Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 63  |  Issue : 1  |  Page : 32--37

The role of tumor budding in colorectal adenocarcinoma: Possible involvement of the intestinal cancer stem cell marker Lgr5


Shaymaa A Sadek1, Dalia M A. Rehim1, Sohaila Fatima2,  
1 Department of Pathology, Minia University, Egypt
2 Department of Pathology, King Khalid University, Abha, Kingdom of Saudi Arabia

Correspondence Address:
Sohaila Fatima
Department of Pathology, King Khalid University, Abha
Kingdom of Saudi Arabia

Abstract

Background: Tumor budding (TB) is a promising prognostic factor in colorectal cancer (CRC) that is independent of tumor-node-metastasis (TNM) staging. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a stem cell marker and a member of the canonical Wnt-signaling cascade. It is involved in colorectal carcinogenesis. However, its role in CRC progression and TB needs to be clarified. Materials and Methods: TB was assessed in both H and E and CK immunostained sections of 92 CRC cases. Associations between TB grade and different clinicopathological parameters were evaluated. Lgr5 expression in CRC cases and its association with TB grade and other clinicopathological features was also evaluated. Results: H and E stained sections revealed low- and high-grade budding in 55 (59.8%) and 37 (40.2%) tumors, respectively, whereas Cytokeratin Immunohistochemistry (CK-IHC) showed low- and high-grade budding in 31 (33.7%) and 61 (66.3%) tumors, respectively. TB grade (in H and E and CK stained sections) was significantly associated with adverse pathological prognostic variables including vascular invasion (P = 0.03 and 0.001), lymph node metastasis (P = 0.001 and 0,001), advanced Dukes (P = 0.000 and 0.000), and TNM (P = 0.001 and 0.000) stages and inversely associated with Tumor infiltrating lymphocytes (TILS) (P = 0.02 and 0.0001) which is known to be a good prognostic indicator. Lgr5 protein was positively expressed in 52.2% (48/92) of the CRCs. Immunoreactivity of Lgr5 was significantly associated with histological grade (P = 0.01), lymph node metastasis (P = 0.002), vascular invasion (P = 0.02), TNM stage (P = 0.000), Dukes stage (P = 0.000), and TILS (P = 0.03). Furthermore, Lgr5 was found to be significantly associated with TB estimated in both H and E and CK stained tumors (P = 0.003 and 0.001 respectively). Conclusion: This study supported the relevance of TB in the assessment of CRC aggressiveness. It also revealed that Lgr5 expression is related to morphologic features in the invasive front of CRC. Lgr5 could have an important role in forming a morphologic feature at the invasive front associated with the aggressiveness of the tumor.



How to cite this article:
Sadek SA, A. Rehim DM, Fatima S. The role of tumor budding in colorectal adenocarcinoma: Possible involvement of the intestinal cancer stem cell marker Lgr5.Indian J Pathol Microbiol 2020;63:32-37


How to cite this URL:
Sadek SA, A. Rehim DM, Fatima S. The role of tumor budding in colorectal adenocarcinoma: Possible involvement of the intestinal cancer stem cell marker Lgr5. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Mar 30 ];63:32-37
Available from: http://www.ijpmonline.org/text.asp?2020/63/1/32/277366


Full Text



 Introduction



Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and leading cause of cancer-related mortality worldwide.[1] The tumor-node-metastasis (TNM) staging system is the primary method for assessing prognosis among cancer patients[2] with significant variation in outcome of patients within the same tumor stage.[3] Tumor budding (TB) represents an important histomorphological indicator of tumor invasion and metastasis.[4] TB is observed at the invasive tumor front, where isolated single or small clusters of tumor cells (up to five cells) become detached from the neoplastic epithelium and migrate into the surrounding stroma at the invasive tumor margin.[5] TB cells have a cancer stem cell (CSC) character because of their potential for migration and re-differentiation locally and at sites of metastasis.[6],[7],[8] CSCs are defined as a small population of tumor cells with the ability to self-renew and potentially promote the formation of tumors. The activation of the Wnt signaling pathway has a close association with CSC.[9] The implication of Lgr5 in colorectal carcinogenesis as a target of Wnt pathway signaling[10] and as an ideal marker of colorectal CSCs has been reported.[11],[12] Expression of putative SC markers such as Lgr5 has been reported to be upregulated at the base of the glandular structures.[13] Cancer therapies could be designed to target CSCs by inducing their differentiation or to eliminate them by inhibiting the maintenance of the stem-cell state.[14] Aim of this study was to assess the intensity of TB and investigate its associations with other clinicopathological features in CRC patients. Furthermore, immunohistochemistry for Lgr5 and its association with clinical-pathologic parameters and TB were evaluated to investigate CSC like features in budding cells as well as CRC cases.

 Materials and Methods



This retrospective study consisted of 92 CRC with available histopathological data of patients diagnosed and treated in our institute between January 2005 and December 2010. The 40 normal colorectal tissues were randomly selected from the 92 cases of CRC as normal controls. Ethical approval for this study was not required by our institution as the experiments carried out did not relate to patients' privacy, impairment, or treatment.

Immunohistochemical analysis

Immunohistochemical staining of Lgr5 and cytokeratin (CK) was carried out.[12] The primary monoclonal rabbit antibody to human Lgr5 was obtained from Abcam, Cambridge, MA, USA and mouse monoclonal antibody to human cytokeratin from Zymed Laboratories, San Francisco, CA, USA. As a negative control, the primary antibody was replaced with normal rabbit serum.

Scoring of TB and peritumoral inflammation

TB was assessed in both H and E and CK immunostained sections according to the invasive front of the tumor scanned at low power to identify region with the highest density of TB. Buds were then counted in one High power field (HPF). The occurrence of ten buds or more was classified as high-grade budding, while low-grade was considered when less than ten buds were counted.[15] Peritumoral inflammation was defined as a reaction with lymphocytic infiltration. It was estimated using a semiquantitative three-tiered system: no or mild inflammation with no or only scattered inflammatory cells, moderate with a marked inflammatory reaction, and severe inflammation with a dense accumulation of lymphocytes.[16]

Lgr5 immunohistochemical score

Semiquantitative immunoreactivity score by two pathologists independently and double-blindly considering both intensity and percentage of immunoreactive cells was done. Consensus was reached by a joint evaluation of the slides in case of discrepancy. The staining intensity was scaled as 0 (no stain), 1 (weak), 2 (moderate), and 3 (strong). The percentage of immunoreactive cells was estimated as 0 (≤5% of total cells), 1 (6-25%), 2 (26-50%), 3 (51-75%), and 4 (>75%). The raw data were converted to the immunohistochemical score (IHS) by multiplying the intensity and the percentage scores for each antibody examined. IHS ranges from 0 to 12. An IHS of 9-12 was considered strong immunoreactivity, 5-8 was considered moderate, 1-4 was considered weak, and 0 was scored as negative.

 Results



Clinicopathological data

[Table 1] summarizes the clinicopathologic characteristics of the 92 CRC cases including 47 males and 45 females with age range 18--73 years and a mean age of 52 ± 4.2 years.{Table 1}

Budding grades by H and E staining and CK-IHC

TB was first evaluated in H and E stained sections [Figure 1]a and [Figure 1]b followed by CK-IHC [Figure 1]c. Among 92 CRC cases, H and E stained sections revealed low- and high-grade budding in 55 and 37 tumors, respectively, whereas CK-IHC showed low- and high-grade budding in 31 and 61 tumors, respectively. A high significant association between TB grades detected by H and E and CK (P < 0.0001) was noted. CK-IHC revealed more detection of TB than H and E staining. Comparing the two assessment methods, [Table 2] demonstrates the changes in the budding grade detected by H and E and CK-IHC. All tumors that showed high-grade budding in H and E stained sections remained high by CK-IHC (high-to-high group). In contrast, only 31 of 55 tumors that showed low-grade budding in H and E stained sections remained low grade with CK-IHC (low-to-low group) and 24 were classified as high-grade (low-to-high group).{Figure 1}{Table 2}

Association of TB with clinicopathological data

High-grade budding (detected by H and E or CK-IHC) was significantly associated with adverse pathological prognostic variables including vascular invasion (P = 0.03 and 0.001), lymph node metastasis (P = 0.001 and 0.001), advanced Dukes (P = 0.000 and 0.000), and TNM (P = 0.001 and 0.000) stages and inversely associated with TILS (P = 0.02 and 0.0001) which is known to be a good prognostic indicator [Table 3]. The current study showed that the high budding grade detected by CK-IHC was more frequently associated with vascular invasion and lymph node metastasis compared to H and E-detected high budding grade (89.2% vs. 57.1% and 91.6% vs. 66.6, respectively).{Table 3}

Lgr5 expression and its association with clinicopathological data and TB

Lgr5 protein was positively expressed in 52.2% (48/92) of the CRCs mostly in the cytoplasm of cancer cells (heterogeneous distribution pattern) with some membrane staining [Figure 2]a and [Figure 2]b. Positive Lgr5 expression was significantly associated with histological grade (P = 0.01), lymph node metastasis (P = 0.002), vascular invasion (P = 0.02), TNM stage (P = 0.000), Dukes stage (P = 0.000), and TILS (P = 0.03) [Table 4]. The present study also examined the relationship between Lgr5 expression and grade of TB in both H and E and CK stained sections to assess its role in tumor invasion. Lgr5 was found to be significantly associated with TB measured in both H and E and CK stained tumors (P = 0.003 and 0.001, respectively).{Figure 2}{Table 4}

 Discussion



Routine histopathological examination of the H and E-stained specimens can easily assess TB and the results are fairly reproducible.[4],[17],[18] TBs cannot be detected easily with H and E staining alone because of the presence of reactive fibroblasts or the presence of inflammatory cells that could mask the buds. The current study assessed TB using H and E staining and CK-IHC to be more accurate and objective. Scoring of TB on H and E and CK stained sections revealed moderate agreement between both assessments. A tendency to underestimate TB on H and E sections was observed in some cases that were classified as bud low on H and E sections while estimated as bud high based on CK-IHC, using cut-off of ten buds for both methods. The present work found that CK-IHC was helpful in the detection of single tumor cells or small clusters of cells embedded in fibrous stroma. This was in agreement with studies that demonstrated significantly higher budding counts with CK-IHC than in H and E.[19],[20] However, a modest improvement in interobserver variability in TB with CK-IHC has been observed.[21]

Some authors suggest that the assessment of budding should be limited to H and E because of the cost and impracticality of performing IHC in routine cases, whereas others claim that IHC should be used routinely for better detection of buds and it may require the establishment of a separate cutoff for budding grading rather than that used in H and E.[22] It is important to verify the presence of a nucleus for the identification of a TB to avoid overcounting of CK positive microvesicles and membrane fragment.[23]

TB represents two malignant features: cellular discohesion and active invasion. The presence of buds has been considered to be characteristic feature of aggressive CRC.[24],[25] The present study detected that TB grade shows a strong association with clinicopathologically aggressive parameters including lymph node invasion, vascular invasion, and tumor stage. Consistent with previous reports,[25] a significant inverse association between TB grade and peritumoral lymphocytic infiltration (PTL) was identified. The absence of budding in association with dense PTL might be explained by the immune destruction of budding cells.

The current study detected positive Lgr5 expression in 52.2% of cases consistent with two previous studies[12],[26] which was significantly associated with aggressive features such as high tumor grade, lymph node metastasis, vascular invasion, advanced TNM and Duke's stages, and low TILS. However, the relationship between Lgr5 and TB has not been fully investigated in previous literature. The molecular mechanisms and genetic events triggering TB remain incompletely understood. The present study detected relatively/significantly higher Lgr5 expression scores and TB at the invasive front than in the central portions of the tumors. Moreover, Lgr5 scores at the invasive front were significantly higher in tumors with high budding scores. This finding was consistent with study by Gao et al. It supports the concept that Lgr5 expressing tumor cells, i.e., stem cell like become detached as so called TB from the primary tumor site and migrate to the surrounding stroma and the metastatic sites as described by Baker et al.

Application of this promising parameter is hampered by the lack of a standardized scoring system and sufficient evidence of reproducibility. Development of an internationally accepted scoring system to rapidly and reproducibly identify CRC specimens with prognostically significant levels of TB is challenging. Finding the right balance between accuracy, reproducibility, and practicality is crucial. Recent multicenter studies have begun to address these issues.[3],[27]

 Conclusion



This study supported the relevance of TB in the assessment of CRC aggressiveness. Lgr5 could have an important role in forming a morphologic feature at the invasive front associated with the aggressiveness of the tumor. Elucidation of LGR5 functions and the mechanism of its regulation may provide better understanding of colorectal tumorigenesis and may ultimately lead to the development of novel preventive and therapeutic strategies against CRC. Additional studies are warranted to further investigate the molecular events associated with tumor cells that reside in the invasive tumor front and budding areas, which will lead to a better understanding of CRC invasion and metastasis as well as potential targeted therapeutic strategies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.
2Sobin LH, Gospodarowicz MK, Wittekind Ch, editors. TNM Classification of Malignant Tumours. Chichester, West Sussex, UK, Hoboken, NJ: Wiley-Blackwell; 2010.
3Puppa G, Senore C, Sheahan K, Vieth M, Lugli A, Zlobec I, et al. Diagnostic reproducibility of tumour budding in colorectal cancer: A multicentre, multinational study using virtual microscopy. Histopathology 2012;61:562-75.
4Lugli A, Karamitopoulou E, Zlobec I. Tumour budding: A promising parameter in colorectal cancer. Br J Cancer 2012;106:1713-7.
5Prall F. Tumour budding in CRC. Histopathology 2007;50:151-62.
6Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T. Opinion: Migrating cancer stem cells - An integrated concept of malignant tumour progression. Nat Rev Cancer 2005;5:744-9.
7Yusra, Semba S, Yokozaki H. Biological significance of TB at the invasive front of human CRC cells. Int J Oncol 2012;41:201-10.
8Karamitopoulou E. TB cells, cancer stem cells and epithelial-mesenchymal transition-type cells in pancreatic cancer. Front Oncol 2012;2:209.
9Katoh M. Network of WNT and other regulatory signaling cascades in pluripotent stem cells and cancer stem cells. Curr Pharm Biotechnol 2011;12:160-70.
10Segditsas S, Sieber O, Deheragoda M, East P, Rowan A, Jeffery R. Putative direct and indirect Wnt targets identified through consistent gene expression changes in APC-mutant intestinal adenomas from humans and mice. Hum Mol Genet 2008;17:24,3864-75.
11Takahashi H, Ishii H, Nishida N, Takemasa I, Mizushima T, Ikeda M, et al. Significance of Lgr5(+ve) cancer stem cells in the colon and rectum. Ann Surg Oncol 2011;18:1166-74.
12Wu XS, Xi HQ, Chen L. Lgr5 is a potential marker of CRC stem cells that correlates with patient survival. World J Surg Oncol 2012;10:244.
13Baker AM, Graham TA, Elia G, Wright NA, Rodriguez-Justo M. Characterization of LGR5 stem cells in colorectal adenomas and carcinomas. Sci Rep 2015;5:8654.
14Massard C, Deutsch E, Soria JC. Tumour stem cell-targeted treatment: Elimination or differentiation. Ann Oncol 2006;17:1620-4.
15Ueno H, Murphy J, Jass JR, Mochizuki H, Talbot IC. Tumour 'budding' as an index to estimate the potential of aggressiveness in rectal cancer. Histopathology 2002;40:127-32.
16Richards CH, Roxburgh CS, Powell AG, Foulis AK, Horgan PG, McMillan DC. The clinical utility of the local inflammatory response in colorectal cancer. Eur J Cancer 2014;50:309-19.
17Hayes BD, Maguire A, Conlon N, Gibbons D, Wang LM, Sheahan K. Reproducibility of the rapid bud count method for assessment of TB in stage II colorectal cancer. Am J Surg Pathol 2010;34:746-8.
18Yamaguchi Y, Ishii G, Kojima M, Yoh K, Otsuka H, Otaki Y, et al. Histopathologic features of the tumor budding in adenocarcinoma of the lung: TB as an index to predict the potential aggressiveness. J Thorac Oncol 2010;5:1361-8.
19Ohtsuki K, Koyama F, Tamura T, Enomoto Y, Fujii H, Mukogawa T. Prognostic value of immunohistochemical analysis of tumor budding in colorectal carcinoma. Anticancer Res 2008;28:1831-6.
20Satoh K, Nimura S, Aoki M, Hamasaki M, Koga K, Iwasaki H, et al. Tumor budding in colorectal carcinoma assessed by cytokeratin immunostaining and budding areas: Possible involvement of c-Met. Cancer Sci 2014;105:1487-95.
21Suzuki A, Togashi K, Nokubi M, Koinuma K, Miyakura Y, Horie H, et al. Evaluation of venous invasion by Elastica van Gieson stain and tumor budding predicts local and distant metastases in patients with T1 stage colorectal cancer. Am J Surg Pathol 2009;33:1601-7.
22Mitrovic B, Schaeffer DF, Riddell RH, Kirsch R. Tumor budding in colorectal carcinoma: Time to take notice. Mod Pathol 2012;25:1315-25.
23Koelzer VH, Zlobec I, Berger MD, Cathomas G, Dawson H, Dirschmid K, et al. Tumor budding in colorectal cancer revisited: Results of a multicenter interobserver study. Virchows Arch 2015466:485-93.
24Shinto E, Mochizuki H, Ueno H, Matsubara O, Jass JR. A novel classification of tumour budding in colorectal cancer based on the presence of cytoplasmic pseudo-fragments around budding foci. Histopathology 2005;47:25-31.
25Steinestel K, Lennerz JK, Eder S, Kraft K, Arndt A. Invasion pattern and histologic features of tumor aggressiveness correlate with MMR protein expression, but are independent of activating KRAS and BRAF mutations in CRC. Virchows Arch 2014;465:155-63.
26Gao FJ, Chen JY, Wu HY, Shi J, Chen M, Fan XS, et al. Lgr5 over-expression is positively related to the tumor progression and HER2 expression in stage pTNM IV colorectal cancer. Int J Clin Exp Pathol 2014;7:1572-9.
27Horcic M, Koelzer VH, Karamitopoulou E, Terracciano L, Puppa G, Zlobec I, et al. Tumor budding score based on 10 high-power fields is a promising basis for a standardized prognostic scoring system in stage II colorectal cancer. Hum Pathol 2013;44:697-705.