Differential Expression of HER2 and SKP2 in Benign and Malignant Colorectal Lesions

Background: Colorectal cancer (CRC) is the fourth most common cancer worldwide. Both HER2 and SKP2 have a carcinogenic role in CRC making them attractive targets for tailored treatment. This work aims to correlate HER2 and SKP2 protein expression as well as HER2 gene amplification with clinicopathological parameters aiming at identifying potential candidates for targeted therapy. Methods: This Study was conducted on 127 paraffin-embedded tissue samples of different colorectal lesions [controls, chronic colitis, ulcerative colitis (UC), hyperplastic polyps (HPs), adenomas and CRCs] to investigate HER2 and SKP2 expression by immunohistochemistry (IHC), Selected CRC cases [equivocal (2+) and positive (3+) by IHC] were further evaluated by ISH (CISH and SISH ) to assess HER2 gene amplification. Results: Chronic colitis, UC, HPs and adenomas were HER2-negative. HER2 positivity (scores 2+ and 3+) was found only in15% of CRCs. Both SISH and CISH showed the same results with high concordance as 66.7% of equivocal and 100% of positive cases showed amplification of HER2 gene. SKP2 positivity was detected in 26.7% and 45% of adenomas and CRCs respectively, while other studied groups were negative. A significant correlation was noted between HER2 and SKP2 expression. Conclusion: A small percent of CRCs exhibited HER2 gene amplification, which would be potential candidates for anti HER2 therapy whereas IHC could be a primary screening test for patient selection. A potential carcinogenic role of SKP2 was suggested by the findings that SKP2 expression was undetectable in normal colonic mucosa but significantly increases from adenoma to carcinoma, hoping adenoma patients to get benefit from targeted therapy.


Differential Expression of HER2 and SKP2 in Benign and Malignant Colorectal Lesions
dietary, familial and hereditary) that become relevant during the different stages of colorectal carcinogenesis (Jemal et al., 2011). Various genetic alterations and distinct molecular phenotypes for each tumor influence patient's prognosis and response to chemotherapy. The choice of treatment depends on the identification of these particular molecular phenotypes. Targeted therapy is more efficient in responders and avoids unnecessary side-effects in nonresponders (Hagan, Orr and Doyle 2013).
HER2 is a well-recognized mediator of the carcinogenic process. It has a role in a wide range of solid tumors, mainly via protein overexpression and/ or gene amplification, thus making HER2 an attractive target for tailored treatment (Sartore-Bianchi et al., 2016;Wakatsuki et al., 2018). Although there have been many reports on the frequency of HER2 over-expression in colon cancer, the role of this oncogene in such cancer is not clearly defined (Erik et al., 2013). The most commonly used method to determine HER2 status is immunohistochemistry (IHC), which is a low-cost technique that can be performed on small samples, even formalin-fixed and paraffin-embedded tissues. Fluorescent in situ hybridization (FISH) is considered the gold standard and can be used to analyze this type of sample. An alternative is provided by the use of other in situ hybridization methods such as silver in situ hybridization (SISH) and chromogen in situ hybridization (CISH) which allows the use of an ordinary light microscope and has shown excellent correlation with results obtained using FISH (Abrahão-Machado et al., 2013). As the accurate assessment of HER2 gene amplification status in CRC appears to be particularly important for patients who might undertake this specific targeted therapy (Zhang et al., 2020).
S-phase kinase-associated protein 2 (SKP2) belongs to the F-box protein family. SKP2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for degradation, including cyclin-dependant kinase inhibitor p27. SKP2 has also been demonstrated to display an oncogenic function since it's over expression has been observed in many human cancers including CRC (Shapira et al., 2005). SKP2 may be a promising therapeutic target for colorectal cancer, and development of SKP2 inhibitors would have a great impact on colorectal cancer therapy (Bochis et al., 2015).
This work aims to correlate HER2 and SKP2 protein expression as well as HER2 gene amplification with clinicopathological parameters aiming at identifying potential candidates for targeted therapy.

Specimens
The material of this study were collected from 127 histologically documented cases with different colonic lesions from archives of Surgical Pathology Departments of Theodor Bilharz Research Institute (TBRI) and Faculty of Medicine, Cairo University, Egypt in the period from January 2015 to October 2017. The specimens were obtained either as endoscopic biopsies (109)

Histopathological Examination
Serial sections were cut from paraffin blocks and stained with hematoxylin and eosin for routine histological examination. CRC classification, grading and staging were carried out following the 2017 AJCC staging system (Jessup et al., 2017). According to TNM classification (Edge et al., 2010), in colectomy specimens (n:18), CRC were classified as 12 specimens in T3 and 6 in T4; 6 in N0, 6 in N1 and 6 cases in N2.

Immunohistochemical (IHC) Technique
Four-µm thick sections from formalin-fixed, paraffin-embedded colonic tissue were cut on charged slides. Antigen retrieval was performed with 10 ml sodium citrate buffer, pH 6.0, at 90°C for 30 min. Sections were incubated in 0.03% hydrogen peroxide for 10 min at room temperature, then in blocking serum (0.04% bovine serum albumin, A2153, Sigma-Aldrich, Shanghai, China), and 0.5% normal goat serum X0907, Dako Corporation, Carpinteria, CA, USA, in PBS) for 30 min at room temperature. Polyclonal antibodies for HER2/neu (A0485 Dako Denmark) and SKP2 (Chongqing Biospes Co., Ltd China) were applied at an optimal dilution of 1:700 and 1:200 respectively and incubated overnight at 4°C. Staining was developed with diaminobenzidine substrate and sections were counterstained with hematoxylin.
For each setting, positive and negative control slides were included. As a negative control, colon biopsy was processed in the above mentioned sequences but PBS was added instead of the primary antibody.
Breast duct carcinoma and high grade prostate carcinoma were used as internal positive controls for HER2 and SKP2 respectively.
SKP2 was expressed as nuclear or cytoplasmic staining. Cases with more than 20% positively stained colon cells were considered SKP2 positive (Ni et al., 2009).

Insitu Hybridization (ISH) technique
Equivocal and positive HER2 cases were evaluated for HER2 gene status by insitu hybridization which were silver in situ hybridization (SISH) and chromogen in situ hybridization (CISH) for detection and confirmation of HER2 gene amplification especially in cytoplasmic expressed cases. In each setting, a case negative for IHC HER2 staining (score 0/1+) was enrolled as a control.

Silver Insitu Hybridization (SISH) technique and interpretation
The BenchMark Series Automated Slide Stainer with HER2 Dual ISH DNA Probe Cocktail, reagents from UltraView Red ISH DIG and UltraView SISH DNP Detection Kits (Roche Tissue Diagnostics, VENTANA Medical Systems, USA) were used. HER2 gene was presented by a black signal, while chromosome 17 was presented by a red signal. Tumor cells were scanned for hot spots by using x20 or x40 objectives and the area with the highest signal was selected. The signals were counted in 20 non-overlapping tumor cell nuclei from each case using x40 or x100 objectives. Small or large clusters were considered to be 6 signals and 12 signals respectively.
HER2 gene amplification was defined as detection of HER2 gene /CEP 17 ratio of ≥2 in 20 tumor nuclei. Normal colon epithelial cells and other adjacent benign cells served as internal controls (Valtorta et al., 2015). test was used. Comparison between percent positive cases and staining intensity were calculated by Chi-Square test. Correlations between variables were studied using Spearmann's correlation test. P value < 0.05 was considered statistically significant.

Results
The study was performed on 105 specimens of colorectal lesions and 22 controls. Patient and groups characteristics are listed in Table 1.
In both conventional and mucinous subtypes of CRC, 6.3% and 9.3% of conventional subtype were of 3+ and 2+ score respectively compared to 12.5% of mucinous cases

Chromogen insitu hybridization (CISH) technique and interpretation
Manual HER2 CISH staining was done in Tissue Culture Lab, National Cancer Institute, Cairo, Egypt using Vortex the ZytoDot 2C SPEC ERBB2/CEN 17 Probe Kit (ZytoVision; Germany). A minimum number of 20 tumor cells per sample were evaluated. A red signal presents chromosome 17 and a green signal presents HER2 gene. Tumor cells were scanned for hot spots by using x20 or x40 objectives and the area with the highest signal was selected. The signals were counted in 20 non-overlapping tumor cell nuclei from each case using x40 or x100 objectives. Small or large clusters were considered to be 6 signals and 12 signals respectively.
HER2 gene amplification was defined as detection of HER2 gene /CEP 17 ratio of ≥2 in 20 tumor nuclei. Normal colon epithelial cells and other adjacent benign cells served as internal controls (Heppner et al., 2014 andHanna et al., 2014).

Statistical Analysis
SPSS software version 23 was used for statistical analysis (IBM Corporation, Armonk, New York, USA). Quantitative data were presented as mean + SD. For comparison of more than three groups; One way ANOVA Regarding tumor depth and nodal metastasis, HER2 positivity, pattern of expression and intensity of expression do not show statistical significant difference in relation to different T and N stages (Table 3).
Evaluated by spearman correlation test, we found significant positive correlation between HER2 and SKP2 immunopositivity among studied cases (r= 0.388, p=0.000).

Discussion
Colorectal cancer (CRC) is characterized by compounding genetic mutations in both oncogenes and tumor-suppressor genes that drive its initiation and promotion under various pathophysiological conditions (Seigel et al., 2011). HER2 mutation is important for clinical treatment and prognosis evaluation in cancer patients and has been found to be a predictive marker to HER2-targeted therapy in breast and gastric cancer (Calhoun and Collins, 2015;Jiang et al., 2018). Several studies evaluating HER 2 in CRC resulted in a large debate    Osako et al., 1998). The current study was conducted on randomly collected archived paraffin blocks of 105 cases with different colonic lesions. In different studied groups, age ranged from 15-77 years with male predominance (51.5%) which was going with Bochis et al., (2017). The majority of CRC patients were males (57.5%) and mean age was 55 years. Two Egyptian studies by Mohamed andTealeb, 2015 andElwy et al., (2012) demonstrated similar results where 70.6% and 60% of their studied patients respectively were males and mean age was 51 and 49.3 years respectively. These results differ from what was reported by Cressey et al., (2006) who detected a higher incidence of CRC in females (63%). Furthermore, studies done by Terzi et al., (2008) and Office for National Statistics, 2011 reported CRC incidence in a higher age than ours (> 60 years). These differences may be due to random collection of cases and different etiologic and predisposing factors for CRC in Egypt compared to other countries.
We found a high statistical significant difference of HER2 expression in CRC group compared with other studied groups which were all negative. This goes with results of Heidari et al., (2017) who found a higher significant difference comparing CRCs to adenomas and normal tissues. Also, Pazurek et al., (2009) found lower expression of HER2 in colon adenoma compared with cancer group by using PCR method.
The food and drug administration (FDA) approved scoring system for breast cancer which is entirely based on membranous HER2 overexpression with strict guidelines to ignore cytoplasmic expression as it does not correlate to any clinical outcome (Walker et al., 2008). In contrast to breast cancer, there is evidence that in CRC; cytoplasmic HER2 could be associated with survival prognosis; as it may be involved in tumor pathogenesis like membranous HER2 in breast cancer (Blok et al., 2013). Intracellular HER2 targeting compounds might be attractive treatment option in one third of CRC patients where cytoplasmic HER2 is actively involved in carcinogenesis of CRC (Seo et al., 2014).
Regarding subtypes of CRC, 5/6 of our positive and equivocal tumors were of conventional subtype but we found no statistically significant relationship between the subtype and HER2 pattern; which matches results of Kruszewski et al., (2010), Elwy et al., (2012) and Mohamed and Tealeb, (2015). In contrast to our results; Shabbir et al., 2016 observed more common membranous HER2 staining in mucinous CRC while more frequent cytoplasmic staining in non-mucinous types.
With respect to grade of differentiation in CRC cases, 4/6 of the positive and equivocal tumors were of low grade and exhibit mainly strong cytoplasmic/membranous expression, however no significant relationship was observed between CRC grades and HER2 pattern, which is consistent with several studies (Elwy et al., 2012;Pappas et al., 2013;Sayadnejad et al., 2017). However, several other studies reported the contrary. Half and his colleagues, 2004 found a significant relation between the cytoplasmic HER2 staining and tumor differentiation. Also, Shabbir et al., (2016) concluded a significant strong association between cytoplasmic HER2 expression and low grades of CRC, as well as between membranous HER2 expression and high grade CRC.
In agreement with Song et al., (2014), Shabbir et al., (2016) and Sayadnejad et al., (2017) we did not find a significant link between HER2 expression and tumor stage or lymph node metastasis (TN stage). On the contrary, Elwy et al., (2012) and Heppner et al., (2014) reported a significant association with higher stages and positive nodal status.
In our study, SISH and CISH for HER2 were applied on 6/40 CRCs. Theses 6 CRC specimens were expressing equivocal (3) and positive (3) staining by IHC. HER2 scoring demonstrated high concordance rates between dual-color SISH and CISH methods as the results of the two techniques were almost identical showing 5 out of these 6 cases i.e 5/40 CRCs (12.5%) proved true for HER2 gene amplification. Our results were not far from studies of Valtorta et al., (2015) and Zhang et al., (2020) who reported that 5% and 5.63% of their CRC patients had HER2 amplification. Heppner et al., (2014) and Seo et al., (2014) stated that HER2 amplification ranged from 1.6% to 6.3%. However, a Korean study by Park et al., (2007) showed that HER2 expression rate was 47%. There are several possible reasons for this discrepancy such as ethnic diversity, technical variability in the IHC performance, sample size, heterogeneity of study population, racial differences, and varied experimental designs (Li et al., 2014;Seo et al., 2014). Another key subject is lack of agreement about whether only membranous, cytoplasmic or both should be considered for evaluation of HER2 overexpression (Pappas et al., 2013).
Researches about the SKP2 signaling suggest that SKP2 targeting may be a very attractive approach to treat human cancers. Chen et al., 2014 confirmed the hypothesis that SKP2 siRNA (small interference RNA) may be a useful therapeutic protocol for the treatment of colon carcinoma.
Our results showed a high statistically significant SKP2 expression in CRC group compared with other studied groups. In our study; 45% of CRC cases were positive to SKP2, this matches results of Li et al., (2004), Ni et al., (2009) and Tian et al., (2013) who found that 50%, 48% and 47.6% of their studied cases respectively expressing SKP2.
No statistically significant relationship was detected between histologic subtypes of CRC and SKP2 immunostaining. This was the same finding of Uddin et al., (2008) on a large tissue microarray of 448 samples of mouse models.
With respect to the grade of CRC tumor differentiation, no significant relationship was observed in relation to SKP2 expression which was consistent with Bochis et al., (2017). On the contrary, Lu et al., (2009) andShen et al., (2018) found SKP2 to be highly associated with histological grade of tumor as SKP2 was highly expressed in poorly differentiated CRCs.
In agreement with Shapira et al., (2005), we concluded that there was no statistically significant link between expression of SKP2 in CRC cases and tumor stage or nodal metastases (TN) stage. In contrast, TNM stage was significantly correlated with the expression of SKP2 in the study of Bochis et al., (2017).
Regarding adenoma group, SKP2 positivity was detected in 26.7%. However, Ni et al., (2009) found SKP2 positivity in only 5% of their adenoma cases. In agreement with our results, Li et al., (2004) and Ni et al., (2009) reported that SKP2expression was sequentially increased from normal mucosa through adenoma to primary carcinoma.
Correlation of SKP2 expression in both types of adenoma (tubular and tubulovillous) and in different grades of associated dysplasia (low and high) showed no statistical significant difference. On the contrary, Li et al., (2004) found a significant increased SKP2 expression from mild through moderate to severe dysplasia in adenomas and these increases were confirmed by Western blot.
In a study done by Lui et al., (2012) on breast invasive carcinomas they found correlation between SKP2 and HER2 positivity. Our results showed a positive correlation between HER2 and SKP2 immunostaining in studied cases. To our knowledge, there is no other published studies examined the correlation between HER2 and SKP2 in benign or malignant colonic lesions.
In conclusion, the prognostic role of HER2 in CRC remains uncertain. Our findings may serve as a basis for future studies on patient selection for HER2 targeted therapy. Although a small percent of CRC patients exhibited HER2 gene amplification, these patients would be potential candidates for anti-HER2 therapy and IHC could be a primary screening test for patient selection. Additionally, more studies on the value of cytoplasmic HER2 expression in CRC must be done as if cytoplasmic HER2 has a pathophysiological role in CRC, intracellular HER2-targeting compounds might be a new treatment choice for these patients.
The potential carcinogenic role of SKP2 was suggested by the findings that SKP2 expression was undetectable in normal colon mucosa but significantly increases from adenoma to carcinoma, hoping these patients to get benefit from targeted therapy.

Funding
This work was financed by TBRI internal project No.113T. Principal investigator: Prof. Dr. Mona Moussa.