Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness.
BACKGROUND
Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like(aCGH) profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1(aCGH) profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like(aCGH) profile.


METHODS
The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-like(aCGH) breast cancers and 45 non-BRCA1-like(aCGH) breast cancers, which had previously been analyzed by aCGH. The BRCA1-like(aCGH) group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-like(aCGH) breast cancers and 32 non-BRCA1-like(aCGH) breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours.


RESULTS
BRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay.


CONCLUSIONS
Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues.
Department of Experimental Therapy, The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
Esther H
EH
Lips
Nadja
N
Laddach
Suvi P
SP
Savola
Marieke A
MA
Vollebergh
Anne M M
AM
Oonk
Alex L T
AL
Imholz
Lodewyk F A
LF
Wessels
Jelle
J
Wesseling
Petra M
PM
Nederlof
Sjoerd
S
Rodenhuis
D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics*; D055028:Comparative Genomic Hybridization; D005260:Female; D018628:Gene Dosage; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011237:Predictive Value of Tests; D011933:Reagent Kits, Diagnostic
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D011933:Reagent Kits, Diagnostic