Colorectal Cancer (CRC): Investigating the Expression of the Suppressor of Fused (SuFu) Gene and Its Relationship with Several Inflammatory Blood-Based Biomarkers

Background: Suppressor of fused (SuFu) is a tumor-suppressor gene that regulates hedgehog signaling. Its involvement in some malignancies is broadly accepted. However, its association with colorectal cancer (CRC) pathogenesis is not clear. Likewise, no study has clearly associated blood-based inflammatory biomarkers with cancer diagnosis/prognosis as yet. Aim: Our goal was to look at SuFu expression levels in CRC patients and its relationship with other clinicopathological factors. Additionally, we looked into the function of a few blood-based biomarkers in CRC and whether or not a combined strategy at the genetic and clinical levels can be applied in CRC. Methods: The investigation included 98 histopathologically confirmed CRC samples and adjacent normal tissues (controls). A colonoscopy was followed by a targeted biopsy for each suspected colon cancer patient. A CT scan and MRI were also performed on every patient with rectal cancer. Real-time polymerase chain reaction and immunohistochemistry (IHC) were used for assessment. A Beckman Coulter DxH900 was used to examine blood parameters. A Beckman Coulter DxI800 was used to identify pretreatment carcinoma embryonic antigens (CEA) and carbohydrate antigens (CA 19–9) in CRC patients. Results: The expression of SuFu was associated with gender, education, passive smoking, tumor grade, perineural invasion (PNI), lymph node metastasis (LNM), node status, stage, vital status, and recurrence (p < 0.05). In the combined analysis, the areas under the curve produced by the platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), and red cell distribution width (RDW) were the greatest (AUCRDW+PLR+NLR = 0.91, 95% CI: 0.86–0.93, p < 0.05). Furthermore, the most severe pathological features were linked to RDW, PLR, NLR, and HPR. SuFu expression, node status, LNM, PNI, and stage all had significant correlations with OS and DFS rates in IHC-based univariate survival analysis (p < 0.05). According to the Cox regression, CA-19.9 had a strong independent predictive link with 3-year DFS (p < 0.05). Conclusion: In CRC, SuFu was downregulated both transcriptionally and translationally, was primarily nucleo-cytoplasmic, and was expressed less in high-grade tumors. In addition, SuFu was linked to a poor overall and disease-free survival rate. It may be possible to use SuFu as a therapeutic target for CRC in the future. However, SuFu expression had no effect on RDW, PLR, NLR, or HPR serum levels.


Introduction
Worldwide, colorectal cancer (CRC) is the second leading cause of death [1]. Currently, surgery remains the primary treatment of choice at early stages but it does not benefit those with advanced cases. Hence, the disease has a poor prognosis. As of today, the standard treatment involves resection of the primary tumor as well as the lymph nodes, followed by adjuvant chemotherapy [2,3]. Like other malignancies, both genetic and modifiable environmental factors have been associated with CRC genesis [4][5][6][7]. Among the various genetic pathways, the hedgehog signaling pathway has been implicated in tumor formation in various organs [8]. However, until recently, investigations and research on the significance of the Hh signaling pathway in the development and progression of CRC have been divided. Its specific role in the genesis, progression, and spread of CRC is unknown. [9,10]. Suppressor of fused (SuFu) is one of the main downregulators of the hedgehog signaling pathway [11]. SuFu-a key tumor-suppressor gene [12] of the hedgehog signaling pathway-has been linked to various cancers [13][14][15]. An earlier study found that patients with gastric cancer had lower levels of SuFu expression [16]. Similarly, Li et al., in their previously conducted study, showed a tumor-suppressive role of SuFu in basal cell carcinoma [17]. However, its association with CRC is not conclusive. A cell line-based study showed that the overexpression of SuFu had a regulatory effect on colon cancer cells, and inhibited cell growth and tumor formation [18].
Additionally, several inflammatory biomarkers have recently been explored for their possible links with cancer as evidence suggests that there are links between inflammation and cancer progression [19,20]. Red cell distribution width (RDW) [21,22], hemoglobinto-platelet ratio (HPR) [23], neutrophil-to-lymphocyte ratio (NLR) [24,25], and platelet-tolymphocyte ratio (PLR) [26,27], for example, are among these markers. NRL, PLR, HPR, and RDW may assist in detecting the early stages of CRC. However, the data regarding their diagnostic value in CRC are not conclusive [28,29]. To date, no concrete work has been conducted regarding the expression status of SuFu in CRC, nor is much known about the utility of inflammatory blood biomarkers in CRC. To evaluate the role of SuFu in CRC development, we looked at its expression status and localization in tumor tissues as well as its possible links with important pathological features associated with metastasis, such as grade, stage, lymphovascular invasion (LVI), perineural invasion (PNI), lymph node metastasis, and distant metastasis (DM). Since there is currently no conclusive evidence linking blood-based inflammatory biomarkers with cancer diagnosis or prognosis, we further explored the potential of blood-based biomarkers as prognostic or diagnostic markers in CRC.

Patient Selection
This study included 98 human histopathologically confirmed CRC samples, as well as nearby normal tissues (controls). Colonoscopy and targeted biopsy were performed on all colon cancer patients. All patients with rectal cancer underwent abdominal CT and pelvic MRI. Between 2 April 2019 and 28 March 2022, samples were collected from patients who had undergone primary surgical resection at the Sher-I-Kashmir Institute of Medical Science (SKIMS) in the Department of General and Minimal Invasive Surgery. As recommended by the College of American Pathologists, the Royal College of Pathologists, the Commission on Cancer of the American College of Surgeons, and the National Cancer Institute [30], the tumor-node-metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC) [31] and the International Union Against Cancer (UICC) [32] was used for the staging of CRC. Although various criteria have been presented [33][34][35], the degree of gland formation is currently the most widely accepted and consistently used criterion for grading colorectal cancer (CRC). According to the TNM classification [36,37], a grade 1 tumor is one that has a high level of differentiation, grade 2 tumors have a moderate level of differentiation, grade 3 tumors have a low level of differentiation, and grade 4 tumors have no differentiation at all. For RNA extraction, samples were stored in RNAlater (Sigma-Aldrich Burlington, VT, USA) and kept at 80 • C for future processing. After receiving permission from the Department of Pathology, formalin-fixed paraffinembedded (FFPE) blocks of CRC tissues and surrounding normal tissues were collected for immunohistochemistry examination (IHC). Tissues from the same CRC patients from whom fresh tissues had been taken at the time of surgery were used for the immunohistochemistry research. Further, the same patients whose tissues had already been employed in mRNA and protein expression underwent routine base-line investigations before surgery, and the data were collected electronically. Various blood parameters such as hemoglobin (HGB), red cell distribution width (RDW), white blood cells (WBC), platelets (PLT), neutrophil, and lymphocyte were recorded. An equal number of healthy controls were selected for blood analysis. A Beckman Coulter DxH900 was used to routinely analyze blood parameters: hemoglobin (HGB), red cell distribution width (RDW), white blood cells (WBC), platelets (PLT), neutrophil, and lymphocyte. NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count (ALC); likewise, PLR was computed by dividing the absolute platelet count by ALC. HPR was calculated as hemoglobin/total number of platelets. Pretreatment CEA and CA-19.9 levels in the CRC group were measured on using a Beckman Coulter DxI800.

Data Collection
A structured questionnaire was administered to collect the data. We gathered information on family history, smoking status, socioeconomic status (SES), lifestyle, education, pesticide exposure, junk food, and intake of fruits and vegetables. Face-to-face interviews were conducted by a single author in order to reduce the interviewer bias. The term "family history" referred to whether or not the patient had a history of cancer in their own family or in a blood relative (both site-specific and other cancers). Regarding smoking status, we did not count how many packs of cigarettes patients smoked daily or weekly (frequency of smoking). We simply documented the patient's statement of whether they smoked or did not smoke. To determine the patient's socioeconomic position, the Kuppuswamy scale and BG Prasad scale [38], which are based on education, occupation, and total income, were employed. Despite being included in the questionnaire, we did not include different food intake measurements in the manuscript since there existed a strong unit measurement bias. Regarding lifestyle activity, sedentary behavior included occupations such as clerk, section officer, engineer, and other indoor occupations that were characterized by less energy expenditure, while active lifestyle occupations were characterized as having more effort and energy expenditure such as police officer, mechanic, farmer, construction worker, etc. However, we did not record any intensity level measurements. Concerning education status, we grouped illiterate, primary, and middle education into one group (lower) and higher secondary, graduate, and above into the higher group. Pesticide exposure was not quantified by us. If the patient had been exposed to pesticides, we merely recorded "yes" or "no" responses from them. Junk foods included those foods which were high in one or more components such as sugar, fat, cholesterol, salt, calories, etc., as described by chapman et al. [39], and usually prepared by deep frying.

Inclusion and Exclusion Criteria
CRC patients treated with surgical resection with a histopathology-confirmed diagnosis were included in the study. Patients with anemia, hematological or systemic diseases, recent blood or platelet transfusions, and chemo-or radiotherapy were excluded, as shown in Figure 1

Inclusion and Exclusion Criteria
CRC patients treated with surgical resection with a histopathology-conf diagnosis were included in the study. Patients with anemia, hematological or sys diseases, recent blood or platelet transfusions, and chemo-or radiotherapy excluded, as shown in Figure 1.

RNA Isolation, and cDNA Synthesis and Real-Time PCR of SuFu
To gain a better understanding of the clinical importance of SuFu express colorectal cancer, we evaluated SuFu expression using a number of clinicopathol and laboratory parameters. RNA was extracted using the Trizol method (Invit Waltham, MA). For RNA, absorbance at A260/280 of 1.9-2 was considered "pure used the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Ltd., Wal MA, USA, #K1622) to synthesize complementary DNA (cDNA) after DNase-I (Q treatment. The reverse transcription of 1 g RNA was performed in a volume of using AMV Reverse Transcriptase and random hexamers. The cycle's thermal setti this experiment were 5 min at 25 °C, 60 min at 42 °C, and lastly, 5 min at 70 °C. PikoReal PCR system (Thermo), quantitative real-time PCR (qRT-PCR) was perfo using SYBR Green Master Mix (Thermo Fisher Scientific Ltd.). Each result

RNA Isolation, and cDNA Synthesis and Real-Time PCR of SuFu
To gain a better understanding of the clinical importance of SuFu expression in colorectal cancer, we evaluated SuFu expression using a number of clinicopathological and laboratory parameters. RNA was extracted using the Trizol method (Invitrogen Waltham, MA). For RNA, absorbance at A260/280 of 1.9-2 was considered "pure". We used the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Ltd., Waltham, MA, USA, #K1622) to synthesize complementary DNA (cDNA) after DNase-I (Qiagen) treatment. The reverse transcription of 1 g RNA was performed in a volume of 20 ul using AMV Reverse Transcriptase and random hexamers. The cycle's thermal settings in this experiment were 5 min at 25 • C, 60 min at 42 • C, and lastly, 5 min at 70 • C. On a PikoReal PCR system (Thermo), quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Master Mix (Thermo Fisher Scientific Ltd., Waltham, MA, USA). Each result was standardized to the housekeeping gene GAPDH, and the experiments were carried out in triplicate. The primers used were as follows: SuFu-F: 5-CGGAGGGGAGAGACCATATT-3, R: 5-CACTTGGCACTGACACCACT-3; GAPDH-F: 5-CACTTGGCACTGACACCACT-3 , R: 5-CTTCACCACCTTCTTGATG-3. For SuFu mRNA expression, the cycle threshold (Ct) was used. Based on Livak and Schmittgen's 2 −∆∆ct method, the relative expression levels were determined. A qRT-PCR reaction mixture was incubated at 95 • C for 3 min, proceeded by 40 cycles of denaturation (15 s at 95 • C), annealing (20 s at 57 • C), and extension (20 s at 72 • C). According to the melt curve study, no non-specific products were generated.

Protein Expression and Localization of SuFu via Immunohistochemistry (IHC)
The protein expression and localization of SuFu via IHC was conducted in the Department of Pathology, SKIMS.

Protocol for IHC
Paraffin blocks were sectioned into 5 m thick tissue sections, and those sections were then mounted on charged poly-L-lysin-coated glass slides (Bio-Optica Milano S.p.a via San Faustino, 58 20134 Milan, Italy LOT #180310). Following deparaffinization in xylene, the slides were rehydrated with ethanol in a graduated sequence of concentrations (100 percent, 95 percent, 90 percent, 80 percent, and 70 percent), followed by distilled water. The sections were covered with hydrogen peroxide (Biocare Medical, Pacheco, CA, USA) to prevent peroxidase activity from occurring naturally, and then, incubated in a humid atmosphere for 15 min. Phosphate-buffered saline (PBS) (pH = 7.4) was then used to wash the sections two to three times. In order to recover antigens, the slides were heated to 95 degrees Celsius in 10 mM citrate buffer (pH 6.0). Then, PBST was used to wash the slides. After that, a protein block (Biocare Medical, USA #BS966G) was added to stop any background tissue staining that was not specific for 15 min. The portions were then washed using PBST (1× PBS with Tween 20). To indicate the borders of the tissue sections, a PAP pen was used to properly dry the slides without disrupting the tissue sections (Abcam, Cambridge, UK). Next, entire sections were incubated with anti-SuFu (HPA008700; 1:200) primary antibody overnight at 4 • C before being washed in PBST the following day. The samples were incubated for 30 to 40 min with goat anti-rabbit secondary antibody that had been HRP-conjugated (MACH 2 Universal HRP-Polymer Detection Kit; Catalog no. #M2U522G). PBST was applied to the sections two to three times. The HRP/DAB Detection IHC kit was used to color the sections (cat. no. BDB2004H; Biocare). The sections were once more PBST-washed three times. Following immunoreactivity, the slides were submerged in distilled water and counterstained with hematoxylin. The sections were then dehydrated in xylene and alcohol and mounted with DPX, and the cover was slipped. A light microscope (181; Olympus, 1 81 Tokyo, Japan) was used to view the slides. In the negative controls, primary antibody was not added to the tissue sections, and phosphate-buffered saline (PBS) was used.

Evaluation of IHC
The evaluation of the IHC slides and images was undertaken by two expert pathologists independently. The intensity of staining was measured using the IHC Profiler plugin for ImageJ software. Comparing adjacent histological normal slides to the tumor slides, staining intensities were determined. The staining intensity was designated as 0 (negative), 1 (weak), 2 (moderate), or 3 (high) and the percentage proportion of cells stained as 0 (0%), 1 (25%), 2 (26-50%), 3 (51-75%), or 4 (>75%). A scoring system (IRS = immunoreactive score) [40] with some modification was used to evaluate the IHC slides. This was achieved by multiplying the staining intensity by the percentage proportion of cells stained. An optimal cut-off score was identified and accordingly set for high and low expression of SuFu in CRC tumors cells. An IRS ≤ 4 was defined as tumors having low SuFu expression.

Follow-Up
A follow-up examination was undertaken once every three months during the first two years following surgery, and once every six months until 3 years after surgery. Patients were followed up in the outpatient department (OPD) or by phone. The patient communication deadline was 2 April 2022. We estimated survival intervals based on both diagnosis and surgery dates.

Ethics
Before surgery, all patients were informed about the study, which was authorized by the SKIMS Ethical Clearance Committee under protocol no: RP 70/2019.

Statistical Analysis
SPSS (v.26), Graph Pad Prism 8, and MedCalc were used for statistical analysis. The Shapiro-Wilk normality test was used to ensure that the data were distributed normally.
Continuous variables with normal distributions were reported as means ± standard deviation (SD), and differences between them were evaluated using the Student's t-test. Mann-Whitney U tests were used to compare groups of data that did not fit the normal distribution. For categorical variables, the Chi-square test was applied. Spearman's correlation was used to examine the relationship between two continuous variables that contradicted the normal distribution assumption, and the Kruskal-Wallis one-way ANOVA test was employed to examine group differences between more than two groups. Receiver-operating characteristic (ROC) curves were utilized to determine the diagnostic value of RDW, PLR, NLR, and HPR, and the Youden index was used to establish the appropriate cut-off value for all of them to distinguish between CRC patients and healthy controls. To compare survival rates between groups, the Kaplan-Meier method and the log-rank test were utilized. For multivariate analysis, the Cox proportional hazard model was used. All tests were two-tailed, with a p-value of <0.05 considered statistically significant.

Patient Characteristics
The expression of SuFu was assessed in 98 histopathologically validated tissues and adjoining normal tissues that had not received chemo or radiotherapy. Table 1 presents the study population's demographic and clinicopathological characteristics. The patients included 57 (58.16%) males and 41 (41.83%) females. The mean age was 57.51 ± 13.9, and 29 (29.59%) were younger than the age of 50 years, whereas 69 (70.40%) patients were older than or equal to 50 years. Among all the subjects, 69 (70.40%) lived in rural areas and 29 (29.59%) lived in urban areas.

SuFu mRNA Expression in CRC
Overall, low expression was seen in 69 (70.40%) of the malignant CRC tumor tissues relative to the adjacent normal tissues. As shown in Figure 2, the average fold change in SuFu was 0.550 ± 0.44.
When comparing the relative mRNA expression of tumor and adjacent normal tissues in terms of ∆ct values, overall, malignant tumors displayed reduced expression, compared to adjacent normal tissues, as shown in Figure 3.

Comparison of SuFu mRNA Expression with Various Clinicopathological and Laboratory Parameters
To gain a better understanding of the clinical importance of SuFu expression in colorectal cancer, we evaluated SuFu expression using a number of clinicopathological and laboratory parameters. The expression of SuFu was significantly associated with gender, education, passive smoking, stage, node status, and recurrence (all parameters have p < 0.05). There was no association with age, blood group, social class, junk food, tumor depth, tumor site, tumor size, and various other parameters listed in Table 2.

Comparison of SuFu mRNA Expression with Various Clinicopathological and Laboratory Parameters
To gain a better understanding of the clinical importance of SuFu expression in colorectal cancer, we evaluated SuFu expression using a number of clinicopathologica and laboratory parameters. The expression of SuFu was significantly associated with gender, education, passive smoking, stage, node status, and recurrence (all parameter have p < 0.05). There was no association with age, blood group, social class, junk food tumor depth, tumor site, tumor size, and various other parameters listed in Table 2.

SuFu Protein Expression and Localization via IHC
Immunohistochemistry was used to evaluate the expression and localization of the SuFu protein. SuFu was downregulated in a higher number of tumor samples (n = 62, 63.2%) than adjacent normal tissues and was predominantly localized in the nucleo-cytoplasm, followed by the nucleus and the cytoplasm. The staining was mostly moderate-to-strong in normal adjacent samples. SuFu was less expressed and downregulated in high-grade tumors. Figure 4 represents the staining pattern of SuFu in CRC tumors and adjacent normal tissues. The nucleo-cytoplasmic, nuclear, cytoplasmic, and mucinous staining patterns for SuFu are illustrated in Figure 5. SuFu expression was significantly correlated with tumor differentiation (grade), tumor invasion depth, stage, PNI, LNM, node status, recurrence, and vital status. The correlation was statistically significant (all parameters had p-value < 0.05). However, there was no association between SuFu expression and other parameters such as tumor site, tumor size, and other variables, as indicated in Table 3.
Further investigation was carried out to determine whether the reduced SuFu expression at the transcriptional and translational levels are related. On comparison, we found that 53 (82.81%) of the malignant tumor tissues that displayed reduced expression at the mRNA level also showed decreased SuFu expression at the protein level. The change was significant. Tumor tissues that displayed low mRNA SuFu expression also expressed low SuFu protein (p < 0.001), as presented in Table 4.

SuFu Protein Expression and Localization via IHC
Immunohistochemistry was used to evaluate the expression and localization of the SuFu protein. SuFu was downregulated in a higher number of tumor samples (n = 62, 63.2%) than adjacent normal tissues and was predominantly localized in the nucleo-cytoplasm, followed by the nucleus and the cytoplasm. The staining was mostly moderate-to-strong in normal adjacent samples. SuFu was less expressed and downregulated in high-grade tumors. Figure 4 represents the staining pattern of SuFu in CRC tumors and adjacent normal tissues. The nucleo-cytoplasmic, nuclear, cytoplasmic, and mucinous staining patterns for SuFu are illustrated in Figure 5. SuFu expression was significantly correlated with tumor differentiation (grade), tumor invasion depth, stage, PNI, LNM, node status, recurrence, and vital status. The correlation was statistically significant (all parameters had p-value < 0.05). However, there was no association between SuFu expression and other parameters such as tumor site, tumor size, and other variables, as indicated in Table 3.

SuFu Protein Expression and Localization via IHC
Immunohistochemistry was used to evaluate the expression and localization of the SuFu protein. SuFu was downregulated in a higher number of tumor samples (n = 62, 63.2%) than adjacent normal tissues and was predominantly localized in the nucleo-cytoplasm, followed by the nucleus and the cytoplasm. The staining was mostly moderate-to-strong in normal adjacent samples. SuFu was less expressed and downregulated in high-grade tumors. Figure 4 represents the staining pattern of SuFu in CRC tumors and adjacent normal tissues. The nucleo-cytoplasmic, nuclear, cytoplasmic, and mucinous staining patterns for SuFu are illustrated in Figure 5. SuFu expression was significantly correlated with tumor differentiation (grade), tumor invasion depth, stage, PNI, LNM, node status, recurrence, and vital status. The correlation was statistically significant (all parameters had p-value < 0.05). However, there was no association between SuFu expression and other parameters such as tumor site, tumor size, and other variables, as indicated in Table 3.

Comparison of Laboratory Parameters between CRC Group and Healthy Controls
The median serum level of CEA and CA-19.9 in CRC patients was 5.60 (IQR: 2.14-17.36) and 20.35 (IQR: 8.95-46.08). The comparison of the two groups is shown in Table 5. The neutrophil, RDW, PLR, NLR, and PLT values were significantly higher in the CRC group than in the healthy controls. The difference was statistically significant (for all parameters, p < 0.05). However, HB, HPR, and lymphocytes were lower in the CRC group compared to the healthy controls. The difference observed was significant (p < 0.05 for all). However, there was no difference in age and WBC count.

Correlation of Laboratory Parameters with Different Clinicopathological Parameters in Patients with CRC
This relationship is summarized in Table 6. RDW showed a significant difference with tumor site, necrosis, node status, perineural invasion (PNI), tumor depth, and stage (p < 0.05 for all). HPR showed a significant difference with necrosis, tumor depth, stage, lymphovascular invasion (LVI), tumor size, and CEA (p < 0.05 for all). PLR showed a significant difference with necrosis and distant metastasis (p < 0.05 for both). NLR showed a significant difference with tumor-associated lymph node response (TALNR) and tumor site (p < 0.05 for both).

Differentiating Colon Cancer from the Normal Healthy Control Group
When differentiating colon cancer from a healthy control group, the laboratory parameters RDW, PLR, NLR, and HPR were assessed for sensitivity, specificity, positive and negative likelihood ratios, and area under the receiver operating characteristic curves (AUC). Youden's index set the cut-off value. Table 7 and Figure 6A show that RDW, PLR, NLR, and HPR had a sensitivity of 73.5%, 79.6%, 59.18%, and 72.45%, respectively, in discriminating colon cancer from healthy controls. RDW had the highest specificity (92.9%). When PLR, NLR, and RDW were combined, the AUC (0.91; 95% CI: 0.86-0.94) was higher than when PLR (AUC = 0.84), NLR (AUC = 0.78), RDW (AUC = 0.87), and HPR (AUC = 0.79), ( Figure 6B) were utilized alone.  Figure 6. (A) Analysis of receiver operating characteristic curves for RDW, NLR, and PLR biomarkers, used alone or together, for differentiating colon cancer patients from healthy controls. Figure 6. (A) Analysis of receiver operating characteristic curves for RDW, NLR, and PLR biomarkers, used alone or together, for differentiating colon cancer patients from healthy controls. (B) Analysis of receiver operating characteristic curve for HPR biomarker used alone for differentiating colon cancer patients from healthy controls. NLR: neutrophil-to-lymphocyte ratio; PLR: Platelet-to-lymphocyte ratio; HPR: hemoglobin-to-platelet ratio; RDW: red cell distribution width. Figure 7A-C, depicts the relationship between PLR and NLR, HPR and RDW, and HPR and PLR. In CRC patients, there was a strong positive correlation between PLR and NLR (r = 0.72, p < 0.001). HPR showed a moderate negative correlation with RDW (r = −0.49, p < 0.001), and a weak negative correlation was found between HPR and PLR (r = −0.30, p < 0.001).

HPR Value's Association with Cancer Stage and Tumor Invasion Depth
To compare HPR values among different cancer stages and tumor invasion depths, a Kruskal-Wallis one-way Analysis of Variance (ANOVA) was employed. As shown in Figure 8A,B, there was a significant association of HPR with stages I and III (p < 0.012) and with T2 and T4 (p < 0.021). Figure 7A-C, depicts the relationship between PLR and NLR, HPR and RDW, and HPR and PLR. In CRC patients, there was a strong positive correlation between PLR and NLR (r = 0.72, p < 0.001). HPR showed a moderate negative correlation with RDW (r = −0.49, p < 0.001), and a weak negative correlation was found between HPR and PLR (r = −0.30, p < 0.001).

HPR Value's Association with Cancer Stage and Tumor Invasion Depth
To compare HPR values among different cancer stages and tumor invasion depths, a Kruskal-Wallis one-way Analysis of Variance (ANOVA) was employed. As shown in Figure 8A,B, there was a significant association of HPR with stages I and III (p < 0.012) and with T2 and T4 (p < 0.021).

HPR Value's Association with Cancer Stage and Tumor Invasion Depth
To compare HPR values among different cancer stages and tumor invasion depths, a Kruskal-Wallis one-way Analysis of Variance (ANOVA) was employed. As shown in Figure 8A,B, there was a significant association of HPR with stages I and III (p < 0.012) and with T2 and T4 (p < 0.021).

Prognostic Analysis of Various Clinicopathological Parameters
A number of pathological parameters were assessed for prognostic significance in CRC by conducting univariate survival analysis with the 3-year disease-free survival (DFS) and overall survival (OS) rates of the patient cohort (Table 8). Stage, PNI, node status, LNM, and SuFu expression were all significantly linked with both 3-year OS and 3-year DFS. However, CA 19-9 exhibited a significant correlation with DFS rate (p < 0.05). The prognosis was not significantly influenced by the tumor grade, tumor depth, lymphovascular invasion (LVI), or tumor site. Figure 9, depicts Kaplan-Meir survival curves, based on IHC for 3-year OS and DFS dependent on SuFu expression, stage, and PNI. Lower suFu expression, PNI positivity, and presence of positive axillary nodes were all associated with the worst OS and DFS rates (p < 0.05). Based on Cox regression analysis (Table 9), a significantly independent predictive association was observed between CA-19.9 protein and 3-year DFS (p < 0.05).
Finally, we correlated various lab inflammatory blood biomarkers with SuFu expression (Table 10). Although RDW, PLR, and NLR values were higher in most of the patients who displayed low SuFu expression, the association was not statistically significant. The HPR values were decreased in most of the patients exhibiting low SuFu expression, but the findings were not significant.

Discussion
In the present study, SuFu mRNA and protein expression were evaluated in histopathologically confirmed tumor tissues and adjacent normal tissues. The findings show that SuFu is downregulated in CRC tumor tissues at both the mRNA and protein levels with predominant nucleo-cytoplasmic localization. Patients with low SuFu expression exhibited poor prognosis. Further high-grade tumors exhibited significantly lower SuFu expression than low-grade tumors.
We found that 70.4% of CRC patients had lower SuFu mRNA expression, which is comparable to previous observations by wang et al. [41] We, for the first time, evaluated SuFu expression in relation to a number of demographic and clinicopathological variables and found that low SuFu expression is associated with late stage, the presence of positive axillary lymph nodes, lower education level, recurrence, female sex, and passive smoking. An earlier study demonstrated that SuFu expression is correlated with tumor invasion depth and tumor diameter [41]. Education is believed to be inversely related to cancer risk [42,43]. Even though a lower education level does not cause cancer at the molecular level, it may impact the risk through behavior, standard of living, and environmental exposure. However, in the current study, a high proportion of participants were from the lower education group. Even though the correlation is statistically significant, it may be coincidental. More studies with larger sample sizes could provide a more accurate picture. Passive smokers exhibited a significantly low expression of SuFu. A previous study conducted on our population by Rafiq et al. [44] linked second-hand smoking with esophageal cancer risk. Moreover, abnormal expression in females, especially in rural areas, could be attributed to poor house design leading to inadequate ventilation, which exposes them to different pollutants such as cooking fumes [45].
IHC analysis revealed that SuFu was downregulated in 63.3% of the malignant tumor tissues relative to adjacent normal tissue samples. The majority of the malignant tumor samples displayed nucleo-cytoplasmic localization of SuFu, followed by the nucleus and the cytoplasm. In a previous study by Tostar et al., SuFu was found to be weakly expressed and predominant in the cytoplasm of tumor muscle cells [46]. SuFu was expressed in normal colon tissues. This is evident, because in species ranging from invertebrates to vertebrates, it plays a crucial part in embryogenesis and adult tissue homeostasis. SuFu has vital functions in mammals, as evidenced by the embryonic lethality of SuFu deletion in mice [47]. However, our results are not in agreement with the findings of Wang et.al, who found no SuFu in normal colonic mucosa in their study [41]. Lower levels of SuFu protein were linked to high-grade tumors, tumor invasion, late stage, node presence, LNM positivity, PNI positivity, CRC recurrence, and vital status. Our findings indicate that SuFu is downregulated and weakly expressed in high-grade tumors in CRC relative to lower-grade tumors. This is the first study that reports the downregulation of SuFu along higher grades in CRC tumors. Interestingly, just one study with a very small sample size reported similar findings in tumors originating from glioblastoma [48]. Based on our results, SuFu has promising prognostic implications. Moreover, the association between low SuFu expression and LNM, PNI, and late-stage CRC suggests that SuFu is associated with the worst pathological features and advanced stages of the disease.
We also found that a low SuFu expression, in addition to shorter and worse overall survival, is associated with poor disease-free survival. Positive axillary lymph nodes, PNI, and LNM showed similar relationships with OS and DFS. Similarly, patients in stage III and IV demonstrated the shortest and worst overall survival and disease-free survival rates. According to our findings, in addition to stage, low SuFu expression, PNI, and LNM are connected to CRC metastases and have a possible prognostic role. According to the Cox regression analysis, the best predictor for DFS was preoperative CA-19.9 levels (RR: 3.7, 95% CI: 1.32-7.11, p < 0.05). Based on these findings, we can say that preoperative CA-19-9 has a significant role in predicting the prognosis for colon cancer patients receiving surgery.
In this study, we found that RDW values were significantly increased in advanced tumor invasion depth (T3 and T4), later stages (III and IV), PNI, necrosis, node status, and tumor site (p < 0.05 for each parameter). No association was found between RDW values and lymph node metastases (LNM), tumor configuration, and tumor size. An earlier study conducted by Shi et.al, reported that elevated RDW is associated with tumor type, tumor invasion depth (T status), clinical stage, and histological type [49]. However, our study did not find any significant differences in RDW values in tumors of the colon or rectum. However, our study is the first study to report that right colon tumors are associated with higher RDW values than left colon tumors. To the best of our knowledge, this is also the first study that has revealed the association of RDW with PNI and necrosis in CRC. Similarly, Elevated PLR was found in DM and necrosis (p < 0.05 for both). A previous study conducted by Hu et al. reported elevated PLR in DM [23], but Mo et al. did not find any association between PLR and DM [50]. A significant association between elevated PLR and necrosis was also noted. This is the first study to reveal the link between necrosis and elevated PLR in CRC. However, the underlying mechanism is still largely unknown. Additionally, earlier studies did not include many clinicopathological data. Further, for the first time, our study also reported elevated NLR in tumors of the transverse colon and in patients where tumor-associated lymph node response (TALNR) was seen (p < 0.05 for both). Additionally, lower HPR values were associated with tumor stage (III and IV), tumor invasion depth (T3 and T4), LVI, necrosis, tumor size (≥4 cm), and high CEA level (p < 0.05 for all). These findings are consistent with the results given by Hu et al. and Mo et al. in their previously conducted studies [23,50].
Further, the diagnostic efficacy of RDW was excellent (AUC = 0.87) (p < 0.05), and was much higher than that reported by Song et al. [21] and Shi et al. [49] in separate studies, suggesting the greater utility of RDW as a diagnostic parameter in our population. The diagnostic value of PLR (AUC = 0.84) and HPR (AUC = 0.79) was also higher than in studies conducted by Mo et al. [50] and Hu et al. [23], respectively. To the best of our understanding, NLR has not been used or tested for diagnostic purposes so far. In our study, NLR yielded moderate diagnostic utility (AUC = 0.78) (p < 0.05). However, upon combining RDW, NLR, and PLR, the diagnostic performance was found to be excellent, and a larger area under the curve was obtained (AUC = 0.91, 95% CI: 0.86-0.93, p < 0.05). Our results suggest that the combined use of RDW, NLR, and PLR may greatly improve the diagnostic efficacy of differentiating CRC from normal healthy controls compared to using them alone. Further, in CRC patients, we conducted a correlation analysis for RDW, NLR, PLR, and HPR. We found a strong positive correlation between PLR and NLR (r = 0.72, p < 0.05), a moderate negative correlation between RDW and HPR (r = −0.49, p < 0.05), and a weak negative correlation between HPR and PLR (r = −0.30, p < 0.05). The exact mechanism for such a relationship is still not clear and no studies that reported such findings are available yet. We further stratified CRC subjects based on stage (I, II, III, and IV) and tumor invasion depth (T1, T2, T3, and T4). Interestingly, we found that HPR was significantly decreased in stage III CRC patients and in advanced tumor invasion depth (T4), when compared with stage I and tumor invasion depth (T2) (p < 0.05 for both).
Lastly, we evaluated whether the mRNA expression of SuFu at the genetic level would affect RDW, PLR, NLR, HPR, and CEA levels. We wanted to identify whether abnormal parameter values and low expression were interconnected. A relationship between them was not established. Both low expression and altered levels of these parameters may play a role in CRC pathogenesis independently. It is nevertheless possible for this study to serve as a platform for future investigations, guiding them toward a combination approach at both the expressional and clinical levels.
Unlike the previous studies mentioned above [50][51][52][53][54], our study was a prospective one. In addition, both blood and tissue samples were included. A proper follow-up procedure was followed. Due to time and resource constraints, we were unable to ascertain the MSS/MSI status of tumors, which was the study's principal shortcoming. This study's survival proportion was affected by the fact that it lasted for only three years.

Conclusions
SuFu was downregulated in CRC tumor tissues at both the mRNA and protein levels. Pathological features such as high-grade tumor, nodes, PNI, and LNM were associated with low SuFu expression. The results of our study indicate that in addition to stage, low SuFu expression, PNI, and LNM have excellent prognostic value. SuFu may be a potential therapeutic target in CRC. Furthermore, RDW, PLR, NLR, and HPR were associated with a range of clinicopathological variables, several of which had not previously been reported. Using RDW, PLR, and NLR together may provide excellent diagnostic ability to differentiate CRC from healthy control groups. In patients with colon cancer who are undergoing surgery, the preoperative CA-19-9 level can be a significant predictor of disease-free survival.