Assessment of Immunohistochemical Expression of CD44 and Osteopontin in Colorectal Carcinoma and colorectal adenoma

doi:10.21203/rs.3.rs-2574213/v1

Background

Colorectal cancer (CRC) is a foremost global health concern and remains one of the major causes of cancer-related morbidity and mortality. It is the third most common cancer in adults after lung cancer and breast cancer worldwide. The theory that cancer originates from a subpopulation of tumor cells, named cancer stem cells (CSC), they have important role of CSC in the initiation and maintenance of the tumor, as well as invasion, metastasis and therapeutic resistance. Among CSC markers, CD44 and OPN are two of the most investigated colorectal CSC markers and their proteins are introduced as the subpopulation with a greater tumorigenicity. This study aiming assessing the immunohistochemical expression of CD44 & OPN in colorectal adenomas & CRCs. And their relation between immunohistochemical expression of CD44 & OPN with tumor differentiation (grading), lympho-vascular invasion, perineural invasion, desmoplasia and TNM stage.

Methods

this is a retrospective descriptive study that included Sixty paraffin embedded blocks from the pathology laboratory, Suez Canal University Hospital. Paraffin blocks included (14 cases of colorectal carcinoma and 18 cases of colorectal adenoma). paraffin blocks reviewed for clinicopathological prognostic factors and stained by CD44 & OPN, monoclonal antibodies by immunohistochemical method.

Results

The CD44 protein was overexpressed in 80% of CRC, while was positive (44.4%) in adenoma this difference was statistically significant. Also, in this study the difference between the expression OPN in CRC and adenomas was statistically insignificant.

Conclusions

CD 44 is highly expressed in large number of CRC (80 of tumors). It is also significantly more expressed in CRC than in adenomas, suggesting a role of CD 44 in CRC tumorigenesis and progression of adenomas into carcinomas. Our study also associated CD 44 overexpression with both late TNM stage and lympho-vascular invasion.

Immunohistochemistry

Colorectal carcinoma

CD44

Osteopontin

Cancer stem cell

Stage of tumor

Colorectal carcinoma is the 3rd most common carcinoma in the world. It is also considered the 3rd most leading cause of death usually due to the presence of liver metastases ⁽¹⁾. In Egypt, it is the seventh among both sex ⁽²⁾.

Colorectal carcinomas are caused by nutritional habits, genetic causes, lifestyle, nutritional habits, and environmental factors. More cases of colorectal cancers are adenocarcinomas. The remaining of colorectal cancers are neuroendocrine carcinoma, malignant carcinoid, squamous cell carcinomas (SCC), lymphoma, and other very rare types ⁽³⁾.

Cancer stem cells (CSCs) are related to the tumor’s initiation ⁽⁴⁾. Numerous studies have shown that CSCs have an important role in colorectal carcinomas ^{(5) & (6)}.

CD44 is a cell surface adhesion marker that is expressed widely in most body cell types, including epithelial cells, leukocytes, tumor cells, and endothelial cells. The CD44 gene is located on chromosome 11 of the human genome ⁽⁷⁾.

CD44 is a marker of stem cells in CRC especially adenocarcinoma. CD44 is a surface adhesion protein which is also part of cell adhesion molecules that plays a role in cell-to-cell adhesion and interactions with the extracellular matrix and has a role in tumor proliferation, occurrence, and spread in which affect the prognosis and patient survival ^{(8) & (9)}.

Osteopontin (OPN) considered as secreted glycophosphoprotein, encoded by the SPP1 gene, located on chromosome 4q13 ⁽¹⁰⁾. It is believed that binding of OPN to integrins regulate neovascularization, tumor cell spreading and, binding of OPN to CD44 receptors controls immune system, adhesion, transformation of normal to cancerous cells and tumor growing process ⁽¹¹⁾. OPN thus influences the tumor growth, for example, resistance to cell death, maintenance of proliferative signaling, evasion of growth suppressors, invasive growth and metastasis, neo-angiogenesis, avoidance of tumor destruction by the immune cells and role of inflammatory reaction ⁽¹²⁾.

Although CD44 and OPN proteins have been tested as adhesion markers in colorectal carcinoma previously. Their prognostic value has not been elucidated clearly ⁽¹³⁾. This study aimed at assessing the immunohistochemical expression of CD44 & OPN in & CRCs. Also, it assessed the relation between immunohistochemical expression of CD44 & OPN with different prognostic parameters in CRC such as: tumor differentiation (grading), lympho-vascular invasion, perineural invasion, desmoplasia and TNM stage. This helps us to use CD44 & OPN in the immunohistochemical panel of colorectal carcinomas as a diagnostic factor.

The aim of this study aimed at assessing the immunohistochemical expression of CD44 & OPN in colorectal adenomas & CRCs. Also, it assessed the relation between immunohistochemical expression of CD44 & OPN with different prognostic parameters in CRC such as: tumor differentiation (grading), lympho-vascular invasion, perineural invasion, desmoplasia and TNM stage.

This study was carried out in the Pathology lab, Faculty of Medicine, Suez Canal University Hospital. It included 60 specimens divided into 42 specimens of CRCs and 18 specimens of colorectal adenomas which was chosen randomly. The sample size was calculated based on the following equation:

$$n={\left[\frac{{Z}_{\propto /2}+{Z}_{\beta }}{{\text{P}}_{1}-{\text{P}}_{2}}\right]}^{2}\left({\text{p}}_{1}{\text{q}}_{1}+{\text{p}}_{2}{\text{q}}_{2}\right)$$

n = required sample size per group

${{Z}}_{\propto /2}$ = 1.96 (The critical value that divides the central 95% of the Z distribution from the 5% in the tail).

${{Z}}_{{\beta }}$ = 0.84 (The critical value that separates the lower 20% of the Z distribution from the upper 80%.

Sections stained by hematoxylin and eosine stain to confirm the first diagnosis. all sections were assessed for the prognostic factors (Grading, lymphovascular invasion, perineural invasion, desmoplasia and TNM Stage).

Immunohistochemical technique: Was manually performed according to the manufacture company in data sheet was manual, sections were cut at 5 microns and mounted on positively charged slides then dried in the oven at 56^o C. Then the sections were deparaffinized by Xylene. The slides were processed through a serial alcohol for rehydration using (Absolute alcohol, 90% Alcohol and 70%) and then rehydrated with distilled water. The slides were rinsed using distilled water for 5 minutes. The slides were placed in peroxidase block for 20 minutes and then rinsed with PBS three times 2 minutes every time. The application of primary antibodies: for CD44, prediluted, ready to use antibody, 100 µl of CD44 was applied to the slides for 30 minutes and for OPN which was titrated using a serial of dilutions to achieve optimal results, with a dilution of 1:50. Also,100 µl of OPN was applied to the slides for 30 minutes, incubated overnight in 4°C with the use of both positive and negative controls, then rinsed with PBS three times 2 minutes every time, then the slides incubated overnight in 4°C. We used the tonsillar tissue as a positive control for CD44 ⁽¹⁴⁾, and for OPN we used the normal gall bladder epithelium ⁽¹⁵⁾, and then rinsed with PBS three times 2 minutes every time. 100µl of HRP (Horseraddish peroxidase) was used for 20 min then rinsed with PBS three times 2 minutes every time. Negative control slide was included in each run by omitting the primary antibody. Sections were completely covered with the working solution of liquid DAB, incubated for 5 minutes at room temperature for full color development. Slides were rinsed using distilled water and counterstained with Hematoxylin for 1 min. Then tissue sections were dehydrated by serial immersion in Coplin jars containing series of graded ethanol, then xylol and mounted with DPX.

Evaluation Of Staining

1-CD 44 expression: The staining of entire tumor-involved area was graded in terms of both extent and intensity ⁽¹⁶⁾. The intensity of the staining was divided into four grades: (0: none, 1: weak, 2: moderate & 3: strong). The extent of staining was divided into five categories: (0: ≤ 5%, 1: 6 − 25%, 2: 26–50%, 3: 51 − 75% & 4: 76 − 100%). Finally, we determined the score by multiplying the intensity and the extent of staining to produce a range of immunostaining scores from (0–12) as (0,1,2,3,4,6,8,9,12), the immunostaining was considered positive when the scores are ≥ 3 scoring of CD44 will divided into (negative= (0–2), mild= (3–5), moderate (6–8) & strong (9–12) ⁽¹⁴⁾.

2-OPN expression: A scoring system related to the extent and intensity of immunostaining of tumor cells was used: The intensity of positive staining was scored as (0: negative, 1: weak, 2: moderate & 3: strong). The extent of positive staining was scored as: (1: (< 10%), 2: (10–50%), 3: (> 50–100%) & 4: (= 100%). The final expression was determined by multiplying the intensity score by extent score, yielding a range 0,1,2,3,4,6,8,9,12. Scoring of OPN was divided into (negative (0–2), mild (3–5), moderate (6–8) strong (9–12) ⁽¹⁷⁾.

The sixty specimens have been examined for expression of CD44 & Osteopontin (OPN). Both markers have been correlated with clinicopathological features of adenoma (type and dysplasia) and CRC (grading, vascular invasion, perineural invasion, desmoplasia and TNM stage). The grade was determined according to WHO 5th edition grading and TNM stage was determined according to AJCC 8th edition.

The age of the sixty cases (adenomas and carcinomas) involved in this study was ranging from 26 to 80 years with mean (age) 57 ± 10.7 (60 ± 11.3 years for male and 49.5 (± 9.5) years for females in CRC) and 52 ± 17.18 (54 ± 18.8 years for male and 49 ± 15.3 for females in colorectal adenomas). The most common age group in carcinomas was those between 40 and 60 years, representing 51% of the study population. Whereas the most common age group in adenomas was that over 60 years representing 38.9% of study population. In this study the predominance sex was male in both adenoma and carcinoma, representing 66.7% in adenomas and 53.7% in CRCs. The distribution of histological subtypes among 18 colorectal adenoma specimens, 11(61.6%) were tubular subtype (Fig. 1a), and only one case (5.5%) was of villous type (Fig. 1b), while 6 (33.3%) were tubulovillous subtype (Fig. 1c). Out of the 18 studied adenomas there were 13 (72.2%) showed low grade dysplasia, while 5 (27.8%) showed high grade dysplasia in this study. In our study most of tumors had cyto-membranous cytoplasmic expression of CD44 (Fig. 2) and cytoplasmic expression (Fig. 3), minor cases had membranous expression. There were (44.4%) of studied colorectal adenomas had positive expression of CD44, whereas there were (81%) of studied CRCs cases had positive expression of CD44 with statistical significance (Table 2). Forty percent of tubular adenomas were positive for CD44 in contrast to tubulovillous; 66.6% were positive. Whereas, the only one studied villous adenoma case was positive (100%); Unfortunately, this relation had no statistically significant. Regarding the grade of dysplasia of adenoma, there was no statistical difference between CD44 expression in different grades of dysplasia (p-value 0.065). According to our results the histologic grade divided into two groups low-grade (grade I-II) (n = 39) representing 90.4% of all study population (Fig. 3) and high-grade (grade III) (n = 3) representing 9.6% of all study population. Unfortunately, the relation between CD44 expression and histologic grade was statistically insignificant (p = 0.488). Although, most of low-grade tumors had positive CD44 expression (representing 80%) of all low-grade tumors while all high grade (100%) had positive CD44 expression. Regarding lympho-vascular invasion (LVI): Positive CD44 expression was more expressed in tumors with positive LVI (95.8%) and their relation was statistically significant (p = 0.049) (Table 3). Regarding, perineural invasion in this study there was no statistically significant relation between CD44 expression (p = 0.669). The relation between immunohistochemical expression of CD44 and desmoplasia had no statistical significance (p = 0.469). In the current study we grouped the four stages of the TNM staging system into two groups (stages I/II together and stages III/IV together) for more clear comparison. The relation between CD44 expression and clustered TNM stage (stages I/II and III/IV) was statistically significant (p < 0.05) (Table 4), with significantly high CD44 expression in tumors with late stages (III/IV) compared with early stage (I/II).

Table 1: The clinicopathological characteristics of the studied colorectal carcinoma specimens:

Features

Number of cases (42)

(%)

Grading

low

High

39

3

93%

7%

Vascular invasion

Positive LVI

Negative LVI

27

15

64.3%

35.7%

Perineural invasion

Free

Involved

35

7

83.4%

16.6%

Desmoplasia

Mild

Moderate

Sever

2

25

15

4.7%

59.5%

35.8%

T stage

T1

T2

T3

T4

8

21

8

5

19%

50%

19%

12%

N stage

N0

N1

N2

28

9

5

66.6%

21.4%

11.9%

Distant metastasis

No

Yes

36

6

85.7%

14.3%

Tumor stage

Stage I

Stage II

Stage III

Stage IV

1

24

12

5

2.3%

57.3%

28.5%

11.9%

Table 2: The frequency of CD44 expression in CRCs & adenomas specimens.

		Cases		P-value
		Adenoma	Carcinoma	0.012*
CD44. Expression Freq. (%)	Negative	10 (55.6%)	8 (19.05%)
	Positive	8 (44.4%)	34 (80.95%)

* Significant p-value chi square test

Table 3: The relation between CD44 expression and lymphovascular invasion by tumor cells of CRC cases. (N=42)

Lympho-vascular Invasion (LVI)	Number	CD44.expression		P-value
		Negative	Positive
Negative	15	7 (41.6%)	11 (59.4%)	*0.049
Positive	27	1 (4.2%)	23 (95.8%)

Fisher's exact test *significant p-value

Table 4: The relation between CD 44 expression with TNM stage of CRC patients. (N=42)

Clustered TNM stage			CD44 expression		P-value
Clustered TNM stage			Negative	Positive	P-value
	I/II	25 (59.5%)	6 (24%)	19 (76%)	*0.05
	I/II
	III/IV	17 (40.5%)	2 (11.9%)	15 (88.1%)
	III/IV

Fisher's exact test * Significant p-value

In the present study revealed 18 cases of adenoma divided into (5 negative & 13 positive cases) according to OPN expression, and 42 CRCs cases divided into 11 negative and 31 positives for OPN (Table 5). Most of studied cases have mainly cytoplasmic expression with minimal cases had cyto-membranous staining. We noticed that the large number of cases in both CRCs and adenomas had positive OPN expression (74% and 72% respectively) (Figs. 4 & 5). There was no statistical difference between the expression of OPN in CRCs and colorectal adenomas (p = value 0.588). Regarding the histologic type of adenoma, there were 6 out of 11 of tubular adenoma were OPN negative and 5 were OPN positive. While the only one villous adenoma in our study was positive for OPN. Also, all tubulovillous type (6/6) were positive (p = 0.167). According to dysplasia of colorectal adenomas all high-grade dysplasia specimens had positive expression for OPN (100%). Their relation was statistically significant (p = 0.023).

In this study, there was 39 low grades including 71% had positive OPN staining and there are 100% of high-grade tumors were OPN positive (Fig. 5), unfortunately, the difference was statistically insignificant (p = 0.619). Eighty percent of tumors with LVI were positive for OPN. While 66.7% of tumor with no LVI were positive for OPN (Table 6). However, the relation between OPN expression and LVI was statistically insignificant (P = 0.177). Regarding perineural invasion all specimens of positive perineural invasion (100%) had positive staining for OPN. While 68.7% of negative perineural invasion had positive OPN staining. The most common degree of desmoplasia in this study was the moderate one (25/42 cases) representing 60% of all study population. 19 cases (≈ 68%) of them having positive expression for OPN. While 2/42 cases showed mild degree of desmoplasia 100% of them were OPN positive. The relation between OPN and desmoplasia was statistically insignificance (p = 0.723). Table (7) showed that there was no statistical significance between low stage tumors (I/II) and high stage tumors (III/IV) regarding OPN expression (p = 0.591).

Table 5

The distribution of OPN expression in CRCs & adenomas.
OPN. expression	Cases		P-value
	Adenoma N = 18	Carcinoma N = 42	0.588
Negative	5 (27.8%)	11 (26.8%)
Positive	13 (72.2%)	31 (73.2%)
chi square test

Table 6

The relation between OPN and lympho-vascular invasion parameter of CRCs. (N = 42)
Lympho-vascular invasion (LVI)	Number	OPN expression				P-value
		Negative		Positive
Negative	8		8 (33.3%)		19 (66.7%)	0.177
Positive	3		3 (20.0%)		12 (80.0%)
Fisher's exact test

Table 7: The relation between OPN expression and TNM stage of CRC patients. (N=42)

Clustered TNM stage	OPN expression			P-value
Clustered TNM stage	Negative		Positive	P-value
I/II (N=25) (59.5%)		6 28.0%	19 72.0%	0.591
I/II (N=25) (59.5%)		6 28.0%	19 72.0%
III/IV (N=17) (40.5%)		5 29.4	12 70.6
III/IV (N=17) (40.5%)		5 29.4	12 70.6

chi square test

Colorectal cancer (CRC) is a foremost global health concern and remains one of the major causes of cancer-related morbidity and mortality in both developed and developing countries ⁽¹⁸⁾. It is the third most common cancer in adults after lung cancer and breast cancer worldwide. Its incidence peaks at age 55–60 years ⁽¹⁹⁾. In Egypt, CRC ranked the sixth, representing about 4% of total cancers in both sexes ⁽²⁰⁾. The incidence rate of colorectal cancer is 5.1% in males and 4.7% in females ⁽²¹⁾.

A variety of CSC markers have been proposed for CRC. Among them, CD44 and OPN are two of the most investigated colorectal CSC markers and CD44+ /OPN + cells are introduced as the subpopulation with a greater colongenic ability and tumor initiation potential ⁽²²⁾.

Our study conducted in 60 cases of CRC & colorectal adenomas to correlate CD44 and OPN immunohistochemical expression with clinicopathological finding such as grade of tumor, lymphovascular invasion, perineural invasion, desmoplasia and TNM stage. Many earlier works studied the association between clinicopathological characteristics of the CRC, with CD44 expression alone or in combination with OPN expression with controversy results. So, here we studied the relation between of CD44 expression and OPN expression, as the two are potential CSC markers with clinicopathological characteristics in CRC.

In the current study we observed that the CD44 was more expressed in CRC (80.95%) than in adenoma (44.4%) and the difference was statistically significant (p = 0.012). This result was similar to ^{(8) & (22)} who found a significant difference between adenoma and CRCs regarding CD44 expression. This might be attributed to different CD44 expression in different stages in pathogenesis in colorectal carcinoma (colonic adenoma-carcinoma sequence) ⁽²⁴⁾. In our study 80.95% of CRC had positive CD44 expression. This was in contrast with ⁽⁸⁾ study that showed that 57.1% of CRC cases had a positive CD44 expression. Also, 51% had a positive CD44 expression reported by ⁽²⁵⁾ study. This different percentage might be attributed to different sample size in our and other studies (42 cases in this study and 250 cases in ⁽⁸⁾ study or may be due to the other studies included different types of colorectal carcinomas (eg: mucinous adenocarcinoma and signet ring adenocarcinoma) rather than adenocarcinoma type (NOS) used in this study ^{(8) & (22)}.

Regarding lympho-vascular invasion in this study there was a significant relation between lympho-vascular invasion and expression of CD44 (p = 0.048). ⁽²⁶⁾ also demonstrated a statistically significant relationship between lympho-vascular invasion and CD44 expression. This may be attributed to tumors with high percentage of CSCs may have the capability to develop tumor cell migration and motility. So subsequently an overexpression of the CSC markers is more expected in advanced cancers with + ve LVI. However, these results were in contrast with ⁽²²⁾ who showed that no statistically significant relation between CD44 expression and the lympho-vascular invasion in CRC (p = 0. 19). Their results agreed with ⁽²⁷⁾ study their p-value was insignificant (p > 0.5). This may be due to the differences in number of cases ⁽²⁵⁾. According to our findings there was a significant relation between CD44 and tumor stage (p = 0.05). There was increase of CD44 expression with late stages than in early stages. Our findings were similar to ⁽²⁶⁾ who also found that there was significant relation between CD44 and the tumor stage. These results accentuate the results of ⁽²⁸⁾ and ⁽²⁹⁾ who also found a significant association between CD44 expression and stage III and IV. However, in ⁽³⁰⁾ study, there was no relation between CD44 expression and stage of the tumor. Also, ⁽⁸⁾ study showed that there was no significant relationship between CD44 and stage. The controversial results of CD44 are common, it is mainly due to different isoforms of the protein and different population upon whom the study was concluded.

Osteopontin (OPN), is a phosphorylated sialic acid–rich non-collagenous bone matrix protein, belonging to small integrin-binding ligand N-linked glycoprotein (SIBLING) family ⁽³¹⁾. Our results showed no statistically significant difference between the expression of OPN in CRCs or colorectal adenomas (p = 0.588) like that of ⁽³²⁾. Suggesting that the OPN is an early event in adenoma carcinoma pathway. This study showed that there was no statistically significant relation between OPN expression and TNM stage of CRCs (p = 0.591. These results were similar to ⁽³³⁾ who demonstrated non-significant statistical difference on estimating the IHC expression of OPN in different CRC stages (p = 0.111). The above-mentioned results were in contrast with ⁽³²⁾ study who showed that the expression level of OPN was significantly correlated with TNM stage (p = 0.0012). ⁽³²⁾ observed high levels of OPN expression could promote tumor progression and cell survival through Akt activation. Moreover, OPN has been proved to regulate cell motility, invasion, and metastasis formation of tumor cells ⁽³²⁾ and ⁽³²⁾.

We evaluate in this study the relation between expression of CD44 & Osteopontin (OPN) in colorectal adenoma and adenocarcinomas (NOS) and their relationship with clinic-pathological prognostic criteria of the disease by using IHC technique. The CD44 protein was overexpressed in 80% of CRC. On the other hand, CD44 expression was positive in about (44.4%) of adenoma. The difference was statistically significant. When we categorized the TNM stage into early and late stages, we found that there was statistical relation between positive CD44 expression and late stages (III &IV). Also, we found there was statistical relation between CD44 expression and tumors with positive lymphovascular invasion. On the other hand, there was no relation between CD44 and Grade, perineural invasion or desmoplasia. Regarding OPN, it was expressed in 74% of CRCs and 72% of adenomas. Also, in this study the difference between the expression OPN in CRC and adenomas was statistically insignificant. Moreover, there was no relation between OPN expression or score and the clinicopathological criteria of CRC (Grade, lymphovascular invasion, perineural invasion, desmoplasia and TNM stage).

CRC

Colorectal carcinoma.

CSC

Cancer stem cells.

DAB

Diaminobenzidine.

HRP

Horseraddish peroxidase.

IHC

Immunohistochemical.

LVI

Lymphovascular invasion.

OPN

Osteopontin.

SIBLING

Sialic acid–rich non-collagenous bone matrix protein, belonging to small integrin-binding ligand N-linked glycoprotein.

SPP-1

Secreted phosphoprotein 1.

Ethics approval and consent to participate Approval from ethical committee of Suez Canal University Faculty of Medicine was obtained. IRB approved waiver request for informed consent due to the following administrative, ethical, and regulatory considerations:

1. The research involves minimal risk. This study involves the IHC on existing tissues and data. These will be deidentified prior to sending them to statistician for further analysis. The results and the data will be only available for these de-identified cases. 2. The research could not be practically carried out without the waiver of consent. Many of the patients are no longer followed, and do not live locally. Thus, we cannot practically contact and obtain a new informed consent in a routine manner for this study. In addition, neither specific results nor general conclusions will be returned to any patients because the significance of such results will still be preliminary and thus will have no meaningful impact on the future care of the patients from whom the biopsy material was obtained. However, no future analyses will be performed on these samples if they represent more than minimal risk, without prior IRB approval.

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No competing interests reported.

Assessment of Immunohistochemical Expression of CD44 and Osteopontin in Colorectal Carcinoma and colorectal adenoma

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Evaluation Of Staining

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1