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\begin{document}

\title{BRCA2 reversion mutation confers resistance to olaparib in breast cancer}



\author[1]{Shinya Yamamoto}%
\author[1]{Kei Kawashima}%
\author[1]{Yoshie Fujiwara}%
\author[1]{Shoko Adachi}%
\author[1]{Kazutaka Narui}%
\author[1]{Chiaki  Hosaka}%
\author[1]{Rina  Takahashi}%
\author[1]{Sho Tsuyuki}%
\author[1]{Makoto  Sugimori}%
\author[1]{Miki  Tanoshima}%
\author[2]{Mahato Sasamoto}%
\author[2]{Masanori Oshi}%
\author[2]{Akimitsu Yamada}%
\author[1]{Chikara  Kunisaki}%
\author[2]{Itaru Endo}%
\affil[1]{Yokohama City University Medical Center}%
\affil[2]{Yokohama City University School of Medicine Graduate School of Medicine}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
A 34-year-old woman with breast cancer and the BRCA2: p.Gln3047Ter was
treated with olaparib. After tumor progression, cancer genomic profiling
testing revealed the BRCA2 p.Gln3047Ter and p.Gln3047Tyr, with 48.9\%
and 0.37\% allele frequency, respectively. These findings shed light on
reversion mutation as a resistance mechanism to olaparib in breast
cancer.%
\end{abstract}%



\sloppy


Case report

\textbf{BRCA2 reversion mutation confers resistance to olaparib in
breast cancer}

Shinya Yamamoto\textsuperscript{1}, Kei
Kawashima\textsuperscript{1}Yoshie Fujiwara\textsuperscript{1}, Shoko
Adachi\textsuperscript{1}, Kazutaka Narui\textsuperscript{1}, Chiaki
Hosaka\textsuperscript{2}, Rina Takahashi\textsuperscript{2}, Sho
Tsuyuki\textsuperscript{3}, Makoto Sugimori\textsuperscript{3}, Miki
Tanoshima\textsuperscript{2}, Mahato Sasamoto\textsuperscript{4},
Masanori Oshi\textsuperscript{4}, Akimitsu Yamada\textsuperscript{4},
Chikara Kunisaki\textsuperscript{3}, Itaru Endo\textsuperscript{4}

\textsuperscript{1} Department of Breast and Thyroid Surgery, Yokohama
City University Medical Center, 4-57 Urafune-cho, Minami-ku, Yokohama
City, Kanagawa 232-0024, Japan

\textsuperscript{2} Department of Clinical Genetics, Yokohama City
University Medical Center, 4-57 Urafune-cho, Minami-ku, Yokohama City,
Kanagawa 232-0024, Japan

\textsuperscript{3} Department of Cancer Genome Medicine, Yokohama City
University Medical Center, 4-57 Urafune-cho, Minami-ku, Yokohama City,
Kanagawa 232-0024, Japan

\textsuperscript{4} Department of Gastroenterological Surgery, Yokohama
City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku,
Yokohama City, Kanagawa 236-0004, Japan

\textbf{Corresponding Author:}

Shinya Yamamoto

Department of Breast and Thyroid Surgery, Yokohama City University
Medical Center, 4-57 Urafune-cho, Minami-ku, Yokohama City, Kanagawa
232-0024, Japan

Email: shinya@rf.catv.ne.jp

Tel: +45-261-5656

Fax: +45 231-1846

\textbf{Data availability statement:} Data sharing is not applicable to
this article as no datasets were generated or analyzed during the
current study.

\textbf{Funding:} none

\textbf{Conflict of Interests}

The authors declare that there are no conflicts of interest regarding
the publication of this article.

\textbf{Ethics Approval} :This study was conducted ethically in
accordance with the World Medical Association Declaration of Helsinki.

\textbf{Patient consent:} Written informed consent was obtained from the
patient to publish this report in accordance with the journal's patient
consent policy.



\textbf{Key Clinical Message}

Reversion mutation was identified as a mechanism underlying resistance
to olaparib in breast cancer using cancer genomic profiling.

\textbf{Abstract}

A 34-year-old woman with breast cancer and the BRCA2: p.Gln3047Ter was
treated with olaparib. After tumor progression, cancer genomic profiling
testing revealed the BRCA2 p.Gln3047Ter and p.Gln3047Tyr, with 48.9\%
and 0.37\% allele frequency, respectively. These findings shed light on
reversion mutation as a resistance mechanism to olaparib in breast
cancer.

\textbf{Keywords}

Breast neoplasms, BRCA, genomics, olaparib

\textbf{Introduction}

The protein-encoded \emph{BRCA} is involved in homologous recombination
repair and plays an important role in DNA double-strand break repair.
PARP plays a vital role in DNA single-strand break
repair.\textsuperscript{1} Therefore, olaparib, a PARP inhibitor, is
effective for tumors with a germline \emph{BRCA} mutation owing to its
synthetic lethality.\textsuperscript{2} The OlympiAD trial demonstrated
the efficacy of olaparib in patients with human epidermal growth factor
receptor type2 (HER2)-negative metastatic breast cancer with a
germline\emph{BRCA} mutation.\textsuperscript{2} The median
progression-free survival was significantly longer in the olaparib group
than in chemotherapy of the physician's choice group (7.0 vs. 4.2
months; P \textless{} 0.001).\textsuperscript{2}

In recent years, several mechanisms of resistance to PARP inhibitors
have been proposed,\textsuperscript{3} including protein alteration in
the homologous recombination pathway, altered expression of a protein in
the DNA replication fork protection pathway,\textsuperscript{4}
epigenetic modifications, restoration of ADP-ribosylation, and
pharmacological alterations.\textsuperscript{3} Importantly, reversion
mutation has been recently reported as a mechanism of resistance to the
PARP inhibitor olaparib.\textsuperscript{5,6}

Herein, we describe a case of a patient with breast cancer with
a\emph{BRCA2} pathogenic variant that was resistant to olaparib and was
suspected to be a reversion mutation based on cancer genomic profiling.

\textbf{Case presentation}

A 34-year-old woman presented to our hospital with a mass in her right
breast, which she noticed after giving birth to her second child. Breast
cancer was diagnosed via core needle biopsy. The tumor was classified as
invasive ductal carcinoma, estrogen receptor (ER) positive, progesterone
receptor (PgR) positive, and HER2 positive. The timeline of the
treatment course is shown in Figure 1. The patient received 8 courses of
docetaxel--pertuzumab--trastuzumab therapy (75
mg/m\textsuperscript{2}docetaxel on day 1, 420 mg/kg pertuzumab on day
1, and 6 mg/kg trastuzumab on day 1 every 3 weeks). Subsequently, right
total mastectomy and axillary lymph node dissection were performed. The
main pathological findings were a residual tumor diameter measuring 53
mm, a number of residual lymph node metastases found in four of 18
nodes, ER and PgR positivity, HER2 negativity, and a histological
response to preoperative therapy of grade 1a. Postoperatively, the
patient received chemotherapy and endocrine therapy
(pertuzumab--trastuzumab, tamoxifen, and a luteinizing hormone-releasing
hormone agonist). Magnetic resonance imaging revealed liver metastases.
After three courses of trastuzumab emtansine (3.6 mg/kg every 3 weeks),
the metastatic lesions had progressed. Subsequently, adriamycin and
cyclophosphamide (AC) therapy (60 mg/m\textsuperscript{2} adriamycin on
day 1 and 600 mg/m\textsuperscript{2} cyclophosphamide on day 1 every 3
weeks) was administered. Considering cardiotoxicity, AC therapy was
completed after 11 courses (total adriamycin dose: 495
mg/m\textsuperscript{2}). Thereafter, lapatinib (1250 mg/kg daily) plus
capecitabin (3600 mg/kg on days 1--14 every 3 weeks) therapy was
administered. After 20 months, the metastatic tumors showed progression
(Figure 2a). \emph{BRCA} genetic testing was performed and a pathogenic
variant was detected. The variant using hg19 as the reference genome was
\emph{BRCA2} : c.9139C\textgreater{}T (p.Gln3047Ter) (Figure 3).
Therefore, olaparib (600 mg/kg daily) was administered. The tumor size
reduced after olaparib administration (Figure 2b); however, progression
of liver metastases was noted after 12 months of olaparib administration
(Figure 2c). Repeated biopsy of the liver metastasis revealed an
ER-positive, PgR-positive, and HER2-negative tumor. Abemaciclib (150 mg
twice daily) plus fulvestrant (500 mg every 4 weeks) therapy was
administered. After 8 months, metastatic lesions progressed. Trastuzumab
deruxtecan (5.4 mg/kg every 3 weeks) was administered. After 4 months,
metastatic lesions progressed; therefore, bevacizumab (10 mg/kg on days
1 and 15 every 4 weeks) plus paclitaxel therapy (90
mg/m\textsuperscript{2} on days 1, 8, and 15 every 4 weeks) was
administered. Cancer genomic profiling (FoundationOne\selectlanguage{ngerman}\textsuperscript{®}
Liquid CDx) was performed during bevacizumab plus paclitaxel therapy.
The sample at the time of surgery was \textgreater{}3 years old, and the
amount of specimen at the time of liver biopsy was insufficient;
therefore, liquid biopsy was performed. Figure 2d shows computed
tomography images of liver metastasis when cancer genomic profiling
testing was performed. The variants detected were \emph{BRCA2}
c.9139C\textgreater{}T (p.Gln3047Ter) and \emph{BRCA2}
c.9139\_9141delinsTAC (p.Gln3047Tyr) mutation, with 48.9\% and 0.37\%
allele frequency, respectively (Figure 3). After 9 months of bevacizumab
plus paclitaxel therapy, metastatic lesions progressed; therefore,
eribulin was administered (1.1 mg/m\textsuperscript{2} on days 1 and 8
every 3 weeks). However, after 3 months, metastatic lesions progressed;
thus, irinotecan (100 mg/m\textsuperscript{2} on days 1, 8, and 15 every
5 weeks) was administered. After one course, the patient's general
condition deteriorated, and the policy of best supportive care was
adopted; 4 months later, the patient died.

\textbf{Discussion}

A reversion mutation is a well-discussed mechanism of resistance to PARP
inhibitors. Waks et al. reported a reversion mutation as the most
commonly observed mechanism of resistance to PARP inhibitors or platinum
chemotherapy in eight patients with metastatic breast cancer
with\emph{BRCA1/2} mutations.\textsuperscript{4} Tobalina et al.
reported the occurrence of reversion mutations in 26.0\% (86/327)
patients with\emph{BRCA1 or BRCA2} mutations, including 27 patients with
breast cancer,\textsuperscript{7} with a higher percentage in
\emph{BRCA2}compared to \emph{BRCA1} (30.7\% vs.
22.0\%).\textsuperscript{7}

Pettitt et al. analyzed and reported 308 homologous recombination gene
reversions associated with a PARP inhibitor or platinum
resistance.\textsuperscript{8} In \emph{BRCA1} , the reversion mutations
occurred throughout the \emph{BRCA1} coding sequence. However,
in\emph{BRCA2} , there were ``hotspots'' and ``deserts.'' The frequency
of reversions 3\selectlanguage{english}' to coding sequence position 7617 (exon 16 onward) was
significantly lower than the expected frequency based on the incidence
dataset. However, numerous reversion mutations in the N-terminal
c.750-775 regions have occurred.\textsuperscript{8} Therefore, our
presented case might be relatively rare. However, as the authors'
indicated, PARP inhibitors are routinely used in clinics and some
reversion mutations will no longer be considered novel enough to be
reported. Therefore, reversion mutations in this area may be
underestimated.

Weigelt et al. reported the detection of reversion mutations using
circulating cell-free DNA in breast and ovarian
cancers.\textsuperscript{9} Tumor tissue was not synchronously
collected; therefore, it was not possible to validate the presence of
the putative reversion mutations in the tumor, which was a limitation in
Weigelt et al.'s study. Similarly, reversion mutation was not confirmed
using tissue biopsy in our case herein. Repeat tumor biopsy from liver
metastases and liquid biopsy to confirm the amplification of newly
detected secondary variants were planned. However, these were not
performed due to the deterioration of the patient's condition and the
fact that no additional treatment would be administered.

In addition, there was a concern in our case. The occurrence of true
reversions to wild-type was previously reported\textsuperscript{8};
however, true reversions are challenging to identify using liquid biopsy
alone. Thus, the prevalence of true reversions may be underestimated,
and it is possible that our case also had a true reversion.

There are several reports on treatment after PARP inhibitor resistance.
Reversions have been predicted to encode immunogenic neopeptides; thus,
immunotherapies may also be an option for direct targeting of the
revertant protein.\textsuperscript{8} As another strategy for acquired
resistance to PARP inhibitors, a cyclin-dependent kinase 12
inhibitor\textsuperscript{10} or WEE1 kinase
inhibitor\textsuperscript{11} can be used. To prove the efficacy of
these treatments, it is important to continue to investigate PARP
inhibitor resistance cases.

\textbf{Conclusion}

We experienced a case in which the possibility of a reversion mutation
was suggested as a mechanism of resistance to olaparib. The amount of
research into these cases has so far been insufficient to elucidate the
mechanism of resistance to olaparib; therefore, reporting each case is
of paramount importance.

\textbf{Authors' Contributions}

SY: collected the clinical data, analyzed the data, wrote the
manuscript, approved the final manuscript

KK, YF, SA, KN, MS. MO, AY, ST, MS, MT: collected the clinical data,
revised the article, approved the final manuscript

CH: collected the clinical data, wrote the manuscript, approved the
final manuscript

RT: collected the clinical data, approved the final manuscript

CK, IE: revised the article, approved the final manuscript

\textbf{Acknowledgments}

None

\textbf{References}

1. Walsh CS. 2015. Two decades beyond BRCA1/2: Homologous recombination,
hereditary cancer risk and a target for ovarian cancer therapy. Gynecol
Oncol 137(2):343-350. doi: 10.1016/j.ygyno.2015.02.017.

2. Robson M, Im SA, Senkus E, et al. Olaparib for metastatic breast
cancer in patients with a germline BRCA mutation. N Engl J Med
377(6):523-533. doi: 10.1056/NEJMoa1706450.

3. Li H, Liu ZY, Wu N, Chen YC, Cheng Q, Wang J. 2020. PARP inhibitor
resistance: The underlying mechanisms and clinical implications. Mol
Cancer 19(1):107. doi: 10.1186/s12943-020-01227-0.

4. Waks AG, Cohen O, Kochupurakkal B, et al. 2020. Reversion and
non-reversion mechanisms of resistance to PARP inhibitor or platinum
chemotherapy in BRCA1/2-mutant metastatic breast cancer. Ann Oncol
31(5):590-598. doi: 10.1016/j.annonc.2020.02.008.

5. Pan JN, Lei L, Ye WW, Wang XJ, Cao WM. 2021. BRCA1 reversion mutation
confers resistance to Olaparib and camrelizumab in a patient with breast
cancer liver metastasis. J Breast Cancer 24(5):474-480. doi:
10.4048/jbc.2021.24.e39.

6. Gornstein EL, Sandefur S, Chung JH, et al. 2018. BRCA2 reversion
mutation associated with acquired resistance to Olaparib in estrogen
receptor-positive breast cancer detected by genomic profiling of tissue
and liquid biopsy. Clin Breast Cancer 18(2):184-188. doi:
10.1016/j.clbc.2017.12.010.

7. Tobalina L, Armenia J, Irving E, O'Connor MJ, Forment JV. 2021. A
meta-analysis of reversion mutations in BRCA genes identifies signatures
of DNA end-joining repair mechanisms driving therapy resistance. Ann
Oncol 32(1):103-112. doi: 10.1016/j.annonc.2020.10.470.

8. Pettitt SJ, Frankum JR, Punta M, et al. 2020. Clinical BRCA1/2
reversion analysis identifies hotspot mutations and predicted
neoantigens associated with therapy resistance. Cancer Discov
10(10):1475-1488. doi: 10.1158/2159-8290.CD-19-1485.

9. Weigelt B, Comino-Mendez I, de Bruijn I, et al. 2017. Diverse BRCA1
and BRCA2 reversion mutations in circulating cell-free DNA of
therapy-resistant breast or ovarian cancer. Clin Cancer Res
23(21):6708-6720. doi: 10.1158/1078-0432.CCR-17-0544.

10. Johnson SF, Cruz C, Greifenberg AK, et al. CDK12 inhibition reverses
de novo and acquired PARP inhibitor resistance in BRCA wild-type and
mutated models of triple-negative breast cancer. Cell Rep
17(9):2367-2381. doi: 10.1016/j.celrep.2016.10.077.

11. Drean A, Williamson CT, Brough R, et al. 2017. Modeling therapy
resistance in BRCA1/2-mutant cancers. Mol Cancer Ther 16(9):2022-2034.
doi: 10.1158/1535-7163.MCT-17-0098.



\textbf{Figure legends}

\textbf{Figure 1 Timeline of the treatment course}

Abbreviations:

TP: trastuzumab and pertuzumab, T-DM1: trastuzumab emtansine, AC:
adriamycin and cyclophosphamide, L+C: lapatinib and capecitabin,
Abema+Flu: abemaciclib and fulvestrant, T-Dxd: trastuzumab deruxtecan,
PTX+Bev: bevacizumab and paclitaxel, CPT-11: irinotecan, BSC: best
supportive care, Bt+Ax: total mastectomy and axillary lymph node
dissection

\textbf{Figure 2 Liver metastasis on computed tomography}

a) Liver metastasis before olaparib administration (Arrows)

b) Liver metastasis has shrunk (Arrows)

c) Liver metastasis has progressed after 12 months of olaparib
administration (Arrows)

d) Liver metastasis (Arrows) at the time of cancer genomic profiling
testing

\textbf{Figure 3 Schema of the BRCA2 mutation}

Middle row: Result of BRCA genetic testing. The following pathogenic
variant in BRCA2 is detected: p.Gln3047Ter.

Lower row: Result of Cancer Genomic Profiling testing. The following new
mutation is identified: p.Gln3047Tyr.

Abbreviations: CGP: cancer genomic profiling testing

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