.

Schwartz M et al. (2007). Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat. 28:205.

Severson, TM et al. (2017). The BRCA1ness signature is associated significantly with response to PARP inhibitor treatment versus control in the I-SPY 2 randomized neoadjuvant setting. Breast Cancer Res. 19:99.

Tibshirani et al. (2002). Diagnosis of multiple cancer types by shrunken centroids of gene expression. PNAS. 99:6567-72.

Turner N et al. (2004). Hallmarks of 'BRCAness' in sporadic cancers. Nat Rev Cancer. 4:814-9. Varga RE et al. (2012). MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for

exclusion of false positives. Anal Biochem. 421:799-801. Vollebergh MA et al. (2011). An aCGH classifier derived from BRCA1-mutated breast cancer and benefit

of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients. Ann Oncol. 22:1561-70.

Wessels LF et al. (2002). Molecular classification of breast carcinomas by comparative genomic hybridization: a specific somatic genetic profile for BRCA1 tumors. Cancer Res. 62:7110-7.

Selected publications using SALSA MLPA Probemix P376 BRCA1ness Akashi-Tanaka S et al. (2015). BRCAness predicts resistance to taxane-containing regimens in triple

negative breast cancer during neoadjuvant chemotherapy. Clin Breast Cancer. 1:80-5. Branham MT et al. (2016). Epigenetic regulation of ID4 in the determination of the BRCAness phenotype

in breast cancer. Breast Cancer Res Treat. 155:13-23. Estal RM et al. (2016). Relationship of immunohistochemistry, copy number aberrations and epigenetic

disorders with BRCAness pattern in hereditary and sporadic breast cancer. Familial Cancer. 15:193-200. Gross E et al. (2016). Identification of BRCA1-like triple-negative breast cancers by quantitative multiplex

ligation-dependent probe amplification (MLPA) analysis of BRCA1-associated chromosomal regions: a validation study. BMC Cancer. 16:811.

Ishikawa et al. (2016). BRCAness is beneficial for indicating triple negative breast cancer patients resistant to taxane. Eur J Surg Oncol. 42:999-1001.

Kosaka Y et al. (2020). BRCAness as an Important Prognostic Marker in Patients with Triple-Negative Breast Cancer Treated with Neoadjuvant Chemotherapy: A Multicenter Retrospective Study. Diagnostics. 10:119.

Kostovska MI et al. (2018). TIMP3 Promoter Methylation Represents an Epigenetic Marker of BRCA1ness Breast Cancer Tumours. Pathol Oncol Res. 24:937-40.

Lips E et al. (2011). Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness. Breast Cancer Res. 13:R107.



Lips E et al. (2013). Triple-negative breast cancer: BRCAness and concordance of clinical features with BRCA1-mutation carriers. Br J Cancer. 108:2172-7.

Mori H et al. (2016). BRCAness as a biomarker for predicting prognosis and response to anthracycline-based adjuvant chemotherapy for patients with triple-negative breast cancer. PLoS One. 11:e0167016.

Mori H et al. (2018). BRCAness Combined With a Family History of Cancer Is Associated With a Poor Prognosis for Breast Cancer Patients With a High Risk of BRCA Mutations. Clin Breast Cancer. 18:e1217-27.

Okuwaki K et al. (2019). Analysis of BRCAness with multiplex ligation-dependent probe amplification using formalin-fixed and paraffin-embedded pancreatic ductal adenocarcinoma tissue obtained via endoscopic ultrasound-guided fine-needle aspiration biopsy. Pancreatology. 19:419-23.

Oonk AM et al. (2012). Clinical correlates of 'BRCAness' in triple-negative breast cancer of patients receiving adjuvant chemotherapy. Ann Oncol. 23:2301-5.

Severson TM et al. (2015). BRCA1-like signature in triple negative breast cancer: molecular and clinical characterization reveals subgroups with therapeutic potential. Mol Oncol. 9:1528-38.

Severson, TM et al. (2017). The BRCA1ness signature is associated significantly with response to PARP inhibitor treatment versus control in the I-SPY 2 randomized neoadjuvant setting. Breast Cancer Res. 19:99.

Tanino H. et al (2016). BRCAness and prognosis in triple-negative breast cancer patients treated with neoadjuvant chemotherapy. PLoS One. 11:e0165721.

Tian T et al. (2019). Evaluation of the BRCAness phenotype and its correlations with clinicopathological features in triple-negative breast cancers. Hum Pathol. 84:231-8.

P376 product history Version Modification B4 One target probe altered, no change in sequence detected. B3 The lengths of two probes have been adusted. B2 Three reference probes have been replaced and the 88, 96 control fragments have been

replaced. B1 Probemix completely restructured compared to version A1. A1 Restricted first release.

Implemented changes in the product description Version B4-01 – 18 March 2021 (04P) - Product description rewritten and adapted to a new template. - Warnings added for two probes at 400 nt and 427 nt that might be more variable under specific experimental conditions - Ligation sites of the probes targeting the BRCA1 and BRCA2 genes updated according to newest version of the NM_ reference sequence. - Various minor textual or layout changes. Version 08 – 30 November 2017 (template T08) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Related products information added. - New references added to articles citing the use of this probemix. - Name of gene GPR98 has been changed to ADGRV1. - Name of gene LOC254312 has been changed to CELF2 region. - Alternative Excel-based analysis method removed from the product description as we recommend to always use Coffalyser.Net for data analysis. - Small textual corrections and changes throughout the document



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