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Correlation between tumor budding, microvascular density, and β-catenin expression in colorectal carcinoma

El Sheikh, Samar A.; Bassam, Amira M.; Ibrahim, Heba A.

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Egyptian Journal of Pathology: July 2016 - Volume 36 - Issue 1 - p 108-114
doi: 10.1097/01.XEJ.0000484382.97776.e5
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Abstract

Introduction

Colorectal cancer (CRC) is the fourth most common cause of cancer mortality worldwide (Zavarhei et al., 2007). In Egypt, the Cancer Pathology Registry of the National Cancer Institute showed that during the years 2003–2004 CRC was the most common among digestive tract malignancies (15.78%) and the fifth among all cancers (4.34%) (Mokhtar et al., 2007). Therefore, identification of molecular markers associated with tumor growth and metastasis is critical to develop appropriate therapeutic interventions (Doger et al., 2006).

Gabbert et al. (1985) observed that at the invasive borders of adenocarcinomas there were small strands of tumor cells projecting from the neoplastic glands. Ultrastructurally, these tumors cells did not elaborate junction complexes, had incomplete desmosomes, and a basement membrane was rudimentary. According to Ueno et al. (2002) budding foci are defined clusters composed of fewer than five undifferentiated cancer cells or single cells at the invasive tumor front. The link between tumor budding and tumor cell migration at the invasive margin by pseudopodia has been proposed by Shinto et al. (2005).

Tumor budding may indicate a high risk for recurrence after surgery and indicates the worst grading pattern. This finding correlates with loss of adhesion molecule expression (Kaihara et al., 2003) and increased metalloproteinase expression corresponding to a more aggressive tumor phenotype (Guzińska-Ustymowicz, 2006). Tumor budding is a risk factor for lymph node metastasis of early-stage (T1) CRC, including occult metastasis (Ishikawa et al., 2008).

β-Catenin is a protein associated with E-cadherin in maintaining cell–cell interactions (Ben-Ze’ev, 1999). β-Catenin is an important component of cell adhesion molecules and signal transduction pathways (Morin, 1999). The oncogenic activation of β-catenin is a result of the inactivation of the tumor suppressor APC gene, or direct mutation of the β-catenin gene, or the activation of the Wnt signaling pathway (Hecht and Kemler, 2000). The association of deregulated Wnt/β-catenin signaling with CRC has been well documented (Polakis, 2007). Among the target genes activated by β-catenin are those known to be involved in the process of invasion and metastasis. These include urokinase-like plasminogen activating receptor, matrilysin, CD44, and laminin 5 (Hlubek et al., 2001).

CD34 is widely used as a marker of vascular endothelial cells. It has also been used as a marker for identification and isolation of hematopoietic stem cells and progenitors in preparation for bone-marrow transplantation and as a marker to identify other tissue-specific stem cells, including muscle satellite cells (Sato et al., 1999). Several studies have noted that microvascular density (MVD) correlates with stage and histological grade of tumor, recurrence, metastasis, and survival in CRC (Grantab et al., 2006).

The aim of the current study was to evaluate tumor budding, and immunohistochemical expression of β-catenin and MVD by CD34 in 50 cases of colorectal carcinoma, and then correlate between tumor budding, expression of β-catenin and MVD, and other clinical and pathological features to determine their possible role in tumor progression and their prognostic value.

Patients and methods

This work included 50 cases of colorectal carcinoma obtained through a collection of archived paraffin blocks. All cases obtained were through colectomy specimens. Data obtained from pathology sheets were age and sex of the patient, site of colorectal carcinoma, size of the tumor, gross appearance, and presence of nodal/distant metastasis.

Serial sections of 4 µm thickness were prepared from each block. One of them was mounted on a glass slide and stained with hematoxylin and eosin (H&E) for histological evaluation. Another two were mounted on charged slides for immunohistochemical staining.

Histological examination

Histopathological examination of H&E-stained slides was performed to determine the histological type and histological grade. Staging was done according to modified Duke’s stage. The depth of tumor invasion, lymph node metastasis, and distant metastasis was also assessed according to TNM staging.

The budding number was determined from counts in one microscopic field ×200 in an area of maximal budding. The degree of budding was classified as negative or positive corresponding to from zero to four and five or more budding foci in one field, respectively, using H&E-stained specimens (Ohtsuki et al., 2008).

Immunohistochemical staining

The sections were deparaffinized in xylene, and then hydrated through a series of graded alcohol (95–70%), distilled water, and PBS (pH 7.5). The slides were then immersed in 10 mm citrate buffer (pH 6) and were twice pretreated in a microwave oven at 800 W for 4 and then 8 min. After a 25 min cooling period, the endogenous peroxidase activity was inhibited by incubation in 3% hydrogen peroxide for 5 min. Antigen retrieval was done by immersing the slides in 10 mm citrate buffer (pH 6) for 10–20 min at 100°C in a microwave followed by cooling at room temperature for 20 min. The tissues were blocked with protein blocking reagent for 30 min to reduce nonspecific staining. After washing with Tris-buffered saline, the sections were incubated with the primary antibody overnight at room temperature. The primary antibody for β-catenin was rabbit polycolonal antibody (Thermo Scientific Company, Massachusetts, USA) and that for CD34 was mouse monoclonal antibody, anti-CD34 clone (QBend10; Genamid Biotechnologies Company, San Francisco, USA). The sections were washed in Tris-buffer and incubated with avidin–biotin–peroxidase system (Dako, California, USA) for 30 min. The excess reagent was tapped off and the slides were washed with PBS and dried. Peroxidase reaction was detected by addition of diaminobenzidine tetrahydrochloride. Two or three drops of streptavidin enzyme label were placed on each slide for a few minutes at room temperature. The excess reagent was tapped off and the slides were washed with PBS and dried. All slides were rinsed well in tap water for 5 min, then slightly counterstained with hematoxylin for 1–2 min, and dehydrated in ascending alcohol.

Evaluation of β-catenin expression

The scoring protocol assigns scores of 1 for loss of cell membrane staining, 1 or 2 for slight or pronounced increase in cytoplasmic staining, respectively, and 1 or 2 for slight or pronounced nuclear staining, respectively, giving a maximum total score of 5. Cases scoring 4 or 5 are regarded as positive for abnormal β-catenin immunoexpression (Jass et al., 2003).

Evaluation of CD34 expression (microvascular density)

MVD was assisted by scanning the entire section at low magnification (×100) to identify the most intense areas of neovascularization within the tumor. After five areas with the highest density of capillaries and small venules were identified, microvessels were counted at high power magnification (×400), and the average of count in five fields was calculated. MVD was quoted as a continuous variable. Cases below the average MVD were considered to be cases with low MVD, whereas cases more than or equal to the average were considered to be cases with high MVD.

Statistical analyses

The data collected were analyzed using SPSS (version 16.0; SPSS Inc., Chicago, Illinois, USA), and the relationship of tumor budding, β-catenin expression, and MVD in colorectal carcinoma to each other and to other clinicopathologic features was ascertained. Determining the P value assessed the significance of results. When P values were less than 0.05 the results were considered to be statistically significant.

Results

This retrospective study was conducted on 50 CRC cases: 23 were male and 27 were female, with a female to male ratio of 1.17 : 1. Their ages ranged from 23 to 86 years with a mean of 49.32±12.84 years. Forty-seven out of 50 (94%) cases studied were older than 40 years and three (6%) were 40 or younger. The most common location of the tumors was the colon, representing 28 (56%) cases (38% in the right colon, 16% in the transverse colon, and 2% in the left colon), whereas 22 (44%) cases were located in the rectum. Twenty-eight (56%) cases showed tumor size more than 5 cm and 22 (44%) cases were less than or equal to 5 cm. Twenty-one (42%) cases manifested as ulcerating tumor, 19 (38%) cases were infiltrating, and 10 (20%) cases were fungating.

Forty (80%) cases were classified histologically as conventional adenocarcinomas, whereas 10 (20%) cases were mucoid adenocarcinoma. As regards the degree of differentiation, 28 (56%) cases were moderately differentiated (grade II), 21 (42%) were poorly differentiated (grade III), and one (2%) was well differentiated (grade I). In the studied cases, carcinomas with modified Duke’s stage C were the most commonly encountered tumors [29 (58%) cases], followed by Duke’s stage B [19 (38%) cases] and Duke’s stage D [two (4%) cases]. Forty-five (90%) cases were stage T3, three (6%) were T4, and two (4%) were T2. Most of the studied cases revealed lymph nodes metastatic deposits (62%).

Our study showed that 31 (62%) cases had tumor buds (Fig. 1), whereas 19 (38%) cases were negative for tumor budding. Membranous expression of β-catenin in normal colonic mucosa was used as internal positive control (Fig. 2). Immunohistochemical assessment of β-catenin expression revealed 33 (66%) negative cases (scores 1, 2, and 3) and 17 (34%) positive cases (scores 4 and 5) (Figs 3–5). The average MVD in our study was 16.16/5 high power fields. Thirty-one (62%) cases showed low MVD (less than average MVD of the studied cases), whereas 19 (38%) cases showed high MVD (Fig. 6).

F1-17
Fig. 1:
Moderately differentiated colonic adenocarcinoma showed tumor budding at the invasive front of the tumor (hematoxylin and eosin, ×400).
F2-17
Fig. 2:
Membranous expression of β-catenin in normal colonic mucosa, used as internal control (IHC, ×100).
F3-17
Fig. 3:
Strong cytoplasmic expression of β-catenin in malignant glands (IHC, ×400).
F4-17
Fig. 4:
Colonic adenocarcinoma showing strong cytoplasmic and nuclear expression of β-catenin (IHC, ×1000).
F5-17
Fig. 5:
Colonic adenocarcinoma showing strong cytoplasmic and nuclear expression of β-catenin (IHC, ×200).
F6-17
Fig. 6:
Moderately differentiated adenocarcinoma associated with high microvascular density (IHC, ×200).

Statistical analysis revealed significant positive correlation between tumor budding and advanced Duck’ stage, positive lymph node metastasis, and increasing stage of lymph node metastasis, whereas no significant correlations were detected with the other studied parameters (Table 1). Statistical analysis revealed nonsignificant correlations between β-catenin expression and other clinicopathological parameters (Table 2). The MVD showed nonsignificant differences with the different clinicopathological variables except for the sex of the patient, as females cases had higher MVD compared with male cases (P=0.029).

T1-17
Table 1:
Correlations between tumor budding and clinicopathological data of the studied colorectal cancer cases
T2-17
Table 2:
Correlations between β-catenin expression and clinicopathological data of the studied colorectal cancer cases

Among the 31 tumor-budding positive cases, 10 cases were associated with β-catenin expression. A nonsignificant correlation was found between β-catenin expression and tumor budding (P=0.344). There was no statistically significant correlation between β-catenin expression and MVD (P=0.77) or between tumor budding and MVD (P=0.895).

Ten out of 50 cases showed positive tumor budding associated with positive expression of β-catenin, nine of which showed positive lymph node metastasis, where a statistically significant correlation was found (P=0.041) (Table 3).

T3-17
Table 3:
Correlations between budding, β-catenin-positive cases, and lymph node metastasis

Discussion

The aim of the current study was to evaluate tumor budding, immunohistochemical expression of β-catenin, and MVD in colorectal carcinoma and then to correlate between them and other clinical and pathological features to determine their possible role in tumor progression and their prognostic value.

In the present study, 54% of the studied patients were female and 46% were male with a female to male ratio of 1.17 : 1. This is similar to what was reported by Cressey et al. (2006), that a higher incidence was seen in female patients (63% of cases). Ages of the patients ranged from 23 to 86 years with a mean of 49.32±12.84 years. Moreover, 94% were older than 40 years. These figures are similar to those reported by researchers from Tanta Cancer Center (Gharbiah, Egypt) (Zeeneldin et al., 2012). However, these figures represent older age groups compared with those studied by researchers on Egyptian patients with CRC. In the study by Soliman et al. (2001) the majority of cases were 40 years old or younger. This difference may be attributed to sample size, sample selection methods, and different geographical areas covering different places in Egypt.

The majority of tumors in the present study were located in the colon, representing 56% of cases (38% of cases in the right colon, 16% in the transverse colon, and 2% in the left colon). The rectum came in the second place, representing 44%. This agrees with the results of Fenoglio-Preiser et al. (2008), who stated that in low-risk countries carcinomas of the right side occur more frequently than carcinomas of the left colon, whereas in high-risk countries colorectal carcinomas more commonly arise in the rectosigmoid region. Regarding the gross patterns of colorectal carcinomas in the current study, 42% were ulcerating, 38% were infiltrating, and 20% were fungating. This is compatible with what was reported by Day et al. (2003) that most CRCs are ulcerating tumors. With respect to histopathological types, 80% were adenocarcinomas and 20% were mucinous adenocarcinomas. These figures are similar to what was reported by Lanza et al. (2011) in which most colorectal carcinomas (85%) were adenocarcinomas, and 10–15% were mucinous adenocarcinomas. Most CRC cases presented with positive lymph node metastasis (62%) and most cases presented with T3 as 90% infiltrated into the subserosa or into nonperitonealized pericolonic or perirectal tissue. According to the modified Duke’s staging system (modified Astler–Coller), 58% of the cases in this study were stage C, 38% were stage B, and 4% were stage D. These results are not in agreement with the results obtained by Sis et al. (2004), in whose study the highest incidence was in stage B (46.4%). This may be explained by delayed onset of seeking medical advice or the prevalence of right-sided tumors in Egypt, which are usually diagnosed at a later onset.

We observed tumor budding in 62% of cases, and it showed a statistically significant correlation with advanced Duke’s stage, positive lymph node metastasis, and increasing stage of lymph node metastasis. Ohtsuki et al. (2008) showed that budding was significantly associated with lymphatic vessel infiltration at the invasive tumor front, positivity for lymph node metastasis, and local recurrence; this suggests that budding is an early event in the phase of lymphatic vessel and venous invasion by tumor cells with subsequent nodal and distant metastasis. Guzińska-Ustymowicz (2006) reported a relationship between tumor budding and expression of matrix metalloproteases (MMP) (MMP-9, MMP-7, MMP-2) or cathepsin B, which could explain the strong association between budding, local deep mural invasion, and lymph node metastasis. Kye et al. (2012) designed a study to identify risk factors for lymph node metastasis in early-stage CRC and found that tumor budding (P=0.047) was the only factor that was significantly associated with lymph node metastasis in submucosal T1 CRC.

Ohtsuki et al. (2008) compared budding assessment in H&E-stained and immunohistochemically stained sections. The number of cases with positive budding was higher with immunostaining assessment. Tanaka et al. (2003) demonstrated the importance of detection of budding using cytokeratin immunohistochemical stains especially if the tumor is accompanied by an inflammatory reaction.

Tumor budding and β-catenin expression revealed a nonsignificant statistical correlation. A study done by Jass et al. (2003) stated that positive findings for β-catenin were more common in cases with budding (P=0.03). This finding indicates that budding is a dynamic process under genetic and epigenetic control and not just the result of architectural disruption caused by a host immune reaction and fibrosis at the tumor margin. Our study showed statistically significant correlation when budding was associated with both β-catenin expression and positive lymph node metastasis. This finding agrees with that of Masaki et al. (2001) and Umemura et al. (2013), in which tumor budding with positive expression of β-catenin was suggested to be a strong risk factor for lymph node metastasis. Therefore, concomitant evaluation of tumor budding with β-catenin expression will lead to modification of therapeutic strategies and avoidance of unnecessary measures in early CRC.

Apart from budding cases, 66% of cases were negative for β-catenin (score 1, 12%; score 2, 34%; and score 3, 20%), whereas 34% were positive for β-catenin (score 4, 20%; and score 5, 14%). No statistically significant correlation was detected between different scores of β-catenin expression and other clinicopathological parameters. Kobayashi et al. (2000) reported that nuclear overexpression of β-catenin was observed in 35% of intramucosal cancers and in 42% of invasive cancers but was not observed in adenomas from either sporadic or familial adenomatous polyposis cases. Wong et al. (2003) found that nuclear β-catenin expression was highly associated with progression of colorectal tissue from normal epithelial tissue, polyps, adenomas, and carcinomas (P=0.0001). They also found that patients with CRC and high nuclear β-catenin expression had a higher incidence of lymph node metastasis (P<0.005). They concluded that assessment of β-catenin immunostaining in colorectal adenomas and polyps is necessary to stratify the risk for those patients of developing subsequent colorectal carcinoma.

The current study showed that 62% of cases showed low MVD (less than average MVD of the studied cases; 16.16), whereas 38% showed high MVD. The MVD showed significant association only with the sex of the patient, as female cases showed higher MVD compared with male cases (P=0.029), but no significant correlation was detected with the other clinicopathological parameters including tumor budding or β-catenin expression. Pity et al. (2013) studied angiogenesis by CD34 in CRC. They demonstrated that CD34 expression had no significant association with the clinicopathologic findings. This suggests that angiogenesis in CRC is possibly not influenced by the degree of tumor dedifferentiation and metastasis (Lazar et al., 2008). This finding was previously demonstrated by Carneiro et al. (2006) on Brazilian patients and by Afrem et al. (2012) on Romanian patients with CRC. In contrast, Lazar et al. (2008) in their study on patients demonstrated a significant correlation of high MVD with advanced stages. Another study conducted by Sharifi et al. (2009) on Iranian patients found a significant correlation between MVD and different grades, thus suggesting that MVD may be valuable in stratifying patients in planning antiangiogenic and antitumor antibody-directed therapy after surgery.

Conclusion

In conclusion, the statistically significant correlation between tumor budding and advanced tumor stage, positive lymph node metastasis, and increasing stage of lymph node metastasis in colorectal carcinoma cases supports the hypothesis that tumor budding acts a prognostic marker in predicting poor outcome of patients with colorectal carcinomas. The study suggests that tumor budding might play a role in the ability of colon cancer cells to invade and metastasize. Thus, proper assessment of tumor budding in early stages of carcinoma may predict the ongoing strategy of surgical treatment to avoid aggressive surgery in budding negative cases. Therefore, it is recommended to apply a scoring system for tumor budding in CRC cases, to be reported in the patient’s pathology report, as it could be useful in predicting outcome in these patients. The statistically significant correlation found when budding was associated positive β-catenin expression with lymph node metastasis supports the potential role of β-catenin in the process of budding and metastasis. To elucidate the possible prognostic significance of β-catenin and MVD in colorectal carcinoma, it will be necessary to carry out similar studies on a larger sample size.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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