TURKISH JOURNAL OF ONCOLOGY

Summary

OBJECTIVE
Maspin is a member of the serpin family that targets proteinases and exhibits characteristics of a tumor suppressor protein. The aim of our study is to evaluate the impact of maspin expression on survival rates in patients diagnosed with locally advanced rectal cancer.

METHODS
A retrospective analysis was conducted on a dataset of 60 patients who had received a histopathological diagnosis of rectal cancer. Maspin expression was assessed using the Ventana Benchmark XT device with automatic immunohistochemical staining via the streptavidin-biotin immunoperoxidase technique. A survival analysis was performed by examining maspin in the specimens of these patients.

RESULTS
In terms of maspin staining, 37 patients (61.7%) were negative, while 23 patients (38.3%) were positive (18 cytoplasmic, 2 nuclear, and 3 both). The 3-year overall survival (OS) was 91.3% in maspin-positive patients and 73% in maspin-negative patients. Clinical T stage was significantly related to OS (p=0.028). Multivariate analysis showed nuclear maspin staining was significantly associated with DFS (p=0.004), and T stage was a significant factor for OS, independent of nuclear maspin expression (p=0.011).

CONCLUSION
Our study underscores the strong link between nuclear maspin staining and disease-free survival in rectal cancer patients, highlighting its promising potential as a prognostic biomarker for future research.

Summary

Introduction

Neoadjuvant chemoradiotherapy (nCRT) is recommended as the standard treatment for locally advanced rectal cancer.[1] Pathological complete response (pCR) rates with chemoradiotherapy (CRT) alone range between 13% and 17%.[2,3] When total neoadjuvant therapy (TNT) is utilized, the pCR rate increases to approximately 27%.[4-6] Recent research supports the efficacy of TNT, demonstrating a significant increase in pCR rates.[7,8] 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.[9] 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.[10-13] 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. [14] Remarkably, it has been observed that maspin expression decreases or even disappears in primary breast cancer cell lines and invasive breast carcinoma. [9] 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.[15] 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.[16]

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.

Introduction

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.

Methods

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).

Results

Discussion

Rectal cancers, which comprise approximately 30% of colorectal malignancies, maintain their pivotal role in management primarily via surgical excision.[17] 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.[18] 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.[19] 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.[20]

In comparing our findings with those of Chang et al.,[16] several notable similarities and differences emerge. Both studies underscore the prognostic significance of maspin expression in rectal cancer. Chang et al.[16] 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.,[16] 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.[16]'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.[21,22] 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.

Discussion

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.

Conclusion