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Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer


Abstract and Figures

Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors. Defects in homologous recombination (HR) are found in some triple negative breast cancers, suggesting they may be sensitive to PARP inhibitors. In this phase II clinical trial of the PARP inhibitor rucaparib, changes in Ki67 levels did not correlate with markers of HR deficiency but HR deficiency was detected in 69% of tumours, indicating that PARP inhibitors may be a useful treatment.
Expression analysis of primary triple negative homologous recombination repair deficient cancers a Association between basal-like and non-basal-like triple negative subtypes, assessed by PAM50, and HRDetect score. p value Fisher’s exact test. n = 20 paired tumour samples. Purple, HRDetect score >0.7; Grey, HRDetect score <0.7. b Change in gene expression on paired tumor biopsies between baseline and end of treatment on rucaparib. Number of genes with a significant change in gene expression (Log fold change >0.5 and false discovery corrected q value < 0.1). Left, categorised by ctDNA suppression or not (CDR15 response <0.25 vs ≥1, n = 8 and n = 3 paired tumour samples respectively). Right, by CDR15 response and HRD score (CDR15 < 0.25 and HRD > 0.7 vs CDR15 ≥ 1 and HRD < 0.7, n = 11 and n = 6 paired tumour samples respectively). c Gene expression changes of PARP1, MKI67, CDKN1A and TMEM173 through treatment, from DESeq2 with false discovery rate (FDR) corrected q value for change. n = 20 paired tumour samples. Magenta lines, BRCA1/2 germline mutation; Blue lines, BRCA1 methylation; Yellow lines, PALB2 germline mutation; Turquoise lines, RAD51C methylation; Grey lines, None/unknown. d Left, Gene set enrichment pathway analysis (GSEA) for gene expression changes through treatment in patients with ctDNA suppression (CDR15 < 0.25, n = 8 paired tumour samples.) on rucaparib. Centre, suppression of G2M checkpoint genes on PARP inhibition, q = 0.006, and right, increased expression of interferon pathway genes on PARP inhibition, q = 0.001. False discovery rate corrected q value for change.
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Homologous recombination DNA repair deciency
and PARP inhibition activity in primary triple
negative breast cancer
Neha Chopra1, Holly Tovey2, Alex Pearson 1, Ros Cutts1, Christy Toms2, Paula Proszek3, Michael Hubank3,
Mitch Dowsett1,4, Andrew Dodson4, Frances Daley1, Divya Kriplani1, Heidi Gevensleben1, Helen Ruth Davies5,6,
Andrea Degasperi 5,6, Rebecca Roylance7, Stephen Chan8, Andrew Tutt1,9, Anthony Skene10, Abigail Evans11,
Judith M. Bliss2, Serena Nik-Zainal 5,6 & Nicholas C. Turner1,12
Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a
subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here,
within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate
the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point,
decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deciency was
identied in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers
with HRDetect mutational signatures of HR deciency had a functional defect in HR, assessed
by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment
there was no association of Ki67 change with HR deciency. In contrast, early circulating
tumor DNA dynamics identied activity of rucaparib, with end of treatment ctDNA levels
suppressed by rucaparib in mutation-signature HR-decient cancers. In ad hoc analysis,
rucaparib induced expression of interferon response genes in HR-decient cancers. The
majority of TNBCs have a defect in DNA repair, identiable by mutational signature analysis,
that may be targetable with PARP inhibitors. OPEN
1Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London CB2 0XZ, United Kingdom. 2Clinical Trials and Statistics Unit,
The Institute of Cancer Research, London, United Kingdom. 3The Centre for Molecular Pathology, The Royal Marsden Hospital, 15 Cotswold Road, Sutton SM2
5NG Surrey, United Kingdom. 4Ralph Lauren Centre for Breast Cancer Research, Royal Marsden Hospital, London, United Kingdom. 5Department of Medical
Genetics, The Clinical School, Box 238, Level 6 Addenbrookes Treatment Centre, Cambridge Biomedical Campus, Cambridge CB2 0QQ, United Kingdom.
6MRC Cancer Unit, Hutchison/MRC Research Centre, University of Cambridge, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, United
Kingdom. 7University College London Hospitals NHS Foundation Trust, NIHR University College London Hospitals Biomedical Research Centre, London, United
Kingdom. 8Nottingham University Hospital Trust (City Campus), Nottingham, United Kingdom. 9Breast Cancer Now Research Unit, Cancer Centre, Guys
Hospital, Kings College London, London, United Kingdom. 10 Royal Bournemouth Hospital, Bournemouth, United Kingdom. 11 Poole Hospital NHS Foundation
Trust, Poole, United Kingdom. 12Breast Unit, The Royal Marsden Hospital, Fulham Road, London, United Kingdom. email:
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Triple negative breast cancer (TNBC) may have diverse
defects in HR DNA repair, through germline mutations in
BRCA1,BRCA2 and PALB2, somatic mutations in BRCA1
and BRCA2, promoter methylation of BRCA1 and RAD51C, and
other as yet to be identied mechanisms14. Over the last decade,
advances in whole-genome sequencing (WGS) have lead to the
identication of mutational processes that leave a characteristic
imprint, a mutational signature on the cancer genome. These
have revolutionalised our understanding of cancer and has the
capability to improve diagnosis and treatment of cancer5,6.
Cancers with defects in HR-based DNA repair have character-
istic chromosomal changes reecting the use of alternative error-
prone repair pathways, including measures of genomic instability;
loss of heterozygosity, telomeric allelic imbalance and large-scale
state transitions to accurately identify BRCA1/2 tumours79,and
their combination to form the HRD Score has allowed identi-
cation of HR-decient tumours (HRD Score >42), independent of
BRCA1/2 deciency within a sporadic TNBC population10.Recent
work has identied WGS signatures of HR deciency with BRCA1/
2decient tumours associated with distinct mutational signatures.
The mutational signatures and chromosomal instability markers of
HR deciency have been aggregated into the HRDetect score,
robustly identifying BRCA1/2 tumours with potential greater
accuracy than indexes such as HRD-score11,12.
Whether mutational signature-based scores such as HRDetect,
can be used to direct therapy in the clinic is unknown, in part as
there is limited direct evidence that cancers classied as HR
decient by these scores have a functional defect in HR. Breast
cancers with BRCA1 and BRCA2 germline mutations are highly
sensitive to PARP inhibitors13,14, which target the underlying HR
DNA repair defect in these cancers. However, no activity was
observed with PARP inhibitors in the treatment of heavily pre-
treated un-selected advanced TNBC15. The extent to which this
PARP inhibitor efcacy may translate to sporadic TNBC is
unknown, as is the best way to identify HR-decient TNBC. To
address these questions, we designed a translational clinical trial,
the RIO trial (EudraCT 2014-003319-12), with the objective of
identifying biomarkers of PARP inhibitor activity in sporadic
Biomarkers of HR deciency in primary TNBC. Patients with
newly diagnosed, treatment naïve TNBC were treated with the
PARP inhibitor rucaparib for 2 weeks prior to surgery or
neoadjuvant chemotherapy. A total of 43 patients were entered
into the trial between August 2015 and August 2017. Blood and
tissue biopsies were taken prior to, and at the end of treatment,
for molecular analysis (Fig. 1a). Within the trial, a subset of
germline BRCA1/2 patients were recruited as a control popula-
tion. The trial prospectively examined three potential biomarkers
of PARP inhibitor activity, a molecular signature of HR deciency
aDid not start treatment (n = 1)
Did not complete 7 days of
treatment (n = 4)
Consent for RIO (n = 43)
Patients with samples
for analysis of
endpoints (n = 38)
Baseline ctDNA level
too low (n = 11)
ctDNA assay failed
(n = 1)
Patients with
paired biopsies (n = 37)
Paired biopsies for Ki67
analysis (n = 26)
Patients with
paired plasma (n = 31)
Assessed for ctDNA
dynamics (n = 19)
Ki67 immunohistochemistry cleaved PARP immunohistochemistry
100 0.4
% change from baseline
Logfold change from baseline
BRCA mutation
BRCA mutated
BRCA wild-type
50% reduction 30% reduction
Biological population
Biological population
Fig. 1 RIO study CONSORT diagram and HRDetect analysis. a RIO study CONSORT diagram. bEffect of rucaparib on Ki67 expression assessed by
immunohistochemistry (IHC). The change in proportion of tumor cells expressing Ki67 between baseline and EOT, in patients that had assessable pairs of
baseline and EOT samples. BRCA mutation cancers had no evidence of decreased Ki67. cEffect of rucaparib on cleaved PARP expression assessed by
immunohistochemistry, as a marker of apoptosis. The change in proportion of tumor cells expressing cleaved PARP between baseline and EOT, in patients
that had assessable pairs of baseline and EOT samples. BRCA mutation cancers had no evidence of increased cleaved PARP expression. Grey bars, BRCA
wild type patients; Blue bars, BRCA germline mutant patients. Orange line, >30% but <50% reduction; Red line, >50% reduction.
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using HRDetect, RAD51 focus formation in a tumor biopsy at the
end of treatment, and BRCA1 methylation. The primary activity
end point was a fall in Ki67 on the end of treatment biopsy, with
circulating tumor DNA dynamics as a prospectively planned
exploratory end point of activity. Patient demographics were as
expected for this population (Table 1). Rucaparib was well tol-
erated with adverse effect prole similar to previous clinical
studies16,17 (Supplementary Table 1).
We assessed rucaparib activity and the relationship with
prospectively planned biomarkers. The primary end point was
assessed in tissue samples, using Ki67 suppression after two weeks
assessed by immunohistochemistry, as a potential biomarker
(Fig. 1b). A drop in Ki67 by 50% in the triple negative patients
without a known BRCA1/2 mutation at trial entry was seen in
12% tumors (95% CI: 2.531.2; n=3/25). In secondary end point
analysis, one additional patient with known BRCA mutation at
trial entry was assessed and did not have a 50% drop in Ki67
(Fig. 1b). No association was observed between Ki67 change with
BRCA1/2 mutated cancers (Fig. 1b). Similarly, no association was
observed with cleaved PARP levels as a marker of apoptosis with
BRCA1/2 mutated cancers (Fig. 1c).
In secondary end point analysis, baseline biopsies of patients
entering the trial, we interrogated the prevalence of HR deciency
in primary TNBC (Fig. 2and Supplementary Table 2). We
performed whole-genome sequencing analysed with the HRDetect
assay11, identifying mutational processes characteristic of HR
deciency in 69% locally assessed TNBC (18/26 with score >0.70,
herewith called HRDetect+ve; Fig. 2a,b and Supplementary Fig. 1a)
as well as an additional control ER-positive cancer with a known
germline BRCA2 mutation. In ad hoc analysis, we individually
determined the mutational status of BRCA1, BRCA2 and PALB2,
and promoter methylation of BRCA1 and RAD51C (Fig. 2a). Of the
HRDetect+ve cancers, 74%(14/19) had a detectable underlying
mutation of BRCA1/2 and PALB2 or gene promoter hypermethy-
lation of BRCA1 or RAD51C. None of the eight HRDetectve
cancers had an underlying genetic/epigenetic defect (p=0.0005,
Fishers exact test; Fig. 2a). A loss of heterozygosity (LOH)/copy-
number-based HRD score was positive in more cancers than
HRDetect, with the HRD score identifying cancers with genomic
instability but without rearrangement signatures and indels at
microhomology. None of the HRD score high but HRDetect low
tumours had detectable pathway aberrations (Supplementary
Fig. 1b), suggesting that HRDetect was more specic.
HRDetect identied all cancers with known HR pathway
defects, as well as additional sporadic cancers with no single
detectable defect (Fig. 2a). In secondary end point analysis, we
next addressed whether HRDetect+ve cancers had an underlying
functional defect in HR DNA repair, using RAD51 focus
formation in the end of treatment (EOT) biopsy. When cells
are exposed to genotoxic agents such as PARP inhibition, RAD51
is recruited to sites of DNA damage and stalled replication forks,
mediating the search for a homologous sequence during HR18,
with RAD51 nuclear foci visible at sites of repair as a hallmark
for HR-mediated repair19. The impaired ability to form RAD51
foci after DNA damage may identify cancers with defective
HR20. We developed a novel immunohistochemistry assay to
assess RAD51 foci, co-staining with geminin (GMNN) to
identify cells in S/G2 phase of the cell cycle and after cytotoxic
treatment; RAD51 score <20% (less than 20% geminin positive
cells having RAD51 foci, RAD51 foci deciency) was assessed to
indicate HR deciency in an independent sample set (Supple-
mentary Fig. 2). Within the RIO trial, RAD51 IHC scores
increased signicantly from baseline to EOT (p=0.0016,
Wilcoxon test), reecting rucaparib induced DNA damage and
RAD51-mediated repair (Fig. 3a). In EOT biopsies, RAD51 foci
deciency was identied in 77% (17/22) locally assessed TNBC,
as well as an ER-positive BRCA2 mutant control cancer (Fig. 3b).
Of the RAD51-decient cancers, 61% (11/18) had an underlying
detectable HR defect compared to none (0/5) of RAD51 foci
procient cancers (p=0.037 Fishers exact test; Fig. 3b). Cancers
with RAD51 foci deciency had signicantly higher HRDetect
scores than tumour samples that were RAD51 foci procient
(n=18, p=0.0146 MannWhitney test; Fig. 3c). HRDetect
therefore identied cancers with a functional defect in HR-based
DNA repair, with functional HR deciency occurring in the
majority of TNBC.
Rucaparib activity assessed by ctDNA dynamics. Circulating
tumour DNA (ctDNA) is released from the tumor, allowing for
serial sampling through the course of treatment21,22. Early
changes in ctDNA dynamics represent an early biomarker of drug
Table 1 RIO study patient demographics.
Age (mean (standard deviation)) 54.6 (13.9)
Age group (years) <40 7 16.3
40-49 13 30.2
50-59 9 20.9
60-69 5 11.6
70+9 20.9
BRCA status Triple negative, known BRCA1/2
carrier at registration
2 4.7
Not TN, known BRCA1/2
mutation carrier at registration
1 2.3
Triple neg, no BRCA mutation 35 81.4
Triple negative, BRCA1/2
mutation identied while on trial
5 11.6
Planned standard
treatment after
Neoadjuvant chemotherapy 32 74.4
Surgical resection 11 25.6
receptor status
ER & PR negativea42 97.7
ER positive & PR negative 1 2.3
Tumour grade
(diagnostic sample)
G1 0 0
G2 12 27.9
G3 24 55.8
Not known 7 16.3
Histological type Inltrating ductal 38 88.4
Inltrating lobular 4 9.3
Mixed ductal & lobular 1 2.3
DCIS present Yes 7 16.3
No 35 81.4
Not known 1 2.3
Tumour size by
<1.5 2 4.7
1.5 1 2.3
>1.5 & 2 10 23.3
>2 & 5 26 60.5
>5 4 9.3
Lymph node
Yes 16 37.2
No 27 62.8
Side of tumour Left 18 41.9
Right 25 58.1
Evidence of metastatic
Yes 0 0
No 43 100
aOne patient was locally assessed as triple negative but central assessment noted weak PgR
score of 3/8 by Allred.
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activity, as cancers that respond to treatment rapidly suppress the
level of ctDNA in plasma2325. Analysis of ctDNA was pro-
spectively planned as an exploratory end point of rucaparib
activity, in part, as Ki67 change has only been validated as an
activity end point in endocrine based therapies2628. To assess
ctDNA in RIO, the primary tumor was sequenced in 35 patients,
with somatic mutations identied in 31 patients, and personalised
digital PCR used to track changes in ctDNA levels between
baseline and end of treatment plasma (EOT). Change in ctDNA
was assessable in the 19 patients with sufciently high baseline
Patient #4, HRDetect <0.7
Patient #27, HRDetect >0.7
aHRDetect positive n = 19
HRDetect negative n = 8
p = 0.0005 Fishers’s exact test
BRCA1/2 germline mutation
BRCA 1 methylation
PALB2 germline mutation
RAD51C methylation
HRDetect score
1 2 3 4 5 6 7 8 9 11 12 14 15 16 17 18 19 20 21 22 23 24 25 26 2713 10
C>A Sig26
4841 substitutions
348 deletions and insertions
14,000 substitutions
1302 deletions and insertions
Copy number
51 rearrangements
Deletion other
Deletion repeat
0 5 10 15 20 25 30 35
370 rearrangements
050 100 150 200
02000 4000 6000 8000 10,000
LOH Gain
Copy number
LOH Gain
50 100 150
Deletion other
Deletion repeat
1000 1500 2000 2500
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Fig. 2 HRDetect analysis. a HRDetect scores were established with whole-genome sequencing in baseline biopsies of 26 patients with untreated primary
triple negative breast cancer (locally assessed) entering the RIO window clinical trial. HRDetect positive cancers (HRDetect score >0.7) were enriched for
inactivating mutations and promoter methylation of HR genes compared to HRDetect negative cancers (p=0.0005, Fishers exact test). Star, an additional
patient with ER-positive breast cancer and BRCA2 germline mutation is shown. 2 stars, locally assessed as TNBC but centrally assessed as PR-positive
breast cancer with BRCA1 methylation. Magenta bars, BRCA1/2 germline mutation; Blue bars, BRCA1 methylation; Yellow bars, PALB2 germline mutation;
Turquoise bars, RAD51C methylation; Grey bar, None/unknown. bExamples of genome plots for a sample with low (top) and high (bottom) HRDetect
scores. The histograms associated with each circos plot show mutation counts for each mutation class: the topmost histogram shows the number of
mutations contributing to each substitution signature; the middle histogram represents indel patterns; and the bottom histogram shows the number of
rearrangements contributing to each rearrangement signature.
RAD51 foci proficient
patient #5
RAD51 foci deficient
patient #14
Baseline EOT
RAD51 score
p = 0.0016, n = 21
p = 0.0146, n = 18
HR Detect score
RAD51 foci
RAD51 foci
RAD51 foci deficient n = 18
p = 0.037, Fisher’s exact test
RAD51 foci proficient n = 5
BRCA1/2 germline mutation
BRCA 1 methylation
PALB2 germline mutation
RAD51C methylation
RAD51 IHC score
40 µm
40 µm
6 8 15 27 14 21 25 22 17 31 18 33 28 39 2 12 24 26 5 4 29 1 9
Fig. 3 Biomarkers of homologous recombination (HR) repair deciency in TNBC. a RAD51 focus assessment in paired baseline and on-treatment
biopsies, p=0.0016 Wilcoxon test. Inset right, example immunohistochemistry images of two cancers, one decient and one procient in RAD51 focus
formation (brown), in nuclei stained for geminin (blue). Scale bars show 40 μm. bRAD51 immunohistochemistry score (>20% RAD51 foci procient), as a
functional assessment of HR prociency, was assessed in end of treatment biopsies after 2 weeks of rucaparib in 25 patients. RAD51 foci decient cancers
are enriched for inactivating mutations and promoter methylation of HR genes compared to RAD51 procient cancers (p=0.037 Fishers exact test). *ER-
positive breast cancer and BRCA2 germline mutation is shown. **Locally assessed TNBC but centrally assessed as PR-positive breast cancer with BRCA1
methylation. Magenta bars, BRCA1/2 germline mutation; Blue bars, BRCA1 methylation; Yellow bars, PALB2 germline mutation; Turquoise bars, RAD51C
methylation; Grey bars, None/unknown. cAssociation between RAD51 foci prociency and HRDetect scores in 18 patients, p=0.0146 MannWhitney
UTest. Line indicates median level.
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ctDNA to assess change (Figs. 1a and 4a and Supplementary
Tables 4 and 5). In contrast to the tumor biopsy-based data, a
substantial proportion of patients suppressed ctDNA after ruca-
parib treatment (Fig. 4a).
Patients with germline BRCA1/2 mutations did not suppress
Ki67, nor induce PARP cleavage, in the end of treatment biopsy
(Fig. 1). In contrast, patients with germline BRCA1/2 mutations
had a greater suppression of ctDNA than patients without
germline mutations (n=19, p=0.021, MannWhitney; Fig. 4b),
validating ctDNA dynamics as a marker of rucaparib activity.
Cancers with decient RAD51 foci formation (n=12, p=0.033,
MannWhitney) and HRDetect+ve cancers had greater ctDNA
suppression (n=15, p=0.027, MannWhitney; Fig. 4b). In ad
hoc analysis patients with suppressed ctDNA after two weeks
rucaparib (ctDNA ratio (CDR) <0.25, methods) were enriched for
germline mutations of BRCA1/2 and PALB2 and gene promoter
methylation of BRCA1 and RAD51C (Fig. 4b). These data
illustrate the potential of ctDNA analysis to transform window
trials, presenting a simple and robust assay of drug activity,
without the potential sampling challenges involved with repeat
biopsies. However, analytical challenges associated with low
plasma DNA levels and low purity tumor samples, will benet
from further technological development.
PARP inhibition induces an interferon response in HR-
decient cancers. Having demonstrated that HRDetect identi-
es sporadic TNBC in which PARP inhibitors have activity, in ad
BRCA1/2 germline mutation
BRCA 1 methylation
PALB2 germline mutation
RAD51C methylation
Normalised mutant copies /mL
Baseline EOT
5, PD within 6 months
aPatient 15
BRCA2, c.36_41TGAAATdelAGAAAins
VIC Fluorescence
FAM Fluorescence
BRCA2 wt
BRCA2 ins
End of treatment
VIC Fluorescence
FAM Fluorescence
BRCA2 wt
p = 0.027 (Mann–Whitney), n = 12
CDR 15 Ratio
RAD51 score vs. CDR15 HR Detect vs. CDR15
p = 0.028 (Mann–Whitney), n = 15
HR Detect
HR Detect
CDR 15 Ratio
p = 0.021 (Mann–Whitney), n = 19
BRCA status vs. CDR15
CDR 15 Ratio
Fig. 4 ctDNA dynamics reveals activity of rucaparib in primary triple negative homologous recombination repair decient cancers. a Change in
circulating tumour DNA (ctDNA) copies/ml between baseline and end of treatment (EOT) after two weeks of rucaparib. The relative change of on-
treatment ctDNA levels (Circulating tumor DNA ratio, CDR15) with HR pathway defects indicated. Right, example digital PCR ctDNA analysis plots.
Magenta lines, BRCA1/2 germline mutation; Blue lines, BRCA1 methylation; Yellow lines, PALB2 germline mutation; Turquoise lines, RAD51C methylation;
Grey lines, None/unknown. bAssociations of ctDNA change on rucaparib at day 15 (CDR15) with left, BRCA1/2 germline mutations (n=19 patients),
middle, RAD51 focus prociency (n=12 patients) and right, HRDetect score (n=15 patients). Centre line, mean; error bars, standard deviation. pvalues
MannWhitney Utest.
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hoc analysis we investigated the mechanisms of PARP inhibition
in these cancers with RNA exome sequencing in 20 paired
tumour samples, baseline and end of treatment. The majority of
tumours were basal-like by PAM50 (75%, 15/20), with HRDetect
scores >0.7 found in 80% (12/15) basal-like TNBC and none (0/4)
of the non-basal TNBC, with one non-basal BRCA1 mutant ER+
tumour having an HRDscore >0.7 (p=0.031 Fishers exact test,
Fig. 5a). Analysis of the immune micro-environment of baseline
samples with CIBERSORT29 suggested higher levels of follicular
helper T cells (p=0.0126, n=20 patients) in HRDetect+ve
cancers along with higher levels of activated macrophages (Sup-
plementary Fig. 3a and b).
Basal n = 15
HR Detect >0.7
HR Detect <0.7
Non-Basal n = 5
ER+ve BRCA1/2
p = 0.031 Fishers’s exact test
Differentially expressed genes —
Good vs Poor response
Differentially expressed genes —
Low CDR15 vs High CDR15
Interferon-α response
Enrichment score
G2M Checkpoint
Enrichment score
Interferon gamma response
Allograft rejection
Interferon alpha response
Inflammatory response
NFkB signalling
IL6 JAK STAT3 signalling
IL2 STAT5 signalling
Epithelial mesenchymal transition
KRas Signalling upregulation
p53 pathway
Apical junction
Xenobiotic metabolism
KRas Signalling downregulation
Reactive oxygen species pathway
Fatty acid metabolism
UV response upregulation
Unfolded protein response
MTORC1 signalling
G2M checkpoint
Cholesterol homeostasis
Protein secretion
MYC targets v2
E2F targets
Oxidative phosphorylation
MYC targets v1
Pathways NES from GSEA
p < 0.05
Normalized enrichment score
BRCA1/2 germline mutation
BRCA 1 methylation
RAD51C methylation
PALB2 germline mutation
Gene expression Log2
Gene expression Log2
Gene expression Log2
Gene expression Log2
q = 0.023
q = 0.027
q = 0.002
q = 0.05
–2 –1 0 1 2 3
50000 15,00010,000
Good responders
Poor responders
High CDR
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Cancers with ctDNA suppression (CDR < 0.25) had more
substantial changes in gene expression than cancers without ctDNA
suppression (CDR > 0.25), with 415 compared to 37 signicantly
differentially expressed genes respectively (Fig. 5b), demonstrating
stability of gene expression through rucaparib treatment in HR-
procient cancers. For individual genes, a signicant decrease in
gene expression was noted in PARP1 mRNA (q=0.023, n=20,
DESeq2 with false discovery rate (FDR) correction), MKI67 mRNA
(Ki67, q=0.027, n=20, DESeq2 FDR correction), as well as
induction in gene expression of both CDKN1A mRNA (p21, q=
0.002, n=20, DESeq2 FDR correction) and TMEM173 mRNA
(STING, q=0.05, n=20, DESeq2 FDR correction; Fig. 5c). In
HRDetect+ve cancers (Supplementary Fig. 4) and cancers with
associated ctDNA suppression (Fig. 5d) pathway analysis demon-
strated signicant gene expression changes in pathways that regulate
proliferation, apoptosis and immune function. There was decreased
expression of G2M checkpoint genes, reecting cell cycle arrest with
rucaparib (Fig. 5d). There was substantial increase in interferon
response genes, which along with TMEM173 mRNA expression,
suggested PARP inhibition activated the cGAScGAMPSTING
pathway in HR-decient cancers (Fig. 5d and Supplementary Fig. 4).
In contrast, HRDetect-ve cancers did not induce interferon response
genes, nor had expression changes of cell cycle arrest, on rucaparib
(Supplementary Fig. 4).
Results from the RIO trial demonstrate that a majority of primary
TNBC have defects in HR-based DNA repair, reinforcing the
companion population-based TNBC study by Staaf et al4. The
primary end point of RIO, suppression of Ki67 in on-treatment
biopsies, was infrequent. In pre-planned secondary analyses, we
show that cancers with HR deciency can be robustly identied
with the mutational-signatures based classier HRDetect (Fig. 2
and Supplementary Fig. 1), which identies cancers with a
functional deciency in HR (Fig. 3b), and with evidence of
activity of PARP inhibitors restricted to these cancers using
ctDNA analysis (Fig. 3c). In ad hoc analysis, HRDetect was more
specic to underlying HR deciency than HRD scores (Supple-
mentary Fig. 1b), suggesting that mutational signature assessment
might be more accurate in identifying cancers that would benet
from platinum chemotherapy or PARP inhibition4,3032.
Induction of RAD51 nuclear foci after neoadjuvant chemotherapy
and PARP inhibition can measure the homologous recombination
functionality in breast cancer biopsies20,3336, with an association to
loss of heterozygosity measures of HR deciency34,37. Studies have
shown that cells with decient BRCA1/2 or other HR proteins, do
not efciently form RAD51 foci which could be used as a marker for
PARP inhibitor sensitivity using FFPE tumour samples35,38,39.The
dynamics of DNA repair alter throughout tumour evolution and a
functional RAD51 assay can be used as a dynamic readout of
tumour HR status at the specic time for treatment decision-
making33,40,41. Current studies are using immunouorescence (IF)
on FFPE samples which can be labour intensive, and here we
develop a RAD51 foci immunohistochemistry (IHC). Our cutoff
<20% for HR deciency is consistent with the RECAP test which has
recently shown to be effective in ascertaining HR deciency in
metastatic breast tumours treated with ionising radiation36.Further
validation of this novel HR decient biomarker is required and if
clinically validated could be a useful tool in the clinic.
One of the main limitations of the study was lower recruitment
into the trial than was anticipated. This was possibly due to
patient preference to start treatment without the possibility of
delaying for short-term trial therapy. Additionally, failure of tis-
sue biopsies containing enough tumour content impacted end
point analysis. We therefore demonstrate the advantage of non-
invasive analysis, and the use of ctDNA as a potentially reliable
and effective surrogate end point to assess response. This will
require further validation.
We demonstrate that PARP inhibitors induce a pro-inam-
matory/interferon response in HR-decient TNBC, likely though
the cGAScGAMPSTING pathway. Consistent with our nd-
ings, PARP inhibition has previously been shown to induce T-cell
recruitment through activation of the cGAS-cGAMP-STING
pathway in a BRCA1-null mouse model of TNBC42. Similarly,
PARP inhibitors have been shown to upregulate interferon
response in TNBC cell lines with BRCA2 depletion43 or mutant
BRCA144. Furthermore, Sceneay et al45 recently demonstrated
using mouse models of TNBC, that immune dysfunction char-
acterised by decreased interferon signalling and decreased antigen
presentation was abrogated by a STING agonists. Together these
ndings underline the potential of exploiting immune dysfunc-
tion in the context of HR deciency, notably in BRCA1/2 mutant
tumours, and TNBC more generally.
Our ndings illustrate the potential of using whole-genome
sequencing mutational signatures to guide cancer treatment,
advocating for clinical trials of PARP inhibitors, potentially in
combination with PD(L)1 targeting immune checkpoint anti-
bodies, in sporadic TNBC with HR deciency.
Study Design. Conducted in 10 hospitals throughout the United Kingdom, the
window study of the PARP inhibitor rucaparib in patients with primary triple
negative or BRCA1/2 related breast cancer (The RIO study; EudraCT 2014-003319-
12, Cancer Research UK trial CRUK/12/034) was a single-group, open-label, phase
II window of opportunity trial assessing rucaparib efcacy in patients with primary
triple negative or BRCA1/2 mutant breast cancer prior to commencing primary
treatment (neoadjuvant chemotherapy or surgery). The trial was co-sponsored by
the Institute of Cancer Research and the Royal Marsden Hospital NHS Foundation
Trust. Ethical approval for The RIO trial was given by the NRES Committee
London - Fulham Research Ethics Committee (REC ID: 14/LO/2181) and
informed consent was obtained from all patients enrolled in the study.
Key eligibility criteria include breast tumour size 2 or <2 cm with cytologically/
histologically conrmed axillary lymph nodes, WHO performance status 02, no
prior history of ipsilateral breast cancer within 5 years and no prior treatment with
PARP inhibitors. Patients received rucaparib 600 mg twice daily for 12 14 days.
Baseline bloods (EDTA and STRECK) and core biopsies (FFPE and RNAlater)
were collected at time of diagnostic biopsy or following trial entry. End of
Fig. 5 Expression analysis of primary triple negative homologous recombination repair decient cancers. a Association between basal-like and non-
basal-like triple negative subtypes, assessed by PAM50, and HRDetect score. pvalue Fishers exact test. n=20 paired tumour samples. Purple, HRDetect
score >0.7; Grey, HRDetect score <0.7. bChange in gene expression on paired tumor biopsies between baseline and end of treatment on rucaparib.
Number of genes with a signicant change in gene expression (Log fold change >0.5 and false discovery corrected qvalue < 0.1). Left, categorised by
ctDNA suppression or not (CDR15 response <0.25 vs 1, n=8 and n=3 paired tumour samples respectively). Right, by CDR15 response and HRD score
(CDR15 < 0.25 and HRD > 0.7 vs CDR15 1 and HRD < 0.7, n=11 and n=6 paired tumour samples respectively). cGene expression changes of PARP1,
MKI67, CDKN1A and TMEM173 through treatment, from DESeq2 with false discovery rate (FDR) corrected qvalue for change. n=20 paired tumour
samples. Magenta lines, BRCA1/2 germline mutation; Blue lines, BRCA1 methylation; Yellow lines, PALB2 germline mutation; Turquoise lines, RAD51C
methylation; Grey lines, None/unknown. dLeft, Gene set enrichment pathway analysis (GSEA) for gene expression changes through treatment in patients
with ctDNA suppression (CDR15 < 0.25, n=8 paired tumour samples.) on rucaparib. Centre, suppression of G2M checkpoint genes on PARP inhibition,
q=0.006, and right, increased expression of interferon pathway genes on PARP inhibition, q=0.001. False discovery rate corrected qvalue for change.
8NATURE COMMUNICATIONS | (2020) 11:2662 | /10.1038/s41467-020-16142-7 |
Content courtesy of Springer Nature, terms of use apply. Rights reserved
treatment bloods (STRECK) and biopsies (FFPE and RNAlater) were taken at
surgery or prior to neo-adjuvant chemotherapy within 2448 h of the last rucaparib
dose. The primary end point was Ki67 response from baseline to end of treatment
dened as a 50% decrease. Secondary endpoints (pre-specied endpoints in the
trial protocol) were association between baseline biomarkers of BRCA1 methylation
and a genomic predictor of HR deciency (HRdetect) with Ki67 response to
rucaparib, apoptosis induction following 1214 days of rucaparib, the proportion
of sporadic TNBC that fail to induce RAD51 foci on end of treatment biopsy
(RAD51 score), safety and tolerability of rucaparib, association of biomarkers with
RAD51 score, the proportion of patients with a change in circulating tumour DNA
levels in response to rucaparib, association between change in circulating tumour
DNA levels with biomarkers, and proportion of patients with BRCA1 and BRCA2
germline mutation related cancers with a Ki67 response to rucaparib and the
proportion that have reduced RAD51 score and increased apoptosis induction.
Pathological complete response was not assessed as an end point, as the two weeks
window was considered to be too short for this end point. Where molecular
analysis was not pre-specied in the trial protocol, this is highlighted as being ad
hoc analsyis.
Ki67 IHC and Scoring. Immunohistochemistry for Ki67 was performed and
scored according to Leung et al46. Tissue sections were deparanised and rehy-
drated prior to antigen retrieval using low pH (pH 6.0) Target Antigen Retrieval
Solution (K8005, Dako UK Ltd). Tissue sections were stained for Ki67 using mouse
monoclonal anti-Ki67 antibody (MIB-1 clone, Dako, M7240) diluted 1:50 in
EnVision Antibody Diluent (K8006, Dako UK Ltd). Sections were washed using
wash buffer (Dako, S3006) before colour development using REAL Kit (Dako,
K5001). Tissues sections were counter stained with hematoxylin and coverslips
mounted using DPX. The percentage of Ki67 positive cells was calculated from
between 200-400 invasive tumour cells according to the method of Leung et al46.A
Ki67 response was determined as a >50% fall in Ki67 at EOT compared to baseline.
Cleaved PARP IHC and scoring. IHC for cleaved PARP (cPARP) was performed
using a rabbit monoclonal antibody specic for PARP cleaved at Asp214. Tissue
sections were deparanised and rehydrated prior to antigen retrieval using High
pH (pH9.0) Target Antigen Retrieval Solution (K8004, Dako UK Ltd), in PT-LINK
(PT101, Dako UK Ltd). Tissue sections were stained for cPARP using rabbit
monoclonal anti-cPARP antibody (Asp214, clone D64E10, Cell Signaling Tech-
nology, #5625) diluted 1:100 in EnVision Antibody Diluent (K8006, Dako UK Ltd).
Sections were washed using wash buffer (Dako,S3006) before colour development
using anti-rabbit link reagent EnVision FLEX LINKER (K8019, Dako UK Ltd).
Tissues sections were counter stained with hematoxylin and coverslips mounted
using DPX. The percentage of cPARP positive cells was calculated from a mini-
mum of 500 invasive tumour cells. If fewer than 500 invasive tumour cells were
present the sample was recorded as insufcient invasive tumour (IIT).
Sample processing. All samples were processed by the central laboratory as part
of the RIO trial. Fresh tumour samples were collected in RNAlatertubes, pro-
cessed within 24 h and stored at -80oC until required for extraction. Baseline
biopsies were sectioned using a cryostat. One section was cut for H&E and
16 sections were cut and stained with Nuclear Fast Red (NFR). A second H&E
section was cut at the end of the series. H&E sections were reviewed and marked by
a pathologist for macro-dissection. If the baseline biopsy did not have tumour the
EOT biopsy (day 1214) was used for sequencing. DNA was extracted using the
Qiagen DNeasy Blood and Tissue kit according to the manufacturers instructions.
DNA was eluted into 200 μl buffer ATE and stored at -20oC before quantication.
DNA was quantied on the Bio-Rad QX-200 ddPCR system running Quantasoft
v1.7, using the RPPH1 (RNaseP, cat# 4403328 ThermoFisher) reference assay to
calculate copies/well and multiplying by the cvalue (3.3 pg), an estima te of the
mass of a single haploid human genome.
Blood collected in STRECK preservation tubes at baseline (day 1 prior to
treatment) and end of treatment were processed within 24 h of sample collection .
Plasma and buffy coat was separated by centrifugation 1600g for 20 min and stored
individually at 80oC until DNA extraction. For plasma extraction, up to 4mls of
archived plasma was extracted using the automated MagMax Cell-Free DNA
Isolation Kit (Thermo Cat # A29319) and ThermoScientic KingFisher Flex
Purication System. DNA was eluted into 100 μl buffer AVE and stored at 20oC.
Buffy coat extraction was performed using the Qiagen DNeasy blood and tissue kit
as per manufacturers instructions. DNA was eluted into 100 μl buffer AE and
stored at 20 °C. Plasma DNA was quantied on the Bio-Rad QX-200 ddPCR
system using the RPPH1 reference assay to calculate copies/well and multiplying by
the cvalue (3.3 pg), an estimate of the mass of a single haploid human genome.
HRDetect Assay. Extracted DNA from fresh tissue with >20% tumour content
and >200 ng quantiable DNA, along with paired buffy coat germline DNA, were
subject to whole-genome sequencing at the Sanger Institute, Cambridge, UK.
A 500-bp insert genomic libraries were constructed according to Illumina
library protocols and 150 bp paired-end sequencing performed on an Illumina
HiSeq X Ten using HCS (v3.5.0) for HiSeq X systems, to an average sequence depth
of 38.5× for both tumour and normal. The resulting reads were aligned to the
reference human genome (GRCh37) using Burrows-Wheeler Aligner (BWA)
(0.7.16a (r1181)). Mutation calling was performed as described previously12.
CaVEMan (Cancer Variants Through Expectation Maximization: http://cancerit. was used for calling somatic substitutions. Indels in the
tumour and normal genomes were called using a modied Pindel version 2.0.
( Structural variants were discovered using a
bespoke algorithm, BRASS (BReakpoint AnalySiS) (
BRASS). All annotation was to Ensembl build 75. Allele-specic copy number
analysis of tumours was performed using ASCAT (v2.1.1) applied to next-
generation whole-genome sequencing data as described previously11,12. Copy
number values and estimates of aberrant tumour cell provided by ASCAT were
input into the CaVEMan substitution algorithm. In addition, ASCAT segmentation
proles were used to establish the presence of copy number changes and loss of
heterozygosity across the BRCA1,BRCA2 and PALB2 genes.
The predominant mutational signatures present in breast cancer have been
identied in a large WGS study involving 560 breast cancers. These comprise
12 substitution signatures and 6 structural rearrangement signatures. The
contributions of these consensus mutational signatures were estimated in the 27
RIO trial WGS samples as described previously12,47. In addition, the contribution
of small insertions and deletion at regions of micro-homology or repeats and HRD
LOH index were estimated7.
Mutational signature contributions for substitution signatures 3 and 8,
rearrangement signatures 3 and 5, deletions at microhomology and HRD LOH
index were calculated for each sample as input in to the weighted model, HRDetect.
The HRDetect algorithm was run as described previously, using the previously
described weights11.
Targeted tissue sequencing. Paired tissue and buffy coat DNA were sent to the
Centre of Molecular Pathology at The Royal Marsden Hospital for sequencing
using a targeted capture-based approach designed to detect mutations and
amplications frequently seen in breast cancer. The targeted panel, ABC-Bio
panel, has been validated in the ABC-Bio (molecular screening for patients with
advanced breast cancer) trial and comprises of 41 genes commonly mutated in
breast cancer48. Libraries were run on a MiSeq (Illumina) using MiSeq Reporter
(MSR v2.5.1). All internally developed code is accessible on request.
AVENIO sequencing. ctDNA samples from six patients from whom adequate
tumour samples were not obtained were sent to Roche for sequencing using the
AVENIO ctDNA targeted tumour proling kit (Roche Sequencing). A total of
4 samples were sequenced on the AVENIO ctDNA targeted kit (17 genes) and
2 samples were run on the AVENIO ctDNA expanded kit (77 genes), using
HighOutput 300 cyc kit on a NextSeq 500 (Illumina) using NextSeq system suite
(v2.2.0). Data was analysed using the AVENIO Oncology analysis software (v1.0.0
and v1.1) available from Roche.
BRCA1 and Rad51C methylation, bisulte sequencing. The promoter region of
BRCA1 and RAD51C was identied using the Eukaryotic Promoter Database
( BRCA1 promoter was amplied with forward-
TATCCC primers. Analysis of BRCA1 methyaltion was pre-planned, and RAD51C
methylation was added ad hoc to BRCA1 methylation prior to association with
activity surrogates, as the potential importance of RAD51C methylation in TNBC
was only recognised after the start of the trial2. The RAD51C promoter was
amplied with forward-TGGTAATTGGTTAGTGTGTGT and reverse-TCCTCA
TCAAATATACACCCTAACT primers. BRCA1 and RAD51C PCR conditions
were optimised for multiplex assay using ThermoFisher Scientic AccuPrime Hi-
delity Taq. Human methylated and unmethylated DNA (Zymo Research, Human
HCT116DKO non-methylated DNA and HCT116DKO methylated DNA) primers
were used as a control.
Extracted DNA from RIO RNA Later samples were subjected to bisulte
sequencing (Zymo Research Methylation Gold spin column kit D5005). Total DNA
input ranged from 10 to 500 ng. Samples were quantied post bisulte sequencing
using Qubit 3.0 uorimeter and subsequently subjected to PCR using
ThermoFisher Scientic AccuPrime Hi-delity Taq at 60 °C for 34 cycles. Samples
were cleaned using Qiagen QIAquick PCR purication kit (ID:28104) and
quantied using Qubit 3.0 uorimeter. Samples were subjected to Illumina
NebNext Ultra II library preparation. Total library input ranged from 10-116 ng
and PCR cycles were adjusted accordingly, and sequenced on the Illumina MiniSeq
platform with Miniseq system suite v1.1. Mean number of reads for BRCA1
amplicon was 36909 (range 9654-60084) and RAD51C amplicon 48879 (range
28404- 71129) with a mean 47% of reads on target for the methylation sequencing
run (range 37-50%).
Bioinformatics analysis of methylation followed a similar workow to previous
studies32. Paired overlapping reads were merged into a single sequence using
ash49 after adaptor trimming using trim-galore (https://www.bioinformatics. Each read was aligned using pairwise
alignment to the BRCA1 or RAD51C amplicons using Biostrings R50 package with
90% identity. Reads with more than 1 mismatch in alignment were additionally
removed. Reads with incomplete bisulphite conversion were removed by
NATURE COMMUNICATIONS | (2020) 11:2662 | /s41467-020-16142-7 | 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved
calculating the unconverted cytosine count at non CG sites as well as reads where
all CG sites in the read were not C or T. Reads were assessed as being methylated
when >90% of CpG sites in the amplicon were methylated. For RAD51C
methylation, 2 sites in the RAD51C amplicon were removed as these were found to
be consistently methylated in all samples.
Immunohistochemistry for RAD51/Geminin (GMNN) double staining. FFPE
samples taken 2448 h after the last dose of rucaparib were cut, deparanised,
rehydrated and stained with hematoxylin and eosin (H&E) and double stained with
GMNN and RAD51. Pre- and post-radiotherapy-induced squamous cell carcinoma
were used as negative and positive controls.
Antigen retrieval was performed and RAD51 primary antibody (mouse
monoclonal, Genetex, GTX70230) was diluted 1/200 in Dako antibody diluent
(K8006) and applied. Slides were incubated with Dako Envision Flex HRP (K8002).
Geminin (GMNN) antibody (rabbit polyclonal, Proteintech 10802-1-AP) diluted
1/1500 in Dako antibody diluent was applied and incubated with Dako Envision
Flex HRP (K8002). Sections incubated with Vector TMB blue (SK4400) and
counterstained with Gills 1 hematoxylin, air dried and dehydrated in xylene before
mounted and cover slipped with Vectamount.
Five random elds at 40× magnication were identied and marked in PathXL.
GMNN staining was identied with blue/green staining and RAD51 was identied
by the presence of brown nuclear foci. Scoring was done by 2 scorers blinded to
each other, time point and clinical details.
The number of tumour cells, GMNN-positive cells and RAD51-positive cells
were counted. Cells with 5 or more RAD51 foci were classied as RAD51 positive.
A minimum of 300 tumour cells and 30 GMNN-positive cells were a minimum
requirement for inclusion. Raw data were collected and the proliferation fraction
((no. of GMNN-positive cells / total number of tumour cells) × 100) and RAD51
score ((no. of RAD51-positive cells / no. of GMNN-positive cells) × 100) was
Circulating tumor DNA analysis. For each trial subject, dPCR assays were
designed for the mutations identied by tissue sequencing according to the method
of Garcia-Murillas et al22. Assays were optimised with temperature gradients and
patients with more than one mutation had multiplex assays optimised. If optimi-
sation was not achieved with multiplexes, a singleplex assay was used.
Mutation analysis was done using ddPCR assays specic for each patients
mutation(s) on a QX200 system (Bio-Rad) running Quantasoft v1.7. 50 μl DNA
(2 ml plasma equivalent) was used divided equally into 2 wells from each time
point. DNA was dried at 60 degrees for 100 min before preparing the PCR
reactions to a volume of 20 μl. Three NTCs and a negative control of the patients
buffy coat DNA were included for each dPCR assay. Only paired samples with at
least 4 positive droplets in baseline samples were analysed for change in ctDNA
levels. The circulating DNA ratio at day 15 (CDR15) was assessed as a ratio of the
ctDNA copies/ml at EOT copies/ml compared to ctDNA copies/ml at baseline.
Where more than one mutation was tracked a weighted mean of ctDNA change
was calculated.
The CDR15 cutoff <0.25 for ctDNA suppression, was pre-specied determined
in a separate study in metastatic breast cancer, that validated this cutoff to predict
progression free survival on cytotoxic paclitaxel therapy25.
RNA sequencing. Paired RNAlatersamples were identied and sectioned using a
cryostat. One section was cut for H&E and 10 sections were cut and stained with
nuclease-free Nuclear Fast Red. A second H&E section was cut at the end of the
series. H&E sections were reviewed and marked by a pathologist for tumour and
assessed for tumour content. NFR stained sections were micro-dissected and RNA
was extracted using the Qiagen RNeasy Mini kit according to the manufacturers
instructions. RNA was eluted twice into separate 50 μl RNA free water and stored
at -80oC before quantication. RNA was quantied using Qubit 3.0 uorimeter
using the QubitRNA HS Assay Kit (Q32852, ThermoFisher Scientic).
Extracted RNA (~1 μg) was sent to Eurons Genetic Services Limited for RNA
Exome sequencing. Total RNA was subjected to RiboZero depletion and Illumina
TruSeq RNA Exome library preparation. Libraries were pooled and sequenced on
an Illumina HiSeq 2500 (v4 chemistry) running HiSeq Control Software (HCS)
v2.2.68. Samples were aligned to the GChr37 genome using STAR aligner (https:// with a mean of 44,117,169 reads per sample (range
14010752-91,470,225). Gene counts were established using htseq (https://github.
com/simon-anders/htseq)52. DeSeq2 (
was used to establish gene-wise normalisation and to look for differential
expression between different sample groups. Gene set enrichment analysis was
carried out using the R package fgsea (
PAM50 and TNBC subtypes were established using AIMS (
10.18129/B9.bioc.AIMS)55 and TNBCtype (
Cibersort ( was run in absolute mode
using normalized gene counts.
Statistics. The study size was determined using a Simon two stage Minimax design
on Ki67 response in patients with sporadic TNBC, with p0 =10% and p1 =25%
Ki67 response rate. With a two-sided alpha 1.6% and 90% power, four Ki67
responders were required in the rst 41 assessable patients to proceed to a full 73
patients. The study would declare inefcacy if <4/41 or <14/73 responses were
observed. An initial futility assessment was also planned after 20 evaluable patients
had completed rucaparib treatment and consideration would be given to stopping
the trial if 0 responses were observed. Additionally, 5, 1.6 and 1.6% two-sided
alphas were allocated to assess rucaparib activity within BRCA1 methylated
tumours, RAD51 foci formation tumours, and genomic classier HRDetect
tumours respectively, for a total study two-sided alpha of 10%. Up to 20 patients
with known BRCA1 or BRCA2 pathogenic germline mutations at the time of trial
entry were recruited as controls for exploratory determination of biomarker end-
points. The study closed to new patients after 43 patients had been recruited on
advice of the IDMC (Fig. 1) due to low recruitment.
Analysis of response data was performed on patients who had taken rucaparib
for 7 days or more. Safety and tolerability are assessed in all patients who received
at least one dose of rucaparib. Response rates and proportions are reported with
95% condence intervals. Associations between biomarkers are analysed using
Fishers exact test or MannWhitney as appropriate. Change in biomarkers
between baseline and day1214 samples are analysed using the Wilcoxon signed
rank test. Analyses were conducted in Stata v13 and GraphPad Prism, with all
analyses reported two-sided.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
Sequencing data from whole-genome sequencing, exome RNA seq, and targeted
sequencing from tumour samples that support the ndings of this study (Figs. 15and
Supplementary Fig. 1, 3 and 4) is deposited in the European Genome-phenome Archive
(EGA), reference EGAS00001004405
Code availability
The code used for the analysis of sequencing data is detailed here with links and
references where appropriate.
Whole-genome sequencing: CaVEMan (Cancer Variants Through Expectation
Maximization:, Pindel version 2.0. (http://cancerit., BRASS (BReakpoint AnalySiS) (
BRASS), and ASCAT (v2.1.1). Annotation was to Ensembl build 75. Code for targetted
tumour sequencing is available upon request. AVENIO ctDNA sequencing: AVENIO
Oncology analysis software (v1.0.0 and v1.1) available from Roche. Bisulte sequencing:
Eukaryotic Promoter Database (, trim-galore (https://, Biostrings R package. RNA
sequencing: STAR aligner (, htseq (https://github.
com/simon-anders/htseq), DeSeq2 (, fgsea
(, AIMS (,
TNBCtype ( and Cibersort (https://cibersort.stanford.
Received: 18 July 2019; Accepted: 3 April 2020;
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This study was funded by the Cridlan Trust, Clovis Oncology Inc., Cancer Research UK
(CRUK; CRUK Ref A15777) core funding to the ICR Clinical Trials and Statistics Unit,
Breast Cancer Now to the Breast Cancer Now Research Centre at The Institute of Cancer
Research, and NHS funding to the NIHR Royal Marsden Biomedical Research Centre.
HRD is funded by a CRUK Grand Challenge Award (C38317/A24043). AD is funded by
a CRUK Pioneer Award. We acknowledge the assistance Katy Jarman, Lynsey Houlton,
Vera Martins, Arjun Naginlal Modi. Sequencing, genomic analyses and personal funding
of SNZ was supported by the Wellcome Trust Intermediate Clinical Fellowship
(WT100183MA), Wellcome Beit Prize, CRUK Advanced Clinician Scientist award
(C60100/A23916) and CRUK Pioneer Award.
Author contributions
N.T. conceived the RIO trial and was the chief investigator. N.T., J.B., C.T., M.D. A.T.
and A.S. formed the RIO protocol development group rening the design. JB oversaw
trial conduct in ICR-CTSU, statistical analysis and data interpretation. N.C. and A.P.
designed and performed translation studies. H.T. and R.C. performed statistical and
bioinformatics analyses. C.T. was RIO study senior trial manager. P.P. and M.H. per-
formed targeted next-generation sequencing experiments. A.D. and F.D. performed
immunohistochemistry experiments. D.K. and H.G. provided pathology support and
NATURE COMMUNICATIONS | (2020) 11:2662 | /s41467-020-16142-7 | 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved
analysed immunohistochemistry. H.R.D., A.D. and S.N.Z. performed whole-genome
sequencing and HRDetect analysis. R.R., S.C., A.T., A.S., and A.E. were principal
investigators at trial sites. N.T., N.C. and A.P. wrote the manuscript and all authors
contributed comments and corrections.
Competing interests
N.T., J.M.B., H.T. and C.T. report research grants and non-nancial support in the form
of study drug provision and distribution from Clovis Oncology Inc., during the conduct
of the study. N.T. reports advisory board Honoraria from AstraZeneca, Bristol-Myers
Squibb, Lilly, Merck Sharpe and Dohme, Novartis, Pzer, Roche/Genentech, Tesaro,
Bicycle Therapeutics and research funding from Astra Zeneca, BioRad, Pzer, Roche/
Genentech and Guardant Health, outside the submitted work. J.M.B. reports grants and
non-nancial support from AstraZeneca, Merck Sharpe & Dohme, Puma Biotechnology
and Janssen-Cilag, grants, non-nancial support and travel support from Pzer and
grants from Medivation, Novartis and Roche outside the submitted work. M.H. reports
Honoraria or research funding from Boehringer Ingelheim, Roche Diagnostics, Bristol
Myers Squibb, Guardant Health, Celgene, Eli Lilley outside of the submitted work. R.R.
reports Honorarium from Pzer. A.T. reports benets from ICRs Inventors Scheme
associated with patents for one of the PARP inhibitors in BRCA1/2 associated cancers.
A.T. also reports Honoraria from Pzer, Vertex, Prime Oncology, Artios, honoraria and
stock in InBiomotion, honoraria and nancial support from AstraZeneca, Medivation,
Myriad Genetics, Merck Serono. S.N.Z. and H.D. are inventors on a patent application
(WO2017191074A1) for HRDetect. S.N.Z. reports advisory board honoraria from Astra
Zeneca and Artios Pharma. M.D. reports advisory board Honoraria from GTx, Radius,
Orion and G1therapeutics, lectures fees from Myriad and Nanostring, research funding
from Pzer and Radius and income from the Institute of Cancer Researchs Rewards for
Inventors Scheme (Abiraterone) outside the submitted work. All other authors declare no
competing interests.
Additional information
Supplementary information is available for this paper at
Correspondence and requests for materials should be addressed to N.C.T.
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... We compared the proportion of the intrinsic BSL subtype among the BSL, TKR, and MSL subgroups and found that the BSL subgroup had the highest probability of intrinsic BSL subtype in both the FUSCC and TCGA data sets ( Figure 5A). HRD represents genomic instability and has been identified as an efficient therapeutic biomarker for TNBC (36). The BSL subgroup constituted 57% of the mutation HRD subtype in HER2-low TNBC samples ( Figure 2G, right). ...
... HRD, which represents genomic instability (36), can be used to identify patients who might benefit from DNA-damaging agents (52) and predict the response to platinum-neoadjuvant chemotherapy (53). HRD scores were higher in the BSL subgroup and could be a potential method to identify BSL subgroups. ...
... We found that RMI1 promoted HR, not NHEJ repair (Fig. 6A, B). Given that RAD51 nuclear foci is a hallmark for HR-mediated repair [36], we further explored how RMI1 influences HR efficiency by examining the RAD51 foci formation. We observed the impaired ability to form RAD51 nuclear foci in RMI1-silenced cells after IR treatment (Fig. 6C, D), suggesting that RMI1 might facilitate loading RAD51 to the DNA damage sites. ...
Full-text available
Ionizing radiation (IR) causes a wide variety of DNA lesions, of which DNA double-stranded breaks (DSBs) are the most deleterious. Homologous recombination (HR) is a crucial route responsible for repairing DSBs. RecQ-mediated genome instability protein 1 (RMI1) is a member of an evolutionarily conserved Bloom syndrome complex, which prevents and resolves aberrant recombination products during HR, thereby promoting genome stability. However, little is known about the role of RMI1 in regulating the cellular response to IR. This study aimed to understand the cellular functions and molecular mechanisms by which RMI1 maintains genomic stability after IR exposure. Here, we showed IR upregulated the RMI1 protein level and induced RMI1 relocation to the DNA damage sites. We also demonstrated that the loss of RMI1 in cells resulted in enhanced levels of DNA damage, sustained cell cycle arrest, and impaired HR repair after IR, leading to reduced cell viability and elevated genome instability. Taken together, our results highlighted the direct roles of RMI1 in response to DNA damage induced by IR and implied that RMI1 might be a new genome safeguard molecule to radiation-induced damage.
... Several studies have demonstrated the prognostic and predictive value of ctDNA for non-mTNBC during or after (neo)adjuvant therapy (Cavallone et al., 2020;Kim et al., 2021;Lin et al., 2021;Ortolan et al., 2021;Riva et al., 2017). Previously, researchers have also been relatively circumscribed, concentrated on evaluating specific copy number variants (CNVs) or ctDNA-based single mutation or clonal evolution or the ctDNA level to predict the prognosis of mTNBC patients and the efficacy of particular treatment regimens (Barroso-Sousa et al., 2022;Chopra et al., 2020;Collier et al., 2021;Stover et al., 2018;Weber et al., 2021;Wongchenko et al., 2020). Even a study showed that the ctDNA level had no prognostic impact on the survival of patients with mTNBC (Madic et al., 2015). ...
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Background Limited data are available on applying circulating tumor DNA (ctDNA) in metastatic triple-negative breast cancer (mTNBC) patients. Here, we investigated the value of ctDNA for predicting the prognosis and monitoring the treatment response in mTNBC patients. Methods We prospectively enrolled 70 Chinese patients with mTNBC who had progressed after ≤2 lines of chemotherapy and collected blood samples to extract ctDNA for 457-gene targeted panel sequencing. Results Patients with ctDNA+, defined by 12 prognosis-relevant mutated genes, had a shorter progression-free survival (PFS) than ctDNA− patients (5.16 months vs . 9.05 months, p=0.001), and ctDNA +was independently associated with a shorter PFS (HR, 95% CI: 2.67, 1.2–5.96; p=0.016) by multivariable analyses. Patients with a higher mutant-allele tumor heterogeneity (MATH) score (≥6.316) or a higher ctDNA fraction (ctDNA%≥0.05) had a significantly shorter PFS than patients with a lower MATH score (5.67 months vs .11.27 months, p=0.007) and patients with a lower ctDNA% (5.45 months vs . 12.17 months, p<0.001), respectively. Positive correlations with treatment response were observed for MATH score ( R =0.24, p=0.014) and ctDNA% ( R =0.3, p=0.002), but not the CEA, CA125, or CA153. Moreover, patients who remained ctDNA +during dynamic monitoring tended to have a shorter PFS than those who did not (3.90 months vs . 6.10 months, p=0.135). Conclusions ctDNA profiling provides insight into the mutational landscape of mTNBC and may reliably predict the prognosis and treatment response of mTNBC patients. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81902713), Natural Science Foundation of Shandong Province (Grant No. ZR2019LZL018), Breast Disease Research Fund of Shandong Provincial Medical Association (Grant No. YXH2020ZX066), the Start-up Fund of Shandong Cancer Hospital (Grant No. 2020-PYB10), Beijing Science and Technology Innovation Fund (Grant No. KC2021-ZZ-0010-1).
PURPOSE Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. METHODS HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial ( identifier: NCT02624973 ). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. RESULTS HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). CONCLUSION The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.
Identification of tumours that have homologous recombination deficiency (HRD) has become of increasing interest following the licensing of PARP inhibitors. Potential methods to assess HRD status include; clinical selection for platinum sensitive disease, mutational/methylation status, genomic scars/signature and functional RAD51 assays. Homologous recombination (HR) is a dynamic process with the potential to evolve over a disease course, particularly in relation to previous treatment. This is one of the major drawbacks of genomic scars/signatures, as they only demonstrate historic HR status. Functional HR assays have the benefit of giving a real time HR status readout and therefore have the potential for clearer identification of patients who may benefit from PARP inhibitors at that specific time point. However, the development of RAD51 foci assays ready for clinical practice has been challenging. Pre-clinical considerations have included; controlling for variation in tumour proliferation, tissue type and whether DNA damage induction is required. Furthermore, the assays require correlation with clinical outcomes, an understanding of how they complement current testing modalities and validation of test performance in large cohorts. Despite these challenges, given the profound benefit from PARP inhibitors seen in those with an HRD phenotype to date, the ongoing development and validation of these functional HR assays remains of high clinical importance.
PURPOSE Triple-negative breast cancer (TNBC) is a heterogeneous disease. We previously showed that homologous recombination deficiency (HRD) and the DNA damage immune response (DDIR) signature are prognostic in TNBC. We hypothesized that these biomarkers reflect related but not completely interdependent biological processes, that their combined use would be prognostic, and that simultaneous assessment of the immunologic microenvironment and susceptibility to DNA damaging therapies might be able to identify subgroups with distinct therapeutic vulnerabilities. METHODS We analyzed the dual DDIR/HRD classification in 341 patients with TNBC treated with adjuvant anthracycline-based chemotherapy on the SWOG S9313 trial and corroborated our findings in The Cancer Genome Atlas breast cancer data set. RESULTS DDIR/HRD classification is highly prognostic in TNBC and identifies biologically and immunologically distinct subgroups. Immune-enriched DDIR+/HRD+ TNBCs have the most favorable prognosis, and DDIR+/HRD– and DDIR–/HRD+ TNBCs have favorable intermediate prognosis, despite the latter being immune-depleted. DDIR–/HRD– TNBCs have the worst prognosis and represent an internally heterogeneous group of immune-depleted chemoresistant tumors. CONCLUSION Our findings propose DDIR/HRD classification as a potentially clinically relevant approach to categorize tumors on the basis of therapeutic vulnerabilities.
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Purpose Approximately 10% to 15% of triple-negative breast cancers (TNBC) have deleterious mutations in BRCA1 and BRCA2 and may benefit from PARP inhibitor treatment. PARP inhibitors may also increase exogenous replication stress and thereby increase sensitivity to inhibitors of ataxia telangiectasia and Rad3-related (ATR) protein. This phase II study examined the activity of the combination of PARP inhibitor, olaparib, and ATR inhibitor, ceralasertib (AZD6738), in patients with advanced TNBC. Patients and Methods Patients with TNBC on most recent biopsy who had received 1 or 2 lines of chemotherapy for advanced disease or had relapsed within 12 months of (neo)adjuvant chemotherapy were eligible. Treatment was olaparib 300 mg twice a day continuously and celarasertib 160 mg on days 1–7 on a 28-day cycle until disease progression. The primary endpoint was confirmed objective response rate (ORR). Tissue and plasma biomarker analyses were preplanned to identify predictors of response. Results 70 evaluable patients were enrolled. Germline BRCA1/2 mutations were present in 10 (14%) patients and 3 (4%) patients had somatic BRCA mutations. The confirmed ORR was 12/70; 17.1% (95% confidence interval, 10.4–25.5). Responses were observed in patients without germline or somatic BRCA1/2 mutations, including patients with mutations in other homologous recombination repair genes and tumors with functional homologous recombination deficiency by RAD51 foci. Conclusions The response rate to olaparib and ceralasertib did not meet prespecified criteria for activity in the overall evaluable population, but responses were observed in patients who would not be expected to respond to olaparib monotherapy.
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Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterisation of a cohort of patients with ABC. Experimental design: Biopsies from patients with advanced breast cancer were sequenced with a 50 gene targeted panel in the Advanced Breast Cancer Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumour DNA (ctDNA) analysis, along with matched primary tumour to assess for acquisition in ABC in a subset of patients. Results: We sequenced 210 ABC samples, demonstrating enrichment compared to primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%) and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER positive cancer cell-line models, loss of NF1 resulted in endocrine therapy resistance, through both ER dependent and independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro Patients with NF1 mutations detected in baseline circulating tumour DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. Conclusions: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.
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Whole-genome sequencing (WGS) brings comprehensive insights to cancer genome interpretation. To explore the clinical value of WGS, we sequenced 254 triple-negative breast cancers (TNBCs) for which associated treatment and outcome data were collected between 2010 and 2015 via the population-based Sweden Cancerome Analysis Network–Breast (SCAN-B) project ( ID:NCT02306096). Applying the HRDetect mutational-signature-based algorithm to classify tumors, 59% were predicted to have homologous-recombination-repair deficiency (HRDetect-high): 67% explained by germline/somatic mutations of BRCA1/BRCA2, BRCA1 promoter hypermethylation, RAD51C hypermethylation or biallelic loss of PALB2. A novel mechanism of BRCA1 abrogation was discovered via germline SINE-VNTR-Alu retrotransposition. HRDetect provided independent prognostic information, with HRDetect-high patients having better outcome on adjuvant chemotherapy for invasive disease-free survival (hazard ratio (HR) = 0.42; 95% confidence interval (CI) = 0.2–0.87) and distant relapse-free interval (HR = 0.31, CI = 0.13–0.76) compared to HRDetect-low, regardless of whether a genetic/epigenetic cause was identified. HRDetect-intermediate, some possessing potentially targetable biological abnormalities, had the poorest outcomes. HRDetect-low cancers also had inadequate outcomes: ~4.7% were mismatch-repair-deficient (another targetable defect, not typically sought) and they were enriched for (but not restricted to) PIK3CA/AKT1 pathway abnormalities. New treatment options need to be considered for now-discernible HRDetect-intermediate and HRDetect-low categories. This population-based study advocates for WGS of TNBC to better inform trial stratification and improve clinical decision-making.
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Heterozygous germline mutations in BRCA2 predispose to breast and ovarian cancer. Contrary to non-cancerous cells, where BRCA2 deletion causes cell cycle arrest or cell death, tumors carrying BRCA2 inactivation continue to proliferate. Here we set out to investigate adaptation to loss of BRCA2 focusing on genome-wide transcriptome alterations. Human cells in which BRCA2 expression is inhibited for 4 or 28 days are subjected to RNA-seq analyses revealing a biphasic response to BRCA2 abrogation. The early, acute response consists of downregulation of genes involved in cell cycle progression, DNA replication and repair and is associated with cell cycle arrest in G1. Surprisingly, the late, chronic response consists predominantly of upregulation of interferon-stimulated genes (ISGs). Activation of the cGAS-STING-STAT pathway detected in these cells further substantiates the concept that BRCA2 abrogation triggers cell-intrinsic immune signaling. Importantly, we find that treatment with PARP inhibitors stimulates the interferon response in cells and tumors lacking BRCA2.
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Background: Dynamic changes in circulating tumour DNA (ctDNA) levels may predict long-term outcome. We utilised samples from a phase I/II randomised trial (BEECH) to assess ctDNA dynamics as a surrogate for progression free survival (PFS) and early predictor of drug efficacy. Patients and methods: Patients with oestrogen receptor positive advanced metastatic breast cancer (ER+ mBC) in the BEECH study, paclitaxel plus placebo versus paclitaxel plus AKT inhibitor capivasertib, had plasma samples collected for ctDNA analysis at baseline and at multiple timepoints in the development cohort (safety run-in, part A) and validation cohort (randomised, part B). Baseline sample ctDNA sequencing identified mutations for longitudinal analysis, and mutation specific digital droplet PCR (ddPCR) assays were utilised to assess change in ctDNA abundance (allele fraction) between baseline and 872 on-treatment samples. Primary objective was to assess whether early suppression of ctDNA, based on pre-defined criteria from the development cohort, independently predicted outcome in the validation cohort. Results: In the development cohort, suppression of ctDNA was apparent after 8 days of treatment (p=0.014), with cycle 2 day 1 (4 weeks) identified as the optimal timepoint to predict PFS from early ctDNA dynamics. In the validation cohort, median PFS was 11.1 months in patients with suppressed ctDNA at 4 weeks and 6.4 months in patients with high ctDNA (HR = 0.20, 95% CI 0.083 - 0.50, p<0.0001). There was no difference in the level of ctDNA suppression between patients randomised to capivasertib or placebo overall (p=0.904) nor in the PIK3CA mutant subpopulation (p=0.071). Clonal haematopoiesis of indeterminate potential (CHIP) was evident in 30% (18/59) baseline samples, although CHIP had no effect on tolerance of chemotherapy nor on PFS. Conclusion: Early on-treatment ctDNA dynamics are a surrogate for PFS. Dynamic ctDNA assessment has the potential to substantially enhance early drug development.
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Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.
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Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor–induced changes in the tumor microenvironment of BRCA1-deficient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8+ T-cell infiltration and activation in vivo, and that CD8+ T-cell depletion severely compromises antitumor efficacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-deficient compared with HR-proficient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinflammatory signaling and is sufficient to abolish olaparib-induced T-cell infiltration in vivo. These findings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. Significance This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8+ T-cell recruitment and antitumor efficacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer. This article is highlighted in the In This Issue feature, p. 681
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The cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I IFN transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defective NSCLC and BRCA1-defective triple-negative breast cancer (TNBC) cells. Mechanistically, PARPi generated cytoplasmic chromatin fragments with characteristics of micronuclei; these were found to activate cGAS/STING, downstream type I IFN signaling, and CCL5 secretion. Importantly, these effects were suppressed in PARP1-null TNBC cells, suggesting that this phenotype resulted from an on-target effect of PARPi on PARP1. PARPi also potentiated IFN-γ-induced PD-L1 expression in NSCLC cell lines and in fresh patient tumor cells; this effect was enhanced in ERCC1-deficient contexts. Our data provide a preclinical rationale for using PARPi as immunomodulatory agents in appropriately molecularly selected populations.
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Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2-related cancers. A test to identify additional HRR-deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2-related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi-sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2-related cancers. © 2018 The Authors. Published under the terms of the CC BY 4.0 license
Immune checkpoint blockade (ICB) therapy, which targets T cell–inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy. Significance These data demonstrate for the first time that age determines the T cell–inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required. This article is highlighted in the In This Issue feature, p. 1143
PURPOSE Biomarkers that predict response to poly (ADP-ribose) polymerase inhibitors (PARPis) are required to detect PARPi sensitivity beyond germline BRCA-mutated (gBRCAm) cancers and PARPi resistance among reverted gBRCAm cancers. Therefore, we previously developed the Repair Capacity (RECAP) test, a functional homologous recombination (HR) assay that exploits the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer tissue. The aim of the current study was to validate the feasibility of this test on histologic biopsy specimens from metastatic breast cancer and to explore the utility of the RECAP test as a predictive tool for treatment with DNA-damaging agents, such as PARPis. METHODS Fresh tissue biopsies from easily accessible metastatic lesions from patients with locally advanced or metastatic breast cancer were irradiated with 5 Gy and cultured for 2 hours followed by detection of RAD51 foci presence (HR proficient) or absence (HR deficient [HRD]). HRD biopsy specimens as well as platinum/PARP-resistant specimens were subjected to BRCA1/2 sequencing. RESULTS RECAP had a success rate of 93% on biopsy specimens from metastatic breast cancer lesions (n = 44). Although HRD was detected in 13 (32%) of 41 specimens, only five showed a gBRCAm. In three patients with gBRCAm, post-treatment RECAP tests showed HR phenotype reversion after in vivo progressive disease on platinum and PARPi treatment, which was explained in one patient by a secondary BRCA1 mutation. CONCLUSION The RECAP test, which reflects real-time HR status regardless of BRCA mutations, is feasible in metastatic breast cancer biopsy specimens. Compared with gBRCA analysis, it may identify twice as many candidates for PARPi treatment.