Hereditary Breast and Ovarian Cancer Molecular Mechanism

and Clinical Practice

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Preface

All cancers are caused by genetic alterations, but not all are hereditary. In the USA
or in Europe, about 5–10% of breast cancer and 10–15% of ovarian cancer are
thought to be hereditary. Nowadays, the incidence of breast cancer in Japan has
rapidly increased up to 100000 cases per year. The proportion of HBOC cases is
assumed to be almost the same in Japan. Therefore, we have established the Japanese
Organization for Hereditary Breast and Ovarian Cancer (JOHBOC) since July 2016.
The mission of the JOHBOC is (1) HBOC data registry, (2) education about HBOC
to medical professionals, patients, and their families, and (3) clinical research
related to the management of HBOC such as screening method including breast
MRI, the significance of risk reducing mastectomy (RRM) or risk reducing salpingo-­
oophorectomy (RRSO), chemoprevention (tamoxifen or AI for prevention of breast
cancer and oral contraceptive for ovarian cancer), and the positioning of new agents
specific to BRCA mutations (PARP inhibitors). In the era of next-generation
sequencing, we may encounter unexpected rare hereditary disease such as
Li-Fraumeni syndrome (P53 mutation) or Cowden disease (PTEN mutation), and
we should not miss them and send them to the specialist of each rare disease. And
one of the hot topics of basic research for HBOC is to detect new genetic alterations
related to carcinogenesis.
This book was originally planned for medical professionals who are interested in
HBOC practice from a variety of aspects. And it was recommended to distribute to
not only Japan but also other countries. Because we anticipate this book will con-
tribute to collaborative works worldwide.
Lastly, we deliver an address of thanks to editors Ms. Machi Sugimoto and Ms.
Kripa Guruprasad of Springer Nature.

Tokyo, Japan Seigo Nakamura

Tokyo, Japan  Daisuke Aoki
Tokyo, Japan  Yoshio Miki

v
Contents

1 History, Advancements, and Future Strategies ��������������������������������������   1

Reiko Yoshida
2 Molecular Basis of BRCA1 and BRCA2: Homologous
Recombination Deficiency and Tissue-Specific Carcinogenesis������������ 15
Tomohiko Ohta and Wenwen Wu
3 Genetic Testing ������������������������������������������������������������������������������������������ 31
Sana Yokoi
4 Variants of Uncertain Significances in Hereditary Breast
and Ovarian Cancer���������������������������������������������������������������������������������� 47
Yuki Yoshino and Natsuko Chiba
5 Genetic Counseling in Hereditary Breast and Ovarian Cancer������������ 65
Mayuko Inuzuka
6 Hereditary Breast Cancer ������������������������������������������������������������������������ 79
Minoru Miyashita and Takanori Ishida
7 Hereditary Ovarian Cancer���������������������������������������������������������������������� 93
Kenta Masuda, Megumi Satake, and Daisuke Aoki
8 Risk-Based Breast Cancer Screening������������������������������������������������������ 107
Wakana Murakami, Mitsuhiro Tozaki, Kelly E. McCann,
and Stephanie Lee-Felker
9 Chemoprevention for Breast Cancer�������������������������������������������������������� 129
Rurina Watanuki, Aiko Nagayama, Tetsu Hayashida,
and Yuko Kitagawa
10 Chemoprevention for Ovarian Cancer���������������������������������������������������� 149
Hiroshi Kobayashi
11 Risk-Reducing Mastectomy (RRM) �������������������������������������������������������� 169
Rina Kotake, Yasue Tsuchida, Kumiko Kida, and Hideko Yamauchi

vii
viii Contents

12 Risk-Reducing Salpingo-oophorectomy (RRSO)������������������������������������ 183

Yusuke Kobayashi and Daisuke Aoki
13 Panel Testing���������������������������������������������������������������������������������������������� 193
Yasuyuki Kojima
14 Germline Findings Through Precision Oncology
for Ovarian Cancer������������������������������������������������������������������������������������ 211
Mika Okazawa-Sakai and Akira Hirasawa
15 Germline Findings from Genetic Testing for Breast Cancer ���������������� 227
Akihiro Sakurai
16 Hereditary Breast and Ovarian Cancer (HBOC) Database
in Japan������������������������������������������������������������������������������������������������������ 243
Masami Arai
17 Ethical Issues: Overview in Genomic Analysis
and Clinical Context���������������������������������������������������������������������������������� 259
Izen Ri and Kaori Muto
18 PARP Inhibitors: Mechanism of Action�������������������������������������������������� 281
Shigeaki Sunada and Yoshio Miki
19 PARPi: Efficacy in Hereditary Breast Cancer���������������������������������������� 293
Akiyo Yoshimura
20 Efficacy of Poly(ADP-Ribose) Polymerase Inhibitors
for Hereditary Ovarian Cancer���������������������������������������������������������������� 313
Koji Matsumoto
History, Advancements, and
Future Strategies 1
Reiko Yoshida

Abstract

Since the recognition of the genetic predisposition to breast and ovarian cancers,
researchers have verified their genetic involvement and causative genes.
Furthermore, treatment strategies and prevention care options to reduce the
overall risk for hereditary cancers have been established based on rapid
advancements in gene sequencing. Owing to the great efforts of our predecessors,
the quality of life of patients diagnosed with hereditary tumors has been
improved. This chapter introduces the history, advancements, and future
strategies on hereditary breast and ovarian cancer (HBOC), which has a high
prevalence of breast and ovarian cancer. In any field of medicine, first, clinical
questions that foresee the truth arise; researchers then seek the truth, and
clinicians deploy their knowledge in the medical field.
Management of hereditary breast and ovarian cancer (HBOC) is a typical
model for other hereditary tumor syndromes.

Keywords

FBOC · HBOC · BRCA1/BRCA2 · Genetic testing · Multi-gene panel · PARP

inhibitor

R. Yoshida (*)
Showa University, Advanced Cancer Translational Research Institute,
Shinagawa-ku, Tokyo, Japan
Division of Cancer Genomics, Cancer Institute, Japanese Foundation for Cancer Research,
Koto-ku, Tokyo, Japan
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2021 1

S. Nakamura et al. (eds.), Hereditary Breast and Ovarian Cancer,
https://doi.org/10.1007/978-981-16-4521-1_1
2 R. Yoshida

1.1 History of Hereditary Breast Cancer (Fig. 1.1)

1.1.1 How It All Started

In 1866, Paul Broca, a French surgeon, was the first to describe a family with a high
prevalence of breast cancer [1]. He tracked the causes of death of 38 people in his
wife’s family for 5 generations from 1788 to 1856 and identified that 10 of the 24
women died of breast cancer. He thus speculated that the predisposition to cancer is
hereditary. In addition, he documented all other types of malignant neoplasms that
included an excess of cancer of the gastrointestinal tract [2]. Jacobsen Oluf, who
was one of the first investigators to question the inheritance of breast cancer as
being solely site-specific, reported an increased frequency of cancer of all parts in
the first-degree relatives in a series of 200 breast cancer patients [3]. In 1971, the
autosomal dominant inheritance of a predisposition to both breast and ovarian
cancers was first described by David E. Anderson [4]. Breast cancer patients with a
family history have been reported to be associated with juvenile-onset, bilateral
breast cancers and ovarian tumors compared with those without a family history.
Since then, several clinical studies on familial breast and ovarian cancers have been
conducted. In 1990, linkage analysis in 23 families of 146 early-onset familial
breast cancers revealed an association with the D17S74 locus (CMM86) on the long
arm of chromosome 17 [5]. This study used the positional cloning method to ana-
lyze DNA of multiple family members, and the authors used gene polymorphism
markers, as well as the information from chromosomal recombination yielding a
logarithm of the likelihood ratio for linkage during meiosis and germ-cell formation.
They were thus able to limit the chromosomal region where the causative gene was
located. Furthermore, in 1994, Yoshio Miki et al. succeeded in cloning BRCA1
using reverse genetics to elucidate its function and determine its complete structure
[6]. However, because there were few male breast cancer patients among BRCA1
mutation carriers, another causative gene is implicated. As with BRCA1, using
linkage analysis of multiple families with breast, ovarian, and male breast cancers,

Limitation of NCCN and

Familial Breast the HBOC NYS/ACMG
Cancer first causative Identification clinical Guideline CIMBA
reported [1] gene on ch17 of BRCA2 published founded

1866 1971 1990 1994 1995 1996 1999 2004 2005 2010

AD inheritance Identification Beginning of BRCA ‘BRCAness’ HBOC prospective

of HBOC first of BRCA1 clinical genetic proposed [25] cohort studies
reported [4] testing provided
(Myriad Genetics)

Fig. 1.1 Timeline of HBOC discovery and clinical setting. AD autosomal dominant, NCCN
National Comprehensive Cancer Network, NYS New York State, ACMG American College of
Medical Genetics, CIMBA Consortium of Investigators of Modifiers of BRCA1/2
1 History, Advancements, and Future Strategies 3

Richard Wooster et al. identified BRCA2 on the long arm of chromosome 13 [7, 8]
in 1995. In 1994, Henry Lynch collectively referred to hereditary ovarian cancer
(HOC), hereditary breast cancer (HBC), and hereditary syndrome that causes both
breast and ovarian cancers as hereditary breast and ovarian cancers (HBOC) [9].

1.1.2 BRCA1 and BRCA2 in the Clinical Setting

After BRCA1 and BRCA2 were identified, they have been immediately applied in
the clinical setting. In 1996, clinical genetic testing for BRCA1 and BRCA2 was
patented by Myriad Genetics and made available worldwide. Various clinical studies
were conducted by multiple research groups to increase the understanding of
BRCA1 and BRCA2 cancers. The Breast Cancer Linkage Consortium, founded in
1989, reported that other cancers, such as prostate cancer, pancreatic cancer, and
melanoma, are associated with BRCA1/BRCA2 mutations [10]. Meanwhile, the
Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), a collaborative
group of researchers in Europe, North and South Americas, Australia, Asia, and
Africa founded in 2005, has described the clinicopathological features of BRCA-­
associated cancers [11], the genotype–phenotype correlations from a prospective
study [12], the characteristics of BRCA male breast cancer [13], and analysis of the
risk of multiple single nucleotide polymorphisms (SNPs) in BRCA mutation carriers
[14]. Moreover, this group has phenotypic data of about 80,000 female and male
BRCA1 and BRCA2 mutant carriers; so far about 43,000 have been genotyped in
the CIMBA project, and an additional 25,000 will have been genotyped in 2020.
Currently, the ClinVar, a database provided by the National Center for
Biotechnology Information, has recognized more than 3400 germline variants of
BRCA1 and 3900 of BRCA2 as pathogenic or likely pathogenic (20/October/2020
assessed). The majority (80%) of which are truncating variants that form immature
stop codons, such as frameshifts and nonsense, whereas missense mutations account
for approximately 10%. Pathogenic missense variants tend to be concentrated in
functionally essential sites, such as the really interesting new gene (RING) finger
domain and BRCA1 C terminus (BRCT) domains of BRCA1 or the regions spanning
the oligosaccharide-binding folds and helical domains of BRCA2 [15]. Abnormal
copy number variants (CNVs) detected by deletion or duplication analysis account
for approximately 10% and vary among populations [16].
A founder mutation is defined by the National Institutes of Health as follows: “A
genetic alteration observed with high frequency in a group that is or was
geographically or culturally isolated, in which one or more of the ancestors was a
carrier of the altered gene.” Particularly, in Ashkenazi Jews, three common mutations
of BRCA1, namely, c.68_69delAG, c.5266dupC, and BRCA2 c.5946delT, account
for 98–99% of the pathogenic variants. Thus, targeted analysis of these three
variants is recommended. However, in other ethnic groups, full-sequence analysis
and CNV analysis are necessary [16]. Various cohort studies and clinical trials have
also been conducted for the medical management for BRCA1and BRCA2 carriers.
Retrospective studies have reported the usefulness of prophylactic surgery and
4 R. Yoshida

breast magnetic resonance imaging (MRI) surveillance [17, 18]. Since 2010, the
findings of prospective cohort studies have provided guidance on improving the
quality of life of patients using these medical interventions [19–21].
With the increasing interest on clinical BRCA genetic testing and the accumula-
tion of information about BRCA1 and BRCA2-mutation carriers, HBOC clinical
guidelines have been established and are widely used globally. Moreover, the
American Society of Clinical Oncology (ASCO) published its own guidelines in
1996, and the National Comprehensive Cancer Network (NCCN) in 1999. In
addition, the New York State and the American College of Medical Genetics (NYS/
ACMG) guidelines were posted on the New York State website in 1999. These
guidelines were developed under the close collaboration of numerous health
professionals, including oncologists, geneticists, genetic counselors, primary-care
physicians, and public health specialists, and contribute to the determination of a
series of HBOC practices: genetic testing criteria, testing methods, interpretation of
test results, and medical management [22].
The association between BRCA mutation location and breast and ovarian cancer
risk has also been reported. In both BRCA1 and BRCA2 carriers, ovarian cancer
cluster regions (OCCR) have been confirmed to be located within or adjacent to
exon 11 [12]. Carriers with pathogenic variants in the OCCR possessed a higher risk
of ovarian cancer, unlike those with pathogenic variants located elsewhere. Similarly,
several breast cancer cluster regions (BCCRs) have been observed in BRCA1 and
BRCA2, respectively, and are associated with a relative increase in breast cancer risk
but a relative decrease in ovarian cancer risk. However, in this previous study, each
hazard ratio for cancer development owing to the difference in mutation locations
was at most 2. Therefore, without additional information, it may be premature to use
correlation between genotype location and cancer risk phenotype for individual risk
assessment and management.
Immediately after cloning of BRCA1 and BRCA2, new findings, including the
role of BRCA in carcinogenesis and the genomic aberrations in BRCA-mutated
cancers, have been reported. DNA repair mechanisms include DNA single-strand
break (dsDNA) repair, double-strand break (dsDNA) repair, base mismatch repair
(MMR), base excision repair (BER), and nucleotide repair (NER). BRCA1 and
BRCA2 are cancer-suppressor genes that maintain genomic stability by repairing
dsDNA via homologous recombination (HR) [23]. In addition to HR repair (HRR),
BRCA1 and BRCA2 regulate centrosome dynamics, chromosome distribution, and
cytokinesis and temporally and spatially stabilize the genome during cell cycle.
Moreover, a hormone-dependent carcinogenic environment is speculated to
contribute to genome instability via the disruption of these BRCA functions and the
accelerated activation of survival signals and the mammary gland cells are converted
to malignant traits [24]. In addition to its DNA-damage repair function, the
involvement of BRCA1 on normal embryogenesis, centrosome replication, spindle
pole synthesis, heterochromatin-satellite RNA expression, estrogen metabolite
synthesis, splicing, brain size regulation, and transcriptional co-activation have
been reported [16]. Because the loss of BRCA function has been associated with HR
defects, the concept “BRCAness” has been proposed in 2004 [25]. It refers to
1 History, Advancements, and Future Strategies 5

sporadic breast cancers exhibiting similar clinicopathological features and charac-

teristic genomic aberrations as BRCA-related cancers; thus, BRCAness can be con-
sidered as a therapeutic biomarker.

1.2 Current Developments (Fig. 1.2)

1.2.1 Multi-gene Panel Testing and Non-BRCA Genes

BRCA1/BRCA2 genetic testing achieved a major transformation in 2013. In the liti-

gation of the BRCA gene patent against Myriad Genetics from 2009 to 2013, the US
Supreme Court ruled in 2013 as follows: because separating that gene from its sur-
rounding genetic material is not an act of invention, isolated human genes cannot be
patented. Furthermore, owing to the development of next-generation sequence
(NGS) technology in 2005 and its plummeting cost since 2007, genetic testing has
become more powerful and generated tremendous data compared with the
conventional Sanger sequence method. Subsequently, multiple genetic testing
companies have started to provide multi-gene panel (MGP) testing, including
BRCA1and BRCA2. In the United States, it has been reported that the number of
MGP testing performed has exceeded that of BRCA alone testing since 2014 [26].
In addition, numerous MGP testings of large cohorts have been developed, resulting
in the accumulation of information on genes that cause breast and ovarian cancer
other than BRCA. Among all breast cancer patients without selection bias, the
BRCA1/BRCA2 mutation detection rate was 4–5%, whereas the total mutation
detection rate of MGP was 6–9%, a 1.4- to 2-fold increase. Similarly, in ovarian
cancer patients without selection bias, the BRCA1/BRCA2 mutation detection rate
was approximately 20%, whereas that of MGP was 26–31%, a 1.5-fold increase
[27–31]. Because “nearly all known HBOC susceptibility genes encode tumor
suppressors that participate in genome stability pathways—in particular HRR, and
to some extent mismatch repair (MMR) and interstrand DNA crosslink repair via
the Fanconi anemia pathway” as reported by Nielsen et al. [32], the detection of

FDA approval of
Development of BRCA patent ‘Precision Medicine’ PARP inhibitor
NGS technology invalidated announced for breast cancer

2005 2009 2013 2014 2015 2016 2017 2018 2019 2020

PARP inhibitor MGP testing FDA approval of Comprehensive

first phase I trial launched PARP inhibitor for approach to cancer
conducted [35] ovarian cancer history for all patients
recommended

Fig. 1.2 Timeline of HBOC development. NGS next-generation sequence, PARP poly ADP ribose
polymerase, MGP multi-gene panel, FDA US Food and Drug Administration
6 R. Yoshida

known susceptibility genes for breast and ovarian cancers other than HR-related
genes is possible via MGP. In addition, hereditary cancer syndromes other than
BRCA1- and BRCA2-related cancers, which could not be identified using one-panel
testing, can be diagnosed using MGP testing. Thus, the utilization of MGP testing
provides opportunities to discover genetic diseases that were not expected from
family and individual medical histories and to take new measures for additional
preventive medical management depending on the constitution of each genetic
disorder. Figure 1.3 shows the results of MGP testing in a Japanese biobank cohort,
including 11 breast cancer-susceptibility genes (BRCA1, BRCA2, PALB2, TP53,
PTEN, CHEK2, NF1, ATM, CDH1, NBN, and STK11) [27]. Compared with MGP
testing results in unaffected group with no familial cancer (the control group), breast
cancer patient group without selection bias, and high-risk group comprising breast
cancer patients meeting the BRCA testing criteria based on NCCN guideline, the
overall mutation detection rate of MGP testing was the highest in the high-risk
group; however, the highest frequency of pathogenic mutations other than BRCA1
and BRCA2 was identified in the control group. Thus, clients except high-risk group
they were more likely to benefit from MGP testing than BRCA testing alone. It has
been suggested that the benefits of MGP testing have been extended to all subjects

a b
%
14 100%
Frequency of Pathogenic Variant
Pathogenic Variant Rate

12
80%
10

8 60%

6
40%
4 8.4%
5.7% 20%
2
0.6%
0 0%
Control Unselected High-Risk Control Unselected High-Risk
Breast Cancer Breast Cancer Breast Cancer Breast Cancer
Patient Patient

BRCA2 ATM NBN

BRCA1 TP53 CDH1
PALB2 PTEN
CHEK2 NF1

Fig. 1.3 The results of MGP testing in a Japanese biobank cohort. The data of the result of MGP
testing including 11 breast cancer-susceptibility genes (BRCA1, BRCA2, PALB2, TP53, PTEN,
CHEK2, NF1, ATM, CDH1, NBN, and STK11) using the Japanese biobank cohort was illustrated
in bar plots. The overall mutation detection rate of MGP testing was the highest in the high-risk
group; however, the highest frequency of pathogenic mutations other than BRCA1 and BRCA2 was
identified in the control group. (a) The result of total pathogenic variant rate between three groups.
(b) The result of frequency of pathogenic variants between three groups. Control group; unaffected
group with no familial cancer, unselected breast cancer patients; breast cancer patient group
without selection bias, high-risk breast cancer group; high-risk group comprising breast cancer
patients meeting the BRCA testing criteria based on NCCN guideline
1 History, Advancements, and Future Strategies 7

considering cancer preventive medicine, not just cancer patients with sus-
pected HBOC.

1.2.2 Target Therapy

In 2005, tumor cells lacking BRCA1 and BRCA2 and key tumor-suppressor proteins
involved in DSB repair via HR were found to be selectively sensitive to small-­
molecule inhibitors of the enzyme poly ADP ribose polymerase (PARP) family of
DNA repair enzymes [33, 34]. Subsequently, a new cancer therapeutic strategy
based on synthetic lethality was conducted in 2009 in the first phase I clinical trial
of this PARP inhibitor in BRCA1- and BRCA2-positive individuals [35]. Then,
clinical trials involving PARP inhibitors targeting ovarian and breast cancers with
germline BRCA mutations showed good results [36, 37]. The PARP inhibitor
olaparib has been approved by the FDA for treatment of metastatic ovarian cancer
with germline BRCA mutations in 2016, and in 2018, it was approved to treat
metastatic breast cancer. Currently, indications for prostate and pancreatic cancers
are being expanded, and those for cancers using characteristic genomic aberrations
representing the HR deficiency (HRD) of tumors as biomarkers, like “HRD score”
[38], are being expanded.
As advances in NGS technology allow more precise analysis of changes in the
cancer genome, it turns out that individual disease causes and cancer status are more
complex than expected; in order to provide realistic medical practice, it is necessary
to divide patients into subgroups, and in 2015, “Precision Medicine” was announced
with the aim of establishing treatment methods and providing preventive medical
care for each subgroup. Cancer medicine using genomic information is rapidly
being developed for this subgrouping. Although the main purpose of these tumor
tissue profiling tests is to select cancer drugs, at the same time, germline pathogenic
variant in 4% to 12% was also found. The detection of pathogenic germline
mutations is often a critical step in initiation of the cascade of genetic testing in
relatives, which can clarify the patient’s own cancer risk and translate into life-­
saving surveillance and risk reduction interventions for family members [39].
Owing to the abovementioned developments, MGP testing, companion diagnos-
tics targeting PARP inhibitors, and cancer genomic medicine, the possibility of
detecting genes other than germline BRCA1 and BRCA2 has increased rapidly in the
clinical setting. In response, the 2013 version of the NCCN guidelines [40] had a
new section titled “Additional genetic mutations associated with breast/ovarian can-
cer risk” in addition to the conventional HBOC syndrome, Li–Fraumeni syndrome,
and Cowden syndrome. It listed 21 genes related to breast and ovarian cancers. In
the 2014 version, a section on “Multi-gene Testing” was released. Particularly, it
stated that when patients meeting the HBOC testing criteria are found negative for
BRCA1 and BRCA2, multi-gene testing should be considered. In its 2015 version,
for 15 genes, including BRCA1 and BRCA2, recommendations, considerations, and
insufficient evidenced medical management for breast MRI surveillance and pro-
phylactic surgery were presented. In 2017, risk and management options for patients
8 R. Yoshida

with breast cancer, ovarian cancer, and other cancers were described in detail,
including high- and moderate-risk genes widely used in MGP testing. Furthermore,
patients with BRCA1 and BRCA2 mutation detected via tumor profiling have been
added to the BRCA testing criteria. Finally, the latest 2020 edition has radically
shifted away from the BRCA gene toward a broad screening of other genes, consis-
tent with current practice. Hereditary pancreatic cancer has also been added to this
guideline, and the two-step approach, “Further Genetic Risk Evaluation” and
“Testing Criteria,” has been changed to “comprehensive approach to cancer history
for all patients” (Fig. 1.4). Moreover, the “BRCA testing criteria” have been changed
to the “High-penetrance Breast and/or Ovarian Cancer Susceptibility Genes Testing
Criteria.” When there are no known pathological gene mutations in relatives, com-
prehensive MGP testing should be performed from the beginning. It suggested that
it is now time to handle and manage beyond BRCA genes in general practice.
However, the use of multiple genes with lower allele frequencies and lower pen-
etrance than BRCA1 and BRCA2 has some challenges. First, this results in an
increased number of variants of uncertain significance (VUSs). As the number of
genes searched by MGP testing increases, the number of VUSs also increases. The
VUS rates of other genes are generally higher than that of BRCA and higher than
pathogenic mutation rates [41]. Second, the variant is examined based on the
ACMG/AMP variant classification guideline [42]; however, because each genetic
testing company makes the final judgment by its own method, some disagreement

Criteria ForFurther Testing criteria Genetic Testing

Genetic Risk Evaluation
BRCA1/2 BRCA1/2 testing
or Multi-Gene Testing

~2019 TP53 testing

TP53
or Multi-Gene Testing

PTEN PTEN testing

or Multi-Gene Testing

Testing criteria Genetic Testing

Breast High-penetrance Breast

Ovarian and/or Ovarian Cancer Multi-Gene Testing
Prostate Susceptibility Genes

Exocrine Pancreatic
2020~ Pancreatic
Cancers All Individuals Multi-Gene Testing

Specific TP53 TP53 or Multi-Gene Testing

All Cancer Syndrome PTEN PTEN or Multi-Gene Testing
Patients

Fig. 1.4 Changes in genetic risk assessment and testing criteria in NCCN guideline. NCCN
guideline 2020 edition has radically shifted away from BRCA genes toward a broad screening of
other genes, consistent with current practice. Hereditary pancreatic cancer has also been added to
this guideline, and the two-step approach, “Further Genetic Risk Evaluation” and “Testing
Criteria,” has been changed to “comprehensive approach to cancer history for all patients”
1 History, Advancements, and Future Strategies 9

on the interpretation of certain variants may exist. To address this, the Evidence-­
Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) [43]
and BRCA ShareTM [44] have been established to provide a critical evaluation of the
risk and assess the clinical importance of VUSs.

1.3 Future Strategies (Fig. 1.5)

Reverse genetics is an approach in molecular genetics that elucidates gene function by

examining the changes in phenotypes via the suppression or enhancement of the gene
expression. On the other hand, forward genetics is the technique of identifying genes
from phenotypes, which existed before the concept of genes was reported. At the time

Past
Current

BRCA Future
Hereditary Cancer
Syndrome

Surgery
Surveillance

Polygenic Risk
Unknown Cancer susceptibility genes

Cancer Target
Therapy

Cancer Preventive
Therapy

Fig. 1.5 HBOC strategy past, current, future. Targets for considering the risk of cancer suscepti-
bility and effective medical practices are expanding
10 R. Yoshida

of the discovery of BRCA1 and BRCA2, many causative genes for hereditary diseases,
including hereditary cancer syndromes, were discovered using reverse genetics.
However, since around 2000, reports of the discovery of the causative genes using
reverse genetics have gradually decreased although there still exist hereditary diseases
of unknown causative genes. Because gene identification by this method has reached
its limit, with the innovative progress of genome sequence analysis technology, the
following new methods have been used to report the discovery of multiple genes.
New methods, including whole-exome sequencing (WES) and whole-genome
sequencing (WGS), can now be utilized at a relatively low cost and achieve fast results.
Currently, WES is used to elucidate the causes of hereditary diseases for which the caus-
ative gene has not been identified. Both WES and WGS are already clinically available.
However, the differences between these new sequencing technologies and MGP testing
should be considered. The main difference is the amount of data generated. Whereas
WGS yields sequence information of all regions in the genome, WES focuses on less
than 2% of the genome. MGP testing selects and searches several to dozens of genes
from more than 20,000 types of genes. WES and MGP testing read only the protein-
coding regions and exclude the promoter or regulatory regions. For example, many
commercially available MGP assays analyze the exon–intron border regions with a
range of 2–5 bp. However, in case of a variant located in the deep intron region affecting
the activity of the target gene, MGP testing and WES could not identify the said variant
[45]. Notably, the required sample amount and cost for analysis do not tremendously
differ among these methods. In addition, the VUS rate is expected to increase using new
methods. Variants found using WES and WGS have indicated that validation using tra-
ditional Sanger sequencing is required [46].
While WES and WGS are expected to find a rare single causative gene that dis-
rupts specific pathways and functions, cancer development is a multifactorial dis-
ease. More recently, polygenic risk score (PRS) that integrates the joint effects of
common genetic variants on disease risk has been developed. PRS is a score that
calculates the overall risk of developing a disease based on the dozens to thousands
of single nucleotide polymorphisms (SNPs) suggested to be associated with each
disease or trait derived from genome-wide association studies (GWAS). While com-
mon variants have small individual effects on disease risk, cumulatively, they can
have large effects—in some individuals, risks equivalent to the strong monogenic
variants such as BRCA1 and BRCA2 [14, 47]. Owing to monogenic mutations and
PRS, the risk of breast cancer by age 75 ranges from 12.7% to 75.7% in BRCA1 and
BRCA2 mutation carriers, whereas that in non-carriers ranges from only 3.3% to
29.6%. Higher PRS correlates with higher risk, whereas the risks of the carrier
group with low PRS and that of the non-carrier group with high PRS are the same.
Thus, the PRS-based approach to patient stratification based on cancer risk may
further improve screening methods and prevention strategies compared with
methods targeting a single gene.
In addition to the development of treatments for cancers following diagnosis,
prophylactic surgery, and the early detection of cancer, evidence for chemoprevention
of HBOC has been established. Although there are limited large prospective clinical
studies involving only women with BRCA mutations who have not developed breast
cancer, reports on breast cancer prevention using selective estrogen-receptor
1 History, Advancements, and Future Strategies 11

modulators (SERMs) are available. The two largest studies (National Surgical
Adjuvant Breast and Bowel Project-P1 (NSABP-P1) [48] and International Breast
cancer Intervention Study-1 (IBIS-1) [49]) found that tamoxifen reduced the
incidence of breast cancer by approximately 40%, and its protective effect extended
beyond the treatment period. Among the 288 cases, there were 8 BRCA1 and 11
BRCA2 mutation carriers [48]. Although it was a result of a small sample size only
for BRCA mutation carriers, a potential reduction in BRCA2- but not BRCA1-­
associated breast cancer was observed following tamoxifen use [50]. In addition, a
meta-analysis report of four case–control studies have analyzed the risk reduction of
CBC due to tamoxifen [51]. In the previous report, although tamoxifen did not
exhibit protective effects in women with BRCA1 mutations who had a high
proportion of triple-negative breast cancer cells, it was protective for women with
BRCA2 mutations. In addition, tamoxifen reduced the risk of contralateral breast
cancer in BRCA1 mutation carriers. However, these studies are not limited to BRCA
and their sample sizes were small. Adverse effects (thrombosis, endometrial cancer,
early menopause, etc.) due to SERMs have also been reported.
Chemoprevention is a promising preventive option for BRCA mutation carriers.
A chemopreventive drug, i.e., a monoclonal antibody (denosumab) targeting
RANKL, has been recently identified. Denosumab is used for the treatment of
osteoporosis and bone metastasis. Various studies have demonstrated that the
progesterone-mediated upregulation of the RANK/RANKL pathway plays a critical
role in mammary epithelial proliferation, mammary stem cell expansion, and
carcinogenesis [52]. In Brca1-mutant mice, the loss of RANKL reduced mammary
tumors and suppressed tumor progression, and its inhibition prevented mammary
tumor development [53]. Moreover, previous studies have reported that the
circulating level of osteoprotegerin (OPG) is significantly lower correlated with
higher progesterone levels in premenopausal BRCA mutation carriers than in non-­
carrier controls. This suggests a significant dysregulation of circulating OPG and
sex hormone levels [54]. A chemopreventive clinical trial of denosumab involving
unaffected BRCA mutation carriers is underway.

1.4 Conclusion

Through the collaboration among experts in many fields, such as basic science,
bioinformatics, statistics, pharmacology, diagnostic imaging, surgery, clinical
medicine, politics, genetics, and genetic clinical practice, increased understanding
of HBOC can be achieved, thereby improving the quality of life of HBOC patients.

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