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Diagnostics 2021, 11, 516. https://doi.org/10.3390/diagnostics11030516 www.mdpi.com/journal/diagnostics
Article
CEA, EpCAM, αvβ6 and uPAR Expression in Rectal Cancer Pa-
tients with a Pathological Complete Response after Neoadju-
vant Therapy
Daan Linders
1,†
, Marion Deken
1,†
, Maxime van der Valk
1
, Willemieke Tummers
1
, Shadhvi Bhairosingh
1
, Dennis
Schaap
2
, Gesina van Lijnschoten
3
, Elham Zonoobi
4
, Peter Kuppen
1
, Cornelis van de Velde
1
, Alexander Vahrmei-
jer
1
, Arantza Farina Sarasqueta
5
, Cornelis Sier
1,6
and Denise Hilling
1,7,
*
1
Department of Surgery, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; d.g.j.lin-
ders@lumc.nl (D.L.); m.m.deken@lumc.nl (M.D.); m.j.m.van_der_valk@lumc.nl (M.v.d.V.); w.s.f.j.tum-
mers@lumc.nl (W.T.); s.bhairosingh@lumc.nl (S.B.); p.j.k.kuppen@lumc.nl (P.K.); c.j.h.van_de_velde@lumc.nl
(C.v.d.V.); a.l.vahrmeijer@lumc.nl (A.V.); c.f.m.sier@lumc.nl (C.S.)
2
Department of Surgery, Catharina Hospital, 5623 EJ Eindhoven, The Netherlands; dennis.schaap@catharina-
ziekenhuis.nl
3
Laboratory of Pathology, Stichting Pathology and Medical Microbiology, 5623 EJ Eindhoven, The Nether-
lands; i.van.lijnschoten@pamm.nl
4
Edinburgh Molecular Imaging LTD, Edinburgh EH16 4UX, UK; e.zonoobi@lumc.nl
5
Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; a.fa-
rina@amsterdamumc.nl
6
Percuros BV, 2333 CL Leiden, The Netherlands
7
Department of Surgical Oncology and Gastrointestinal Surgery, Erasmus MC Cancer Institute, University
Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands
* Correspondence: D.E.Hilling@lumc.nl; Tel.: +31-71-526-2377
† These authors contributed equally to this work.
Abstract: Rectal cancer patients with a complete response after neoadjuvant therapy can be moni-
tored with a watch-and-wait strategy. However, regrowth rates indicate that identification of pa-
tients with a pathological complete response (pCR) remains challenging. Targeted near-infrared
fluorescence endoscopy is a potential tool to improve response evaluation. Promising tumor targets
include carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), integrin
αvβ6, and urokinase-type plasminogen activator receptor (uPAR). To investigate the applicability
of these targets, we analyzed protein expression by immunohistochemistry and quantified these by
a total immunostaining score (TIS) in tissue of rectal cancer patients with a pCR. CEA, EpCAM,
αvβ6, and uPAR expression in the diagnostic biopsy was high (TIS > 6) in, respectively, 100%, 100%,
33%, and 46% of cases. CEA and EpCAM expressions were significantly higher in the diagnostic
biopsy compared with the corresponding tumor bed (p < 0.01). CEA, EpCAM, αvβ6, and uPAR
expressions were low (TIS < 6) in the tumor bed in, respectively, 93%, 95%, 85%, and 62.5% of cases.
Immunohistochemical evaluation shows that CEA and EpCAM could be suitable targets for re-
sponse evaluation after neoadjuvant treatment, since expression of these targets in the primary tu-
mor bed is low compared with the diagnostic biopsy and adjacent pre-existent rectal mucosa in
more than 90% of patients with a pCR.
Keywords: CEA; EpCAM; αvβ6; uPAR; fluorescence imaging; complete response; rectal cancer; tu-
mor targeted; response evaluation; preoperative chemo and radiotherapy
1. Introduction
Curative-intent treatment of locally advanced rectal cancer consists of neoadjuvant
chemoradiation and surgical resection by total mesorectal excision [1,2]. Fifteen to twenty
Citation: Linders, D.; Deken, M; van
der Valk, M.; Tummers, W.;
Bhairosingh, S.; Schaap, D.; van
Lijnschoten, G.; Zonoobi, E.; van de
Velde, C.; Vahrmeijer, A.; et al.; CEA,
EpCAM, αvβ6 and uPAR Expression
in Rectal Cancer Patients with a Patho-
logical Complete Response after Neo-
adjuvant Therapy. Diagnostics 2021, 11
,
516. https://doi.org/10.3390/diagnos-
tics11030516
Academic Editor: Takuji Tanaka
Received: 23 February 2021
Accepted: 11 March 2021
Published: 14 March 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and insti-
tutional affiliations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(http://creativecommons.org/licenses
/by/4.0/).
Diagnostics 2021, 11, 516 2 of 16
percent of rectal cancer patients achieve a pathological complete response (pCR) after ne-
oadjuvant (chemo)radiotherapy [3]. In these patients, an organ-preserving strategy of
watch and wait (W&W) could have been applied to avoid major surgery and its potential
complications [4–9]. However, correct preoperative identification of patients with a pCR
after neoadjuvant therapy by conventional imaging techniques including magnetic reso-
nance imaging (MRI) and endoscopy is challenging [10]. A local regrowth rate of up to
38% indicates that a clinical complete response (cCR) does not always correspond to a
pCR [7]. In those cases, understaging of residual tumor occurred [4,5,7,9]. On the other
hand, MRI is known to overstage disease due to the difficulty of distinguishing fibrosis
and mucin lakes from residual tumor, leading to unnecessary surgery in about 15% of
patients who turned out to have a pCR in the resection specimen [10,11]. Therefore, en-
hanced diagnostic imaging tools are needed to identify a pCR more accurately and hence
select the right patients for a W&W strategy.
Tumor-targeted near-infrared (NIR) fluorescence imaging is a promising technique
that combines the administration of targeted fluorescence contrast agents with the use of
NIR fluorescence light (700–900 nm). It allows for real-time optical imaging by selectively
highlighting cells that express certain molecular targets. Tumor-targeted fluorescence im-
aging during endoscopy could help to identify patients with a pCR, thereby minimizing
under- and overtreatment of rectal cancer patients. Accurate response evaluation using
fluorescence endoscopy requires a target with tumor-specific expression. Preferably, the
protein of choice has (1) an exclusive upregulation by tumor or tumor-related stromal cells
compared with surrounding pre-existent mucosa, (2) expression that is not influenced by
neoadjuvant therapy, and (3) no expression in the tumor bed if there is no residual tumor.
Various cancer-associated cell membrane proteins are currently under extensive investi-
gation for tumor-targeted fluorescence imaging [12,13]. Promising targets in rectal cancer
include carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5, from
here on to be referred to as CEA), epithelial cell adhesion molecule (EpCAM), αvβ6 integ-
rin, and urokinase-type plasminogen activator receptor (uPAR) [14–17].
The cell membrane-bound glycoprotein CEA is involved in the regulation of adhe-
sion, growth, and differentiation of epithelial cells, and is overexpressed on tumor cells of
various origins, including colorectal cancer [13]. CEA has already been used as a target
for fluorescence contrast agents in preclinical studies and recent clinical phase I/II trials
[15,18,19]. CEA-targeted NIR imaging during colorectal cancer surgery provides an en-
hanced macroscopic differentiation between tumor and normal tissue. On the microscopic
level, the pattern of the fluorescence signal in colon cancer tissue is consistent with CEA
expression as measured using immunohistochemistry [15]. EpCAM is a transmembrane
glycoprotein that plays a role in cell-to-cell interactions and adhesions [20]. The expression
of EpCAM is highly upregulated in nearly all epithelial malignancies, such as colorectal
adenocarcinoma [21–23]. Preclinical studies have demonstrated the feasibility of an Ep-
CAM-specific fluorescence contrast agent for tumor visualization [14,24,25]. Integrin αvβ6
is overexpressed by malignant epithelial cells and activated endothelial cells and mediates
adhesion to the basement membrane [26]. Integrin αvβ6 is overexpressed in colorectal
adenocarcinoma and its upregulation is associated with increased metastatic potential
and reduced survival [27]. In a preclinical study, an αvβ6-specific fluorescence contrast
agent was developed and validated for imaging of pancreatic ductal adenocarcinoma [17].
uPAR, the cell membrane-bound receptor of urokinase-type plasminogen activator, is in-
volved in tissue remodeling, cell signaling, and proliferation, and is overexpressed in most
colorectal adenocarcinomas, largely due to upregulation by tumor-associated stromal
cells [28–30]. Preclinical research showed the potential use of different uPAR-targeted flu-
orescence contrast agents in NIR fluorescence tumor imaging [16,31,32]. A phase I clinical
trial has demonstrated the safe use and feasibility of uPAR-targeted positron emission
tomography in different cancer types [33].
Expression of CEA, EpCAM, αvβ6, and uPAR is known to be upregulated by the
majority of rectal cancer (associated) cells [20,26,29,34]. Previous immunohistochemical
Diagnostics 2021, 11, 516 3 of 16
data demonstrated that CEA and EpCAM expression in rectal cancer cells and adjacent
pre-existent mucosa does not change after neoadjuvant therapy. It showed that these pro-
teins are still overexpressed in patients with a partial or no response after neoadjuvant
therapy [35]. These findings suggest CEA and EpCAM are suitable targets for accurate
response evaluation after neoadjuvant therapy using fluorescence endoscopy. The current
study is a continuation of this research to further evaluate which of the rectal cancer-as-
sociated membrane proteins CEA, EpCAM, αvβ6, and uPAR is the most useful indicator
of absence of residual vital cancer cells after neoadjuvant therapy. To this end, we ana-
lyzed protein expression by immunohistochemistry in resected primary tumor beds and
corresponding diagnostic biopsies of rectal cancer patients with a pathological complete
response after neoadjuvant therapy and compared both. The protein expression levels in
the tumor bed were also compared to adjacent pre-existent rectal mucosa.
2. Material and Methods
2.1. Human Rectal Cancer Tissue Samples
Available formalin-fixed paraffin-embedded (FFPE) tissue blocks of diagnostic bi-
opsy and resection specimens of the primary tumor from 56 patients who underwent sur-
gical resection of locally advanced rectal cancer between 2008 and 2015 and had achieved
a pCR after neoadjuvant chemo- and/or radiation therapy were collected from the Labor-
atory of Pathology (Stichting Pathology and Medical Microbiology), associated to the
Catharina Hospital Eindhoven, The Netherlands (Figure 1).
Figure 1. Overview of included tissues. Notes: Described are the number of included tissues. Fifty-
six patients were selected. Twelve patients were excluded. From fifteen of the remaining 44 pa-
tients, FFPE tissue blocks of both diagnostic biopsy and resection specimen of the corresponding
Diagnostics 2021, 11, 516 4 of 16
primary tumor were available. From 29 patients, only tissue blocks of the resection specimen were
available. Abbreviations: RC, rectal cancer; pCR, pathological complete response; FFPE, formalin-
fixed paraffin-embedded.
Twelve of fifty-six patients were excluded because no tissue was available (n = 3),
there was no pathological complete response upon revision of the pathology (n = 5), or
tissue was of poor quality (n = 4). From 15 of the remaining 44 patients, tissue blocks of
both the diagnostic biopsy and the resection specimen of the corresponding primary tu-
mor were available. From the remaining 29 patients, only tissue blocks of the resection
specimen were available.
Medical records and pathology reports were retrospectively reviewed. A representa-
tive FFPE tissue block of the primary resection specimen of each patient, and the diagnos-
tic biopsy when available, was chosen by a board-certified gastrointestinal pathologist
(AFS). Tumor bed was selected as the area in the specimen with reactive changes as a
result of the neoadjuvant therapy, mainly ulceration, acellular mucinous pools, and fibro-
sis. Ideally, the selected slides were representative of the tumor bed in relation to adjacent
pre-existent rectal mucosa.
All patients had given informed consent for retrospective use of their archived tis-
sues. All samples were nonidentifiable and used in accordance with the code for proper
secondary use of human tissue as prescribed by the Dutch Federation of Medical Scientific
Societies and conformed to a protocol that had been reviewed and approved by the insti-
tutional review board of the Leiden University Medical Center (LUMC). This study was
conducted in accordance with the Declaration of Helsinki.
2.2. Immunohistochemistry (IHC)
After cutting the FFPE blocks into 4 μm sections, these were mounted on adhesive
slides (Starfrost, Waldemar Knittel Glasbearbeitungs GmbH, Braunschweig, Germany),
deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. Sub-
sequently, slides were rinsed with demineralized water, and endogenous peroxidase was
blocked with 0.3% hydrogen peroxidase (Merck Millipore, Darmstadt, Germany) for 20
min at room temperature. Slides were rinsed with demineralized water, and antigen re-
trieval was performed in the PT Link (Agilent, Santa Clara, CA, USA), using either Target
Retrieval Solution pH 6.0 (Agilent, Santa Clara, CA, USA) at 95 °C or 0.4% pepsin at 37 °C
for 10 min (Table A1). After rinsing with phosphate-buffered saline (PBS, pH 7.4), slides
were stained overnight at room temperature with primary antibodies, diluted in 1% bo-
vine serum albumin/PBS, against CEACAM5 (clone CI-P83-1, SantaCruz Biotechnology,
200 μg/mL, dilution 1:1000), EpCAM (clone MOC31, Acris Antibodies, 0.64 mg/mL, dilu-
tion 1:10000), αvβ6 (clone 6.2A1, Biogen Idec, 0.5 μg/mL, not diluted), and uPAR (clone
ATN-617, Monopar Therapeutics Inc., 0.48 mg/mL, dilution 1:400). After three PBS wash-
ing steps, the slides were incubated with a horseradish peroxidase (HRP)-labelled second-
ary antibody against mouse (EnVision, Agilent, Santa Clara, USA) for 30 min at room
temperature. Binding of the antibody was visualized using 3,3’-diaminobenzidine (Ag-
ilent, Santa Clara, USA). All slides were counterstained with hematoxylin for 10–15 s, de-
hydrated at 37 °C, and mounted with pertex. Slides were scanned using the Philips Intel-
liSite Pathology Solution (Philips Electronics, Eindhoven, The Netherlands).
2.3. Scoring Method
All diagnostic biopsies and primary resection specimens from patients with rectal
cancer who had achieved a pCR after neoadjuvant therapy were scored for expression of
CEA, EpCAM, αvβ6, and uPAR. Not all tissues have been scored for all four markers due
to incidental poor slide quality. The total immunostaining score (TIS) was calculated by
multiplying the proportion score (PS) by the intensity score (IS) [22]. The PS represented
the percentage of positively stained cells and ranged between 0 and 4 (0 = none; 1 < 10%;
2 = 10%–50%; 3 = 51%–80%; 4 > 80%). The IS represented the intensity of the stained cells
Diagnostics 2021, 11, 516 5 of 16
and could range between 0 and 3 (0 = no staining; 1 = weak; 2 = moderate; 3 = strong).
Subgroups were defined based on the calculated TIS: 0, no expression; 1–5, weak expres-
sion; 6–8, moderate expression; 9–12, intense expression. For dichotomization of sub-
groups, TIS 0–5 (no to weak expression) was regarded as low expression, TIS 6–12 (mod-
erate-to-intense expression) as high expression. IHC staining scoring was performed by
two independent observers (AFS and WT). The observers were blinded for the origin of
the tissues. The weighted Kappa was 0.90. In case of disagreement, the mean of the two
observed total immunostaining scores, rounded upwards, was used.
2.4. Statistical Analysis
Statistical analyses were performed using SPSS version 23.0 software (SPSS, IBM Cor-
poration, NY, USA) and GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA).
For each patient, differences in expression levels between tumor tissue in the diagnostic
biopsy and tumor bed in the corresponding resection specimen were calculated using the
Wilcoxon signed-rank test. This test was also used to calculate differences in expression
levels between tumor bed and adjacent pre-existent rectal mucosa in the resection speci-
men per patient. A Kruskal–Wallis test was used to determine the differences in tumor
bed-to-pre-existent rectal mucosa protein expression ratio between all four biomarkers. In
all tests, results were considered statistically significant at the level of p < 0.05.
3. Results
Patient and tumor characteristics are summarized in Table 1. Median time to surgery
was 11 weeks. For 36 patients, the neoadjuvant therapy consisted of radiotherapy with a
total dose of 50 Gray in 25 fractions in combination with capecitabine. Four patients re-
ceived short-course radiotherapy with a total dose of 25 Gray in five fractions, followed
by an extended waiting period (median time to surgery of these four patients was 18
weeks). Another four patients participated in a clinical trial (RAPIDO trial, NCT01558921)
and received radiotherapy with a total dose of 25 Gray in five fractions, followed by a
median of six courses capecitabine and oxaliplatin, with or without bevacizumab.
Table 1. Patient and tumor characteristics.
Biopsy and Resec-
tion (n = 15)
Only Resec-
tion (n = 29)
All Pa-
tients (n =
44)
Age at surgery, median years (range) 71 (48–88) 61 (28–78) 66 (28–88)
Gender
Male 10 13 23
Female 5 16 21
Tumor type
Adenocarcinoma 14 24 38
Tubulovillous adenoma with dysplasia 1 1 2
Unknown 0 4 4
Tumor size, median cm (range) 5 (3–12) 5 (2–15) 5 (2–15)
Type of neoadjuvant therapy
25 × 2 Gy + capecitabine 12 24 36
5 × 5 Gy 2 2 4
5 × 5 Gy + capecitabine, oxaliplatin, bevacizumab 0 2 2
5 × 5 Gy + capecitabine, oxaliplatin 1 1 2
Type of surgery
Low anterior resection 7 15 22
Abdominoperineal resection 6 12 18
Transanal endoscopic microsurgery 1 2 3
Diagnostics 2021, 11, 516 6 of 16
Hartmann's procedure 1 0 1
Clinical stage
Tumor stage, n
cTx 5 7 12
cT2 2 1 3
cT3 7 9 16
cT4 1 12 13
Nodal stage, n
cNx 5 7 12
cN0 2 5 7
cN1 5 3 8
cN2 3 14 17
Metastatic stage, n
cMx 6 7 13
cM0 9 20 29
cM1 0 2 2
Pathologic stage
Tumor stage, n
pT0 15 29 44
Nodal stage, n
pN0 14 29 43
pN1 1 0 1
Metastatic stage, n
pM0 15 27 42
pM1 0 2 2
Time between neoadjuvant therapy and surgery, me-
dian weeks (range) 11 (6–57)
Abbreviations: Gy, Gray; n, number of patients; c, clinical; p, pathological.
3.1. CEA, EpCAM, αvβ6, and uPAR Expression
Figure 2 shows representative CEA, EpCAM, αvβ6, and uPAR stained tissue slides
of a diagnostic biopsy and corresponding primary resection specimen derived from one
patient. On tumor cells (Figure 2A), CEA expression was highest on the apical membrane.
EpCAM and αvβ6 showed a membranous, circumferential staining pattern. In the re-
sected primary tumor bed (Figure 2B), target expression was predominantly absent, due
to the lack of epithelial cells. In some patients, CEA, EpCAM, and αvβ6 showed nonspe-
cific staining in fibrosis, necrotic areas, and acellular mucin lakes. uPAR was expressed by
(cancer-associated) fibroblasts in the diagnostic biopsies and resected primary tumor
beds. CEA, αvβ6, and in particular EpCAM were to some extent positive in pre-existent
rectal mucosa.
Diagnostics 2021, 11, 516 7 of 16
Figure 2. Representative images of CEA, EpCAM, αvβ6, and uPAR expression in a diagnostic bi-
opsy and corresponding primary resection specimen derived from one patient with rectal cancer
with a pCR after neoadjuvant therapy. (A) Diagnostic biopsy (magnification 5×, black bar = 200
μm). (B) Corresponding primary resection specimen (magnification 1×, black bar = 1 mm). The
arrow indicates the location of the tumor bed. The zoom contains a magnification of the transition
area of tumor bed to adjacent pre-existent rectal mucosa (magnification 20×). Abbreviations: HE,
hematoxylin–eosin; CEA, carcinoembryonic antigen; EpCAM, epithelial cell adhesion molecule;
αvβ6, integrin αvβ6; uPAR, urokinase-type plasminogen activator receptor; pCR, pathological
complete response.
Total immunostaining scores (TIS) of all four targets in all scored tissues are summa-
rized in Table 2. Figure 3 shows the percentages of diagnostic biopsies and resection spec-
imens with a high (TIS 6–12) and low (TIS 0–5) expression of each marker. High TIS was
seen in 100% of the diagnostic biopsies for CEA, 100% for EpCAM, 33% for αvβ6, and 46%
for uPAR. Conversely, there was low or no expression of CEA in 93% of the resection
specimens, of EpCAM in 95%, of αvβ6 in 85%, and of uPAR in 62.5%. In adjacent pre-
existent rectal mucosa, defined as healthy rectal mucosa exposed to neoadjuvant chemo-
radiation therapy, targets had a high expression in 80% (CEA), 95% (EpCAM), 52% (αvβ6),
Diagnostics 2021, 11, 516 8 of 16
and 3% (uPAR). In Table A2, the number of patients for which all corresponding tissues
have been scored are summarized per marker.
Table 2. Total immunostaining scores (TIS) of all stained tissues.
Total Immunostaining Score (TIS) on Biopsy, pCR Tumor Bed, and Pre-existent Rectal Mucosa
n (%)
No Expres-
sion (TIS =
0)
Weak Expres-
sion (TIS =
1–5)
Moderate Ex-
pression (TIS =
6–8)
Intense Expression
(TIS = 9–12)
Total (n) Tis-
sues
CEA
Biopsy 0 (0%) 0 (0%) 0 (0%) 15 (100%) 15
Tumor bed 30 (75%) 7 (18%) 0 (0%) 3 (7%) 40
Pre-existent
rectal mu-
cosa
2 (5%) 6 (15%) 10 (25%) 22 (55%) 40
EpCAM
Biopsy 0 (0%) 0 (0%) 1 (7%) 14 (93%) 15
Tumor bed 33 (85%) 4 (10%) 0 (0%) 2 (5%) 39
Pre-existent
rectal mu-
cosa
1 (2.5%) 1 (2.5%) 0 (0%) 39 (95%) 41
αvβ6
Biopsy 4 (27%) 6 (40%) 4 (27%) 1 (6%) 15
Tumor bed 28 (70%) 6 (15%) 2 (5%) 4 (10%) 40
Pre-existent
rectal mu-
cosa
1 (3%) 19 (45%) 11 (26%) 11 (26%) 42
uPAR
Biopsy 0 (0%) 7 (54%) 3 (23%) 3 (23%) 13
Tumor bed 5 (12.5%) 20 (50%) 8 (20%) 7 (17.5%) 40
Pre-existent
rectal mu-
cosa
10 (24%) 30 (73%) 0 (0%) 1 (3%) 41
Notes: Shown are the number (and percentage) of all stained tissues with a certain total
immunostaining score (TIS), per target and per tissue type. Subgroups were defined based on the
TIS: 0, no expression; 1–5, weak expression; 6–8, moderate expression; 9–12, intense expression.
Tissue types were diagnostic biopsy, pCR tumor bed in the resection specimen, and adjacent pre-
existent rectal mucosa in the resection specimen. Abbreviations: TIS, total immunostaining score;
CEA, carcinoembryonic antigen; EpCAM, epithelial cell adhesion molecule; αvβ6, integrin αvβ6;
uPAR, urokinase-type plasminogen activator receptor.
Diagnostics 2021, 11, 516 9 of 16
Figure 3. High and low expression in the diagnostic biopsy and resected pCR tumor bed. Notes:
Shown are the percentages of diagnostic biopsies and resected pCR tumor beds with a high (TIS 6–
12) and low (TIS 0–5) expression of each marker. The total number of scored biopsies and tumor
beds was respectively 15 and 40 for CEA, 15 and 39 for EpCAM, 15 and 40 for αvβ6, and 13 and 40
for uPAR. Abbreviations: TIS, total immunostaining score; CEA, carcinoembryonic antigen; Ep-
CAM, epithelial cell adhesion molecule; αvβ6, integrin αvβ6; uPAR, urokinase-type plasminogen
activator receptor; pCR, pathologic complete response.
3.2. Comparison of Protein Expression in Tumor Tissue in Diagnostic Biopsy and Corresponding
Tumor Bed after Neoadjuvant Therapy
From fifteen patients, both the diagnostic biopsy and the corresponding resection
specimen were available. Figure 4 shows the TIS of CEA, EpCAM, αvβ6, and uPAR in the
diagnostic biopsy compared to the tumor bed in the corresponding resection specimen for
each patient. CEA and EpCAM expression were significantly higher in the biopsies than
in the tumor bed of the corresponding resection specimens (p < 0.01 for CEA and EpCAM).
αvβ6 and uPAR expression was not significantly higher in the biopsies (respectively p =
0.082 and p = 0.246). Median TIS in the diagnostic biopsies was respectively 12 (range 11–
12) for CEA, 12 (range 6–12) for EpCAM, 4 (range 0–12) for αvβ6, and 4 (range 3–11) for
uPAR. Median TIS in the tumor bed was respectively 0 (range 0–12) for CEA, 0 (range 0–
12) for EpCAM, 6 (range 0–12) for αvβ6, and 1 (range 0–12) for uPAR.
Figure 4. Target expression in tumor tissue in the diagnostic biopsy compared to the pCR tumor
bed in the corresponding resection specimen. Notes: Shown is the target expression (as TIS) in the
diagnostic biopsy compared to the pCR tumor bed in the corresponding resection specimen, per
patient and per target. Every line represents one or more patients. The number of patients with a
certain expression score (TIS) in the biopsy and tumor bed is indicated next to the corresponding
lines. A horizontal line indicates the same level of expression between biopsy and tumor bed. A
descending or ascending line indicates respectively a higher or lower expression in the biopsy
compared with the tumor bed. The asterisks (*) indicate a significantly higher expression in the
diagnostic biopsy compared with the corresponding pCR tumor bed in the resection specimen.
Diagnostics 2021, 11, 516 10 of 16
Abbreviations: TIS, total immunostaining score; CEA, carcinoembryonic antigen; EpCAM, epithe-
lial cell adhesion molecule; αvβ6, integrin αvβ6; uPAR, urokinase-type plasminogen activator
receptor; pCR, pathological complete response.
3.3. Comparison of Protein Expression in Tumor Bed and Adjacent Pre-existent Rectal Mucosa
Figure 5 shows the TIS of CEA, EpCAM, αvβ6, and uPAR for each patient in the
tumor bed compared to adjacent pre-existent rectal mucosa in the resection specimen.
CEA, EpCAM, and αvβ6 expression was significantly lower in the tumor bed compared
with adjacent pre-existent mucosa (p < 0.01 for CEA, EpCAM, and αvβ6). uPAR expres-
sion was significantly higher in the tumor bed compared with adjacent pre-existent mu-
cosa (p < 0.05). Median TIS in the tumor bed and adjacent pre-existent mucosa was respec-
tively 0 (range 0–12) and 9 (range 0–12) for CEA, 0 (range 0–12) and 12 (range 0–12) for
EpCAM, 0 (range 0–12) and 6 (range 0–12) for αvβ6, and 3.5 (range 0–12) and 1 (range 0–
12) for uPAR. A significant difference in the tumor bed-to-pre-existent mucosa expression
ratio between biomarkers was found, with the lowest rank for EpCAM, followed by CEA,
αvβ6, and uPAR (p < 0.01).
Figure 5. Target expression in the tumor bed compared to adjacent pre-existent rectal mucosa in
resection specimens of pathological complete responders. Notes: Shown is the target expression
(as TIS) in the pCR tumor bed compared to adjacent pre-existent rectal mucosa in the resection
specimen, per patient and per target. Every line represents one or more patients. A horizontal line
indicates the same level of expression in tumor bed and adjacent pre-existent mucosa. A descend-
ing or ascending line indicates respectively a higher or lower expression in the tumor bed com-
pared with the adjacent pre-existent mucosa. The arrows indicate the number of tumor beds that
show a decreased or enhanced expression compared with adjacent pre-existent mucosa; = refers to
equal expression scores. The asterisks (*) indicate a significantly lower expression in the tumor bed
compared with adjacent pre-existent rectal mucosa. Abbreviations: TIS, total immunostaining
score; CEA, carcinoembryonic antigen; EpCAM, epithelial cell adhesion molecule; αvβ6, integrin
Diagnostics 2021, 11, 516 11 of 16
αvβ6; uPAR, urokinase-type plasminogen activator receptor; pCR, pathological complete re-
sponse.
4. Discussion
Response evaluation after neoadjuvant therapy using targeted NIR fluorescence en-
doscopy has the potential for more accurate selection of rectal cancer patients for a W&W
strategy, avoiding both unnecessary surgery and local regrowth. The present study inves-
tigated the expression of four promising biomarkers in the diagnostic biopsy, correspond-
ing resected tumor bed, and adjacent pre-existent mucosa of rectal cancer patients with a
pCR after neoadjuvant treatment. Our study demonstrates that the targets CEA and Ep-
CAM are absent or have low expression in the tumor bed of nearly all rectal cancer pa-
tients with a pCR. Our data also confirm that CEA and EpCAM expression is significantly
higher in tumor tissue of the diagnostic biopsy compared with the corresponding resected
pCR tumor bed. Furthermore, we show that CEA, EpCAM, and αvβ6 expression is signif-
icantly lower in the pCR tumor bed compared with adjacent pre-existent mucosa. In con-
trast, uPAR is highly expressed in the pCR tumor bed of a significant number of patients
and is not upregulated in the diagnostic biopsies compared with the corresponding pCR
tumor bed. In addition, uPAR expression is not significantly lower in the pCR tumor bed
compared with adjacent pre-existent mucosa.
Even though the membrane proteins αvβ6 and uPAR are associated with rectal can-
cer, their relatively high expression in the tumor bed of patients with a pCR makes these
biomarkers less suitable as a target for response evaluation. A possible explanation for
this high expression in the tumor bed after neoadjuvant therapy is that αvβ6 and uPAR
both play an important role in tissue remodeling and wound healing [28,36].
Our findings indicate that the cell adhesion molecules CEA and EpCAM could be
used as targets for response evaluation using NIR fluorescence endoscopy. A prerequisite
for this technique is a tumor target with a significant different expression between tumor
and healthy tissue, whose expression is not influenced by neoadjuvant therapy and is ab-
sent when there is no residual tumor. Previous research has shown that CEA and EpCAM
expression is upregulated in rectal cancer cells compared with adjacent pre-existent rectal
mucosa [20,34]. A previous study by our group has demonstrated that CEA and EpCAM
are still overexpressed in patients with a partial or no response after neoadjuvant therapy
[20,34,35]. In that study, CEA and EpCAM expression after neoadjuvant therapy was high
(TIS > 6) in respectively 93% and 100% of the partial- and nonresponders [35]. The current
study demonstrates that CEA and EpCAM have an absent or low expression (TIS < 6) in
the tumor bed of nearly all patients with a pCR after neoadjuvant therapy. Therefore, both
CEA and EpCAM could be suitable targets for response evaluation using fluorescence
endoscopy. Both the current and a previous study by our group show that the median
CEA expression measured by immunohistochemistry in pre-existent rectal mucosa is
lower compared with EpCAM [35]. This would, in theory, favor CEA above EpCAM as a
target. However, both studies have been carried out in small groups of patients and might
be underpowered to draw this conclusion.
Although these results are promising, the present study has some limitations. The
main drawbacks are the small number of patients from whom both the biopsy and resec-
tion specimen were available and the use of semiquantitative IHC to measure protein ex-
pression. Validated antibodies and a previously evaluated scoring method were used to
minimize variability of the performed IHC [22]. Still, differences in staining intensities
between immunohistochemical studies could be observed due to the use of different an-
tibody clones against the same target. Moreover, the degree of correlation between CEA,
EpCAM, αvβ6, and uPAR expression measured by IHC in FFPE material and in vivo ex-
pression as measured by fluorescence signal intensity has yet to be elucidated in clinical
trials. However, clinical trials investigating c-Met-targeted fluorescence endoscopy have
demonstrated an excellent correlation between the fluorescence signal intensity measured
in vivo and the expression of the target measured with IHC [37,38], indicating the clinical
Diagnostics 2021, 11, 516 12 of 16
relevance of IHC expression data of potential targets. Although the expression of CEA
and EpCAM has already been demonstrated in patients without a pCR after neoadjuvant
therapy [35], future studies should include these patients as a control group to directly
compare marker expression between patients with and without a pCR.
In the treatment of rectal cancer, the application of targeted fluorescence contrast
agents in combination with an endoscope equipped with NIR fluorescence light has the
potential to correctly identify patients with a complete response after neoadjuvant ther-
apy. This is illustrated by the fact that fluorescence endoscopy with a topical or intrave-
nously applied contrast agent enables visualization of neoplastic lesions that are visible
with conventional endoscopy, as well as additional neoplastic lesions that are missed by
conventional endoscopy alone [39–42]. An ongoing clinical trial investigates the use of
vascular endothelial growth factor (VEGF)-targeted fluorescence endoscopy for response
evaluation in rectal cancer patients following neoadjuvant therapy (ClinicalTrials.gov
Identifier: NCT01972373). A previous pilot study indicated that, like CEA and EpCAM,
expression of the rectal cancer-associated membrane protein VEGF is absent in the tumor
bed of patients with a pCR. It also demonstrated that VEGF-targeted fluorescence endos-
copy has a higher sensitivity to detect residual tumor compared with MRI combined with
conventional endoscopy [43]. This improved sensitivity demonstrates the possible value
of fluorescence endoscopy for a better identification of residual tumor, decreasing under-
staging and local regrowth. Furthermore, the expression of CEA and EpCAM, in contrast
to VEGF expression, is not influenced by neoadjuvant therapy, possibly making these
markers even more suitable for response evaluation [44].
Possible limitations of fluorescence endoscopy for response evaluation could be
false-positive results due to nonspecific positivity in, for instance, mucin lakes and fibrosis
or false-negatives due to complete submucosal localization of residual tumor or isolated
metastatic lymph nodes. However, a totally submucosal residual or recurrent tumor after
neoadjuvant therapy, with no tumor reaching the mucosa, is rare, occurring in only 1% of
cases [45]. Furthermore, NIR light penetrates tissues by up to ~8 mm, probably enabling
detection of the vast majority of submucosal-located residual tumors [43,46].
The use of fluorescence-labeled contrast agents targeting CEA and EpCAM has been
shown to be safe and feasible for tumor imaging in humans and is the subject of extensive
investigation. In colorectal and pancreatic cancer surgery, the intraoperative use of a flu-
orochrome-labeled anti-CEA monoclonal antibody, SGM-101, provides an enhanced dif-
ferentiation between tumor and normal tissue [19]. It seems to facilitate the detection of
additional neoplastic lesions, changing the final treatment strategy in 35% of colorectal
patients [15]. Recently, two phase III randomized controlled trials were initiated, further
investigating the intraoperative use of SGM-101 in colorectal cancer surgery (ClinicalTri-
als.gov Identifier: NCT03659448 and Netherlands Trial Register, ID NL7653). An ongoing
phase I clinical dose escalation and optimization trial explores the intraoperative use of
an EpCAM-specific fluorescence agent in esophageal, gastric, and rectosigmoid cancer
(Netherlands Trial Register, ID NL7363). Since these fluorescence contrast agents against
CEA and EpCAM have been introduced in the clinic and have proven to be safe and ef-
fective, rapid implementation of these agents in NIR fluorescence endoscopy is possible.
However, future trials regarding the clinical application of these markers in NIR fluores-
cence endoscopy require development of additional, target-specific fluorescence endo-
scopes and an optimal contrast agent dose. Therefore, future research should focus on
dose escalation and the added value of this technique in correctly classifying rectal cancer
patients with a complete response after neoadjuvant therapy.
5. Conclusions
CEA and EpCAM seem to be suitable targets for response evaluation in rectal cancer
using NIR fluorescence endoscopy, since immunohistochemical evaluation shows that ex-
pression of these markers in the tumor bed is low compared with the diagnostic biopsy
and adjacent pre-existent rectal mucosa in nearly all (>90%) patients with a pCR.
Diagnostics 2021, 11, 516 13 of 16
Author Contributions: Conceptualization, M.V., W.T., and D.H.; methodology, D.L., M.D., C.S.,
M.V., W.T., A.V., and D.H.; investigation, W.T., S.B., D.S., E.Z., P.K., G.L., and A.F.S.; data curation,
W.T., S.B., D.S., E.Z., P.K., G.L., and A.F.S; validation, W.T., S.B., D.S., E.Z., P.K., G.L., and A.F.S;
writing—original draft preparation, D.L, M.D., C.S., and D.H.; writing—review and editing, D.L,
M.D., M.V., W.T., A.F.S., A.V., C.S., and D.H.; supervision, A.V., C.V., and D.H.; funding acquisition,
D.H. All authors have read and agreed to the published version of the manuscript.
Funding: Funding for this study was obtained by the Bas Mulder Award granted to DH by the Alpe
d’HuZes Foundation and Dutch Cancer Society (grant UL2015-7966), and by the Dutch Cancer So-
ciety (grant UL2015-8089). Funding was also obtained from the European Commission under two
Marie Skłodowska-Curie Action awards: H2020-MSCA-RISE-2019 (Project number: 872860 -
PRISAR2) and H2020-MSCA-ITN-2019 (Project number: 857894 - CAST).
Institutional Review Board Statement: All patients had given informed consent for retrospective
use of their archived tissues. All samples were nonidentifiable and used in accordance with the code
for proper secondary use of human tissue as prescribed by the Dutch Federation of Medical Scien-
tific Societies and conformed to a protocol that had been reviewed and approved by the institutional
review board of the LUMC. This study was conducted in accordance with the Declaration of Hel-
sinki.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Anti-uPAR was kindly provided by A. P. Mazar.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Primary antibodies used.
Antibody Company Clone
Stock
Concentration
Dilutio
n Antigen Retrieval
Anti-
CEACAM5
SantaCruz
Biotechnology
Cl-P83-
1 0.2 μg/mL 1/1000
Dako PT Link Target Retrieval
Solution, pH 6.0
Anti-EpCAM Acris Antibodies MOC31 0.64 mg/mL 1/10000 Dako PT Link Target Retrieval
Solution, pH 6.0
Anti-αvβ6 Biogen Idec 6.2A1 0.5 μg/mL - 0.4% Pepsin
Anti-uPAR Monopar
Therapeutics Inc
ATN61
7 0.48 mg/mL 1/400 Dako PT Link Target Retrieval
Solution, pH 6.0
Abbreviations: CEACAM5, carcinoembryonic antigen-related cell adhesion molecule 5; EpCAM,
epithelial cell adhesion molecule; αvβ6, integrin αvβ6; uPAR, urokinase-type plasminogen activator
receptor.
Table A2. Available stained tissue “pairs” per marker.
Biopsy and Resection (n) Tumor Bed and Normal Epithelium (n)
CEA 14 37
EpCAM 14 37
αvβ6 14 38
uPAR 12 38
Notes: Not all tissues have been scored for all four markers due to incidental poor slide quality.
Therefore, not all fifteen biopsies had a corresponding resection specimen for every marker, and not
all 44 resection specimens had both tumor bed and pre-existent rectal mucosa scored. Listed are the
number of patients per marker of which both a biopsy and primary resection specimen or both tumor
bed and pre-existent mucosa in the resection specimen were scored. Abbreviations: CEA,
carcinoembryonic antigen; EpCAM, epithelial cell adhesion molecule; αvβ6, integrin αvβ6; uPAR,
urokinase-type plasminogen activator receptor; n, number of patients.
Diagnostics 2021, 11, 516 14 of 16
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