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Colorectal cancer (CRC) is the third most common
diagnosed cancer among men and women and the second leading cause of cancer
death in USA 1. Worldwide, more than 1 million people get colorectal cancer
annually 2.

At the time of diagnosis, over 20% of patients present
with metastatic (stage IV) colorectal cancer, and up to 25% of these patients
will have isolated liver metastasis that is potentially resectable. Five year
survival outcomes

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are now exceeding 50% in the lesions which undergo
curative resection 3. Survival rates for early detected stages are about 5
times that of late stage Cancer. Colorectal cancer arises from adenomatous
polyps in 80% of cases making this cancer suitable for screening 4.

The continuous and
bilateral molecular interaction between normal epithelial cells and stromal
cells   is disrupted by several factors
secreted by tumor cells or by stromal cells under the influence of tumor cells 5-9. One such important factor is the matrix metalloproteinase
(MMPs). MMPs have an important role in tumor progression and in defining the
role of stromal microenvironment in invasiveness and metastatic   potential 10. 

CD10 is a matrix metallopeptidases (MMPs) involved in
carcinogenesis via the release of bioactive molecules that stimulate invasion,
extracellular matrix (ECM) degradation, inhibition of apoptosis, and promotion
of angiogenesis and immune response modulation 11.

CD10 is a zinc-dependent
peptidase (metalloproteinase) that is commonly expressed in bone marrow
lymphoid stem cells, pro-B lymphoblasts, mature neutrophils, various subtypes
of lymphoma, renal cell carcinoma and endometrial stromal sarcoma cells.
CD10-positive cells have also been reported in the stroma of prostate 12, breast13,14, colorectal15, and lung carcinomas16.

In colorectal cancer tissue, CD 10 has been detected
in tumor cells, tumor associated fibroblasts and infiltrating inflammatory
cells 17. Several investigators have reported association between CD 10
expression in CRC and increased invasiveness, lymph node involvement, liver
metastasis and poor prognosis 17-19. 
However, only few studies have been carried out to determine the
potential role of CD10 in colon cancer and its precursor lesions (colorectal
adenomas) 20.

We aimed in this study to investigate the
immunohistochemical expression of CD10 in tumor and stromal cells of colorectal
cancer and premalignant lesions (colorectal adenomas). In addition, the
correlation between CD10 expression in colorectal cancer and various
clinicopathologic factors is studied.

MATERIALS
AND METHODS

Study group

This work
included 50 cases of archived paraffin blocks of colorectal adenomas (n=20) and
colorectal carcinomas (n=30) obtained through colonoscopic biopsies and
colectomy specimens respectively during the period from January 2013 till
February 2015, from the Pathology department, Faculty of medicine, Cairo
University.

Histopathological
Evaluation

1.     Formalin fixed and paraffin embedded blocks were
sectioned at 5mm thickness
and examined microscopically using H&E stain.

2.     According to the grade of dysplasia, adenomas were
classified into two groups, low grade (mild-moderate dysplasia) and high grade
adenomas (marked dysplasia). Low-grade dysplasia consists of stratified
dysplastic epithelium that retains its columnar shape. The nuclei are spindle
or oval shaped. The stratified nuclei tend to remain in the basal epithelium
extending no more than three quarters of the height of the epithelium. There is
minimal nuclear hyperchromasia. High-grade dysplasia is present when the nuclei
consistently come to the surface of the epithelium, loss of the columnar shape
with cellular rounding, increasing nuclear:cytoplasmic ratios, nuclear
irregularity, loss of polarity, development of cellular pleomorphism, and
heaping up of cells 21.

3.    
Colorectal
carcinomas were examined microscopically to evaluate the histopathologic type,
grade, stage and, nodal metastasis. All colorectal carcinomas (n=30) were
adenocarcinoma. Mucinous adenocarcinoma was excluded from the study. The tumors
were staged according to American Joint Committee on Cancer
(AJCC) Tumor Node Metastasis (TNM) system (2010) and graded according to the World Health
Organization criteria 22), 23.

Immunohistochemicalstaining

Each
paraffin block was cut at 5mm thickand
taken for the study. The slides   were deparaffinized in xylene, then were rehydrated through a series of
graded alcohols then were twicelymicrowave-treated for 4 and 8
minutes   in 10 mm sodium citrate buffer
(pH 6.0). Endogenous peroxidase activity was blocked with 3% H2O2 for 15 min,
followed by washing withTris-buffered
saline (TBS).The sections
were then incubated with monoclonal mouse CD10 antibody clone 56C6
(Genemed, South San Francisco, CA) at 1:50 dilution for one hour at room
temperature.

Sections were again washed in TBS and incubated with
avidin-biotin-peroxidase system (DAKO) for 30 minutes. Finally the
diaminobenzidine was used as a chromogen and hematoxylin as a counterstain.Positive control was obtained
from tonsils, which exhibited strong intensity of CD10 immunostaining.

Evaluation
of immunohistochemical expression of CD10

The
immunostaining of CD10 was evaluated in tumor cells (tCD10)and stromal cells
(sCD10). The immunostaining of tCD10 was expressed as fine to coarse cy­toplasmic
granules. Positive tCD10 was considered if more than 10% of tumor cell stained
positive for CD10.The extensity of sCD10 was graded according to a 4-point
scale based on the percentage of positively stained area: 0 (< 10 % positive tumor cells), +1 (10-25% positive tumor cells), +2(25 to 50% positive tumor cells), +3(>50% positive tumor cells). For
statistical analysis purpose, the cases were divided into two groups: negative
group (0, 1) and the positive group (2, 3) 20.

Statistical
Analysis

SPSS version
18.0 (Statistical Product for services solutions) was  used for data analysis and the probability value
(P value) of less than 0.05 was chosen to represent statistical significance.
The chi-square test was used for evaluation of p value.

 

A total of 50 tissue samples from colorectal adenomas
(n=20) and, colorectal carcinomas (n=30) were studied. The mean age of the
studied patients with colorectal carcinoma was 54.87 years, ranging from 29 to
78 years with median age 50 years. The age of colorectal adenoma patients
ranged from 25 to 82 years.

The
clinicopathologic features of colorectal adenoma and colorectal carcinoma
patients are shown in (table 1) and (table 2) respectively.

CD 10 immunostaining
was observed in colorectal adenomas and carcinomas as brown, fine to coarse
granular cytoplasmic staining in both tumor and stromal cells. Immunohistochemical
expression of CD10 in tumor cells (tCD10) progressively increased from20%
( 2 out of 10) in low grade adenomas 
(Fig 1), to 50% (5 out of 10) in high
grade adenomas(Fig 2),reaching
80% (24 out of 30) cases
in invasive CRC(Fig 3),and that was
statistically significant , (P=0.002). Invasive CRC
and high grade adenoma cases showed higher CD10 immunohistochemical expression in stromal
cells 57% (17 out of 30) and 60% (6 out of 10)cases respectively, than low grade adenoma cases 30% (3 out of 10), however no statistically significant correlation was detected. The correlation
between tCD10 and sCD10 immunohistochemical expression in colorectal adenomas
and carcinomas are summarized in (table3).

The tCD10 and
sCD10immunohistochemical expression in 30 cases of invasive colorectal
carcinoma were studied and correlated with clinicopathologic variables as shown
in(table 4).CD10 immunohistochemical expression in tumor cells negatively
correlated with the depth of tumor invasion as 100% of T2 cases (4 out of 4)
and 83% of T3 cases (20 out of 24) showed positive tCD10 expression and 100 %
of T4 cases (2 out of 2) showed negative tCD10 immunohistochemical expression, (p=0.01).

No significant correlation was detected between tCD10 and sCD10
expression in invasive CRC and other clinicopathologic variables.

 

 

 

 

 

 

 

 

CD10 is an important molecule involved in
integrating signals from either the cell environment or the intracellular compartment
by cleaving peptides through enzymatic activity and through intracellular
signaling pathways that interfere with other major signaling pathways. It is
thus obvious that CD10 expression derangement is associated with the
development or progression of different tumort ypes24.

In the current study we examined CD10 immunoreactivity
in both tumor cells and stromal cells in thirty cases of colorectal carcinoma
and twenty cases of colorectal adenoma. The correlation between CD10
immunohistochemical expression and different clinicopathologic factors was
analyzed.

In this work, CD10immunohistochemical expression in tumor cells (tCD10) progressively increased from  20% (2 cases out of 10 cases) in low grade
adenomas to 50% (5 cases out of 10 cases) in high grade adenomas and
reached  80% (24 cases out of 30 cases)
in invasive CRC and that was statistically significant ( P value< 0.05 ). These findings coincide with the results obtained by other studies.Janget al.20, who stated that tCD10immunohistochemical expression significantly increased from 14% in low grade adenomas (3 cases out of 22 cases), to 22% in high grade adenomas (6 cases out of 27 cases) and 44% in invasive CRC (14 cases out of 32 cases) and this support the involvement of CD10 in progression and carcinogenesis of colorectal carcinoma.  Likewise, Wang et al.25, reported that there was progression in tCD10immunohistochemical expression from 0.8% in low grade adenomas to 9.1% in high grade adenomas and 40% in invasive CRC. Also, this is in agreement with what was reported by Iwase et al.26and  Kogaet al.27and Hirano et al.28, that tCD10immunohistochemical expression was reported more frequently in invasive phenotype rather than adenomas. Stromal cells showed CD10immunohistochemical expression in both adenoma and carcinoma. Fifty-seven percent (17 cases out of 30 cases) of CRC showed positive sCD10immunohistochemical expression while only forty-five percent (9 cases out of 20 cases) of adenoma showed positive sCD10immunohistochemical expression. Invasive CRC and high grade adenoma cases showed higher CD10 immunohistochemical expression in stromal cells 57% and 60% respectively than low grade adenomas which represented only 30%. However, no statistically significant correlation was detected. Ogawa et al.15similarly reported higher sCD10immunohistochemical expression in invasive CRC (79%) and high grade adenoma (70.6%) than low grade adenomas which represented only 21.9%and,also reported that sCD10immunohistochemical expression was similar in high grade adenomas and carcinomas, and its expression is induced at the step in which low grade adenomas (mild to moderate dysplasia) become high grade adenomas (severe dysplasia). Jang et al.20also reported progression of expression of sCD10immunohistochemical expression from low grade adenoma (41%) to high grade adenoma (70%) to invasive CRC (88%) and that was statistically significant. In the current study, CD10 immunohistochemical expression in tumor cells inversely correlated with the depth of tumor invasion, 100% of cases in T2 and (83%) of cases in T3 showed positive tCD10 immunohistochemical expression and 100% of cases in T4 showed negative tCD10 expression,with statistically significant relationship, these findings suggest that tCD10 is involved in invasiveness   potential in early CRC. In contrast, no significant correlation was reported between tCD10 and depth of tumor invasion in the study done by Jang et al.20. No significant correlation was detected in the current study between tCD10 and sCD10 immunohistochemical expression and clinicopathologic variables as age, sex, histologic grade, lymph node status and, stage in CRC cases.This is most probably due to the low number of cases enrolled in the present study. CONCLUSIONS In conclusion, the progression of tCD10 expression from low grade adenoma to high grade adenoma to invasive CRC and  the higher percentages  of sCD10 expression in CRC and high grade adenomas  compared to low grade adenoma  as demonstrated in this study supports the role of CD10 as potential biomarker in colorectal carcinogenesis along the adenoma carcinoma sequence. In addition, the   significant inverse correlation between tCD10 expression and  depth of tumor invasion in CRC suggests that CD10 is involved in invasiveness potential in early colorectal cancer. Thus analysis combining CD10 expression in tumor and stromal cells in CRC and premalignant lesions may be one of the quite useful predictors of development and early progression of CRC and more future studies on larger sample size to validate our results are recommended

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