Introduction
Neoadjuvant chemoradiotherapy (nCRT) is recommended
as the standard treatment for locally advanced
rectal cancer.[] Pathological complete response (pCR)
rates with chemoradiotherapy (CRT) alone range between
13% and 17%.[,] When total neoadjuvant therapy
(TNT) is utilized, the pCR rate increases to approximately 27%.[-] Recent research supports the efficacy
of TNT, demonstrating a significant increase in pCR
rates.[,] Significant numbers of patients are unable to
achieve pCR, highlighting the importance of intensifying
treatments. Therefore, it is essential to identify which
groups are unlikely to respond. Even though a great deal
of research has been conducted on molecular markers,
clinical tumor characteristics, and biological factors to predict how a tumor will respond to chemoradiotherapy
before surgery in patients with rectal cancer, no definitive
conclusions have been reached.[] Therefore, it
is essential to identify the group unlikely to respond.
Maspin, also known as a mammary serine protease
inhibitor, is a member of the serpin family that targets
proteinases and exhibits characteristics of a tumor
suppressor protein. This protein has a variety of functional
capabilities, including inhibition of invasion, enhancement
of apoptosis, and modulation of urokinase
plasminogen activator (uPA) and tissue plasminogen
activator activities.[-] Maspin, also known as
SERPINB5, is a type II tumor suppressor that is mostly
found in normal breast myoepithelial cells as well as in
the prostate, epidermis, lung, and corneal stromal cells.
[] Remarkably, it has been observed that maspin
expression decreases or even disappears in primary
breast cancer cell lines and invasive breast carcinoma.
[] Nonetheless, some studies have proposed an apparently
contradictory function for maspin. In advanced
stages of diseases such as inflammatory bowel disease
and gastric and colorectal malignancies, elevated
maspin expression has been detected, suggesting that
the tissue of origin of the tumor significantly influences
the role of maspin in cancer progression.[] Notably,
the role of the SERPINB5 gene in the prognosis of rectal
cancer has only been examined once in the existing
literature, with our study being the first to examine
maspin expression in this context.[]
The aim of our study is to evaluate the effects of
maspin expression on survival in patients diagnosed
with locally advanced rectal cancer. Our secondary objective
is to assess the pathologic response rates to nCRT.
Methods
Following approval from the Gazi University Ethics
Committee, a retrospective analysis was performed on a
dataset consisting of 60 patients who had received a histopathological
diagnosis of rectal cancer. These patients
presented to the Medical Oncology Department of Ankara
Gazi University Faculty of Medicine between January
2009 and January 2018. People with adenocarcinoma
of locally advanced rectal cancer who were staged as
T3 or T4 or had metastases in their pelvic lymph nodes
were included in the study. This was confirmed by pelvic
magnetic resonance imaging (MRI) staging.
The inclusion criteria for the study consisted of
patients who were 18 years of age or older and had a
planned neoadjuvant chemoradiotherapy treatment.
The exclusion criteria for this study included several factors: individuals who had been diagnosed with anal
squamous cell carcinoma; those who had undergone
preoperative or postoperative pathology at a facility
outside of the study center; individuals with known immunosuppression;
those who did not have a pelvic MRI;
individuals with distant metastases; those who had previously
received chemotherapy in addition to neoadjuvant
chemoradiotherapy (nCRT); those who received
neoadjuvant short-course radiation therapy; patients
who declined to undergo surgery; and individuals who
opted for a "watch and wait" management approach.
Demographic information, clinical characteristics,
Eastern Clinical Oncology Group performance scores
(ECOG), and pathological data were gathered. In addition,
we documented the specific chemotherapy and
radiotherapy treatment protocols administered to the
patients. Prior to the initiation of chemoradiotherapy
(CRT), baseline assessments were conducted. The assessments
conducted encompassed the quantification
of serum carcinoembryonic antigen (CEA) and carbohydrate
antigen 19-9 (CA 19-9), a comprehensive
analysis of blood composition, and an extensive array
of biochemical tests on the serum. The administration
of a cumulative dose of 45 Gy was carried out over a
span of 5 weeks, consisting of 25 fractions, in accordance
with the established protocol. This treatment
was accompanied by the use of either capecitabine or
5-fluorouracil as the chemotherapeutic agent.
Tissue Sampling
Preoperative tissue specimens from the selected patients
were retrieved from the archive. The pathologist
had no information about the patients. These hematoxylin
and eosin-stained preparations were re-examined
by a single pathologist. Tumor blocks, where tumor
cell concentration was highest and necrosis was minimal,
were identified and selected for further analysis.
Sections of 4 micrometers in thickness were prepared
from the formalin-fixed, paraffin-embedded tissues
and placed onto positively charged slides.
To assess maspin expression, staining was conducted
on the Ventana Benchmark XT device using the
automatic immunohistochemical staining method, employing
the streptavidin-biotin triple indirect immunoperoxidase
technique. The Ultraview Universal DAB
Detection Kit was used in conjunction with the maspin
antibody (polyclonal, Invitrogen, 1/200 dilution). Normal
breast tissue served as the positive control. For expression
prevalence, tissues with staining of 10% and
above were classified as maspin-positive staining (Fig.
1). The Modified Ryan Tumor Regression Grade was employed in the pathological response assessment. Patients
with a tumor regression grade of 0 or 1 were categorized
as good responders, while patients with grades
of 2 or 3 were categorized as poor responders.
Fig. 1. (a, b) Cytoplasmic expression of maspin (×100 and ×200, respectively) (c) Solely nuclear expression of maspin
(×200) (d) Both cytoplasmic and nuclear expression (×200) (Tissues with 10% or more staining in terms of expression
prevalence were considered maspin positive staining).
Statistical Analysis
Statistical analyses were performed using the Statistical
Package for the Social Sciences (SPSS) program.
For categorical data, frequencies were reported, while
for continuous data-based on the distribution-either
means±standard deviations or medians (with
minimum-maximum ranges) were presented. The Kolmogorov-Smirnov test was planned to evaluate the normality
of data distribution. For variables conforming to
a normal distribution, parametric tests (Independent
Sample T-Test) were employed, while non-parametric
tests (Chi-Square, Mann-Whitney U Test) were used
for variables not conforming to normal distribution.
Univariate analyses concerning parameters affecting
survival times were conducted using the Log-Rank
test in Kaplan-Meier analysis. Multivariate analyses
with parameters affecting survival times were performed
via Cox Regression analysis. The threshold for
statistical significance in this study was set at p?0.05.
Results
A total of 60 patients were included in the study. The
demographic and clinical characteristics of the patients
are shown in Table 1. With a median age of 65.6 years
(range: 29-91 years), the cohort consisted of 41 men (68.3%) and 19 women (31.7%). In 24 of these patients
(40%), the tumor was located in the lower rectum
(less than 5 cm from the anal verge). At the time of
diagnosis, 54 (90%) of the patients had a clinical T3
or T4 tumor. Clinical nodal involvement was found in
48 patients (80%). Maspin staining was negative in 37
patients (61.7%) and positive in 23 patients (38.3%).
Cytoplasmic maspin staining was seen in 18 of these
patients (30%), nuclear staining in 2 (3.3%), and both
cytoplasmic and nuclear staining in 3 (5%).
Table 1 Demographic and clinical characteristics of patients
A pathologically complete response was observed
in 10 patients (16.7%). No pathological complete
response was detected in any of the 5 patients with
nuclear maspin positivity (p=0.296). Among the 21
patients with cytoplasmic maspin staining, 2 (9.5%)
had a pathological complete response (p=0.276). It
was determined that 25 of the patients had a good
response according to TRG. The tumor stage (ypT)
and nodal stage (ypN) after neoadjuvant therapy are
presented in Table 2.
Table 2 Pathological outcomes of patients operated after
neoadjuvant chemoradiotherapy
While DFS in maspin-positive patients was 56.9
months (min-max: 38.8-75.0 months) (p=0.485).
In maspin-positive patients, the 1-year, 2-year,
and 3-year disease-free survival (DFS) rates were
found to be 95.6%, 69.4%, and 65%, respectively. In
maspin-negative patients, these rates were 83.8%,
78.4%, and 65%, respectively (Fig. 2). In patients with
poor tumor regression grade, DFS was 46.4 months
(min-max: 13.4?79.3 months) (p=0.013). On univariate
analysis, poor TRG response, high CA-19 level,
and nuclear maspin positivity were found to negatively
impact DFS (Table 3).
Fig. 2. Maspin expression and disease-free survival.
Table 3 Univariate and multivariate analyzes to determine the parameters affecting
disease-free survival
The 3-year overall survival was significantly higher
in the maspin-positive group at 91.3% compared
to 73% in the maspin-negative group (Fig. 3). Median
overall survival (mOS) was not reached in the entire
patient group. There was a significant association
between clinical T stage and overall survival (OS)
(p=0.028). For overall survival, the median tumor regression
grade was not reached in either good or poor
responders (p=0.052) (Table 4).
Fig. 3. Maspin expression and overall survival.
Table 4 Univariate and multivariateanalyzes to determine the parameters affecting overall
survival
In multivariate analysis, only nuclear maspin staining
was found to be significantly associated with DFS
(p=0.004, HR=2.982, 95% CI=1.052?8.450) (Table 3).
Although only clinical T stage was found to be significant for overall survival in univariate analyses, nuclear
maspin staining was statistically significant for DFS
and was therefore included in the multivariate analysis.
In addition, multivariate analysis identified clinical
T stage as a significant factor associated with mOS
independent of nuclear maspin expression (p=0.011,
HR=3.452, 95% CI=1.330?8.962).
Discussion
Rectal cancers, which comprise approximately 30% of
colorectal malignancies, maintain their pivotal role in
management primarily via surgical excision.[] Responses
to neoadjuvant therapy in rectal cancer are heterogeneous.
There is an ongoing search for biomarkers
to aid in the selection of the right treatment for the
right patient. Extant literature suggests maspin's potential
oncogenic role in colorectal neoplasms, linking
it with unfavorable prognostic markers.[] However,
given maspin's characteristics, such as inhibition of invasion
and enhancement of apoptosis, in this study, we
evaluated the relationship between maspin expression
and neoadjuvant therapy. While we identified a relationship
between nuclear maspin staining and DFS, we
did not find a correlation with pCR or OS.
In our study, we employed the Modified Ryan Tumor
Regression Grade (TRG) System to evaluate the pathological
response subsequent to neoadjuvant therapy. A notable association was identified between TRG and
disease-free survival. Among the cohort, 19 of the 35 patients
(54%) exhibited tumor regression grades of 2 and 3 (indicative of a poor response). A research study led by
Huh et al.[] in 2019, which encompassed 639 patients
over a 56.7-month follow-up span, unveiled a significant
correlation between the 5-year overall survival and disease-
free survival rates in relation to TRG. Specifically,
the 5-year disease-free survival was discerned to be 93%
for TRG 0 and 1, and 68% for TRG 2?3. Such revelations
underscore the importance of TRG as an autonomous
prognostic determinant for survival among rectal cancer
patients undergoing neoadjuvant therapy.
Our multivariate survival analysis illuminated a significant
association between nuclear maspin staining in
patients and disease-free survival, wherein recurrence
was observed in 100% of these patients. Conversely, no
consequential association emerged regarding overall
survival among those with nuclear maspin staining. A
comprehensive review of existing literature manifests
that while cytoplasmic maspin staining is often correlated
with a favorable prognosis in tissues exhibiting high tumor
density, nuclear maspin staining is linked with markers
of poor prognosis such as lymphovascular and perineural
invasion, lymph node metastasis, enhanced tumor
aggressiveness, and diminished survival durations.[]
In comparing our findings with those of Chang
et al.,[] several notable similarities and differences emerge. Both studies underscore the prognostic significance
of maspin expression in rectal cancer. Chang
et al.[] focused on SERPINB5 expression and its association
with chemoradiotherapy (CCRT) response
and overall prognosis. Their results indicated that SERPINB5
overexpression was linked to a poor response to
CCRT, as well as reduced disease-specific survival, local
recurrence-free survival, and metastasis-free survival.
Similarly, our study identified a significant relationship
between nuclear maspin staining and disease-free survival
(DFS), with poor tumor regression grade (TRG)
and high CA-19 levels also negatively impacting DFS.
However, unlike Chang et al.,[] who reported SERPINB5'
s influence on various survival metrics, our data
did not show a significant correlation between maspin
staining and overall survival (OS) in univariate analysis.
Instead, clinical T stage emerged as a significant factor
for OS in multivariate analysis. Notably, our study
found no pathological complete response in patients
with nuclear maspin positivity, aligning with Chang et
al.[]'s findings on SERPINB5's association with adverse
outcomes. This comparison highlights the potential
of maspin as a prognostic biomarker in rectal cancer
and underscores the need for further research to clarify
its role in treatment response and long-term survival.
According to the literature, the impact of maspin
on prognosis has been investigated in various cancer
types. Our study is one of the most important studies in the literature evaluating the prognostic significance
of maspin in rectal cancer.[,] Currently, total neoadjuvant
therapy (TNT) has become the standard in the preoperative treatment of rectal cancer. Our data
does not include patients who received TNT. The prognostic
value of maspin in patients who have received
TNT is a subject that needs further investigation.
The limitations of our study include the small number
of patients included in our center, its reliance on
retrospective data, and the inclusion of only patients
followed in our clinic. Additionally, the lack of literature
data on this topic makes the interpretation of results
difficult. Considering the aggregate data, discerning
the precise role of maspin, an exclusive member of
the serpin family, in oncology remains complex. Furthermore,
understanding its response to neoadjuvant
treatment and its implications for both disease-free
and overall survival is challenging.
Conclusion
Our study underscores the crucial link between nuclear
maspin staining and disease-free survival in rectal cancer
patients, highlighting its potential as a prognostic indicator.
To pave the way for potential maspin-centric therapeutic
interventions, especially for patients predisposed
to heightened risk of rectal cancer, a comprehensive understanding
of maspin's molecular mechanisms within
the oncogenic processes of rectal cancer is paramount.
Ethics Committee Approval: The study was approved by
the Gazi University Clinical Research Ethics Committee (no:
53, date: 25/01/2021).
Authorship contributions: Concept - G.A.E., N.Ö.; Design
- G.A.E., O.S., B.Ö.; Supervision - O.S., N.Ö., B.Ö.;
Materials - G.A.E., B.Ö.; Data collection and/or processing
- G.A.E., B.Ö.; Data analysis and/or interpretation - G.A.E.,
O.S.; Literature search - G.A.E., O.S., N.Ö.; Writing - G.A.E.,
O.S.; Critical review - O.S., B.Ö., N.Ö.
Conflict of Interest: All authors declared no conflict of interest.
Use of AI for Writing Assistance: No AI technologies utilized.
Financial Support: None declared.
Peer-review: Externally peer-reviewed.
References
Glynne-Jones R, Wyrwicz L, Tiret E, Brown G, Rödel
C, Cervantes A, et al. Rectal cancer: ESMO Clinical
Practice Guidelines for diagnosis, treatment and follow-
up. Ann Oncol 2017;28:22-40.
Sauer R, Becker H, Hohenberger W, Rödel C, Wittekind
C, Fietkau R, et al. Preoperative versus postoperative
chemoradiotherapy for rectal cancer. N Engl J
Med 2004;351(17):1731-40.
Ulusoy C, Kamalı GH, Nikolovski A. Analysis of survival
in complete pathological response after longcourse
chemoradiotherapy in patients with advanced
rectal cancer. Curr Oncol 2023;30(1):1054-64.
Al-Sukhni E, Attwood K, Mattson DM, Gabriel E,
Nurkin SJ. Predictors of pathologic complete response
following neoadjuvant chemoradiotherapy for rectal
cancer. Ann Surg Oncol 2016;23(4):1177-86.
Hasan S, Renz P, Wegner RE, Finley G, Raj M, Monga D,
et al. Microsatellite instability (MSI) as an independent
predictor of pathologic complete response (pCR) in locally
advanced rectal cancer: A national cancer database
(NCDB) analysis. Ann Surg 2020;271(4):716-23.
Díaz-González JA, Calvo FA, Cortés J, García-Sabrido
JL, Gómez-Espí M, Del Valle E, et al. Prognostic factors
for disease-free survival in patients with T3-4 or
N+ rectal cancer treated with preoperative chemoradiation
therapy, surgery, and intraoperative irradiation.
Int J Radiat Oncol Biol Phys 2006;64(4):1122-8.
Nilsson PJ, van Etten B, Hospers GA, Påhlman L, van
de Velde CJ, Beets-Tan RG, et al. Short-course radiotherapy
followed by neo-adjuvant chemotherapy in locally
advanced rectal cancer-the RAPIDO trial. BMC
Cancer 2013;13:279.
Conroy T, Lamfichekh N, Etienne PL, Rio E, Francois
E, Mesgouez-Nebout N, et al. Total neoadjuvant
therapy with mFOLFIRINOX versus preoperative
chemoradiation in patients with locally advanced rectal
cancer: Final results of PRODIGE 23 phase III trial,
a UNICANCER GI trial. J Clin Oncol 2020;38:4007.
Park YA, Sohn SK, Seong J, Baik SH, Lee KY, Kim NK,
et al. Serum CEA as a predictor for the response to
preoperative chemoradiation in rectal cancer. J Surg
Oncol 2006;93(2):145-50.
Zhang M, Sheng S, Maass N, Sager R. mMaspin: The
mouse homolog of a human tumor suppressor gene
inhibits mammary tumor invasion and motility. Mol
Med 1997;3(1):49-59.
Zou Z, Anisowicz A, Hendrix MJ, Thor A, Neveu
M, Sheng S, et al. Maspin, a serpin with tumor-suppressing
activity in human mammary epithelial cells.
Science 1994;263(5146):526-9.
Sheng SJ, Pemberton PA, Sager R. Production, purification,
and characterization of recombinant maspin
proteins. J Biol Chem 1995;269:30988-93.
Sheng S, Carey J, Seftor EA, Dias L, Hendrix MJ, Sager
R. Maspin acts at the cell membrane to inhibit invasion
and motility of mammary and prostatic cancer
cells. Proc Natl Acad Sci U S A 1996;93(21):11669-74.
Bailey CM, Khalkhali-Ellis Z, Seftor EA, Hendrix
MJ. Biological functions of maspin. J Cell Physiol
2006;209(3):617-24.
Schaefer JS, Zhang M. Targeting maspin in endothelial
cells to induce cell apoptosis. Expert Opin Ther Targets
2006;10(3):401-8.
Chang IW, Liu KW, Ragunanan M, He HL, Shiue YL,
Yu SC. SERPINB5 expression: Association with CCRT
response and prognostic value in rectal cancer. Int J
Med Sci 2018;15(4):376-84.
Oronsky B, Reid T, Larson C, Knox SJ. Locally advanced
rectal cancer: The past, present, and future.
Semin Oncol 2020;47(1):85-92.
Payne CM, Holubec H, Crowley-Skillicorn C, Nguyen
H, Bernstein H, Wilcox G, et al. Maspin is a deoxycholate-
inducible, anti-apoptotic stress-response protein
differentially expressed during colon carcinogenesis.
Clin Exp Gastroenterol 2011;4:239-53.
Huh JW, Kim HC, Kim SH, Park YA, Cho YB, Yun
SH, et al. Tumor regression grade as a clinically useful
outcome predictor in patients with rectal cancer
after preoperative chemoradiotherapy. Surgery
2019;165(3):579-85.
Banias L, Gurzu S, Kovacs Z, Bara T, Bara T Jr, Jung I.
Nuclear maspin expression: A biomarker for budding
assessment in colorectal cancer specimens. Pathol Res
Pract 2017;213(9):1227-30.
Fokas E, Schlenska-Lange A, Polat B, Klautke G, Grabenbauer
GG, Fietkau R, et al. Chemoradiotherapy plus induction
or consolidation chemotherapy as total neoadjuvant
therapy for patients with locally advanced rectal
cancer: Long-term results of the CAO/ARO/AIO-12
randomized clinical trial. JAMA Oncol 2022;8:e215445.
Fokas E, Allagauer M, Polat B, Klautke G, Grabenbauer
GG, Fietkau R, et al. Randomized phase II trial
of chemoradiotherapy plus induction or consolidation
chemotherapy as total neoadjuvant therapy for locally
advanced rectal cancer: CAO/ARO/AIO-12. J Clin
Oncol 2019;37(34):3212-22.
