functional ex vivo assay reveals homologous …variant calling was performed by comparison to the...

Precision Medicine and Imaging

Functional Ex Vivo Assay Reveals HomologousRecombination Deficiency in Breast CancerBeyond BRCA Gene DefectsTitia G. Meijer1, Nicole S.Verkaik1, Anieta M. Sieuwerts2, Job van Riet3,4, Kishan A.T. Naipal1,Carolien H.M. van Deurzen5, Michael A. den Bakker6, Hein F.B.M. Sleddens5,Hendrikus-Jan Dubbink5, T. Dorine den Toom5,Winand N.M. Dinjens5, Esther Lips7,Petra M. Nederlof7, Marcel Smid2, Harmen J.G. van de Werken3,4, Roland Kanaar1,John W.M. Martens2, Agnes Jager2, and Dik C. van Gent1

Abstract

Purpose: Tumors of germline BRCA1/2 mutated carriersshow homologous recombination (HR) deficiency (HRD),resulting in impaired DNA double-strand break (DSB) repairand high sensitivity to PARP inhibitors. Although this therapyis expected to be effective beyond germline BRCA1/2mutatedcarriers, a robust validated test todetectHRD tumors is lacking.In this study, we therefore evaluated a functional HR assayexploiting the formation of RAD51 foci in proliferating cellsafter ex vivo irradiation of fresh breast cancer tissue: the recom-bination REpair CAPacity (RECAP) test.

Experimental Design: Fresh samples of 170 primary breastcancer were analyzed using the RECAP test. The molecularexplanation for theHRDphenotypewas investigatedby explor-ing BRCA deficiencies, mutational signatures, tumor-infiltrat-ing lymphocytes (TIL), and microsatellite instability (MSI).

Results: RECAP was completed successfully in 125 of 170samples (74%). Twenty-four tumors showed HRD (19%),whereas six tumors were HR intermediate (HRi; 5%). HRDwas explained by BRCA deficiencies (mutations, promoterhypermethylation, deletions) in 16 cases, whereas seven HRDtumors were non-BRCA related. HRD tumors showed anincreased incidence of high TIL counts (P ¼ 0.023) comparedwithHR proficient (HRP) tumors andMSI wasmore frequent-ly observed in the HRD group (2/20, 10%) than expected inbreast cancer (1%; P ¼ 0.017).

Conclusions: RECAP is a robust functional HR assay detect-ing both BRCA1/2-deficient and BRCA1/2-proficient HRDtumors. Functional assessment of HR in a pseudo-diagnosticsetting is achievable and produces robust and interpretableresults. Clin Cancer Res; 24(24); 6277–87. �2018 AACR.

IntroductionBreast cancer is the most common malignancy in women with

the secondhighest cancer-relatedmortality rate (1). Approximate-ly 3% of all breast cancer cases are due to germline mutations inBRCA1/2 (2), and in triple-negative breast cancers (TNBC) thispercentage is even 10% to 20% (3). The BRCA proteins play animportant role in the homologous recombination (HR) pathway,

the error-free DNA double-strand break (DSB) repair pathwaythat operates during the S- and G2-phase of the cell cycle. HRdeficiency (HRD) leading to impaired DNA DSB repair is fre-quently caused by, but not limited to, defects in BRCA1/2 (4).

Therapies specifically targeting tumor cells with impaired HRcapacity are PARP inhibitors (PARPi), as well as classical che-motherapies such as platinum-derivates and alkylating agents (5).PARPi causes persistence of single-strand DNA breaks (SSB) bytrapping PARP1 onDNA, whereas platinum-derivates cause DNAinterstrand crosslinks. Both types of lesions result in replicationfork stalling and/or collapse, frequently leading toDSBs that needHR for their repair (5). The targeted approach of PARPi kills tumorcells lacking HR, whereas normal cells remain unharmed, due totheir normal DSB repair capacity, a phenomenon often referred toas synthetic lethality. Recently, FDA approval was granted for theuse ofOlaparib in germline BRCAmutated breast cancer based onthe results of the Olympiad trial (6).

Although evidence is emerging that the use of PARPi could beextended beyond germline BRCA1/2mutated cancers to sporadiccancerswith BRCA-like features, a gold standard test for predictingresponse to treatments targeting HR is not yet available (7).Several different HRD tests exist, mostly based on genomicpatterns or transcriptional predictors of BRCAness (8–12).

These genomic tests measure the accumulation of mutationsand chromosomal aberrations over time, but not necessarilyreflect the real-time HR status. Beyond mutational status, several

1Department ofMolecular Genetics andOncode Institute, ErasmusMCUniversityMedical Center Rotterdam, the Netherlands. 2Department of Medical Oncology,ErasmusMCCancer Institute, ErasmusMCUniversity Medical Center Rotterdam,the Netherlands. 3Erasmus MC University Medical Center Rotterdam, CancerComputational Biology Center, Rotterdam, the Netherlands. 4Department ofUrology, Erasmus MC University Medical Center Rotterdam, the Netherlands.5Department of Pathology, Erasmus MC University Medical Center Rotterdam,the Netherlands. 6Maasstad ziekenhuis, Rotterdam, the Netherlands. 7Depart-ment of Pathology, Netherlands Cancer Institute, Amsterdam, the Netherlands.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corrected online March 27, 2019.

Corresponding Author: Dik C. van Gent, Erasmus MC University MedicalCenter Rotterdam and Oncode Institute, P.O. Box 2040, Rotterdam 3000 CA,the Netherlands. Phone: 31 10 704 39 32; Fax: 31 10 704 10 03; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-18-0063

�2018 American Association for Cancer Research.

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other factors influence tumor behavior and therapy response,such as epigenetic changes and the microenvironment of thetumor cells. Independent of the underlying cause, the down-stream effect of HR impairment (phenotype) can be assessedfunctionally. A functional diagnostic assay therefore has thepotential for more precisely detecting patients who may benefitfrom PARPi than genomic assays.

A functional HRD assay was first described by Graeser et al.,assessing RAD51 focus formation, a marker of HR competence,in tumor biopsies obtained 24 hours after in vivo anthracyclinetreatment (13). This provided the first evidence that RAD51focus formation can serve as a predictive biomarker. Toenhance clinical utility of this biomarker, test outcomes shouldbe available before start of treatment. Therefore, we developedthe homologous recombination REpair CAPacity (RECAP) testexploiting the formation of RAD51 foci in proliferating cellsafter ex vivo irradiation of fresh breast cancer tissue, providing areal-time HR status of the tumor (14). The aim of this study wasto validate the RECAP test in an extensive cohort of primarybreast cancers and provide evidence that this functional test isachievable in a pseudo-clinical setting. Additionally, thoroughmolecular characterization of the HRD phenotype is per-formed, proving that HRD tumors encompass more than onlyBRCA deficiencies.

Materials and MethodsPrimary breast cancer specimens

Residual fresh breast cancer tissue was prospectively collectedfrom lumpectomy of the breast or mastectomy specimens in theErasmus MC Cancer Institute, Haven hospital, and Maasstadhospital in Rotterdam, the Netherlands, between 1 October2011 and 1 September 2016. The first 41 patients were alsoincluded in our previous cohort (14). After macroscopic evalu-ation of the surgical specimen by trained pathologists, residualtumor tissue was collected for our research purposes according tothe "Code of proper secondary use of human tissue in the Nether-lands" established by the Dutch Federation of Medical ScientificSocieties and approved by the local Medical Ethical committees(MEC-11-098). Patients who had objected to secondary use of

residual tumor material for research purposes were not includedin this study. Patients with ductal carcinoma in situ (DCIS) only orpatients receiving neo-adjuvant chemotherapy were excluded.

RECAP testObtained tissue samples were immediately transferred into

customized breast tissue culture medium, as described in Naipalet al. (14). Processing of samples was performed within 4 hoursafter the tissue was resected. Microscopic analysis of hematoxylinand eosin (HE) stained sections was performed to determinepresence of invasive carcinoma. The RECAP test, a functionalassay exploiting the formation of RAD51 foci in proliferatingcells after ex vivo irradiation of fresh breast cancer tissue, wasperformed and results were analyzed as described previously(14). In brief, presence of RAD51 foci was determined in S–G2

cells only, which stain positive for Geminin. At least 30 Gemininexpressing cells were counted per tumor sample. A cell wasconsidered RAD51 positive when at least five RAD51 foci couldbe detected. Based on previous experiments with patient-derivedxenograft (PDX) models with known BRCA status, tumors wereclassified as HR proficient (HRP), HR deficient (HRD), or inter-mediate (HRi) when more than 50%, less than 20% or between20% and 50% of geminin positive cells showed �5 RAD51 foci,respectively.

Workflow of molecular characterization of the HRDphenotype

To unravel the possible molecular mechanism underlying theHRD phenotype, several molecular tests were performed retro-spectively (Fig. 2; Supplementary Fig. S1). As no DNA could beobtained for one HRD sample, we conducted the analyses for 23HRD and six HRi samples. First, BRCA sequencing and BRCA1promoter methylation analysis was performed in HRD and HRisamples, as well as in all TNBC (n ¼ 5), ER/PR�HER2þ (n ¼ 2),and 21 ER/PRþ HRP tumors (total n ¼ 28; Supplementary Fig.S1). TheHRD andHRi tumors withoutmolecular explanation fortheir phenotype were subjected to BRCA1 and BRCA2 MLPAanalysis to identify large genomic rearrangements (LGR), as LGRsare not usually identified by targeted sequencing. In addition tothis targeted approach, morphologic examination of TILs andwhole exome sequencing (WES) was performed on a selection oftumors to further exploremolecular aspects connected to theHRDphenotype.

DNA isolationIsolation of DNA from 30 mm fresh frozen tissue section

samples was performed using the NucleoSpin Tissue Kit(Macherey-Nagel) according to the manufacturer's instructions.Quantity and quality checks of isolated DNA were performedusing theMultiNAmicrochip electrophoresis system (Shimadzu'sHertogenbosch), Nanodrop 2000-v.1 (Thermo Fisher Scientific),and Qubit (Thermo Fisher Scientific).

BRCA1/2 analysesIon semiconductor sequencing on the Ion Torrent Personal

S5XL was performed according to manufacturer's instructions(Thermo Fisher Scientific). Adapter-ligated libraries were con-structed using the AmpliSeq Library Kit 2.0 with ampliconsdesigned targeting BRCA1/2 and TP53. Generation of sequencereads, trimming adapter sequences, filtering, and removal ofpoor signal-profile reads was performed via the Ion Torrent

Translational Relevance

The functional RECAP test assesses HR capacity in freshtissue samples. This is a very accurate method for diagnosingHRD tumors, because the read-out is a dynamic process ratherthan a static genomic status. The main clinical implication ofthis study is that functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust andinterpretable results. Selection of tumors based on the HRDphenotype, instead of germline BRCAmutations, can identify50% more HRD tumors. Breast cancers with HRD phenotypeshowed an increased incidence of high TILs andmicrosatelliteinstability (MSI). This observation provides a basis to studywhether these specific subgroups of patients with breast cancerwould benefit not only fromPARP inhibitors (PARPi) but alsoimmunotherapy. Clinical trials have been initiated toevaluate the predictive value of the RECAP test for in vivoresponse to PARPi.

Meijer et al.

Clin Cancer Res; 24(24) December 15, 2018 Clinical Cancer Research6278




platform-specific pipeline software Torrent Suite v5.2.2. Initialvariant calling was performed by comparison to the referencegenome hg19 (build 37) using the "Torrent Variant Callerv5.2.0.3400 plug-in from the Torrent Suite Software. All BRCA2variants were validated by Sanger sequencing and pathogenicitywas evaluated using interactive Biosoftware Alamut Visualv.2.7.2. BRCA1 promoter methylation was analyzed as previ-ously described (15). Multiplex ligation-dependent probeamplification (MLPA) analysis of BRCA1 and BRCA2 wasundertaken to identify large rearrangements using the SALSAMLPA Kit P002B, and for confirmation of observed abnormal-ities, the SALSA MLPA Kit P087 was used (MRC Holland).Analyses were performed according to the manufacturer'sinstruction; products were run on an ABI automated sequencer(ABI 3730XL), and the data were analyzed by Genemarkerversion 2.7.0 (Softgenetics).

In situ detection of BRCA1 RNAIn situ detection of BRCA1 mRNA was performed using RNA-

Scope (Advanced Cell Diagnostics) on the automated VentanaDiscovery Ultra system (Ventana Medical Systems). BRCA1 andpositive control peptidylprolyl isomerase B (PPIB) probes (prod-uct codes: 485479 and 313909) were purchased from the samecompany. RNAscope analysis was performed according to man-ufacturer's instructions using the reagent kit (VS Reagent Kit320600;AdvancedCellDiagnostics)onproteinaseK(0.1%,5minat 37 �C)-treated paraffin sections (4 mm).

Exome sequencingDNA libraries for Illumina sequencing were generated using

standard protocols (Illumina) and subsequently sequenced in anIllumina HiSeq 2500 system by GATC-biotech. Exome-targetingwas performed using the Sureselect v5 (v6 for tumors M077,M209, M211) methods using standard protocols (Agilent Tech-nologies). DNA libraries were whole-exome sequenced (2 �125bp) using the HiSeq v4 paired-end sequencing protocol toa minimum depth base coverage of 90� for tumor samples and60� for matched normal. Sequence reads were mapped againsthuman reference genome GRCh37 using Burrows–WheelerAligner (v0.7.12) with default settings (16). Sequence readsoriginating from multiple lanes were merged after alignmentusing Samtools (v1.5) prior to further analysis (17). Sequenceduplicates were marked using PicardTools (v1.129; ref. 18).Somatic variant calling was performed by Mutect2 (v3.7) usingamatched-normal designwhile utilizing the dbSNP (v149, hg19)and COSMIC (v80, hg19) databases and using default settings(19–21). Variant annotation was performed by ANNOVAR (22).Heuristic filtering removed variants not passing all standardMutect2 post-calling filters. Sequence data have been depositedat the European Genome-phenome Archive (EGA, http://www.ebi.ac.uk/ega/), which is hosted by the EBI, under accessionnumber EGAD00001003929.

Mutational signaturesFor each somatic variant, its trinucleotide context was derived

from the human reference genomeGRCh37 and enumerated intoamutational spectrummatrixMij (i¼96; number of trinucleotidecontexts; j ¼ number of samples) using the MutationalPatterns Rpackage (v1.4.0) in the R statistical platform (23). Multi-allelicand InDel variants were not included in this analysis. The 30consensus mutational signatures, as established by Alexandrov

and colleagues, (matrix Sij; i¼96; number of trinucleotidemotifs;j ¼ number of signatures) were downloaded from COSMIC (asvisited on 8-11-2017; ref. 24). Per sample, a constrained linearcombination of the 30 validated mutational signatures wasconstructed, which reconstructs the sample-specific mutationalspectrum, using nonnegative least squares regression imple-mented in the R package pracma (v1.9.3). Signatures withlower relative contribution than 3% were summarized into a"Filtered" category.

MSI analysis, MMR protein IHC, and MLH1 promotormethylation assay

These analyses were performed as previously described (25).

Tumor-infiltrating lymphocytesTumor-infiltrating lymphocytes (TIL) were scored on HE

stained sections, according to the consensus by the InternationalTILs Working Group 2014 (26).

Statistical analysisStatistical analyseswere all two-sided andperformedusing IBM

SPSS statistics v21. Significance was calculated by Fisher exact testfor categorical data, by Mann–Whitney test for continuous data,and by exact binomial test for the incidence of MSI. P-values of<0.05 were considered significant.

ResultsEx vivo functional RECAP test

A total of 170 samples were subjected to the RECAP test(Supplementary Fig. S1; ref. 14). In 125 of 170 (74%) primarybreast cancer tissues RECAP was completed successfully (Fig. 1).In all cases, the reason for failure (n¼ 45)was lack of proliferatingtumor cells. No differences in clinicopathologic characteristicsbetween tumors that yielded successful versus nonsuccessful testswere observed (Supplementary Table S1). The first 41 patientswere also included in our previous cohort. Here, we show thatexecution of a functional assay in a pseudo-diagnostic setting isachievable and validate the findings from the earlier cohort (11%HRD in cohort 1 vs. 19%HRD in cohort 2, P¼ 0.339). In total, weidentified 95 (76%) HRP, 24 (19%) HRD, and 6 (5%) HRisamples (Fig. 1). BothHRi andHRD tumorsweremore frequentlyTN (P < 0.001) and Bloom and Richardson (B&R) grade 3(P < 0.001) than HRP tumors. Also, HRD tumors had a largersize (P ¼ 0.050) and were never B&R grade 1 (SupplementaryTable S2).

Identification of BRCA defects in HRD breast cancersPathogenic BRCA2mutations were found in six (6/23 ¼ 26%)

HRD samples, but not in the tested HRP samples (Table 1). In sixtumors (twoHRD, oneHRi, and threeHRP),BRCA2 variants weredetected that were classified as benign. We did not identify anyBRCA1 point mutations. Next, BRCA1 promoter hypermethyla-tion was detected in six (6/23 ¼ 26%) HRD tumors and in oneHRi tumor, but not in testedHRP samples (Table 1). Interestingly,all six HRD tumors displaying BRCA1 promoter hypermethyla-tion were TNBCs (Table 1; Supplementary Table S3). Vice versa, ofthe sixHRD tumors harboring a pathogenic BRCA2mutation, fiveshowed hormone receptor positivity.

Thus, 12/23 HRD and 1/6 HRi samples were explained bya BRCA2 mutation or BRCA1 promoter hypermethylation. Sub-sequently, we proceeded with an MLPA analysis for BRCA1 and

Functional Assay Reveals HRD in Breast Cancer Beyond BRCA

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BRCA2 on the 16 HRD and HRi samples that remained unex-plained to identify possible LGRs. Large BRCA1 deletions werefound in four samples and BRCA2 deletions in two samples(Table 1), of which one tumor harbored both a BRCA1 andBRCA2 deletion. Two tumors [M248 (HRD) and M112 (HRi)]showed extensive chromosomal instability as they containeda mosaic BRCA1 deletion (meaning the deletion was present ina subclone of the tumor) and a BRCA2 duplication (Table 1). Intotal, BRCA defects have thus been identified in 16/23 HRD and2/6 HRi samples.

Silencing of BRCA1 was validated in tumors with BRCA1promoter hypermethylation (n ¼ 7) and BRCA1 LGRs (n ¼ 4)by RNAscope in situ RNA hybridization (SupplementaryFig. S2; Table 1). All tumors displaying BRCA1 promoterhypermethylation showed absence of BRCA1 RNA, except fortwo heterogeneous samples that contained BRCA1 positive andnegative areas (Supplementary Fig. S2). As expected, the BRCA1deletion in tumor M094 led to a total absence of BRCA1mRNA(Supplementary Fig. S2). The mosaic BRCA1 deletions did notresult in complete BRCA1 silencing (Supplementary Fig. S2).

M1

41

M1

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BRCA2 mutation

BRCA1 promotor methylation

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MSI

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ER+

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32

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56

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06

M2

11

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70

M0

93

M0

77

M2

60

M2

71

RECAP negative

RECAP intermediate

Yes

No

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MSI mutational signature

Not performed

Figure 2.

Characteristics of HRD and HRi tumors. Overview of molecular alterations and TIL counts in each HRD or HRi tumor (NB, data for one HRD tumor is absent,because DNA was unavailable). ER, estrogen receptor. High TILs were defined as >10% TILs.

Figure 1.

RECAP test results: 19% of primary breast cancersshowed HRD. A, Schematic representation ofthe primary breast cancers obtained forRECAP testing. B, Percentage of RAD51 foci positivetumor cells among geminin-expressing nuclei in the125 successful tests. A total of 95 breast cancersamples were HRP (>50% Geminin positive cells withRAD51 foci), 24 were HRD (<20% Geminin positivecells with RAD51 foci), and six samples were HRi(>20%/<50%Geminin positive cellswith RAD51 foci).Black dots indicate TNBCs.

Meijer et al.





Neither did the BRCA1 deletion in M232, however thistumor also harbored a BRCA2 deletion that can explain theHRD phenotype.

After thorough analysis of the BRCA genes using severaltechniques, 13/23 HRD and 1/6 HRi samples could beexplained by deficiencies in BRCA. Moreover, 3/23 HRD(M131, M277, and M248) and 1/6 HRi (M112) tumors alsoharbored BRCA deficiencies (BRCA1 promoter methylation ormosaic BRCA1 deletions), which explain the HRi and partiallythe HRD phenotype, as these tumors showed heterogeneousBRCA1 mRNA expression. Finally, 7/23 HRD and 4/6 HRitumors did not show any BRCA defects and therefore remainedunexplained (Fig. 2). To further characterize these HRD tumors,functional features correlating with the HRD phenotype weredetermined.

HRD tumors show more tumor infiltrating lymphocytes thanHRP tumors

Recently, a subgroup of patients with TNBCwas identified whoshowed good response to immune checkpoint inhibition throughprogrammed death-ligand 1 (PD-L1) blockade (27, 28). Thissubgroup was characterized by having >10% TILs and high CD8lymphocyte counts in the tumor centers (28). Because 11/17TNBCs in our study showed HRD, we hypothesized that theRECAP test might select for a specific subgroup of patients withTNBC who might benefit from PD-L1 therapy. We found thatsignificantly more HRD tumors (6/22) had >10% TILs than HRPtumors (0/28) (P¼ 0.004; Fig. 3). Also, tumors with BRCAdefectsshowed more frequently >10% TILs compared with non-BRCAtumors (P ¼ 0.001), which was also true for TNBCs comparedwith ER/PRþ tumors (P ¼ 0.026).

Table 1. Overview of samples with BRCA defects

BRCA2 mutationBRCA1 promotorhypermethylation(score)

BRCA1 mRNARNAscope BRCA LGRs Tumor % RECAP statusSample Mutation Pathogenicity

M057 c.517G>Cp.G173R

Pathogenic $ >50% Negative (HRD)

P002 c.7617þ1G>T Pathogenic 60% Negative (HRD)M096 c.7285G>T

p.E2429XPathogenic $ 50–70% Negative (HRD)

M188 c.3846_3847delp.T1282fs

Pathogenic Macrodissected(FFPE)

Negative (HRD)

M231 c.755_758delp.D252fs

Pathogenic 65% Negative (HRD)

M275 c.3269delTp.M1090fs

Pathogenic 33% Negative (HRD)

M213 c.1708A>Cp.N570H

Benign >50% Positive (HRP)

M114 c.6829T>Cp.L2277L

Benign 40% Positive (HRP)

P9 c.3445A>Gp.M1149V

Benign 50% Positive (HRP)

M211 c.5054C>Tp.S1685L

Benign � (0.02) Positive >50 Negative (HRD)

M106 c.6347A>Gp.H2116R

Benign � (0.01) Positive 50–70 Negative (HRD)

P001 þ (NA) Negative — Negative (HRD)M028 þ (NA) Negative — Negative (HRD)M119 þ (0.56) Negative 50–70 Negative (HRD)M131 þ (0.82) Heterogeneous >70 Negative (HRD)M182 þ (0.45) Negative >50 Negative (HRD)M277 þ (0.24) Heterogeneous 20 Negative (HRD)M141 þ (0.29) Negative 50 Intermediate (HRi)M094 � (0.01) Negative BRCA1 deletion 50–70 Negative (HRD)M232 � (0.02) Positive BRCA 1 þ 2 deletion >70 Negative (HRD)M248 � (0.01) Positive Mosaic BRCA1 deletion,

BRCA2 duplication50–70 Negative (HRD)

M112 c.6935A>Tp.D2312V

Benign � (0.01) Positive Mosaic BRCA1 deletion,BRCA2 duplication

50 Intermediate (HRi)

M156 � (0.01) Positive BRCA2 deletion >70 Negative (HRD)M093 � (0.01) Positive WT >70 Negative (HRD)M260 � (0.01) Positive WT 30 Negative (HRD)M270 � (0.01) Positive WT 50–70 Negative (HRD)M271 � (0.01) Positive WT 30 Negative (HRD)M077 � (0.01) Positive WT >50 Negative (HRD)M253 � (0.01) Positive WT 50 Intermediate (HRi)M209 � (0.01) Positive WT 50 Intermediate (HRi)M055 � (0.01) Positive WT 50–70 Intermediate (HRi)M278 � (0.01) 10 Intermediate (HRi)

NOTE: Methylation score between 0.0 and 1.0, cut-off for presence of promotor hypermethylation is >0.2. $ Mutations were somatic.






HRD tumors show mutational signatures related to BRCAdeficiencies and microsatellite instability

Next, WES was performed to determine the molecular land-scape ofHRD tumors. A selection ofHRD (n¼ 8) andHRi (n¼ 3)tumors with BRCA1/2 mutations/deletions, BRCA1 promoterhypermethylation, and BRCA WT tumors and one HRP tumorwere subjected to WES. First, mutational load was determined inthese tumors and high mutational load did not correlate withhigh numbers of TILs. Second, we did not identify commonlymutated genes other than BRCA1/2, which might explain thefunctional HR defect. Third, WES data were used to identifymutational signatures which are specific combinations of muta-tions that arise due to a certain underlying mutational or DNArepair processes (29).

Mutational signatures were derived from the WES data fromHRD/HRi tumors of which matching normal DNA wasavailable to filter out germline variants (n ¼ 10) to explore novelmechanisms related to or underlying the HRD phenotype.Because discussion in the field exists that mutational signaturescan only be faithfully obtained from WGS instead of WES data,we first carried out a pilot experiment comparing mutationalsignature analysis using all somatic mutations in five BrC WGSdatasets (30) and filtered these WGS datasets to only containsomatic mutations on exonic regions. Both methods resulted insimilar distributions of the mutational signatures (Supplemen-tary Fig. S3).

Mutational signature 3 is related to failure of DSB repair by HRand associated with germline and somatic BRCA1/2 defects in

breast, pancreatic, and ovarian cancers (31). Signature 3 waspresent in 6 of 10 analyzed samples (M94, M95, M119, M131,M141, and M221; Fig. 4). APOBEC-related mutagenesis (pre-dominantly C>G or C>T substitutions in TCA or TCT motifs) iscaptured in signatures 2 and 13,which arise through activity of theAID/APOBEC family. BRCA-related signatures could also beidentified to a lesser extent in samples (M211 and M094) havinga high mutational burden of signatures 2 and 13.

Microsatellite instability in HRD breast cancersOne tumor (M077) showed a high mutational load and

high contributions of signatures 15, 20, and 26, which arerelated to microsatellite instability (MSI) and mismatchrepair (MMR) deficiency (http://cancer.sanger.ac.uk/cosmic/signatures). MSI is a common feature in endometrial andgastro-intestinal cancers, caused by either germline (Lynchsyndrome) or somatic mutations in one of the MMR genesand/or promoter hypermethylation (32, 33). Presence of MSIin tumor M077 was confirmed using pentaplex PCR and IHCfor the MMR proteins, which showed absence of MLH1 andPMS2, caused by MLH1 promoter methylation (Supplemen-tary Fig. S4).

A set of 44 tumors (20 HRD, 6 HRi, 18 HRP) was subjected toMSI analysis by pentaplex PCR and IHC for the MMR proteins.One HRD tumor (M188) of which mutational signatures werenot available, also showed MSI. MSI was never detected inany HRP tumors (Fig. 5). Tumor M188 showed absence ofMSH2 and MSH6, caused by a homozygous deletion of MSH2

A

B P = 0.004 P = 0.026

<1% TILs 15% TILs 70% TILs

100 µm

P = 0.001

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ILs

TNBC0

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ILs

Figure 3.

HRD tumors more frequently showed >10% tumor infiltrating lymphocytes than HRP tumors. A, HE sections were scored for stromal TILs. Examples ofsamples with <1%, 15%, and 70% TILs are shown respectively. B, More HRD tumors (6/22) had >10% TILs than HRP tumors (0/28; P ¼ 0.004). Also, tumorswith BRCA defects showed more frequently >10% TILs compared with non-BRCA tumors (P ¼ 0.001), which was also true for TNBCs compared toER/PRþ tumors (P ¼ 0.026). Significance was calculated by Fisher exact test.

Meijer et al.




http://cancer.sanger.ac.uk/cosmic/signatures

http://cancer.sanger.ac.uk/cosmic/signatures


(Supplementary Fig. S4). The two MSI BrCs (M077 and M188)were TN and BRCA WT and ER positive and BRCA2 mutated,respectively (Fig. 2). The incidence of MSI within the HRDgroup (2/20, 10%) is significantly higher than the incidence ofMSI in the unselected breast cancer population (1%; P ¼ 0.017;refs. 34, 35).

DiscussionHere, a unique series of fresh primary breast cancer tissues

(n ¼ 125) has been analyzed for HRD using the functionalRECAP test. This first large validation study describes thatfunctional assessment of HR in a pseudo-diagnostic setting isachievable and produces robust and interpretable results formost patients (74%). We found that the percentage of HRDtumors detected by the RECAP test is similar in this largercohort, as compared with our previous report. Therefore, bothcohorts were combined to achieve more power to thoroughlyinvestigate the molecular mechanism underlying the HRDphenotype. Sixteen HRD samples showed deficiencies in

BRCA1/2 (BRCA mutations, deletions, or promoter hyper-methylation), whereas seven HRD tumors were non-BRCArelated, demonstrating that HRD tumors encompass morethan only BRCA-deficient tumors.

Several different HRD tests have been designed to identifyHRD tumors in addition to the BRCA mutated or promotermethylated tumors to enlarge the population of patients withbreast cancer that could benefit from treatments targeting theHR pathway. For example, the BRCAness classifier, which isbased on specific genomic patterns derived from copy numberdata of BRCA1/2 mutated breast cancers that also occur insporadic cancers (10, 11) and the Myriad MyChoice HRD test,which is a combined score of three different structural chro-mosomal aberrations [telomeric allelic imbalance (TAI), large-scale transition (LST), and loss of heterozygosity (LOH);ref. 36]. Both the BRCAness classifier and MyChoice are robust,easily applicable in the clinic, and have also been validated topredict in vivo response to high dose chemotherapy and neo-adjuvant platinum-based therapy, respectively, in patients withTNBC (37, 38). As opposed to the neo-adjuvant setting, the

0

25

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75

100

0

1,000

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Figure 4.

HRD and HRi tumors showed mutationalsignatures related to BRCA and MSI. Relative andabsolute contribution of the mutational signatures inWES datasets is depicted, and total numbers ofsingle-nucleotide mutations and percentage ofTILs in the sample.






MyChoice HRD test did not predict response to PARPi therapyin platinum-sensitive recurrent ovarian cancer (39). Thesegenomic HRD tests have the drawback that they do not deter-mine the real-time HR status, also it remains unclear whetherall HRD cases are identified. In theory, the functional RECAPtest can also detect reversion of the HRD phenotype in BRCA-deficient tumors, that have been treated with various DNAdamaging chemotherapies that may have induced resistance.Moreover, the BRCAness classifier focuses on TNBC only,whereas the RECAP test identifies HRD independent of hor-monal status. More recently, a HRD test based on severalgenomic signatures has been published, HRDetect (40). Thistest has recently been shown to predict in vivo response toplatinum-based therapies in advanced breast cancer (41).HRDetect relies on whole genome sequencing and is thereforemore expensive and has a longer turnaround time for biopsyresults, hampering its clinical implementation. Furthermore,the tumor cell percentage needs to be above 50% for reliableresults, which is not a prerequisite for the RECAP test. How-ever, HRDetect has the advantage that it can be performedon frozen material, whereas the RECAP test requires freshmaterial. The percentage of non-BRCA HRD tumors (approx-imately 33%) detected by the HRDetect and the RECAP test arequite comparable, although it remains elusive whether these

tests identify the same tumors, therefore comparison of severalHRD tests within the same patient cohort is required.

The major strength of this study is that functional diagnos-tics have been applied to a unprecedented large collection oftumors. The advantage of the RECAP test over genetic tests, isits functional character for exploring the HR phenotype ratherthan the static nature of genomic tests. Also, the RECAP test isfeasible in samples with low tumor percentage, because themicroscopic read-out allows differentiation between tumorand stromal cells. The RECAP test has a high success rate andresults are available within one week after the biopsy proce-dure. In this study, reproducibility and robustness of theRECAP test is validated in an independent set of 129 tumors.For the molecular analyses to unravel the mechanisms under-lying the HRD phenotype, the 41 samples included in ourprevious publication were also included, to achieve morepower. The main limitation of this study is that althoughprospective trials evaluating the predictive value of RECAP forin vivo patient response to PARPi have been initiated, resultsare not yet available. However, previously a functional RAD51test performed on biopsies obtained from patients 24 hoursafter start of therapy correlated with response to anthracycline-based therapies, indicating that functional assessment of HRcan have predictive value for therapy response (13).

B

M077

MSH2 MSH6 MLH1 PMS2

100 µm

M188

A MSIn = 2

HRDn = 20

HRPn = 18

HRin = 6

Figure 5.

Two HRD breast tumors showed MSI. A, MSI was found in two HRD tumors, but not in any HRP or HRi tumors. B, IHC staining of MLH1, PMS2, MSH2, and MSH6.

Meijer et al.





Among the spectrum of BRCA defects, we have not identifiedany BRCA1mutations. This is somewhat remarkable but could bedue to a selection bias, as the Erasmus Medical Center is special-ized in hereditary BrC and all patients with TNBC are tested,therefore most families with hereditary BRCA1 mutations havebeen identified and carriers are offered strict screening programsor undergo prophylactic surgery in The Netherlands (42). Also, inthis study there is a selection bias for tumor size, as the tumorshould be large enough to provide residual material withoutcompromising standard diagnostic procedures. Since BRCAmutation carriers are offered strict screening programs, tumorsare often identified at an early stage and residual material is notavailable for the RECAP test. Also, many BRCA1mutation carrierswith TNBC are treated with neoadjuvant chemotherapy, whichwas an exclusion criterion for this study.

Clinical consequences of BRCA1 promoter methylation areunclear (43). In the current study, BRCA1 promoter methyl-ation resulted in absence of BRCA1 RNA in four samples, butin two tumors there was still heterogeneous expression ofBRCA1 RNA (Supplementary Fig. S2). In these tumors, thepercentage of cells with RAD51 foci was 1% and 2%, respec-tively. The discrepancy between the very low HRD score andheterogeneous BRCA1 RNA could be explained by samplingfrom different areas of the tumor, since the tumor sample forthe RECAP test was irradiated and an unirradiated tumorsample was used for molecular analyses. This sampling erroris not limited to this study, but also occurs in regular diag-nostics when biopsies are obtained from a certain region of aheterogeneous tumor. Tumor heterogeneity in BRCA1 promot-er methylated tumors is very important for clinical decisionson PARPi use. If subsequent studies reveal that thisphenomenon is observed in a large fraction of these tumors,PARPi may not be very effective in tumors with BRCA1 pro-moter methylation.

We identified 6 HRi BrCs in the current cohort. Only in one ofthe HRi tumors, distinct areas of RAD51 negative and RAD51positive tumor cells were observed, suggesting clonal heteroge-neity. The other HRi tumors all showed interspersed RAD51negative as well as RAD51 positive tumor cells. As a BRCA defectwas found in 2/6 HRi tumors, they biologically resemble HRDtumors. However, whether HRi tumors benefit from PARPi treat-ment remains to be elucidated.

Mutational signature analyses were performed to explorenovel mechanisms related to or underlying the HRD pheno-type. One HRD tumor that showed a large contribution of threesignatures related to MSI and MMR deficiency proved to betruly MSI. Using pentaplex PCR and IHC, MSI was discovered intwo HRD (2/20, 10%) but not in HRP or HRi tumors. Thisincidence is much higher than in the unselected breast cancerpopulation (1%; refs. 34, 35), suggesting that the RECAP testmay also identify MSI tumors. The relation between MSI andHRD as well as the order in which tumors develop thesedeficiencies remains unclear and future research is required.We hypothesize that either MSI tumors acquire HRD over timedue to accumulation of mutations in genes involved in HR(44), or HRD tumors acquire MSI at a later stage of tumordevelopment as a compensatory mechanism, to lower replica-tion fork instability by not repairing mismatches but rathercontinuing DNA replication. As MSI tumors have many neo-antigens, PD-L1 blockade therapy showed antitumor activity in

phase I trials (45). Recently, a first report of a patient with MSIBrC showing a profound response to PD-L1 blockade waspublished (46). Moreover, tumors with high numbers of TILsare generally more sensitive to immunotherapy (47). Interest-ingly, in our cohort, the two MSI tumors comprise a differentsubset of HRD tumors than the ones with high TILs. Thus, theRECAP test identifies not only tumors with BRCA defects (n ¼16), but also a subgroup of breast cancers that might respondwell to immunotherapy due to either MSI (n ¼ 2) or high TILcounts (n ¼ 6).

The RECAP test is a robust functional HR assay, detecting bothBRCA1/2-deficient and BRCA1/2-proficient HRD tumors. Func-tional assessment of HR in a pseudo-diagnostic setting is achiev-able and produces robust and interpretable results. Clinical trialsevaluating the predictive value of the RECAP test for in vivoresponse to PARPi have been initiated.

Disclosure of Potential Conflicts of InterestH.J.Dubbink reports receiving commercial research grants fromAstraZeneca.

W.N.M.Dinjens reports receiving speakers bureau honoraria fromRoche, and isa consultant/advisory board member for Amgen. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: T.G. Meijer, N.S. Verkaik, K.A.T. Naipal, A. Jager,D.C. van GentDevelopment of methodology: W.N.M. Dinjens, P.M. Nederlof,H.J G. van de Werken, J.W.M. Martens, A. Jager, D.C. van GentAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.G. Meijer, N.S. Verkaik, A.M. Sieuwerts,K.A.T. Naipal, C.H.M. van Deurzen, M.A. den Bakker, H.-J. Dubbink,T.D. den Toom, W.N.M. Dinjens, E. Lips, P.M. Nederlof, A. JagerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.G. Meijer, J. van Riet, H.-J. Dubbink,T.D. den Toom, W.N.M. Dinjens, E. Lips, P.M. Nederlof, M. Smid,H.J G. van de Werken, J.W.M. Martens, A. Jager, D.C. van GentWriting, review, and/or revision of the manuscript: T.G. Meijer, N.S. Verkaik,A.M. Sieuwerts, K.A.T. Naipal, C.H.M. van Deurzen, H.-J. Dubbink,W.N.M. Dinjens, E. Lips, P.M. Nederlof, M. Smid, H.J G. van de Werken,R. Kanaar, J.W.M. Martens, A. Jager, D.C. van GentAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.M. Sieuwerts, J. van Riet, H.F.B.M. Sleddens,A. JagerStudy supervision: R. Kanaar, A. Jager, D.C. van Gent

AcknowledgmentsThe authors thank many colleagues from the Departments of Molecular

Genetics, Medical Oncology, and Pathology at Erasmus MC as well as fromthe Maasstad Hospital, who contributed to the collection of patient material.We thank Lindsey Oudijk for her assistance with the TILs scoring. We thankRonald van Marion for expert technical assistance. D.C. van Gent, A. Jager,and R. Kanaar have received funding from the Dutch Cancer Society (Alped'Huzes grant number EMCR 2014-7048 and grant number EMCR 2008-4045). This work is part of the Oncode Institute, which is partly financed bythe Dutch Cancer Society and was funded by the gravitation programCancerGenomiCs.nl from the Netherlands Organisation for ScientificResearch (NWO).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 8, 2018; revised May 17, 2018; accepted August 17, 2018;published first August 23, 2018.






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Correction

Correction: Functional Ex Vivo Assay RevealsHomologous Recombination Deficiency inBreast Cancer Beyond BRCA Gene DefectsTitia G. Meijer, Nicole S. Verkaik, Anieta M. Sieuwerts,Job van Riet, Kishan A.T. Naipal, Carolien H.M. van Deurzen,Michael A. den Bakker, Hein F.B.M. Sleddens,Hendrikus-Jan Dubbink, T. Dorine den Toom,Winand N.M. Dinjens, Esther Lips, Petra M. Nederlof,Marcel Smid, Harmen J.G. van de Werken, Roland Kanaar,John W.M. Martens, Agnes Jager, and Dik C. van Gent

In the original version of this article (1), 28 of 170 tumors were misclassified. Theauthors have updated the article text, Fig. 1, and Supplementary Table S3 to accuratelyreflect the decrease in positive tests from 148 to 125. The error has been corrected inthe latest online HTML and PDF versions of the article. The authors regret this error.

Reference1. Meijer TG, Verkaik NS, Sieuwerts AM, van Riet J, Naipal KAT, van Deurzen CHM, et al. Functional

ex vivo assay reveals homologous recombination deficiency in breast cancer beyond BRCA genedefects. Clin Cancer Res 2018;24:6277–87.

Published first May 1, 2019.doi: 10.1158/1078-0432.CCR-19-0936�2019 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 2935

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-19-0936&domain=pdf&date_stamp=2019-4-9

2018;24:6277-6287. Published OnlineFirst August 23, 2018.Clin Cancer Res Titia G. Meijer, Nicole S. Verkaik, Anieta M. Sieuwerts, et al. Deficiency in Breast Cancer Beyond BRCA Gene Defects

Assay Reveals Homologous RecombinationEx VivoFunctional

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