Introduction
Breast cancer comes first as the cancer-related cause
of death in the female population; despite the effective
screening programs which result in early diagnosis.
The survival rate is high when the disease is diagnosed in the early stage; however, 20-30% of local breast cancer
cases will be progressed to the metastatic stage.
[] The progressive potential of the disease is based
on disease and patient characteristics such as age, receptor
status, and tumoral invasiveness. Aggressive
disease courses in patients with worse pathological characteristics underlines the demand for enhanced
methods to predict the patients who are a candidate for
aggressive and novel targeted strategies for treatment.
Elevated plasma CA-15-3 levels are tested frequently
for surveillance of recurrent breast cancer; however, its
correlation with patient characteristics and prediction
of treatment modalities are not validated and routine
analysis does not recommend in current guidelines.
[] At present, the most commonly used markers to
guide the treatment options are hormone receptor and
HER2-neu status on pathological material. However,
disease courses are different even in patients with the
same histologic properties.[] There is a need for discovering
new biomarkers to explain these differences.
Serum biomarkers are extensively investigated in
cancer patients to close the gaps in diagnostic and predictive
markers. Nectins are a kind of immunoglobulin-
like homo-heterophilic cell adhesion proteins that
maintain intercellular adherence and tight junctions.[]
There are four nectin proteins that described; nectin-1
and -2 are mainly found in adult somatic tissues,
nectin-3 is expressed mainly in reproductive organs
such as testes and placenta, and also nectin-4 expression
is naturally limited to the placenta.[-] On the other
hand, binding characteristics are different for each
nectin subgroup.[] There have been reports of expression
of nectin-4 in ductal breast carcinoma and lung
adenocarcinomas.[-] Nectin-2 is less frequently
studied in oncologic patients; Oshima et al.[] have
been shown the elevated expression in breast cancer tissues.
However, in previous studies showing diagnostic
biomarker potential of nectin-2 and nectin-4; the argument
was not firmly made that nectin-2 and -4 could be
therapeutic targets, or predicting prognostic and clinical
properties of the disease.[,] With this study,
we aimed to define whether overexpression of nectin
proteins and mRNA remnants in plasma correlates with
related clinical properties of patients with breast cancer.
Methods
Study Population and Design
The study group included 60 breast cancer patients who
were receiving treatment at the Istanbul University,
Institute of Oncology, between 2017 and 2018. All patients
were diagnosed pathologically or radiologically.
The disease was staged by physician according to the
American Joint Committee on Cancer staging system.
All volunteers are informed and then signed consent
form for the study and ethical approval was obtained
from Istanbul Medical Faculty Ethical Committee.
Venous blood samples were obtained from patients
before any treatment was given, then clotted for 10 min
before centrifuged. The collected serum samples stored
at -20°C until analysis after the centrifugation (10 min
4000 rpm) at the room temperature.
Evaluation of Serum Nectin-2 and Nectin-4 Levels
Serum nectin-2 and nectin-4 protein levels were determined
by enzyme-linked immunosorbent assay
(ELISA) method (Shanghai Sunred Biological Technology
Co. Ltd.). Serum samples/standards, biotinylated
Fab monoclonal capture antibody, and streptavidin
horseradish peroxidase conjugates were, respectively,
added to the wells which are pre-coated antibody. During
the 1 h incubation at 37°C, the antigen-antibody
complexes formed. After this incubation, the unbound
material was washed away and the colorless chromogen
solution was added and again incubated at 37°C for 10
min (protect from light) for the conversion of the colorless
solution to a blue solution. Since the enzyme interacts
with its substrate, a color reaction occurred in
direct proportion to the antigen concentration in the
sample. This color reaction was stopped by the addition
of an acidic stop solution and the blue color turned
yellow. The colored reaction product was measured using
an automated ELISA reader (ChroMate® 4300) at
450 nm. The concentrations of the samples were determined
with the help of the standard curve drawn with
standards of known concentration and the results were
expressed as ng/L.
Quantification of Nectin-2 and Nectin-4 mRNA Expression
in Serum
Nectin-2- and nectin-4-specific mRNAs in serum
samples of patients" total RNA were isolated using a
monophasic phenol and guanidine thiocyanate solution.
Two hundred microliters of sample, 800 ?l of
RNA isolation solution, and 200 ?l chloroform are
mixed. The mixture is incubated on ice for 5 min and
centrifuged at 11,000 rpm for 15 min at 4°C. The RNA
phase is transferred to a 550 ?l propanol-containing
tube. After centrifugation at 11,000 rpm for 10 min at
4°C, RNA is washed with 75% alcohol, dried at room
temperature, and transfused in 20 µl RNase-free water.
cDNA synthesis was performed according to the
procedure of a commercial kit (Roche, Mannheim,
Germany). cDNA provides superior components that
ensure total RNA templates. Nectin-2 and nectin-4
gene expression in serum were measured semi-quantitatively
using GAPDH (housekeeping gen) and
SYBR Green. Real-time polymerase chain reaction (RT-PCR) process was performed using LightCycler
480 Instrument. RT-PCR components and conditions
which have been used are explained in Table 1a and
1b. Probe library was used in primer selection. The
confirmation of the cycle product for each molecule
was carried out with melting curve analysis. Instrument
measurable threshold value of fluorescence level
in RT-PCR reaction the cycle it passes is called Ct
(cycle threshold). According to the Ct value obtained,
2-??Ct method is used.
Table 1a: Real-Time PCR Components
Table 1b: Real-Time PCR Conditions
Statistical Analysis
SPSS software was used for recording and analyzing the
data (SPSS-21, Chicago, IL, USA). The Mann-Whitney
U-test was applied by examining the conformity of
variables to a normal distribution using visual and analytical
methods (Kolmogorov-Smirnov/Shapiro-Wilk
tests). P<0.05 was accepted as statistically significant.
Results
The median age of the study population was 53 (range:
24?71) years. Twenty-eight patients were in the premenopausal
period. The tumor was located on the right
side in 27 patients. The mean tumor size was 30.21±17.32
mm. Tumor pathology in 44 patients was pathologically
reported as invasive ductal carcinoma. The number of
patients with Grade 3 was 32. De novo distant metastasis
was detected in four patients. Forty-one patients were
in the luminal group. Lymph node involvement was detected
in 25 patients. The clinical and pathological characteristics
of patients are summarized in Table 2.
Table 2: The clinical and pathological characteristics of
patients
When the menopausal status and tumor localization
of the patients included in the study were compared,
no significant difference was found between
the nectin-2 and nectin-4 levels. When the pathological
characteristics of the patients were compared, the
patients who were showed poor prognostic disease
characteristics such as larger tumor size, lymph node
positivity, the presence of lymphovascular invasion,
presence of necrosis, and histological Grade 3 have
higher serum levels of nectin protein levels than the
data showing good prognosis; however, statistically
insignificant. The nectin-4 expression level was statistically
significantly higher in those with Ki-67 ?30
and those with positive distant metastasis compared
to the other group. In addition, nectin-2 expression
was higher in patients with Grade 3 tumors which also
found statistically significant. The results of nectin level
measurements are shown in Table 3.
Table 3: Distribution of Nectin-2 and 4 levels
Discussion
In this study, increased serum mRNA expression of
nectin-2 and nectin-4 has been detected in patients
with breast carcinoma which have pathological worse characteristics. Nectin-2 mRNA expression level is
elevated in Grade 3 tumors. Nectin-4 mRNA expression
level is elevated in metastatic patients and patients
with high pathological Ki-67 levels. However, we could
not able to show any statistically significant difference
between serum protein levels and clinicopathologic
properties of the disease. Using nectin-4 for the active
follow-up of breast cancer patients might help to early
detection of metastatic patients. Until now, there is no
established biomarker to use for active follow-up of
breast cancer patients. Current guidelines do not recommend
ordering serum analysis of any biomarker
even for CA-15-3. This study could be a starting point
to research the usefulness of nectin molecules as a
biomarker for breast cancer surveillance.
The loss of function of tight connections that control
cell-cell adhesion and intracellular permeability causes
the spread of cancer cells and metastasis. Nectins,
which are calcium-independent immunoglobulin-like
cell adhesion molecules, are in a relationship with cadherin
in various intercellular associations, sometimes
independently, and sometimes in cooperation with
the afadine molecule.[,] All nectin molecules except
nectin-4 are normally expressed in adult epithelial,
endothelial, hematopoietic, and neuronal tissues.
Although nectin-4 is expressed during embryogenesis,
not detectable in adult tissues or serum.[] Nectin-2-
mediated cell adhesion has been implicated in the formation
of cadherin-induced adherence complexes and
the formation of a claudin bound tight linkage complex in epithelial cells. Overexpression of nectins is related
to cancer and there is evidence that tight links are necessary
to cell growth regulation.
Overexpression of nectin-2 and nectin-4 in cancerous
cells has been detected and has been found associated
with poor prognosis in previous reports. Nectin-2
is a less frequently studied subgroup of the nectins
in cancer patients. Diagnostic and prognostic value
of nectin-2 in colorectal cancer,[] pancreatic cancer,[] gallbladder carcinoma,[] esophageal cancer,[] and lung cancer[] has been studied earlier.
Until now, only Oshima et al.[] evaluated nectin-2 on
ovarian cancer and breast cancer tissues pathologically
using gene expression profile analysis and immunohistochemistry.
They also evaluated various in vitro cancer
cell cultures and found elevated levels of nectin-2.
They hypothesized nectin-2 might be a treatment target
since anti-nectin-2 antibodies have resulted in antigen-
dependent cell death.[] However, ensuing studies
have not evolved in this era. In our study, nectin-2
is evaluated in serum and found to be related to Grade
3 tumors. Although this study requires validation,
nectin-2 might be evaluated for prognostic and treatment
predictive biomarker in further researches based
on this pilot study. There is evidence that nectin-2
may serve as new immunogenic therapies. T-cell immunoglobulin
and ITIM domain (TIGIT) recognize
nectin-like adhesion molecules, specifically nectin-2,
and therefore play a critical role in the innate immune
response to malignant transformation.[]
High expression of nectin-4 in ductal breast, lung,
and pancreatic cancers and its relation with a poor
prognosis has been shown.[,] Moreover, according
to another study with ovarian carcinoma patients, the
expression of nectin-4 could be related to resistance to
chemotherapeutic agents.[] Lattanzio et al.[] studied
nectin-4 expression in their cohort of node-negative
early breast cancer. The presence of nectin-4 on
cell membrane of tumor cells was associated with poor
metastasis-free survival in patients with luminal-A tumors.
The high serum level of nectin-4 in metastatic
patients in our study suggests that it can be used as a
serum marker to detect early metastasis in patients by
contributing to this study. In addition, we have found
that the patients with higher Ki-67 level show high
nectin-4 level that also correlates with the metastatic
predisposition of the tumor.
There are several limitations to this study that must
be mentioned. First of all, this is a cross-sectional study
that the survival results of the patients were not noted.
Furthermore, there is not a normal control group, and the study cohort is categorized by medians of the patients"
nectin-2 and nectin-4 serum levels. On the
other hand, the main strength of the study is the comprehensive
evaluation of serum nectin-2 and nectin-4
levels with two different methods; RT-PCR and ELISA.
Furthermore, although tissue studies are available in
the literature with this biomarker, synchronous serum
analyses of these two markers in breast cancer are rare.
Conclusion
Nectin-4 mRNA is overexpressed in the patients
with high Ki-67 levels and metastasis. Furthermore,
nectin-2 mRNA is higher in the serum of patients with
Grade 3 tumors. Further studies are recommended to
determine the impact of nectin-2 and nectin-4 as a
surveillance marker. Studies that are designed prospectively
which contain serial measurement of serum levels
of these biomarkers might give appropriate results
to change the practice.
Peer-review: Externally peer-reviewed.
Conflict of Interest: The authors declare that they have no
conflict of interest.
Ethics Committee Approval: The study was approved by
the Istanbul Medical Faculty Clinical Research Ethics Committee
(No: 18, Date: 28/10/2016).
Financial Support: The study is funded by the Scientific
Research Projects Unit of Istanbul University with project
number of 24545.
Authorship contributions: Concept - S.B., S.V.; Design
- S.B., S.V., M.S.; Supervision - S.B., S.V., M.Z.Ü; Materials
- M.S., S.B., S.V.; Data collection and/or processing - N.A.,
M.Z.Ü.; Data analysis and/or interpretation - N.A., M.Z.Ü.;
Literature search - N.A., M.S., S.B.; Writing - N.A., S.B., S.V.,
M.S.; Critical review - N.A., M.S., M.Z.Ü., S.B., S.V.
References
Early Breast Cancer Trialists" Collaborative Group
(EBCTCG). Effects of chemotherapy and hormonal
therapy for early breast cancer on recurrence and 15-
year survival: an overview of the randomised trials.
Lancet 2005;365(9472):1687-717.
Park YH, Senkus-Konefka E, Im SA, Pentheroudakis
G, Saji S, Gupta S, et al. Pan-Asian adapted ESMO
clinical practice guidelines for the management of patients
with early breast cancer: A KSMO-ESMO initiative
endorsed by CSCO, ISMPO, JSMO, MOS, SSO
and TOS. Ann Oncol 2020;31(4):451-69.
Makki J. Diversity of breast carcinoma: histological subtypes and clinical relevance. Clin Med Insights
Pathol 2015;8:23-31.
Sakisaka T, Takai Y. Biology and pathology of nectins
and nectin-like molecules. Curr Opin Cell Biol
2004;16(5):513-21.
Takai Y, Nakanishi H. Nectin and afadin: novel
organizers of intercellular junctions. J Cell Sci
2003;116(1):17-27.
Irie K, Shimizu K, Sakisaka T, Ikeda W, Takai Y. Roles
and modes of action of nectins in cell-cell adhesion.
Semin Cell Dev Biol 2004;15(6):643?56.
Nakanishi H, Takai Y. Roles of nectins in cell adhesion,
migration and polarization. Biol Chem
2004;385(10):885-92.
Reymond N, Fabre S, Lecocq E, Adelaide J, Dubreuil
P, Lopez M. Nectin4/PRR4, a new afadin-associated
member of the nectin family that trans-interacts with
nectin1/PRR1 through v domain interaction. J Biol
Chem 2001;276(46):43205-15.
Takano A, Ishikawa N, Nishino R, Masuda K, Yasui
W, Inai K, et al. identification of nectin-4 oncoprotein
as a diagnostic and therapeutic target for lung cancer.
Cancer Res 2009;69(16):6694-703.
Fabre-Lafay S, Garrido-Urbani S, Reymond N, Goncalves
A, Dubreuil P, Lopez M. Nectin-4, a new serological
breast cancer marker, is a substrate for tumor
necrosis factor-alpha-converting enzyme (TACE)/
ADAM-17. J Biol Chem 2005;280(20):19543-50.
Oshima T, Sato S, Kato J, Ito Y, Watanabe T, Tsuji I, et
al. Nectin-2 is a potential target for antibody therapy
of breast and ovarian cancers. Mol Cancer 2013;12:60.
Fabre-Lafay S, Monville F, Garrido-Urbani S, Berruyer-
Pouyet C, Ginestier C, Reymond N, et al. Nectin-4
is a new histological and serological tumor associated
marker for breast cancer. BMC Cancer 2007;7:73.
Kanzaki N, Ogita H, Komura H, Ozaki M, Sakamoto
Y, Majima T, et al. Involvement of the nectin-afadin
complex in pdgf-induced cell survival. J Cell Sci
2008;121(12):2008-17.
Ikeda W, Nakanishi H, Miyoshi J, Mandai K, Ishizaki H, Tanaka M, et al. Afadin: a key molecule essential
for structural organization of cell-cell junctions of
polarized epithelia during embryogenesis. J Cell Biol
1999;146(5):1117-32.
Noyce RS, Bondre DG, Ha MN, Lin LT, Sisson G, Tsao
MS, et al. Tumor cell marker PVRL4 (Nectin 4) is an
epithelial cell receptor for measles virus. PLoS Pathog
2011;7(8):e1002240.
Karabulut M, Gunaldi M, Alis H, Afsar CU, Karabulut
S, Serilmez M, et al. Serum nectin-2 levels are diagnostic
and prognostic in patients with colorectal carcinoma.
Clin Transl Oncol 2016;18(2):160-71.
Liang S, Yang Z, Li D, Miao X, Yang L, Zou Q, et al. The
clinical and pathological significance of nectin-2 and
DDX3 expression in pancreatic ductal adenocarcinomas.
Dis Markers 2015;2015:379568.
Miao X, Yang ZL, Xiong L, Zou Q, Yuan Y, Li J, et al.
Nectin-2 and DDX3 are biomarkers for metastasis and
poor prognosis of squamous cell/adenosquamous carcinomas
and adenocarcinoma of gallbladder. Int J Clin
Exp Pathol 2013;6(2):179-90.
Li M, Qiao D, Pu J, Wang W, Zhu W, Liu H. Elevated
Nectin-2 expression is involved in esophageal squamous
cell carcinoma by promoting cell migration and
invasion. Oncol Lett 2018;15(4):4731-6.
Erturk K, Karaman S, Dagoglu N, Serilmez M, Duranyildiz
D, Tas F. Serum Nectin-2 and Nectin-4 are
diagnostic in lung cancer: which is superior? Wien
Klin Wochenschr 2019;131(17?18):419-26.
Deuss FA, Gully BS, Rossjohn J, Berry R. Recognition
of Nectin-2 by the natural killer cell receptor T cell
immunoglobulin and ITIM domain (TIGIT). J Biol
Chem 2017;292(27):11413-22.
Lattanzio R, Ghasemi R, Brancati F, Sorda RL, Tinari N,
Perracchio L, et al. Membranous Nectin-4 expression
is a risk factor for distant relapse of T1-T2, N0 luminal-
a early breast cancer. Oncogenesis 2014;3(9):e118.
Derycke MS, Pambuccian SE, Gilks CB, Kalloger SE,
Ghidouche A, Lopez M, et al. Nectin 4 overexpression
in ovarian cancer tissues and serum: potential role as a
serum biomarker. Am J Clin Pathol 2010;134(5):835-45.
