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CONTRIBUTORS

Gaurav Agarwal
Department of Endocrine Surgery, Sanjay Gandhi Post Graduate Institute of Medical
Sciences, Lucknow, Uttar Pradesh, India
Mark Awuku
Department of Pediatrics, Schulich School of Medicine & Dentistry—Windsor Program,
Western University, Windsor, Ontario, Canada
John Crown
Department of Medical Oncology, St. Vincent’s University Hospital, Dublin, Ireland
Michael J. Duffy
UCD School of Medicine and Medical Science, Conway Institute, University College
Dublin, and UCD Clinical Research Centre, St. Vincent’s University Hospital, Dublin,
Ireland
Guido Filler
Department of Pediatrics; Department of Medicine, and Department of Pathology &
Laboratory Medicine, Schulich School of Medicine & Dentistry, Western University,
London, Ontario, Canada
Carmen E. Georgescu
Department of Endocrinology, University of Medicine and Pharmacy “Iuliu-Hatieganu”,
Cluj-Napoca, Romania
Ioana R. Ilie
Department of Endocrinology, University of Medicine and Pharmacy “Iuliu-Hatieganu”,
Cluj-Napoca, Romania
Surendra Kumar
Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow, Uttar Pradesh, India
Giuseppe Lippi
Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma,
Italy
Laudelino Lopes
Department of Obstetrics and Gynecology, Schulich School of Medicine & Dentistry,
Western University, London, Ontario, Canada
Enda W. McDermott
UCD School of Medicine and Medical Science, Conway Institute, University College
Dublin, Dublin, Ireland
Sophie E. McGrath
Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, United
Kingdom

ix
x Contributors

Agnieszka Michael
Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, United
Kingdom
Balraj Mittal
Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow, Uttar Pradesh, India
Rama Devi Mittal
Department of Urology and Renal Transplant, Sanjay Gandhi Post Graduate Institute of
Medical Sciences, Lucknow, Uttar Pradesh, India
Richard Morgan
Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, United
Kingdom
Hardev Pandha
Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey, United
Kingdom
Chao-Ke Tang
Life Science Research Center, Key Laboratory for Atherosclerology of Hunan Province,
Molecular Target New Drug Discovery and Cooperative Innovation Center of Hunan
Province, University of South China, Hengyang, PR China
Sonam Tulsyan
Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow, Uttar Pradesh, India
Siun Walsh
UCD School of Medicine and Medical Science, Conway Institute, University College
Dublin, Dublin, Ireland
Xiao-Hua Yu
Life Science Research Center, Key Laboratory for Atherosclerology of Hunan Province,
Molecular Target New Drug Discovery and Cooperative Innovation Center of Hunan
Province, University of South China, Hengyang, PR China
Xi-Long Zheng
Department of Biochemistry and Molecular Biology, The Libin Cardiovascular Institute of
Alberta, Cumming School of Medicine, The University of Calgary, Health Sciences Center,
Calgary, Alberta, Canada
PREFACE

The fourth volume of the Advances in Clinical Chemistry series for 2015 is
presented.
In Chapter 1, breast cancer biomarkers are reviewed. Emphasis is placed
on their importance in identification of malignancy especially within high-
risk families, prognosis, evaluation of therapy, postoperative surveillance,
and monitoring therapy in advanced disease. In Chapter 2, polycystic ovary
syndrome is highlighted. This syndrome is one of the most common endo-
crinopathies in women, but is difficult to detect due to its complexity and
high heterogeneity. New methods for detection involving epigenetics and
miRNA profiling are explored. In Chapter 3, markers for diagnosis of pros-
tate cancer, a common malignancy in males, is reviewed. Emphasis is placed
on novel markers to identify early disease at limited disease stage. In
Chapter 4, the cytochrome P450 system is discussed with respect to their
impact on anticancer treatment and outcomes. Polymorphisms within this
complex gene family can have tremendous impact on treatment outcomes.
The role of cytochrome P450 inhibitors and their potential impact on the
pharmacokinetics of anticancer therapies are explored. In Chapter 5, novel
markers to assess renal function in children are presented. The importance of
correctly providing a current state measurement is vital to ensure that drugs
are cleared appropriately, especially in these vulnerable patients. In
Chapter 6, the concept of hemolysis index is revisited as an important
preanalytical tool. This chapter highlights the need for agreement between
laboratories on a standardized approach in the interpretation of this quality
measure. In Chapter 7, activation of peroxisome proliferator-activated
receptor α is explored. This unique nuclear receptor plays an important role
in lipid metabolism and mitigates atherosclerosis by blocking macrophage
foam cell formation, vascular inflammation, vascular smooth muscle cell
proliferation and migration, plaque instability, and thrombogenicity.
I thank each contributor of Volume 71 and to my colleagues who
provided peer review. I thank Shellie Bryant and Roshmi Joy for their
editorial support.

xi
xii Preface

I hope the fourth volume for 2015 will be enjoyed. Your comments and
feedback are always appreciated.
I would like to dedicate Volume 71 to our upcoming trip to the Bound-
ary Waters.
GREGORY S. MAKOWSKI
 CHAPTER ONE

Biomarkers in Breast Cancer:

Where Are We and Where Are
We Going?
Michael J. Duffy*,†,1, Siun Walsh*, Enda W. McDermott*, John Crown{
*UCD School of Medicine and Medical Science, Conway Institute, University College Dublin, Dublin,
Ireland
†
UCD Clinical Research Centre, St. Vincent’s University Hospital, Dublin, Ireland
{
Department of Medical Oncology, St. Vincent’s University Hospital, Dublin, Ireland
1
Corresponding author: e-mail address: [email protected]

Contents
1. Use of Biomarkers in the Identification of Women at Increased Risk of Developing
Breast Cancer (Risk Assessment) 2
2. Use of Biomarkers in Determining Prognosis 4
2.1 uPA and PAI-1 5
2.2 Oncotype DX 5
2.3 MammaPrint 6
2.4 Other Multigene Signatures 6
2.5 CA 15-3 8
3. Use of Biomarkers in Guiding Treatment 8
3.1 Estrogen Receptor for Predicting Response to Endocrine Therapy 9
3.2 HER2 for Predicting Response to Anti-HER2 Therapy 11
4. Use of Biomarkers in the Postoperative Follow-Up of Asymptomatic Patients
Following Curative Surgery 12
5. Use of Biomarkers in Monitoring Therapy in Patients with Metastasis 13
6. Emerging Biomarkers for Breast Cancer 14
6.1 Circulating Tumor Cells 14
6.2 Circulating Tumor-Derived DNA 15
6.3 MicroRNAs 16
7. Conclusion 17
Acknowledgments 17
References 17

Abstract
Biomarkers play an important role in the detection and management of patients with
breast cancer. Thus, BRCA1/2 mutation testing is used for risk assessment in families with
a high prevalence of breast and ovarian cancer. Following a diagnosis of breast cancer,
measurement of multi-analyte profiles such as uPA/PAI-1 or Oncotype DX may be used
for determining prognosis and identifying lymph node-negative patients who may be

Advances in Clinical Chemistry, Volume 71 # 2015 Elsevier Inc. 1

ISSN 0065-2423 All rights reserved.
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2 Michael J. Duffy et al.

spared from having to receive adjuvant chemotherapy. Other -gene tests such as the
PAM50 ROR, Breast Cancer Index, and EndoPredict have been reported to predict the
development of late recurrences and thus may be of value in selecting patients for
extended hormone therapy. Mandatory assays include estrogen receptors for identifi-
cation of endocrine-sensitive cancers and HER2 in selecting patients for treatment with
anti-HER2 therapy (e.g., trastuzumab, lapatinib, pertuzumab, and ado-trastuzumab
emtansine). Finally, serum biomarkers such as CA 15-3 or CEA may be used in monitor-
ing therapy in patients with advanced disease receiving systemic therapy. Promising
new biomarkers undergoing evaluation include circulating tumor cells and circulating
tumor-derived DNA.

1. USE OF BIOMARKERS IN THE IDENTIFICATION OF

WOMEN AT INCREASED RISK OF DEVELOPING BREAST
CANCER (RISK ASSESSMENT)
Biomarkers are playing an increasingly important role in the detection
and management of patients with several different cancer types, including
breast cancer [1,2]. For breast cancer, biomarkers are particularly useful in
the identification of individuals at increased risk of developing the malig-
nancy within high-risk families, determining prognosis at the time of initial
diagnosis, identifying the most appropriate systemic therapy, postoperative
surveillance, and monitoring therapy in advanced disease. The aim of this
chapter is to critically review the role of biomarkers in these different set-
tings. In addition, we will review new biomarkers emerging for breast can-
cer and speculate on their likely integration into routine use.
It has been known for decades that certain families have an increased risk
of breast cancer. Although defects in several different genes are known to
predispose to breast cancer, the best characterized are BRCA1 and BRCA2
[3]. Both BRCA1 and BRCA2 are tumor suppressor genes involved in the
repair of breaks in double-stranded DNA. These genes thus play a critical
role in maintaining integrity of DNA. Although both genes are involved
in DNA repair, their roles are distinct and nonoverlapping.
In a large prospective study, the cumulative risk of developing breast can-
cer by age 70 years was 60% for BRCA1 carriers and 55% for BRCA2 car-
riers [4]. In the same study, the corresponding risks for the development of
ovarian cancer were 59% and 16.5% for BRCA1 and BRCA2 carriers,
respectively. Interindividual variation in risk is thought to be due to the loca-
tion and type of mutation as well as environmental factors. Pathogenic muta-
tions in BRCA2 but apparently not in BRCA1 can also increase
susceptibility to pancreatic and prostate cancers [3].
BRCA1/2 germline testing is thus now a common practice for risk assess-
ment in families with a high prevalence of breast or ovarian cancer. According
Biomarkers in Breast Cancer 3

to the U.S. Preventive Services Task Force (USPSTF) guidelines [5], primary
care providers should “screen women who have family members with breast,
ovarian, tubal, or peritoneal cancer with one of several screening tools
designed to identify a family history that may be associated with an increased
risk for potentially harmful mutations in breast cancer susceptibility genes
(BRCA1 or BRCA2).” It was furthermore recommended that those with
positive findings should undergo genetic counseling. BRCA genetic testing
was not recommended for women without a family history of the disease.
Potential benefits of undergoing germline BRCA testing for inherited
breast cancer susceptibility include a more accurate risk assessment for the
individual as well as their family, with the possibility of early cancer detec-
tion or indeed prevention. Individuals found to be mutation carriers should
be advised to consider the options both for decreasing the risk of breast can-
cer and for early detection. These include regular surveillance with mam-
mography and magnetic resonance imaging (MRI), prophylactic bilateral
mastectomy, oophorectomy, or administration of prophylactic tamoxifen
or prophylactic raloxifene [6,7]. Currently, the USPSTF recommends
administration of prophylactic tamoxifen or prophylactic raloxifene to
women at high risk of breast cancer and have a low risk of suffering from
adverse medication effects [5].
Although BRCA1 and BRCA2 are the best characterized and the most
prevalent breast cancer susceptibility genes, pathogenic mutation in these
genes is believed to be responsible for only approximately 15–25% of breast
cancers with a hereditary component [8]. Other genes implicated in confer-
ring an increased susceptibility to breast cancer are listed in Table 1. As
inherited defects in these non-BRCA genes appear to be rare, routine

Table 1 Breast Cancer Susceptibility Genes and Their

Penetrance
Gene Penetrance
BRCA1/2 High
TP53 (p53) High
LKB1 High
PTEN High
CHEK2 Moderate
ATM Moderate
PALB Moderate
4 Michael J. Duffy et al.

genetic testing for them is not widely carried out at present. However, in the
near future, it is likely that testing for breast cancer genetic susceptibility will
involve panels of genes or whole-exome sequencing, rather than investigat-
ing individual genes such as BRCA1 or BRCA2. The advantage of multi-
gene testing is that for some individuals it may save time and reduce costs
compared to sequential testing of one or a small number of genes. Interpre-
tation of results from gene panel, however, is presently complicated due to
lack of data on penetrance of the different mutations and the increased like-
lihood of finding alteration of unknown clinical significance. Furthermore,
guidelines for the management of subjects found to harbor many of these
alterations are not presently available [8].

2. USE OF BIOMARKERS IN DETERMINING PROGNOSIS

Following a diagnosis of primary invasive breast cancer, the most
urgent questions to be addressed are: what is the prognosis and should adju-
vant systemic therapy (e.g., chemotherapy) be administered? Thus, the
accurate determination of prognosis at the time of diagnosis of breast cancer
is clearly essential for optimum patient management, especially to avoid
overtreatment of nonaggressive disease and undertreatment of aggressive
forms. Traditionally, pathological and clinical criteria such as tumor size,
tumor grade, number of lymph nodes with metastasis, patient age, and
patient morbidity status were used for this purpose. Of these factors, the
number of lymph nodes with metastasis has been the most widely employed.
However, with the advent of screening mammography in recent years, more
than 50% of women diagnosed with breast cancer present with lymph node-
negative disease. Furthermore, most of these women are cured of their breast
cancer using surgery and radiotherapy and are therefore unlikely to benefit
from adjuvant chemotherapy. Despite this high cure rate with local treat-
ment, most lymph node-negative patients currently receive adjuvant che-
motherapy. Thus, currently, the main requirement for prognostic
biomarkers in breast cancer is to aid the differentiation of newly diagnosed
lymph node-negative patients who are cured by surgery and radiotherapy
from those women who might benefit from adjuvant chemotherapy.
Over the last 20 years, an enormous amount of work has been carried out
to address this issue and hundreds of putative biomarkers have been pro-
posed for predicting outcome in women with newly diagnosed breast cancer
[9]. Most of these studies, however, were of low evidence [10] due to inad-
equate design, low numbers of patients, failure to show independent
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Biomarkers in Breast Cancer 5

prognostic value, inappropriate clinical validation and failure to demonstrate

clinical utility [9]. Among the most widely investigated are the tumor tissue
biomarkers, urokinase plasminogen activator (uPA)/PAI-1, gene-
expression profiles (e.g., Oncotype DX, MammaPrint), Ki-67, and the
serum biomarkers, CA 15-3 and CEA [2,9].

2.1 uPA and PAI-1

uPA and PAI-1 are the only biomarkers validated for breast cancer using
level 1 evidence data [10]. Indeed, uniquely for cancer prognostic bio-
markers, these biomarkers have undergone validation in both a randomized
prospective trial and a pooled analysis of individual patient data [11–14].
These high-level studies have confirmed the independent prognostic benefit
of uPA and PAI-1 in lymph node-negative breast cancer. Furthermore, the
prospective randomized trial also showed that high-risk lymph node-
negative patients, as identified by high levels of uPA/PAI-1, benefited from
adjuvant chemotherapy [12,14].
According to the American Society of Clinical Oncology (ASCO)
guidelines on biomarkers in breast cancer [15], uPA/PAI-1 “may be used
for the determination of prognosis in patients with newly diagnosed,
node-negative breast cancer. Low levels of both markers are associated with
a sufficiently low risk of recurrence, especially in hormone receptor-positive
women who will receive adjuvant endocrine therapy, that chemotherapy
will only contribute minimal additional benefit.” The guidelines further
added that cyclophosphamide-methotrexate-5-fluorouracil (CMF)-based
adjuvant chemotherapy “provides substantial benefit, compared with obser-
vation alone, in patients with high risk of recurrence as determined by high
levels of uPA and PAI-1.” Other expert panels recommending measurement
of uPA/PAI-1 for determining prognosis in newly diagnosed patients with
invasive breast cancer include the National Academy of Clinical Biochem-
istry (NACB) USA [16] and the European Group on Tumor Markers
(EGTM) [17].
Although extensively validated, uPA and PAI-1 are not widely used in
the clinic, the reason being that testing for these biomarkers requires fresh or
freshly frozen tissue.

2.2 Oncotype DX
Although less extensively validated than uPA and PAI-1, Oncotype DX is
more widely used for clinical purposes. Indeed, Oncotype DX was one of
6 Michael J. Duffy et al.

the first multi-gene tests recommended for determining prognosis in any can-
cer [15–18]. According to the ASCO guidelines [15], Oncotype DX can be
used to predict the risk of recurrence in estrogen receptor (ER)-positive,
lymph node-negative breast cancer patients treated with adjuvant tamoxifen.
In particular, Oncotype DX may be used to identify those women who are
likely to obtain the most therapeutic benefit from adjuvant tamoxifen and
thus may not require adjuvant chemotherapy [19,20]. Additionally, patients
with high recurrence scores were reported to derive relatively more benefit
from adjuvant chemotherapy than from tamoxifen [21,22].
Currently, Oncotype DX is undergoing validation in two large random-
ized prospective trials: the Trial Assigning IndividuaLized Options for
Treatment (Rx) (TAILORx) and Rx for Positive Node, Endocrine
Responsive Breast Cancer (RxPONDER) trial. TAILORx is a randomized
phase III trial study involving women who have node-negative, estrogen
receptor-positive breast cancer that aims to determine whether endocrine
therapy alone or endocrine therapy plus chemotherapy is better for women
who have an Oncotype DX intermediate recurrence score [23]. In contrast
to the TAILORx trial, the RxPONDER trial involves ER-positive patients
with one to three positive lymph nodes [24]. The primary aim of this trial is
to establish the potential benefit of adjuvant chemotherapy in patients with
lymph node-positive breast cancer with a low Oncotype DX score.

2.3 MammaPrint
Another widely investigated multi-gene prognostic test is MammaPrint
[25–29]. Although MammaPrint has been approved by the U.S. Food
and Drug Administration (FDA), for determining outcome in lymph
node-negative breast cancer patients with tumors <5 cm in diameter, it is
not yet widely recommended for clinical use by expert panels. This situa-
tion, however, may change following the publication of the findings from
the ongoing MINDACT randomized clinical trial. The main aim of this
trial, which includes breast cancer patients with negative or one to three pos-
itive nodes, is to confirm if women with a low-risk gene profile by
MammaPrint and high risk by clinical and pathological criteria can avoid
chemotherapy, without affecting outcome.

2.4 Other Multigene Signatures

Several other multi-gene/multiprotein signatures have been shown to have
prognostic value in breast cancer (Table 2) [30,31]. Unlike Oncotype DX
Biomarkers in Breast Cancer 7

Table 2 Some of the Multigene/Multiprotein Tests for Predicting Outcome in Patients

with Breast Cancer
Commercial Validation Recommended/
Test Test Company in RPT Approved
uPA/PAI-1 Yes American Yes ASCO, NACB,
Diagnostica/ EGTM
Sekisui
Oncotype Yes Genomic Health Ongoing ASCO, NCCN,
DX ESMO, NACB
MammaPrint Yes Agendia Ongoing ESMO, FDA
EndoPredict Yes Sividon No CE mark
Diagnostics
Mammostrat Yes Clarient No No
BCI Yes bioTheranostics No No
Curebest Yes Sysmex No No
95GC Breast
Rotterdam No None No No
signature
ProSigna Yes NanoString Ongoing FDAa, CE mark
(PAM50) Technologies
IHC4 No None No No
RPT, randomized prospective trial; ASCO, American Society of Clinical Oncology; NACB, National
Academy of Clinical Biochemistry USA; EGTM, European Group on Tumor Markers; NCCN,
National Comprehensive Cancer Network; ESMO, European Society of Medical Oncology; FDA,
Food and Drug Administration; CE mark, Conformité Européenne (CE); BCI, Breast Cancer Index.
The CE marking is the manufacturer’s declaration that the product meets the requirements of the
applicable European Community directives.
a
510(k) cleared by FDA.
Data reviewed from Refs. [30,31].

and MammaPrint, some of these tests (PAM50 ROR, EndoPredict, and

Breast Cancer Index) were reported to predict the development of late
metastases [32–35]. These signatures may thus have utility in the selection
of ER-positive patients for extended endocrine treatment such as 10 years
of tamoxifen or 5 years of tamoxifen followed by 5 years of an aromatase
inhibitor [36–38]. At the same time, these tests could identify those women
that do not need the prolonged treatment, thus saving on costs and potential
toxicity.
8 Michael J. Duffy et al.

2.5 CA 15-3
All of the above prognostic biomarkers require tumor tissue for their mea-
surement, thus necessitating biopsy or surgery. Indeed, most of the research
on prognostic biomarkers in breast cancer has investigated tumor tissues.
Clearly, a validated serum-based prognostic biomarker would be of value,
especially if it provided independent information in lymph node-negative
patients. One of the best investigated serum-based prognostic biomarker is
CA 15-3 which detects the soluble moiety of the MUC1 protein [39].
Indeed, a multiplicity of studies has shown that elevated concentration of
serum CA 15-3 at initial presentation is associated with adverse outcome [39].
A likely reason why high preoperative CA 15-3 levels in patients with breast
cancer predict poor outcome is that the biomarker signal micrometastases or
occult metastases that are not clinically or radiologically evident.
Apart from the EGTM [17], expert panels do not at present recommend
measurement of CA 15-3 for determining prognosis in patients with early
breast cancer. However, since the biomarker is relatively easy and cheap
to determine, a preoperative CA 15-3 level might be combined with exis-
ting prognostic factors in planning the optimum management of patients
with newly diagnosed breast cancer [32]. In any event, elevated levels at ini-
tial presentation should prompt an investigation for possible metastatic
disease.
Other serum-based biomarkers shown to have prognostic significance in
breast cancer include CEA, soluble HER2, TPA, and TPS (for review, see
Refs. [39,40]). Like CA 15-3, these biomarkers are not widely used in deter-
mining prognosis in patients with breast cancer.

3. USE OF BIOMARKERS IN GUIDING TREATMENT

A wide variety of systemic treatments are available for patients with
breast cancer, including chemotherapy drugs, hormone treatments, and
anti-HER2 agents (Table 3). With such a broad range of treatment options,
it is important that every patient receives the most appropriate regime. For-
tunately, predictive biomarkers are available for two of the three main forms
of treatment, i.e., ER and progesterone receptor (PR) for selecting for hor-
mone therapy and HER2 gene amplification/overexpression for identifying
women likely to benefit from anti-HER2 therapy. Despite intensive efforts,
there are no validated predictive biomarkers for identifying response to a
specific chemotherapy agent over another.
Biomarkers in Breast Cancer 9

Table 3 Systematic Treatments Used for Breast Cancer

Hormone Cytotoxic Anti-HER2
Tamoxifen Cyclophosphamide/ Trastuzumab
methotrexate/fluorouracil
Anastrozole Platinum salts Lapatinib
Letrozole Anthracyclines Pertuzumab
Exemestane Taxanes Ado-trastuzumab
emtansine
Fulvestrant Vinorelbine Neratiniba
Oophorectomy Eribulin Afatiniba
LH–RH agonists Capecitabine
List of cytotoxic agents is not exhaustive.
a
Undergoing clinical trials.

3.1 Estrogen Receptor for Predicting Response to Endocrine

Therapy
ER is predominantly a nuclear protein existing in two main forms, ERα and
ERβ [41]. Although both forms of ER bind estradiol and the tamoxifen
metabolite, endoxifen, they appear to mediate different biological activities.
In particular, while ERα enhances breast cancer cell proliferation, ERβ has
been reported to inhibit proliferation [41,42]. Currently, a validated clinical
role has only been established for ERα which will be referred to as ER in this
chapter. While the original ligand-binding ER assays are likely to have
detected both ERα and ERβ, the current immunohistochemistry measure-
ments detect only ERα.
Studies carried several decades ago showed that only approximately 30%
of patients with metastatic breast cancer responded to the then available hor-
mone therapy (oophorectomy, adrenalectomy, or high-dose steroids) [41].
However, if the ER protein was present in the primary tumor, 50–60% of
patients benefited. In contrast, patients negative for ER were found to rarely
respond to hormone treatment [41]. Although ER is still used to predict
response to hormone therapy in advanced cancer, currently its main
application is in identifying patients with early breast cancer that are sensitive
to tamoxifen or an aromatase inhibitor. In this setting, the administration of
adjuvant tamoxifen for approximately 5 years to patients with ER-positive
invasive breast cancer was found to decrease recurrence rates by almost 50%
10 Michael J. Duffy et al.

and mortality by about 30% [43]. As in advanced disease, ER-negative

patients failed to benefit from adjuvant tamoxifen.
More recently, administration of tamoxifen to ER-positive patients for
10 years was shown to be superior to treatment for 5 years [36,37]. Similarly,
administration of an aromatase inhibitor to postmenopausal women for
5 years has been shown to be superior to treatment with tamoxifen for
the same period of time [44–48]. For premenopausal ER-positive patients,
a recent phase III study concluded that the addition of ovarian suppression to
tamoxifen failed to provide a significant benefit in the overall study popu-
lation. However, for patients who were deemed to be at sufficient risk for
recurrence to receive adjuvant chemotherapy and who remained
premenopausal, the combination of ovarian suppression and tamoxifen
was found to enhance outcome [49].
Because of its critical importance in planning treatment, measurement of
ER on all newly diagnoses patients with invasive breast cancer is now uni-
versally recommended [15–18] and indeed, mandatory. However, as the ER
status may change between a primary tumor and a subsequently formed met-
astatic lesion [50], measurement should also be performed on the metastatic
lesion, where feasible. Detailed guidelines for performing ER immunohis-
tochemistry assays have been published [51].
An ongoing debate in reporting ER concentration relates to the cutoff
point for defining positivity. According to the ASCO/College of American
Pathologists (CAPs) guidelines, the presence of immunoreactive ER in 1%
of the tumor nuclei should be regarded as positive [51]. Some recent studies,
however, have suggested that patients with ER staining in 1–10% of tumor
nuclei behave more like ER-negative than ER-positive cancers [52,53].
Thus, Gloyeske et al. [52] showed that cancers with 1–10% positive nuclei
exhibited specific morphological and biochemical features more frequently
found in ER-negative than ER-positive tumors such as necrosis, inflamma-
tory infiltrate, and absence of PRs. In another study, patients with tumors
having 1–10% nuclei staining exhibited a significantly worse survival than
did patients with >10% nuclei staining, even when adjusted for tumor grade
and tumor stage [53]. Indeed, survival rates were similar for patients with
1–10% nuclei staining and those totally negative for ER. These findings sug-
gest that further research is necessary to establish the optimum cutoff point
for ER in detecting endocrine-sensitive breast cancers.
PR is frequently measured alongside ER. Although the predictive
potential of PR for response to adjuvant endocrine therapy is not clear [43],
several different studies have shown that PR is independently prognostic in
Biomarkers in Breast Cancer 11

breast cancer, i.e., high levels are generally associated with a favorable out-
come [54–58]. Because of its prognostic role, most published guidelines rec-
ommend simultaneous measurement of ER and PR [15–18]. Like ER, PR
is also measured using immunohistochemistry [51]. However, as with ER,
further work is necessary to establish the optimum cutoff point for PR [56].
It is important to state that ER and PR are not the only biomarkers used
in guiding therapy in patients with breast cancer. Approximately, 7% of
ER-positive patients are HER2 positive. These ER-positive and HER2-
positive patients not only receive hormone therapy but are also candidates
for anti-HER2 treatment, see below.

3.2 HER2 for Predicting Response to Anti-HER2 Therapy

A further mandatory therapy predictive biomarker for breast cancer is HER2
which is used for identifying patients likely to benefit from anti-HER2
therapy [59]. While ER is present in >80% of breast cancer, HER2 gene
amplification/overexpression is detectable in only approximately 20% of
cases. HER2 protein overexpression is mainly measured by immunohisto-
chemistry, while gene amplification is mostly determined by in situ hybridi-
zation (ISH), e.g., fluorescent ISH.
Prior to the availability of anti-HER2 treatment, HER2 gene
amplification/overexpression was associated with adverse outcome in
patients with breast cancer and indeed used as a biomarker for determining
prognosis. However, at present, the main reason for determining HER2 sta-
tus is to select patients who are likely to benefit from anti-HER2 treatment
in the neo-adjuvant, adjuvant, and advanced disease settings. The use of
anti-HER2 therapy has dramatically improved the outcome of patients with
HER2 overexpression. Indeed, the outcome for patients with HER2-
positive disease that receive anti-HER2 therapy now appears to be similar
or better than for those with HER2-negative disease [60].
Currently, four forms of anti-HER2 therapies are available for treatment
of women with HER2-positive breast cancer, trastuzumab (Herceptin),
lapatinib (Tykerb), pertuzumab (Perjeta), and ado-trastuzumab emtansine
(T-DM1; Kadcycla) [59]. Based on available data, it appears that HER2
gene amplification/overexpression is necessary to achieve response to all
of these anti-HER2 agents. According to the recent ASCO guidelines for
use of anti-HER2 therapy in patients with metastatic breast cancer, first-line
treatment for HER2-positive cases should be trastuzumab, pertuzumab, and
taxane, unless the patient has a contraindication to taxanes [61]. For those
12 Michael J. Duffy et al.

who progress during or after first-line anti-HER2 treatment, T-DM1

should be administered [61]. In the adjuvant setting, trastuzumab (in com-
bination with chemotherapy) is the main form of anti-HER2 therapy ini-
tially administered. As with ER, detailed guidelines for performing
HER2 assays have been published [62].

4. USE OF BIOMARKERS IN THE POSTOPERATIVE

FOLLOW-UP OF ASYMPTOMATIC PATIENTS
FOLLOWING CURATIVE SURGERY
Intensive surveillance following treatment with curative-intent sur-
gery is now a common practice in patients with several different cancer
types, including breast cancer. This practice is based on the widely held
belief that early detection of recurrent/metastatic disease as a result of regular
testing, followed by the early commencement of treatment in the event of a
biomarker increase, results in a better outcome, compared to starting treat-
ment when disease recurrence is clinically detectable. While early treatment
in this setting may result in better outcome, high-level evidence for this is
lacking in breast cancer. Furthermore, the use of regular testing of asymp-
tomatic women during surveillance may lead to false-positive results and
additional testing. Clearly, therefore, active surveillance is not only costly
but increases patient anxiety and may result in harm, e.g., radiation exposure
from imaging [63].
Despite the lack of evidence for clinical utility, many centers measure
serial levels of serum biomarkers during the follow-up of asymptomatic
patients who have undergone curative surgery for breast cancer. The most
widely used biomarkers in this setting are CA 15-3 and CEA [40,64,65].
Other biomarkers that may be used include TPA, TPS, CA 125, and the
shed form of HER2 [40,64,65]. In recent years, a number of studies have
addressed the value of whole-body imaging using MRI or FDG-PET/CT
for the early detection of recurrence in asymptomatic patients with increas-
ing biomarker levels. In one of these studies, Gioia et al. [66] measured CA
15-3, CEA, and CA 125 at 6-week intervals. Out of 813 patients monitored,
44 exhibited previously defined biomarker increases and were subjected
whole-body MRI or PT/CT scan. Of the 44 patients, 29 (66%) were found
to have metastasis, 6 (14%) had secondary malignancies other than breast
cancer, while 9 (20%) were found to have no evidence of malignancy.
Another study found tumor recurrences using PET/CT scans in 65% of
patients exhibiting an increase in serum biomarker levels [67].
Biomarkers in Breast Cancer 13

Despite the ability of serum biomarkers to detect early recurrences in

some patients who have undergone curative surgery for breast cancer, most
guidelines including those published by ASCO, National Comprehensive
Cancer Network (NCCN), and European Society of Medical Oncology
(ESMO) are opposed to the use of regular biomarker testing during
surveillance of asymptomatic women, following a diagnosis of breast cancer
[15,18,68]. The reason these organizations are opposed to biomarker
measurement in this setting is the lack of evidence from prospective ran-
domized trial that the practice enhances patient outcome. It should be stated,
however, that a large prospective randomized trials comparing follow-up
with and without modern biomarker measurement have not been
carried out.
In contrast to most organizations, the EGTM panel recommends mea-
surement of “serial CA 15.3 and CEA for the early detection of recurrence in
patients with breast cancer and no evidence of disease” [17]. According to
this group, biomarkers should be measured every 2–4 months during the
initial 5 years after diagnosis, then every 6 months for the next 3 years,
and at yearly intervals thereafter. It should be stated that this recommenda-
tion is based on a consensus of expert opinion rather than on high-level
evidence.

5. USE OF BIOMARKERS IN MONITORING THERAPY

IN PATIENTS WITH METASTASIS
While therapy predictive biomarkers indicate upfront the likelihood
of an initial response to a specific treatment, most patients sooner or later
develop resistance and tumor progression. In order to determine real-time
response to a systemic treatment, measurement of serial serum biomarker
measurements is a low cost and relative minimally invasive approach. The
most widely used biomarker for monitoring response to treatment in
advanced breast cancer is CA 15-3, although CEA, TPA, and TPS are also
used [32,64,65]. In general, serum biomarker levels increase with therapy
resistance and tumor progression. In contrast, biomarkers tend to decrease
with response to therapy and tumor regression. Although in most patients,
changes in serial levels of biomarker parallel tumor progression/regression,
this does not always occur [32]. Clearly, therefore, CA 15-3 or indeed any
other serum biomarker cannot be used alone for monitoring therapy in
patients who have advanced breast cancer that can be evaluated by conven-
tional imaging.
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