Summary
Objectives:
Colorectal cancer (CRC) is the third most common cancer worldwide and is the second leading cause of cancer-related deaths. Serine protease inhibitor Kazal-type 1 (SPINK1) is found to be related to poor prognostic criteria and shortened overall survival of some malignancies such as liver, breast, lung, pancreatic and renal cancers. SPINK1 can be a potential biomarker for early detection and prediction of immune checkpoint blockade treatment response. Therefore, this study aimed to examine the possible role of SPINK1 in colorectal carcinogenesis and its prognostic ability.
Methods:
This study used paraffin blocks of patients diagnosed with colorectal adenoma, primary CRC and available positive lymph node metastases. Specimens were obtained between April 2018 and June 2022 at the Department of Pathology, Faculty of Medicine, Minia University, Egypt. Immunohistochemistry was done for SPINK1 antibody and appropriate statistical analysis of results was performed.
Results:
A total of 70 archival samples of CRC, 18 of colorectal adenoma and 20 of metastatic lymph nodes were used in this study. In a normal colon, there was a negative to weak SPINK1 immunostaining. High cytoplasmic immunostaining was seen in 57.1% of patients while low immunostaining in 42.9% of CRC. SPINK1 immunostaining was statistically associated with tumour grade (P = 0.024), stage (P <0.001), nodal status (P = 0.010), lymph node ratio (P = 0.044), lymphovascular invasion (P <0.001), tumour necrosis (P <0.001) and tumour infiltrating lymphocytes (P <0.001). No statistically significant association was found between SPINK1 and patient gender, age, tumour site, tumour size, histological subtype, perineural invasion, margin status and adenoma. A statistically significant association was detected between SPINK1 immunostaining in CRC and adenomas (P = 0.019) and between CRC and associated nodal metastasis (P = 0.013).
Conclusion:
SPINK1 immunostaining is increased in CRC and associated with poor prognostic criteria and is significantly associated with immunoactivity in adenoma and associated nodal metastasis.
Keywords: SPINK1, Colorectal Cancer, Immunohistochemistry, Prognosis, Egypt
Advances in Knowledge
The article has the potential for adding data that will help in understating the carcinogenesis of colorectal carcinoma by providing a relation between SPINK1 expression with tumour infiltrating lymphocytes and nodal metastasis.
Application to Patient Care
The studied molecule could be helpful for applying targeted therapy in colorectal carcinoma and stratification of patients for personalised oncology.
1. Introduction
Colorectal cancer (CRC) is the second most common cause of cancer-related fatalities globally and the third most common type of cancer. GLOBACAN estimates that CRC accounts for approximately 10% of all cancer cases and 9.4% of cancer-related fatalities.1 Approximately, 6.5% of all malignant tumours in Egypt are thought to be CRC. In the National Cancer Institute registry at Cairo University in Egypt, CRC rose to the sixth most frequently reported tumour in the years 2002–2003 with 4.2% prevalence in men and 3.8% in females of the total population.2
The serine protease inhibitor Kazal-type (SPINK) family (SPINK1 to 14) is made up of a class of serine protease inhibitors. Pancreatic acinar cells release SPINK1 as the initial defence against trypsinogen activation too soon. SPINK1 controls stomach protection, tissue healing and normal pancreatic development.3,4 SPINK1 had been correlated with poor prognostic criteria in pancreatic, liver, lung, renal and gastric malignancies.5,6 On the other hand, ovarian cancer progression-free survival as well as urothelial cancer prognosis are expected to be good.7,8 There is a need for identification of novel biomarkers in CRC that could have a significant clinical impact and therapeutic role. The prognostic significance of SPINK1 in CRC is debatable; some researchers had shown SPINK1 association with advanced stage and nodal metastases;9,10 other researchers have reported its association with a favourable outcome.11,12
The hypothesis of the current study was to test the possible role of SPINK1 in colorectal carcinogenesis and its prognostic importance. Therefore, this study aimed to examine the SPINK1 immunostaining in a subset of CRC and its relationship with clinicopathological and prognostic parameters and its potential role as prognostic marker in colorectal carcinogenesis.
2. Methods
The study was performed on paraffin blocks of patients diagnosed with colorectal adenoma, primary CRC and available positive lymph node metastases. Normal colonic mucosa adjacent to the tumour was included whenever available. Specimens were obtained according to availability of clinicopathological data between April 2018 and June 2022 were included if the paraffin blocks were of good quality. Patients underwent hemicolectomy without neoadjuvant therapy. A total of 5 5-micrometer cuts were prepared to perform haematoxylin and eosin staining and immunohistochemistry. Patients' age was categorised into 2 groups: ≤55 or >55 years old using the median as the cut-off value.13,14 Lymph node ratio (LNR) was calculated and classified as LNR = 0 (no lymph nodes involved), LNR = 1 (ratio of 0.01–0.17), LNR = 2 (ratio: 0.18–0.41), LNR = 3 (ratio: 0.42–0.69) and LNR = 4 (ratio: >0.70). LNR was categorised as low (score 0, 1 and 2) and high (score 3 and 4).15
Immunostaining was performed using the avidin biotin-peroxidase complex technique. Polyclonal rabbit SPINK1 antibody (100μ concentrated, Sigma Laboratories, Tucson, Arizona, USA) was used in a dilution of 1:100. Diaminobenzidine chromogen detection system was used for immunostaining (Sigma Laboratories). Subsequently, slides were washed, counterstained with haematoxylin and mounted. Negative control and positive control slides were included where appropriate.
Sections were evaluated independently without knowledge of the clinicopathological characteristics of patients by 2 independent pathologists (ON and WG). A semiquantitative method based on intensity and percentage scoring was used. Estimated proportion of positively stained tumour cells (score as 0: none, 1: <10%, 2: 10–50% and 3: >50%). Also, intensity score was assigned a score of 0 = none, 1 = weak, 2 = moderate and 3 = strong. The proportion and intensity scores were then added to obtain a total score, with a range of 0–6. For further analysis, results were divided into 2 groups where scores 0, 1, 2 and 3 were considered as low immunostaining and scores 4, 5 and 6 were considered as high immunostaining.10,16
The association between two categorical variables were tested by using Chi-square/fisher exact tests. Non-parametric Chi-square test was used to test variance along one variable. Multivariate logistic regression test was used to test prognostic significance. Statistical Package for Social Sciences (SPSS) software, Version 28 (IBM Corp., Armonk, New York, USA) was used and the 2-sided statistical significance was determined at P value of ≤0.05.
3. Results
A total of 70 archival samples of primary CRC, 18 of colorectal adenoma and 20 of metastatic lymph nodes (20 out of 38 patients reported with nodal metastasis) were used in this study; 35 normal colonic mucosa samples adjacent to the tumour were also included [Table 1]. In normal colonic epithelium, SPINK1 immunostaining was negative to weak which was mostly confined to the base of the crypt. High immunostaining was detected in 6/18 (33.3%) of adenomas while 12/18 (66.7%) showed low immunostaining. In CRC, SPINK1 immunostaining was identified in the cytoplasm of malignant cells and high immunostaining was identified in 40/70 (57.1%) of tumours while 30/70 (42.9%) of tumours showed low immunostaining. SPINK1 immunostaining in normal, adenoma, CRC and nodal metastasis are shown in Fig. 1.
Table 1.
Clinicopathological parameters of colorectal cancer patients (N = 70).
| Parameter | n (%) |
|---|---|
| Gender | |
| Male | 41 (58.6) |
| Female | 29 (41.4) |
| Age group in years | |
| ≤55 | 31 (44.3) |
| >55 | 39 (55.7) |
| Tumour site * | |
| Left colon and rectum | 42 (60) |
| Right colon | 28 (40) |
| Tumour size in cm | |
| <5 | 30 (42.9) |
| ≥5 | 40 (57.1) |
| Histological type | |
| Conventional adenocarcinoma | 41 (58.6) |
| Mucinous carcinoma | 24 (34.3) |
| Signet ring carcinoma | 5 (7.1) |
| Tumour grade | |
| Low grade | 51 (72.9) |
| High grade | 19 (27.1) |
| Primary tumour | |
| pT1 | 8 (11.4) |
| pT2 | 25 (35.7) |
| pT3 | 30 (42.9) |
| pT4 | 7 (10) |
| Nodal metastasis | |
| Negative | 32 (45.7) |
| Positive | 38 (54.3) |
| Lymph node ratio | |
| Low score | 55 (78.6) |
| High score | 15 (21.4) |
| Lymphovascular invasion | |
| Negative | 37 (52.9) |
| Positive | 33 (47.1) |
| Perineural invasion | |
| Negative | 55 (78.6) |
| Positive | 15 (21.4) |
| Tumour necrosis | |
| Low score | 40 (57.1) |
| High score | 30 (42.9) |
| Tumour infiltrating lymphocytes | |
| Low infiltrate | 40 (57.1) |
| High Infiltrate | 30 (42.9) |
| Margin status | |
| Free | 50 (71.4) |
| Infiltrated | 20 (28.6) |
| Associated adenoma | |
| Present | 18 (25.7) |
| Absent | 52 (74.3) |
T1 = tumour invades submucosa; T2 = tumour invades muscularis propria; T3 = tumour invades through the muscularis propria into the subserosa or into non-peritonealised pericolic or perirectal tissues; T4 = tumour directly invades other organs or structures, and/or perforates visceral peritoneum.
Right sided tumours include caecum, ascending colon up to hepatic flexure. Left sided tumours include splenic flexure, descending colon, sigmoid colon and rectum.
Fig. 1.
SPINK1 immunostaining of (A normal colonic mucosa at × 100 magnification showing weak staining at base of crypts and absent staining in apical portion, (B a villous adenoma at × 200 magnification showing high SPINK1 immunostaining, (C a conventional colorectal adenocarcinoma at × 200 magnification showing high SPINK1 immunostaining and (D metastatic deposits in a lymph node at × 200 magnification showing high SPINK1 immunostaining.
Regarding clinicopathological data, SPINK1 immunostaining in CRC was statistically associated with tumour grade (P = 0.024), tumour stage (P <0.001), nodal status (P = 0.010), LNR (P = 0.044), lymphovascular invasion (LVI) (P <0.001), tumour necrosis (P <0.001) and tumour infiltrating lymphocytes (TILs) (P <0.001). No statistically significant association was found between SPINK1 and patient gender (P = 0.234), patient age (P = 0.187), tumour site (P = 0.622), tumour size (P = 0.944), histological subtype (P = 0.526), perineural invasion (PNI) (P = 0.400), margin status (P = 0.819) and adenoma (P = 0.875) [Table 2].
Table 2.
The association between SPINK1 immunostaining in colorectal cancer and clinicopathological data.
| SPINK1 immunostaining | |||
|---|---|---|---|
|
|
|||
| Parameter | Low (n = 30) | High (n = 40) | P value |
| Gender | |||
| Male | 20 (48.8) | 21(51.2) | 0.234* |
| Female | 10 (34.5) | 19 (65.5) | |
| Age in years | |||
| ≤55 | 16 (51.6) | 15 (48.4) | 0.187* |
| >55 | 14 (35.9) | 25 (64.1) | |
| Tumour site † | |||
| Left colon and rectum | 19 (45.2) | 23 (54.8) | 0.622* |
| Right colon | 11 (39.3) | 17 (60.7) | |
| Tumour size in cm | |||
| <5 | 13 (43.3) | 17 (56.7) | 0.944* |
| ≥5 | 17 (42.5) | 23 (57.5) | |
| Histological type | |||
| Conventional adenocarcinoma | 19 (46.3) | 22 (53.7) | 0.526* |
| Mucinous carcinoma | 10 (41.7) | 14 (58.3) | |
| Signet ring carcinoma | 1 (20) | 4(80) | |
| Tumour grade | |||
| Low grade | 26 (51) | 25 (49) | 0.024* |
| High grade | 4 (21.1) | 15 (78.9) | |
| Primary tumour | |||
| Early pT stage (pT1 and pT2) | 22 (66.7) | 11 (33.3 | <0.001* |
| Advanced pT stage (pT3 and pT4) | 8 (21.6) | 29 (78.4) | |
| Nodal metastasis | |||
| Negative | 19 (59.4) | 13 (40.6) | 0.010* |
| Positive | 11 (28.9) | 27 (71.1) | |
| Lymph node ratio | |||
| Low score | 27 (49.1) | 28 (50.9) | 0.044* |
| High score | 3 (20) | 12 (80) | |
| Lymphovascular invasion | |||
| Negative | 24 (64.9) | 13 (35.1) | <0.001* |
| Positive | 6 (18.2) | 27 (81.8) | |
| Perineural invasion | |||
| Negative | 25 (45.5) | 30 (54.5) | 0.400* |
| Positive | 5 (33.3) | 10 (66.7) | |
| Tumour necrosis | |||
| Low score | 25 (62.5) | 15 (37.5) | <0.001* |
| High score | 5 (16.7) | 25 (83.3) | |
| Tumour infiltrating lymphocytes | |||
| Low infiltrate | 8 (20) | 32 (80) | <0.001* |
| High infiltrate | 22 (73.3) | 8 (26.7) | |
| Margin status | |||
| Free | 21 (42) | 29 (58) | 0.819 |
| Infiltrated | 9 (45) | 11 (55) | |
| Associated adenoma | |||
| Absent | 22 (42.3) | 30 (57.7) | 0.875 |
| Present | 8 (44.4) | 10 (55.6) | |
T1 = tumour invades submucosa; T2 = tumour invades muscularis propria; T3 = tumour invades through the muscularis propria into the subserosa or into non-peritonealised pericolic or perirectal tissues; T4 = tumour directly invades other organs or structures, and/or perforates visceral peritoneum.
Using Chi-square/fisher exact tests.
Right sided tumours include caecum, ascending colon up to hepatic flexure. Left sided tumours include splenic flexure, descending colon, sigmoid colon and rectum.
There is a statistically significant association between SPINK1 immunostaining in CRC and adenoma (P = 0.019) [Table 3]. High SPINK1 immunostaining was more in adenoma with high grade dysplasia and large-sized adenoma and (62.5% and P = 0.019 each). However, it was not associated with neither site nor type of adenoma (P = 0.137 and P = 0.543, respectively).
Table 3.
Association between SPINK1 immunostaining in colorectal cancer and adenoma (n = 18).
| Immunostaining in adenoma | |||
|---|---|---|---|
| Immunostaining in carcinomas |
|
||
| Low (n = 10) | High (n = 8) | P value* | |
| Low (n = 12) | 9 (75) | 3 (25) | 0.019 |
| High (n = 6) | 1 (16.7) | 5 (83.3) | |
Using Chi-square/fisher exact tests.
With regards to SPINK1 immunostaining in CRC and corresponding available nodal metastasis (20 out of 38), there is a statistically significant association (P = 0.013) as all nodal metastasis with high immunostaining showed high immunostaining in primary carcinoma, and vice versa for low immunostaining [Table 4].
Table 4.
Association between immunostaining of SPINK1 in colorectal cancer and associated nodal metastasis (n = 20).
| Immunostaining in carcinomas | |||
|---|---|---|---|
| Immunostaining in LNs |
|
||
| Low (n = 4) | High (n = 16) | P value* | |
| Negative (n = 9) | 4 (44.4) | 5 (55.6) | 0.013 |
| Positive (n = 11) | 0 (0) | 11 (100) | |
Using Chi-square/fisher exact tests.
In multivariate analysis, high SPINK1 immunostaining in CRC was an independent predictor of tumour grade (P = 0.026), tumour stage (P = 0.006), tumour necrosis (P = 0.010) and TILs (P = 0.010) [Table 5].
Table 5.
Prognostic factors prediction by high SPINK1 immunostaining.
| Parameter | Adjusted OR (95% CI) | P value* |
|---|---|---|
| Tumour grade | 4.806 (1.202–19.218) | 0.026* |
| Tumour staging | 1.77 (0.052–0.604) | 0.006* |
| Tumour Necrosis | 4.031 (1.075–15.119) | 0.039* |
| TILs | 0.189 (0.053–0.675) | 0.010* |
OR = odds ratio; CI = confidence interval.
Multivariate logistic regression test.
4. Discussion
CRC ranks among the third most common cancers and is one of the leading causes of cancer-related deaths globally.1 Approximately half of the CRC patients with KRAS and BRAF mutations acquire resistance to anti-epidermal growth factor receptor (EGFR) drugs, highlighting the necessity for additional targeted therapies. Therefore, the identification of other useful biomarkers that could have a significant clinical impact and guide the therapeutic treatment options in CRC is required.9 SPINK1 could be a potential biomarker for the early detection, targeted therapy and prediction of immune checkpoint blockade treatment response. Attenuation of SPINK1 expression in CRC may promote the efficacy of radiotherapy by decreasing effects of anti-apoptosis.9,17 SPINK1 is thought to encourage proliferation, migration, invasion, chemo-resistance and radiation resistance in CRC.11 SPINK1 was proposed to serve as a growth factor in inflammatory tissue repair. If left unchecked, it would promote cell proliferation and malignancy. Acute-phase reactions are induced by an IL6-responsive element found in the SPINK1 gene. IL-6 stimulates the STAT3 pathway, which in turn promotes SPINK1.18 SPINK1 immunostaining was seen in the base aspect of normal colonic crypt, which is an important site for proliferation of the epithelium. Accordingly, SPINK1 may have a crucial role in the proliferation.10 In addition, SPINK1 promotes cell proliferation by activating PI3K/AKT1 signalling pathway which is known to drive cell proliferation and invasion.19 Also, SPINK1 in cancer cells is found to have a relation with miRNAs as miR-32 which promotes cell proliferation and invasion.17
In this study, cytoplasmic SPINK1 immunostaining was identified in dysplastic and malignant cells while normal colonic cells showed negative to weak staining and was localised to crypt base. The subcellular localisation is consistent with previous studies.10,19 In the current study, high SPINK1 immunostaining was more than low immunostaining in CRC; this was in accordance with another study.20 However, high SPINK1 immunostaining has been reported as less than low immunostaining.9 Such a discrepancy may be due to the different antibody clones used, differing sample size and differing scoring system which rely only on the percentage of positive cells. High SPINK1 level in tumour cells is related to its competition with EGF for binding to EGFR triggering subsequent signalling pathway; EGFR-extracellular signal-regulated kinase-cyclin-dependant kinase 4/6-E2F2 (a member of family of transcription factors in higher eukaryotes) axis is involved in the proliferation, differentiation, migration and apoptosis of cancer cells.6
The current study found that high SPINK1 immunostaining was associated with high tumour grade, which is similar to a previous study.7 However, another study showed significant association with low tumour grade.11 This may be related to the administration of neoadjuvant chemotherapy in the study group. On the other hand, a study reported no significant association between grade and SPINK1 expression.9 This may be due to a difference in number of patients included in their study, where more than 90% of patients had a low tumour grade. SPINK1 was found to be related to dedifferentiation by mediating pathways allowing cellular plasticity and cancer stemness.6
In terms of tumour stage, the current study's findings were in line with different reported studies that found positive association of SPINK1 immunostaining and advanced stages.10,21 Other research showed insignificant association.9,11 This can be as a result of a different number of patients included in each stage and that all patients in these studies received neoadjuvant chemotherapy. SPINK1 is thought to encourage proliferation, migration and invasion of cancer which can be the cause of its association to advanced tumour stage.9,22
This study found a significant relation between high SPINK1 immunoactivity and positive nodal metastasis which is supported by a previous study's results.10 However, such an association was not reported in another study.21 This difference could be explained by using a different scoring system which depended on percentage of positive cells only. Considering LVI, some researchers showed the same findings as the current study as substantial association was revealed between high SPINK1 immunostaining and presence of LVI; others reported no conclusive link.9 This may be due to a difference in scoring system as it depends on the percentage of positive cells only or staining intensity only.
The association of high SPINK1 nodal metastasis and LVI may be explained by the ability of SPINK1 to induce epithelial-to-endothelial transition, as supported by the upregulation of CD31 and CD34 (vascular lineage markers indicative of neoangiogenesis induced by stromal SPINK1).8 In addition, the expression of SPINK1 is invariably associated with expression of tumour-associated trypsin, which activates several matrix metalloproteinases. These have been shown to mediate cancer invasion.9,19
The current study found no asoociation between SPINK1 immunostaining in CRC and the occurrence of adenoma. This negative result may be attrituable to the small number of adenoma tested in this study. However, there was a significant association between SPINK1 immunostaining and adenoma with high grade dysplasia. This supports the role of SPINK1 in inducing tumour dedifferentiation. The relation between SPINK1 and large adenoma size may be a result of allowing cellular proliferation and its anti-apoptotic role.9
SPINK1 immunostaining had an independent relationship to low TILs score. SPINK1 is suggested to produce immunosuppressive substances so immune cells become polarised into immunosuppressive phenotypes such as helper T cell 2 and bone marrow-derived inhibitory cells. Also, SPINK1 is suggested to activate PI3K/AKT pathway leading to decreased number of TILs.17,23 This study demonstrated significant association between SPINK1 immunostaining and high necrosis score. Hypoxic tumour microenvironment causes an increase in secreted SPINK1 levels by HIF-1. Also, DNA damage mediates the expression of SPINK1 through NF-κB.17 In the current study, there was no significant association between SPINK1 immunoactivity and patient's gender, age, tumour size, tumour site, histological subtypes, PNI and margin status. The current study's results are in accordance with studies done by others.9,10,21
Multivariate analysis revealed that SPINK1 was an independent predictor of tumour grade, tumour stage, TILs and tumour necrosis. These findings support the prognostic role of SPINK1 in CRC.
The limitations of this study include limited financial funding for testing more CRC, limited follow-up data for survival analysis and availability of CRC patients with liver metastasis and their follow-up data.
5. Conclusion
To the best of the authors' knowledge, this is the first study to present an association between SPINK1 and LNR, TILs and tumour necrosis. SPINK1 immunostaining was related to poor prognostic features in CRC of high tumour grade, advanced stage, nodal metastasis, LVI, high LNR, high necrosis score and low TILs score. These results suggest that SPINK1 may have a role in colorectal carcinogenesis, especially adenoma-carcinoma pathway, or may contribute in resistance to therapy. Further studies on a large number of CRC along with nodal metastasis and distant metastasis are required for more elucidation of SPINK1 role in CRC. Molecular investigations are recommended to detect the relation between SPINK1 and genetic markers of primary CRC for detection of the possibility of anti-SPINK1 targeted therapy.
Authors' Contribution
Ola Nasser: Conceptualisation, Data curation, Formal analysis, Investigation, Methodology, Visualisation, Writing – original draft. Wafaey Gomaa: Conceptualisation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualisation, Writing (original draft), Writing (review & editing). Wafaa Aref: Conceptualisation, Supervision, Validation, Writing (review & editing). Rabab Moussa: Conceptualisation, Supervision, Validation, Writing (review & editing).
Ethics Statement
The study was approved by the institutional review board, faculty of medicine, Minia university, Egypt number 469/10/2023.
Conflict of Interest
The authors declare no conflicts of interest.
Funding
No funding was received for this study.
Data Availability
Data is available upon reasonable request from the corresponding author.
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Associated Data
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Data Availability Statement
Data is available upon reasonable request from the corresponding author.