review articles

Homologous Recombination
Deficiency in Breast Cancer:
A Clinical Review

BRCA1 and BRCA2 germline mutation–associated breast cancers are known to be deficient in the
abstract

process of homologous recombination and often respond favorably to drugs targeting this
important DNA repair pathway. There is emerging evidence that a significant proportion of
patients with BRCA1/BRCA2 wild-type breast cancer are also deficient in homologous re-
combination, and it is hypothesized that these patients may derive similar benefit from drugs
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targeting this pathway. Current research has focused on the development of a companion di-
Copyright © 2024 American Society of Clinical Oncology. All rights reserved.

agnostic to identify these sporadic BRCA-like tumors. This review outlines the various ap-
proaches that researchers have taken to predict homologous recombination deficiency as part of
correlative biomarker work in various studies and clinical trials in breast cancer. As some of these
tests of homologous recombination deficiency move closer to clinical use, understanding the
approach and limitations of each is of relevance to clinicians who treat patients with breast cancer.
Precis Oncol 00. © 2017 by American Society of Clinical Oncology

INTRODUCTION The role of BRCA1/BRCA2 in double-strand DNA

Wendie D. den Brok
Kasmintan A. Schrader
Sophie Sun
Anna V. Tinker
Eric Yang Zhao
Samuel Aparicio
Karen A. Gelmon
Wendie D. den Brok,
Sophie Sun, Anna V.
Tinker, Samuel Aparicio,
and Karen A. Gelmon, BC
Cancer Agency; and
Kasmintan A. Schrader
and Eric Yang Zhao,
University of British
Columbia, Vancouver,
British Columbia, Canada.
Corresponding author:
Wendie den Brok, MD,
FRCPC, Department of
Medical Oncology,
BC Cancer Agency, 600
West 10th Ave, Vancouver,
British Columbia, Canada
V5Z 4E6; e-mail:
wdenbrok@bccancer.bc.ca.
DNA repair pathways represent a tightly con-
trolled network protecting cells from exogenous
and endogenous DNA damage. These pathways
are frequently aberrant in cancer cells, leading to
the accumulation of DNA damage and genomic
instability. The BRCA1 and BRCA2 genes were
discovered in 19941 and 1995,2 respectively, and
are associated with hereditary breast and ovarian
cancers. BRCA1 and BRCA2 play an integral role
in the process of DNA repair as part of the Fanconi
anemia/BRCA DNA damage response pathway.
This highly conserved pathway is involved in
double-strand DNA break repair by the process
of homologous recombination (HR). The base
excision repair pathway is a second highly con-
served repair process involved in single-strand
DNA breaks. Poly (ADP-ribose) polymerase
(PARP) enzymes play an important role in this
pathway. Recent evidence demonstrates that
PARP is important for resolving stalled repli-
cation forks, and its inhibition during base ex-
cision repair requires BRCA-dependent HR to
resolve.3,4 Targeting DNA damage response
pathways has emerged as an attractive strategy
to destabilize tumor genomic integrity and
trigger genomic catastrophe and cell death in a
tumor-specific fashion.
repair via HR has been well documented,5 and
there is mounting evidence that breast cancers
arising in BRCA1/BRCA2 germline mutation car-
riers respond favorably to therapies that target
DNA repair pathways, such as platinum salts and
PARP inhibitors (PARPis).6-8 Hereditary BRCA1/
BRCA2 mutations account for up to 7% of all
unselected breast cancers,9 increasing to 11% to
15% of unselected individuals with triple-negative
breast cancer (TNBC: estrogen receptor [ER],
progesterone receptor [PR], human epidermal
growth factor receptor 2 [HER2] –negative) and
as high as 50% with a strong family history.10-12
BRCA1 mutations are most commonly associated
with the TNBC clinical subtype, whereas BRCA2
mutations are more frequently associated with ER-
positive tumors. Some sporadic (ie, BRCA1/BRCA2
wild-type [WT]) breast cancers also harbor defects
involving the HR pathway and respond similarly to
platinum salts. It is also hypothesized that some
sporadic breast cancers with defects in the HR
pathway may benefit from the addition of PARPis
to standard therapies. These sporadic breast tumors
are commonly referred to as being BRCA-like and
are often associated with TNBC. It is estimated that
up to 40%13 of familial and sporadic breast cancers
are HR deficient (HRD).

1 ascopubs.org/journal/po JCO™ Precision Oncology

© 2017 by American Society of Clinical Oncology

 A Mutations
Normal TTA GAG TGT
Tumor TTA A AG TGT
B Promoter methylation
D TAI
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Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
Fig 1. Genomic
mechanisms associated
with homologous
recombination (HR)
deficiency (HRD). (A)
Somatic mutations,
including substitutions and
small insertions/deletions
in key HR-related genes,
such as BRCA1 and BRCA2,
can be associated with
HRD. (B) Genes important
to HR can also be silenced
epigenetically through
promoter methylation,
resulting in repressed
expression. (C) Frequent
copy number alteration
(CNA) is a hallmark of
HRD and can be observed
as regional or whole-
chromosome deletions/
amplifications. (D)
Quantification of large-
scale structural variants has
been used as an indicator of
an HRD phenotype. These
include telomeric allelic
imbalance (TAI; large
allelic imbalances
extending into a telomere),
large-scale transition (LST;
number of transitions
between large regions of
differing allelic states), and
loss of heterozygosity
(LOH; large regions
displaying somatic loss of
one haplotype, which can
be copy variable as in
deletion LOH or copy
neutral).
2 ascopubs.org/journal/po JCO™ Precision Oncology
C Frequent CNA
Substitution Deletion Insertion Normal
TTA GAG TG GAG TGT C
TTA GTG GAG AC T GTC
BRCA1
LST
Normal Tumor Normal Tumor
Loss Gain Neutral
Current research has focused on the develop-
ment of a companion diagnostic to identify
sporadic BRCA-like tumors that would allow
clinicians to identify those patients who may
benefit from drugs targeting DNA repair path-
ways and to spare those who are unlikely to
benefit. This review outlines the methods cur-
rently available for identifying HRD tumors,
with an emphasis on studies and clinical trials in
breast cancer, and discusses potential clinical
implications.
APPROACH TO HRD
Tests of HRD focus on either the detection of
the underlying driver mutations responsible for
the HR defect or the resultant mutational land-
scape of deficient HR inferred by nonspecific
collateral damage to the genome (Fig 1). Driver
germline (inherited) or somatic (acquired) mu-
tations may take the form of sequence or struc-
tural variants that generally result in loss of
function or aberrant functioning of BRCA1/
BRCA2 or other genes encoding members of
the HR pathway. Epigenetic changes, such as
BRCA1 promoter methylation, can also occur
somatically. 14
TESTS OF DRIVER MUTATIONS
Sequence variants (or mutations) include substitu-
tions, deletions, or insertions of nucleotides , 1 kb
(Fig 1A). Those that occur within genes may
result in pathogenic protein abnormalities. The
genes and their protein products involved in
HR are numerous, and their interactions are
complex.
Tumor
Regional Whole-chromosome
CNAs amplifications
LOH
Normal Tumor
Deletion Copy neutral
Germline Mutations in HR-Associated
Genes
In addition to germline BRCA1/BRCA2 muta-
tions, clinical genetic testing panels now include a
number of proposed breast cancer predisposition
genes, although not all of these genes have de-
finitively been shown to increase breast cancer
risk. Other hereditary predisposition genes in-
volved in HR that are proven to be moderate to
high risk include PALB2, ATM, and CHEK2.
More recently, BARD1 and RAD51D have been
shown to increase breast cancer risk, whereas
some genes (NBN, MRE11A, RAD50, RAD51C,
BRIP1) are unlikely or confirmed not to increase
breast cancer risk.15 Interestingly, some of these
HR genes do increase the risk of ovarian cancer,
suggesting that the underlying biology of breast
and ovarian cancer is different despite the im-
portance of the role of HRD in both of these
malignancies.
Although considered BRCA-like, it is not clear if
breast cancers arising from these germline mu-
tations are as sensitive to DNA-damaging ther-
apies as BRCA1/BRCA2-mutated breast cancers
in large part as a consequence of the rarity and
relative recent discovery of these mutations.16
Incidence and accurate risk estimates are emerg-
ing for non-BRCA germline mutations, but the
prognosis and response to anticancer therapy
remain unknown.15,17
Somatic Mutations in HR Genes
Somatic mutations may also arise in genes in-
volved in HR. Somatic mutations in BRCA1/
BRCA2 occur in approximately 2.5% of all
 sporadic breast cancers.18 It is hypothesized that
somatic BRCA1/BRCA2-mutated breast cancers
will respond similarly to DNA-damaging thera-
pies, but it is not definitively known if germline
and somatic BRCA1/BRCA2 mutations are bio-
logically equivalent. As in ovarian cancer, where
somatic mutations in BRCA1/BRCA2 occur in up
to 7% of patients,19 somatic BRCA1/BRCA2 mu-
tations constitute up to 3% of unselected patients
with breast cancer20 and likely contribute to the
BRCA-like phenotype, where DNA-damaging
therapy may also be effective. Next-generation
sequencing studies18,21 continue to expand the list
of genes involved in breast cancer, and this list
includes HR genes. The extent to which these HR
genes drive tumorigenesis continues to be ex-
plored, but it is likely that they too contribute
to the BRCA-like phenotype.
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Looking beyond germline BRCA1/BRCA2 muta-
Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
tions has implications in terms of choosing pa-
tients who stand to benefit from DNA-damaging
therapies, most notably PARPis. How this can be
achieved is not certain. For example, in ovarian
cancer, the PARPi niraparib resulted in a signif-
icant increase in progression-free survival irre-
spective of BRCA1/BRCA2 mutation status or
HRD status (by Myriad’s myChoice HRD, Salt
Lake City, UT) for patients with recurrent and
highly platinum-sensitive disease.22 Unlike in
ovarian cancer, PARPis in non–BRCA1/BRCA2-
mutated breast cancer have shown little activity to
date, likely highlighting a different underlying
biology, although patient selection may also
be a factor. It is possible that HRD status will
be a better predictor of PARPi response in breast
cancer. Early-phase clinical trials of PARPis in
metastatic TNBC and BRCA-WT HER2-negative
breast cancer are underway, with correlative
HRD assessment (ClinicalTrials.gov identi-
fiers: NCT02401347 and NCT00707707).
Gene Panels of HRD
As researchers began to understand the complex
nature of HRD, with contributions beyond
BRCA1/BRCA2 function, the focus shifted to
characterization of BRCA1-associated gene ex-
pression patterns to identify sets of differently
expressed genes. An early approach used a 69-
gene DNA repair microarray signature to analyze
tissue from 105 patients with sporadic TNBC
treated with neoadjuvant chemotherapy (fluoro-
uracil, epirubicin, cyclophosphamide; doxorubi-
cin, cyclophosphamide [AC], or taxane-based
chemotherapy) and found that the signature was
associated with higher pathologic complete
3 ascopubs.org/journal/po JCO™ Precision Oncology
response (pCR) rates in patients receiving
anthracycline-based treatment but relative resis-
tance to taxane-based treatment.23 The adaptive
Investigation of Serial Studies to Predict Your
Therapeutic Response through Imaging and Mo-
lecular Analysis I (I-SPY II) clinical trial24 assessed
two DNA repair deficiency signatures in patients
treated with the combination of chemotherapy
plus carboplatin and the PARPi veliparib (V/C)
versus standard chemotherapy in an attempt to
predict which patients harbor HRD: a 77-gene
BRCAness gene expression signature and a PARPi-7
signature (ie, 7-gene signature). Seventy-seven of
the 115 HER2-negative cohort were predicted to
have DNA repair deficiency by one of these sig-
natures or by the presence of BRCA1/BRCA2
germline mutations (56 of 59 TNBC and 21 of
56 ER positive). Of those treated on the V/C arm,
22 of 38 patients with TNBC and five of 13 patients
who were ER positive achieved a pCR. Although
the results of these biomarkers are promising, they
are exploratory. The concordance of the two sig-
natures was moderate at 64% (k = 0.29). Thus, the
genes involved in HR are not yet well defined, and
equal weighting may overestimate HRD, as seen
in a study of ovarian cancer.25 Similarly, predictive
model systems based on an empirical set of
genes differentially expressed in BRCA1/BRCA2-
mutated breast cancers are unlikely to clearly define
HRD. These caveats represent major limitations to
the HR panels currently being developed.
Epigenetic Changes
BRCA1 promoter methylation (PM), which is
unique to BRCA1 and mutually exclusive of germ-
line BRCA1 mutations,26 has also been implicated
in HRD (Fig 1B). Within breast cancer subtypes,
BRCA1 PM has been found to be almost exclusive
to TNBC.27
BRCA1 PM in sporadic TNBC has been reported
in two studies.28,29 The first was a retrospective
analysis on archival tumor samples from 39 pa-
tients receiving anthracycline-based chemother-
apy for stage I to III TNBC.28 BRCA1 PM was
identified in 30% of tumors and was associated
with significantly lower recurrence-free survival
(RFS) and overall survival (OS) after adjusting for
stage, a finding supported in a recent meta-analysis.30
The second study, a phase II clinical trial of 30
patients with stage II or III TNBC treated with
neoadjuvant platinum-based chemotherapy (car-
boplatin, docetaxel, erlotinib), assessed BRCA1
PM, low BRCA1 mRNA expression levels, and
germline BRCA1 mutation (30%, 15%, and
8%, respectively).29 Those patients with BRCA1
 deficiency as a result of any of these causes had
significantly improved OS compared with those
without BRCA1 deficiency. The importance of
BRCA1 PM alone in this study is difficult to in-
terpret, and results may be explained by defi-
ciencies in BRCA1 as a result of germline
mutations or by mRNA expression. Currently,
the role of BRCA1 PM in HRD remains
controversial.
Copy Number Aberrations
Copy number aberration/alteration (CNA) refers
to acquired changes in copy number of genes in
tissue, such as tumor, whereas copy number var-
iant (CNV) refers to changes in copy number of
genes in the germline, affecting all cells in an
individual. CNVs may also be reported in cancer,
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although they are usually qualified with the term
Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
acquired as compared with constitutional, so as to
differentiate between the somatic and germline
settings. In contrast to entire chromosome num-
ber gains or losses (ie, aneuploidy), CNA/CNVs
are on a much smaller, generally submicroscopic,
scale, with the size of DNA copy-number alter-
ations (gain or loss) being . 1 kb in length (Fig
1C). Although the extent to which CNAs contrib-
ute to tumorigenesis is not entirely known, some
of the well-established driver events in cancer are
CNAs (eg, Myc, HER2, Cyclin D1). Further-
more, an increased burden of CNAs is associated
with higher genomic instability and subsequent
malignant transformation.31 Using DNA micro-
array technologies, such as array comparative
genomic hybridization (aCGH), entire genome
imbalances can be identified. This technique com-
pares test DNA and control DNA, allowing the
relative fluorescent intensity to be quantified, pro-
viding information on relative copy number se-
quences in the test genome versus the control
genome.32
Clinically, aCGH was first reported using a clas-
sifier of BRCA1-like tumors on the basis of CNA
in BRCA1-mutated breast cancers.33 This study
included 230 patients with stage III HER2-
negative breast cancer randomly assigned to ad-
juvant high-dose platinum-based or anthracycline-
based chemotherapy. Eighteen percent of patients
had BRCA1-like disease, and those treated with
high-dose platinum had improved RFS (hazard
ratio [HR], 0.12; 95% CI, 0.04 to 0.43), whereas
patients with non–BRCA1-like disease treated with
high-dose platinum did not (HR, 0.78; 95% CI,
0.50 to 1.20). The difference between the two
groups was statistically significant (test for interac-
tion, P = .006). In a subset of TNBC tumors (n = 60),
4 ascopubs.org/journal/po JCO™ Precision Oncology
only eight of 13 known germline BRCA1 or BRCA2
mutations were inferred using this method. In
another study of adjuvant high-dose ifosfamide,
epirubicin, and carboplatin chemotherapy versus
standard chemotherapy in 117 patients with high-
risk breast cancer (stage II or III, node positive), the
aCGH classifier identified 11 of 68 (19%) as having
BRCA1-like disease in the high-dose alkylator-
based arm and five of 49 (10%) as having
BRCA1-like disease in the standard treatment
arm. 34 BRCA1-like tumors treated with high-
dose platinum-based chemotherapy had better
disease-free survival (HR, 0.05; 95% CI, 0.01 to
0.38), distant disease-free survival (HR, 0.06;
95% CI ,0.01 to 0.43), and OS (HR, 0.15; 95%
CI, 0.03 to 0.83). All were statistically significant,
and test for interaction was positive.
Studies of non–platinum-containing regimens
have not shown a benefit in patients with
BRCA1-like signatures identified by this method.
A study in patients with TNBC treated with
adjuvant AC; FEC; docetaxel, doxorubicin, cyclo-
phosphamide; or cyclophosphamide, methotrex-
ate, fluorouracil chemotherapy found that 65%
had BRCA1-like disease but with no statistically
significant difference in 5-year RFS in any of the
treatment arms.35 Similarly, a neoadjuvant study
of dose-dense AC or capecitabine/docetaxel in
163 HER2-negative breast cancers showed tu-
mors identified as BRCA1-like by this method
were not predictive of chemotherapy response
even though present in 57% of patients with
TNBC.36
These studies suggest that BRCA1-like tumors, as
defined by aCGH, are most commonly associated
with TNBC, and platinum-containing regimens
are beneficial in those with a BRCA1-like signa-
ture, whereas non–platinum-containing regimens
fail to improve responses over those without a
BRCA1-like signature. However, the above stud-
ies used unusual regimens of high-dose platinum
and intensified alkylating agents that are not stan-
dard of care, and the response seen in the BRCA1-
like group may be due to intensified alkylator
therapy. The aCGH classifier also failed to iden-
tify some patients with BRCA1/BRCA2 germline
mutations, making it a promising but incomplete
test of HRD.
Structural Rearrangements
Inversions, translocations, and recombination
change the location or orientation of a DNA
sequence.14 Translocations result in the ex-
change of DNA between nonhomologous re-
gions of DNA. Inversions result in the change
 of orientation of a segment of DNA. Recombi-
nation results in exchange of DNA between
homologous regions of DNA, and this structural
rearrangement may lead to loss of heterozygosity
(LOH), a gross chromosomal event that results in
the loss of entire genes (eg, BRCA1, BRCA2).
Two types of acquired LOH are important to
note: deletion LOH, where there is a copy num-
ber loss; and copy number–neutral LOH, where
the absolute copy number remains the same
(Fig 1D). Both deletion and copy number–
neutral LOH, as with CNAs, lead to allelic im-
balance that can be inferred by studying single-
nucleotide common variation across the genome
(single-nucleotide polymorphism [SNP] analy-
sis); this may be in the form of other types of
DNA microarrays that may be fully or partially
based on SNP probes across the genome.
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Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
Three tests of structural rearrangements have
come to the forefront: telomeric allelic imbalance
(TAI), large-scale transition (LST), and LOH.
TAI was developed using a SNP genotype array
platform37 to detect the number of chromosomal
regions with allelic imbalance extending to the
subtelomere, a common genomic abnormality
that leads to an unequal contribution of maternal
and paternal DNA sequence but does not neces-
sarily change overall DNA copy number. Break-
points of TAI regions were nonrandom and
enriched for CNVs, which results in an imbalance
and then leads to HRD, which may result in
platinum sensitivity in the way that BRCA1-
associated cancer responds. Allelic imbalance
was the best predictor of cisplatin sensitivity after
identifying associations between a variety of sub-
chromosomal abnormalities and cisplatin sensi-
tivity, and TAI predicted higher rates of pCR in 79
patients enrolled in the neoadjuvant Cisplatin-1
(monotherapy) and Cisplatin-2 (cisplatin plus
bevacizumab) TNBC trials.
LST measures chromosomal breaks between
adjacent chromosomal regions of at least 10
Mb. LST was developed using SNP array anal-
ysis on 65 patients with basal-like (an intrinsic
breast cancer subtype that overlaps with ap-
proximately 80% of TNBC) breast cancer to
define BRCA1-associated genomic instabil-
ity.38 LST was predictive of BRCA1 status
and identified 85% of proven BRCA1-inactivated
cases. It was then validated in 55 primary basal-
like breast cancers and 17 basal-like breast
cancer–derived cell lines. The signature pre-
dicted BRCA1/BRCA2 inactivation in basal-
like cancers with 100% sensitivity and 90%
specificity.
5 ascopubs.org/journal/po JCO™ Precision Oncology
LOH measures the number of LOH regions . 15
Mb and less than a whole chromosome and was
recognized as a discriminatory assay in two in-
dependent data sets of ovarian tumors.39 LOH was
more frequently identified in tumors with defec-
tive BRCA1/BRCA2 and detected HRD regardless
of etiology. The first clinical trial in TNBC to
assess HRD-LOH was a phase II neoadjuvant trial
of gemcitabine, carboplatin, and iniparib (a weak
PARPi) in 80 patients with stage I to IIIA TNBC
or germline BRCA1/BRCA2 mutation–associated
breast cancer with pCR the study end point.40 The
mean HRD-LOH score was higher in responders
even when patients with germline BRCA1/BRCA2
mutations were removed. Contemporary to this
study was the single-arm phase II Translational
Breast Cancer Research Consortium (TBCRC)
study, where both HRD-LOH and HRD-LST
were studied in an exploratory analysis assessing
response to single-agent cisplatin or carboplatin in
86 patients with metastatic TNBC.41 This study
showed those patients with a BRCA1 or BRCA2-
like genomic instability signature (ie, BRCA1/
BRCA2 WT) could discriminate responders and
nonresponders (mean HRD-LOH/HRD-LST,
12.68 for responders v 5.11 for nonresponders).
A number of recent studies have incorporated all
three HRD scores (ie, HRD-LOH, HRD-LST,
HRD-TAI). The combination of all three scores
has been shown to correlate with BRCA1/BRCA2
deficiency in all breast cancer subtypes and cap-
tures more BRCA1/BRCA2 deficiency informa-
tion than the individual scores.27 In the GeparSixto
study42 of neoadjuvant paclitaxel/liposomal doxo-
rubicin with or without carboplatin, the un-
weighted sums of LOH, TAI, and LST, in
addition to BRCA1/BRCA2 mutation in the pri-
mary tumor (N = 193), found HRD in 70.5% of
TNBC tumors; 60.3% without a BRCA mutation
in the primary tumor were HRD high. HRD-high
tumors were more likely to achieve a pCR than
HRD low (55.9% v 29.8%; P = .001), and carbo-
platin increased pCR rates from 45.2% to 64.9%
(P = .025) in HRD-high tumors but not in HRD-
low tumors. At the 2015 San Antonio Breast Cancer
Symposium, two studies using the three HRD
scores, Myriad Genetics myChoice HRD, were
reported. The first pooled patients with TNBC
from a number of studies (n = 267) and used the
assay to predict those with HR deficiency (either by
HRD score or BRCA1/BRCA2 germline muta-
tion).43 Patients had been treated with various neo-
adjuvant platinum-containing regimens (carboplatin,
gemcitabine, iniparib, or cisplatin with or without
bevacizumab or carboplatin, eribulin, or carboplatin,
 nanoparticle albumin-bound paclitaxel with or with-
out vorinostat). Patients with tumors predicted to
have HRD were more likely to achieve a pCR than
those predicted to have normal HR function (44% v
8%; P , .01). The second study used the myChoice
HRD assay to predict pCR in 48 patients with HER2-
negative breast cancer treated with neoadjuvant car-
boplatin, nanoparticle albumin-bound paclitaxel with
or without vorinostat.44 In the exploratory biomarker
analysis of the myChoice HRD assay, higher pCR
rates were seen in patients with tumors predicted to
have HRD than those that were not (50% v 7.7%;
P = .002) in the overall cohort. In a subgroup analysis
of patients without germline BRCA1/BRCA2 muta-
tions (25 ER-positive, 15 TNBC), pCR rates
remained higher among those with a high HRD
score (64.3% v 7.7%; P , .001).
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In the metastatic setting, however, results of the
Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
myChoice HRD assay have not been consistent
with those in the neoadjuvant or early breast
cancer setting. In the phase III Treating to New
Targets (TNT) study, patients with metastatic
TNBC were treated with carboplatin or doce-
taxel.8 The dichotomized score did not select
those patients who would be sensitive to carbo-
platin versus docetaxel. The response rate was
38.2% in patients with HRD-high disease treated
with carboplatin versus 42.6% in patients with
HRD-high disease treated with docetaxel; re-
sponse rate was 29.2% in patients with HRD-
low disease treated with carboplatin, versus 34.7%
in patients with HRD-low disease treated with
docetaxel. Neither was statistically significant.
However, it is important to note that archival
tissue from the primary tumor was used to perform
the myChoice HRD assay and does not necessarily
represent the underlying biology of advanced
disease, where treatment-resistant clones emerge
representing different underlying tumor charac-
teristics compared with disease at an earlier stage.
Although studied as a correlative biomarker in the
metastatic TNT trial, the myChoice HRD assay
has effectively only been studied on tissue from
early or locally advanced disease. There is a pau-
city of published data on the ability of this assay to
discriminate between HR-defective tumors and
HR-intact tumors on the basis of tissue from a
metastatic lesion. Herein lies a possible explana-
tion for the discrepancy of findings from the TNT
trial, in that the assay may be measuring a scar of
what has been but does not necessarily reflect what
is occurring in the cell at present. It is known that
tumors that display the presence of HRD or scar
may go on to revert to a phenotype whereby they
regain the ability to perform HR.45 Finally, in the
6 ascopubs.org/journal/po JCO™ Precision Oncology
case of ovarian cancer, the myChoice HRD assay
was insufficiently sensitive to accurately predict
treatment benefit from the PARPi nirapirib.22
FUNCTIONAL MEASURES OF HR
PATHWAY DEFICIENCY
In contrast to genomic tests that characterize the
mutations in HR pathway genes or characterize
the mutational landscapes of HRD tumors, func-
tional measures of HR pathway deficiency provide
the most direct evidence of an HR pathway defect.
However, functional assays of HRD are difficult to
perform clinically and cannot be performed on
formalin-fixed archival specimens. To determine
HR function, viable cells first need to be subjected
to DNA-damaging insults (eg, radiation) followed
by assessment of HR.
Formation of RAD51 foci is a functional, or live,
assessment of a cell’s ability to perform HR. One
study showed that low RAD51 scores were more
common in TNBC and associated with high grade
and high Ki-67 and strongly predicted pCR.46 In
this study of sporadic breast cancers receiving
anthracycline-based chemotherapy, 68 patients
underwent biopsies 24 hours post chemotherapy
with RAD51 focus formation assessed by im-
munofluorescence. In another study, 54 HER2-
negative fresh primary breast tumors underwent
ex vivo irradiation followed by analysis of the
formation of ionizing radiation–induced foci of
RAD51, and those with impaired foci formation
underwent further genetic and epigenetic analy-
sis.47 Again, this HR defect was significantly as-
sociated with TNBC, but only five of 45 tumors
with sufficient numbers of proliferating tumor
cells were RAD51-formation deficient. More re-
cently, functional assessment of RAD51 foci for-
mation in response to ex vivo irradiation coupled
with whole-exome sequencing to determine struc-
tural genomic alterations (ie, genomic scar) showed
that the combination of deficient RAD51 foci for-
mation and elevated LST or DNA repair mutational
panel may identify genetic alterations of an HR gene
as a result of somatic or germline mutation.48
None of these studies reported outcomes, and
they are limited by small numbers, but functional
assessment of HR is promising and further stud-
ies are warranted.
DISCUSSION
The ability to consistently measure clinically
meaningful deficiencies in HR has proven a dif-
ficult task. In this review, we discuss several studies
spanning over the last two decades that have used
different methodologies in an attempt to identify
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7
Table 1. Studies and Trials Involving Tests of HRD in Breast Cancer
Study/Trial,
Test for HR Deficiency First Author Tissue Type N Primary Outcome Treatment Patient Population Main Results
BRCA1-associated Rodriguez23 Retrospective 105 pCR Neoadjuvant AC, FEC, TNBC Defective DNA repair associated
expression pattern taxane-based with higher pCR rates to
Archival frozen, FFPE
using 69-gene LDA by anthracyclines and relative taxane
qRT-PCR resistance
77-gene BRCAness gene van t’ Veer24 Exploratory analysis in an 115 pCR Neoadjuvant standard HER2-negative locally DNA repair deficiency in 77 patients
expression signature adaptive chemotherapy v advanced (38% of ER-positive and 95% of
plus PARPi-7 randomization trial veliparib, carboplatin, triple-negative)
signature (I-SPY II) chemotherapy
Fresh tissue DNA repair deficiency associated
with higher rates of pCR in V/C
group
BRCA1 insufficiency by Sharma28 Retrospective 30 42 months RFS, OS Neoadjuvant carboplatin, Stage II-III TNBC BRCA1 insufficiency associated with
BRCA1, BRCA2 docetaxel, erlotinib better 42-month OS and RFS
FFPE
mutation, BRCA1 PM,
BRCA1 mRNA
BRCA1 PM Sharma29 Retrospective 39 RFS, OS Neoadjuvant/adjuvant Stage I-III TNBC BRCA1 PM in 30% and associated
chemotherapy (90% with worse RFS, OS
FFPE
anthracycline, 69%
taxane)
BRCA1-like aCGH Vollebergh33 Retrospective 230 RFS, OS Adjuvant HD-PB v Stage III, HER2-negative 18% BRCA1-like; BRCA1-like
classifier standard treated with HD-PB had
FFPE
anthracycline-based improved RFS; no benefit in
chemotherapy non–BRCA1-like treated with
HD-PB
BRCA1-like aCGH Schouten33 Retrospective 117 DFS, DDFS, OS Adjuvant high-dose High-risk stage II-III, BRCA1-like associated with TNBC
based on copy number ifosfamide, epirubicin, any biomarker status
FFPE BRCA1-like treated with high-dose
profiles carboplatin v standard
regimen had better DFS, DDFS,
chemotherapy
and OS
No benefit in BRCA1-like negative.
35
BRCA1-like aCGH Oonk Retrospective 101 5-year RFS Adjuvant AC, FEC, TNBC 65% were BRCA1-like. No
classifier by MLPA TAC, CMF difference in 5-year RFS
FFPE
BRCA1- and BRCA2- Lips36 Retrospective 163 pCR Neoadjuvant dose-dense HER2-negative BRCA1 dysfunction frequent in
like aCGH classifier AC TNBC cohort but no difference in
response to ddAC in BRCA1-like
v non–BRCA1-like
BRCA1 PM, BRCA1 Pretreatment snap frozen BRCA2-like frequent in ER-positive
mRNA, EMSY cohort and associated with better
amplification response to treatment
(Continued on following page)

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8
Table 1. Studies and Trials Involving Tests of HRD in Breast Cancer (Continued)
Study/Trial,
Test for HR Deficiency First Author Tissue Type N Primary Outcome Treatment Patient Population Main Results
TAI Birkbak37 Correlative study of two 79 pCR Neoadjuvant cisplatin- TNBC TAI predicted cisplatin sensitivity
clinical trials based chemotherapy and pCR rates in TNBC
Pretreatment biopsy
LST Popova38 Retrospective 65 Preclinical Postsurgery BLC, triple marker Signature predicted BRCA1,
negative BRCA2 inactivation in BLC with
Fresh frozen
100% sensitivity and 90%
specificity
HRD-LOH Telli40 Correlative study in 80 pCR Neoadjuvant Stage I-IIIA TNBC or Mean HRD-LOH higher in
a phase II trial gemcitabine, gBRCA responders independent of BRCA
(PrECOG 0105) carboplatin, iniparib mutation status
Pretreatment core biopsy
HRD-LOH and Isakoff41 Exploratory analysis in 86 ORR Single-agent carboplatin Metastatic TNBC ORR 54% in gBRCA
HRD-LST a phase II trial or cisplatin
(TBCRC009)
FFPE Mean HRD-LOH/HRD-LST
higher in responders without
gBRCA mutation
HRD-LOH, HRD- Von Minckwitz42 Correlative study in 193 pCR Neoadjuvant paclitaxel/ Stage II-III TNBC 70.5% were HR deficient; 60.3%
LST, HRD-TAI, and a phase II trial liposomal doxorubicin without tumor BRCA1/BRCA2
BRCA1/2 mutation in (GeparSixto) with or without were HR deficient
primary tumor carboplatin
FFPE HRD high more likely to achieve
pCR than HRD low. Carboplatin
increased pCR in HRD high but
not in HRD low
HRD (HRD-LOH, Tutt8 Correlative study in 390 ORR Carboplatin v docetaxel Metastatic/recurrent Dichotomized HRD score did not
HRD-LST, a phase III trial (TNT) locally advanced select sensitivity to carboplatin
HRD-TAI) TNBC over docetaxel
Archival tissue from the
primary tumor
HRD (HRD-LOH, Timms27 Retrospective 215 Frequency of BRCA1/2 Not specified Any biomarker status gBRCA1, BRCA2 seen in all
HRD-LST, defects in different subtypes
HRD-TAI) breast cancer subtypes
Archival tissue from BRCA1 PM exclusive to TNBC
commercial vendors
HRD mean score captured BRCA1,
BRCA2 deficiency information
not captured on individual scores
or clinical variables
HRD (HRD-LOH, Telli43 Pooled analysis of six 267 pCR Neoadjuvant carboplatin, TNBC and known HR 63% were HR deficient
HRD-LST, phase II trials with gemcitabine, iniparib; deficiency based on
HRD-TAI) HRD score known cisplatin with or HRD score or tumor
without bevacizumab; BRCA mutation
(Continued on following page)

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9
Table 1. Studies and Trials Involving Tests of HRD in Breast Cancer (Continued)
Study/Trial,
Test for HR Deficiency First Author Tissue Type
HRD (HRD-LOH, Connolly44 Exploratory correlative
HRD-LST, study in a placebo
HRD-TAI) controlled trial
(TBCRC008)
Baseline tumor tissue
RAD51 score (functional) Graeser46 Retrospective
FFPE (tissue obtained 24
hours after first cycle of
chemotherapy and
xenografted.
Xenografts were
irradiated with tumors
removed 6 hours later
and formalin fixed)
RAD51 score (functional) Naipal47 Correlative study in
a phase II trial
Fresh tissue
RAD51 foci formation Powell48 Exploratory study
(functional) combined
with whole-exome
sequencing (LST,
LOH, TAI and
Fresh tissue
BRCA1/BRCA2
mutational signature)
N Primary Outcome Treatment
carboplatin, eribulin;
carboplatin, NAB-
paclitaxel with or
without vorinostat
48 pCR Neoadjuvant carboplatin, TNBC or ER-positive
NAB-paclitaxel with or
without vorinostat
68 RAD51 levels, pCR Neoadjuvant
anthracycline-based
chemotherapy
45 RAD51 foci after ex vivo Postsurgery (no
radiation neoadjuvant
treatment)
29 RAD51 recruitment after Not specified
ex vivo radiation,
evidence of alterations
in HR genes
Patient Population Main Results
HRD high had a significant fivefold
increase in pCR compared with
nondeficient tumors
46% of tumors were HR deficient.
Higher HRD score corresponded
to significantly higher pCR rates
in overall cohort. Trend toward
higher pCR rates in HRD-high
scores for ER-positive, TNBC
subgroups with no difference
between treatment arms
Sporadic primary breast Low RAD51 score present in 26% of
cancer, any biomarker patients and more common in
status TNBC, higher grade, and higher
Ki-67
Low RAD51 significantly predicted
pCR
Any biomarker status 5/45 samples were RAD51 deficient
and strongly associated with
TNBC
Sporadic breast cancer, 45% were RAD51 deficient. LST
any biomarker status and BRCA1/BRCA2 mutations
signature was associated with
RAD51 deficiency
8 of 9 samples with HRD (by RAD51
and genomic scar) had germline or
somatic alteration of both alleles in
known HR gene

Abbreviations: AC, doxorubicin, cyclophosphamide; aCGH, array comparative genomic hybridization; BLC, basal-like breast carcinoma; CMF, cyclophosphamide, methotrexate, fluorouracil; ddAC, dose-dense adriamycin and
cyclophosphamide; DDFS, distant disease-free survival; DFS, disease-free survival; ER, estrogen receptor; FEC, fluorouracil, epirubicin, cyclophosphamide; FFPE, formalin-fixed paraffin-embedded; gBRCA, germline BRCA; HD-PB,
high-dose platinum based; HER2, human epidermal growth factor receptor 2; HR, homologous recombination; HRD, homologous recombination deficiency; I-SPY, Investigation of Serial Studies to Predict Your Therapeutic Response
through Imaging and Molecular Analysis; Ki-67, protein encoded by the MKI67 gene; LDA, linear disciminate analysis; LOH, loss of heterozygosity; LST, large-scale transition; MLPA, multiplex ligation-dependent probe
amplification; NAB, nanoparticle albumin-bound; ORR, overall response rate; OS, overall survival; PARPi, poly (ADP-ribose) polymerase inhibitor; pCR, pathologic complete response; PM, promoter methylation; qRT-PCR,
quantitative reverse-transcriptase polymerase chain reaction; RFS, recurrence-free survival; TAC, docetaxel, doxorubicin, cyclophosphamide; TAI, telomeric allelic imbalance; TBCRC, Translational Breast Cancer Research
Consortium; TNBC, triple-negative breast cancer; TNT, Treating to New Targets; V/C, veliparib plus chemotherapy

ascopubs.org/journal/po JCO™ Precision Oncology

 deficiencies in HR (Table 1). Although the pri-
mary focus is on TNBC, the role of HRD in many
other tumor types is emerging as an important
target. BRCA1 and BRCA2 mutations are the
archetype of HR-deficient tumors and have
provided insight into other causes of HRD. Devel-
oping a reliable biomarker will be key to identifying
patients with HRD tumors who may benefit from
HRD-targeted therapies.
Studies attempting to use gene panels known to
be involved in HR make sense mechanistically, but
it is clear that not all genes are equal in the process,
and basing a score on a composite of these genes
may overestimate HRD. Similarly, identifying
BRCA1 PM has not consistently been shown to
be useful in defining HRD tumors, as highlighted
in the study where patients with either germline
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BRCA1/BRCA2 mutations, low BRCA1 mRNA
expression, or BRCA1 PM were analyzed together.
Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
Although platinum-based neoadjuvant chemo-
therapy resulted in improved OS for the entire
cohort, the impact BRCA1 PM had on clinical
outcomes remains unclear.
The aCGH classifier was able to show that pa-
tients with BRCA-like tumors had significantly
better RFS and OS when treated with high-dose
platinum-based chemotherapy versus conven-
tional anthracycline-based chemotherapy. How-
ever, in the subset of patients with TNBC, the
aCGH classifier failed to identify nearly 40%
of patients with a germline BRCA1/BRCA2
mutation, and the experimental high-dose alkylator
Fig 2. Circos plot
summarizing large-scale
somatic findings of whole-
genome sequencing of
breast tumor DNA
compared with normal
DNA from blood. In
addition to cataloging
somatic point mutations
and small insertions/
deletions, the use of whole-
genome sequencing
enables characterization of
large-scale copy-number
variation, loss of
heterozygosity, and
structural variation. This
enables precise and
massively parallel
assessment of molecular
phenotypes of homologous
recombination deficiency.
10
chemotherapy regimens used in this study are not
standard and confound the findings of this study.
More recent tests of HRD that are based on SNP
analysis have been combined in various ways, such
as the Myriad Genetics myChoice HRD assay
(HRD-TAI, HRD-LOH, HRD-LST). This as-
say is the most developed for clinical use, with
promising results in platinum-based neoadjuvant
clinical trials of breast cancer with the primary end
point of pCR. Even when patients with known
germline BRCA1/BRCA2 were removed from anal-
ysis, those with high scores had better rates of pCR.
In the metastatic setting, the myChoice HRD assay
failed to select those patients who may benefit from
platinum-based therapy in the TNT study. How-
ever, the assay was performed on archival primary
tumor tissue and may not be representative of HRD
status of metastatic tumors. This discrepancy sup-
ports emerging evidence that early-stage breast can-
cer and advanced disease are different entities
whereby treatment-resistant clones emerge in ad-
vanced disease, resulting in different tumor charac-
teristics that may involve reversions from an HR-
deficient state to an HR-efficient state. Thus, the
approach to identify biomarkers will need to take
temporal tumor evolution into consideration.
HRD assays that have been developed to date and
that focus on the genotype are similar in that they
measure a scar, a record of what the genome has
been. It is known that BRCA1/BRCA2-deficient
cells can over time regain their ability to perform
HR, and current biomarkers of HRD are limited
Copy gains
Copy losses
Loss of heterozygosity
Structural variants
Genome
Transcriptome
ascopubs.org/journal/po JCO™ Precision Oncology
 in identifying such tumors. A direct functional
measure is needed to assess the changing HRD
status of tumors in real time. However, the feasi-
bility of performing such assays is challenging in
the clinical setting because it requires fresh tissue
biopsies recently exposed to DNA-damaging in-
sults. Patient-derived xenografts represent rea-
sonable models of the human breast tumor49
that could be used to explore real-time functional
HR versus measures of HR by other methods
described in this review and provide further in-
sight into understanding HRD.
Ultimately, whole-genome sequencing can provide
direct or supportive evidence of almost all the in-
formation that each of the above assays is intending
to measure (Fig 2). This approach, combined with
BRCA mutation status, accurately detected all classes
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of genomic alterations in the Assessment of Ruca-
Copyright © 2024 American Society of Clinical Oncology. All rights reserved.
parib in Ovarian Cancer: Phase 2 Clinical Trial
(ARIEL2) trial of ovarian cancer,50 and whole-
genome sequencing of breast cancers has identified
three signatures that seem to be associated with
HRD.18 However, until the cost of whole-genome
sequencing decreases, scientists and clinicians are left
to find less-expensive alternatives. To date, the dif-
ferent categories of HRD assays have not been di-
rectly compared (eg, Myriad myChoice HRD,
aCGH classifier, mutation panels). Several novel
drug therapies targeting defects in HR are currently
in development and clinical trials. These studies
represent a unique opportunity to prospectively de-
velop, compare, and validate HRD biomarkers to
select patients who will most benefit from
HRD-targeted therapies.
AUTHOR CONTRIBUTIONS
Conception and design: Wendie D. den Brok, Kasmintan A.
Schrader, Sophie Sun, Eric Yang Zhao, Samuel Aparicio,
Karen A. Gelmon
Collection and assembly of data: Wendie D. den Brok,
Sophie Sun, Karen A. Gelmon
Data analysis and interpretation: Wendie D. den Brok,
Sophie Sun, Anna V. Tinker, Karen A. Gelmon
Manuscript writing: All authors
Final approval of manuscript: All authors
Accountable for all aspects of the work: All authors
AUTHORS’ DISCLOSURES OF
POTENTIAL CONFLICTS OF INTEREST
The following represents disclosure information provided by
authors of this manuscript. All relationships are considered
compensated. Relationships are self-held unless noted. I =
Immediate Family Member, Inst = My Institution. Relation-
ships may not relate to the subject matter of this manuscript. For
more information about ASCO’s conflict of interest policy,
please refer to www.asco.org/rwc or po.ascopubs.org/site/ifc.
11
META-SUMMARY
There is evidence that HRD assays have pre-
dictive value for response to platinum and pos-
sibly PARPis in combination with standard
therapy. Head-to-head comparisons of the dif-
ferent HRD assays have not been performed,
and it is not clear which assays or combination of
assays is the best predictor of HRD. A major
limitation of the specificity of these assays is
their ability to detect only if HRD has occurred
but not if it is still occurring. Whole-genome
sequencing encompasses the abilities of each
HRD assay.
SEARCH STRATEGY AND SELECTION
CRITERIA
A literature search conducted in Ovid MED-
LINE and Embase databases using the key-
words breast neoplasms, brcaness, BRCA1,
BRCA2, and homologous recombination defi-
ciency was performed on February 11, 2016,
without limiting the earliest date of publica-
tions. The ASCO (2014 and 2015) and San
Antonio Breast Cancer Symposium (2014 to
2016) databases were searched at the title and
abstract levels using the search terms breast
cancer, BRCA1, BRCA2, and homologous re-
combination deficiency. Only articles published
in English were reviewed. The final reference
list was generated based on originality and rel-
evance to the broad scope of this review.
DOI: https://doi.org/10.1200/PO.16.00031
Published online on ascopubs.org/journal/po on June 19, 2017.
Wendie D. den Brok
No relationship to disclose
Kasmintan A. Schrader
No relationship to disclose
Sophie Sun
No relationship to disclose
Anna V. Tinker
No relationship to disclose
Eric Yang Zhao
No relationship to disclose
Samuel Aparicio
No relationship to disclose
Karen A. Gelmon
No relationship to disclose
ACKNOWLEDGMENTS
We thank the BC Cancer Agency and Michael Smith Genome
Sciences Centre’s Personalized Onco-genomics Program.
ascopubs.org/journal/po JCO™ Precision Oncology
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