| Abstract|| |
Objective: To evaluate the association of tumor budding (TB) with prognostic histomorphological parameters in oral squamous cell carcinoma (OSCC) and to investigate the correlation of TB intensity with epithelial to mesenchymal transition (EMT).
Material and Method: A total of 200 cases diagnosed as OSCC were selected and their TB status was reviewed using Hematoxylin and eosin (H and E) and Immunohistochemistry (IHC). Correlation with histomorphological prognostic parameters was done. Also, IHC for Vimentin and E-cadherin was performed to look for EMT.
Results: On H and E examination, TB was observed in 154/200 (77%). About 88/154 (57.14%) cases showed a high TB (>5 TB/10 hpf) which increased to 100/154 (64.9%) cases on IHC staining. The intensity of TB was significantly associated with tumor grade and depth of invasion. It was also significantly associated with reduced expression for E-Cadherin and upregulation of Vimentin establishing a pathogenetic correlation between the TB and EMT.
Conclusion: Therefore, our results suggest that TB is associated with poor prognosis and histologically represents EMT in OSCC which further adds to the aggressiveness of the tumor.
Keywords: Epithelial mesenchymal transition, histomorphological prognostic markers, oral squamous cell carcinoma, tumor budding
|How to cite this article:|
Yadav K, Singh T, Varma K, Bhargava M, Misra V. Evaluation of tumor budding and its correlation with histomorphological prognostic markers in oral squamous cell carcinoma and its association with the epithelial-mesenchymal transition process. Indian J Pathol Microbiol 2023;66:3-8
|How to cite this URL:|
Yadav K, Singh T, Varma K, Bhargava M, Misra V. Evaluation of tumor budding and its correlation with histomorphological prognostic markers in oral squamous cell carcinoma and its association with the epithelial-mesenchymal transition process. Indian J Pathol Microbiol [serial online] 2023 [cited 2023 Mar 23];66:3-8. Available from: https://www.ijpmonline.org/text.asp?2023/66/1/3/367941
| Introduction|| |
Oral squamous cell carcinoma (OSCC) is one of the most commonly encountered malignancies of the oral cavity in the head and neck region with more than 3,00,000 new cases being diagnosed every year and 1,45,000 cancer-related deaths. The incidence of oral cancer is highest in India and other Southeast Asian countries and smokeless tobacco consumption has emerged as the major risk factor in this region.
Despite advances in diagnosis and treatment, 5 year survival rate of OSCC is very poor. Many histopathological prognostic indicators (tumor grade, lymphovascular invasion, depth of invasion, lymph node metastasis) have been evaluated. However, none have proved very reliable, hence there was a need to identify newer prognostic markers, which are feasible and can be incorporated in routine histomorphological reporting.
An emerging and promising prognostic histomorphological marker for tumor invasion and metastasis is tumor budding (TB). It is described as isolated tumors cells or in small clusters (<5 cells) present in the stroma along the invasive margin of the tumor.,, Its role has already been established as an independent prognostic factor in colorectal, lung, and breast carcinoma. However, very few studies highlight its role in OSCC. TB is considered as a histomorphological marker of loss of cellular adhesion, and thus, it is closely associated with epithelial-mesenchymal transition (EMT), which is the hallmark of invasion and metastasis. The present study was thus undertaken to assess the prognostic significance of TB in OSCC. Also its association with EMT was evaluated to unravel its pathogenetic mechanism.
| Material and Methods|| |
The study included 200 cases of OSCC from different sites of oral mucosa including buccal mucosa, tongue, oropharynx diagnosed over a period of 2 years. Complete resected specimens of all patients were obtained [Figure 1]a. Standard protocol for surgical grossing of the specimens was followed. Histopathological examination was done in detail.
|Figure 1: (a) Gross picture of right hemimandibulectomy showing ulcerative growth in buccal mucosa. (b) Showing high tumor budding activity (H&E, 400×). (c) Showing broad pushing margins with finger-like projections (POI 2) (H&E, 100×). (d) Showing depth of invasion (H&E, 40×). (e) Showing heavy host lymphocyte response and obscuration of tumor budding. (H&E, x 100×). (f) Showing high tumor budding activity (IHC, 100×). (g) Showing strong expression of vimentin in tumor buds (IHC, 400×). (h) Showing absence of E-cadherin in tumor buds (IHC, 400×)|
Click here to view
Clinicopathological profile of patients has been shown in [Table 1].
All sections from 200 cases were reviewed independently by two pathologists. TB was identified at the invasive front of the tumor in scanner view (4× objective). Tumor buds (1-5 cell clusters) were identified in low power (10× objective). The number of tumor buds were counted in 10 high power fields (HPF) (40× objective) on conventional H and E sections and IHC using Cytokeratin AE1/AE3 in all the cases.
IHC for Cytokeratin AE1/AE3 using monoclonal mouse Anti- antibody (BIO-SB: USA: Prediluted antibody) was done using standard procedure. Tumor cells showing brown cytoplasmic staining were taken as positive. Mimickers of tumor buds like inflammatory cells, macrophages, multinucleated giant cells, fibroblasts, endothelial cells, smooth muscle cells, and artifacts were excluded.
Grading of TB
Grading for TB was done both on HE and IHC. Low budding was defined as TB ≤5 TB/10 hpf and high budding as >5 TB/10 hpf. TB intensity i.e., high and low was calculated. A comparison between TB intensity seen on conventional HE sections and IHC was done as shown in [Table 2]. It was found to be higher on IHC and this was taken as a gold standard.
Comparison of TB intensity with newer histomorphological prognostic markers
Further conventional, as well as newer prognostic markers, were evaluated i.e., degree of differentiation, lymph node involvement, lymphovascular invasion (LVI), host lymphocyte response (HLR), stromal response (SR), TNM stage, depth of invasion (DOI), and worst pattern of invasion (WPOI). The tumors were graded histologically into well, moderately, and poorly differentiated according to their degree of differentiation. Tumor invasion within arterial, venous, or lymphatic channels qualified for LVI. HLR was evaluated at the invasive tumor front and graded semi-quantitatively as none, mild, moderate, and heavy. The stromal response was graded as loose, desmoplastic, and hyalinized.
DOI was measured as the distance between the lowest part of the adjacent normal mucosa and the lowest part of the tumor using a slide caliper. The invasive tumor front was evaluated for the pattern of invasion (POI) and TB. The POI was determined as described in the literature previously and classified into five patterns. Pattern 1 was defined as broad, pushing margin of the tumor with a smooth outline. Pattern 2 was defined as broad, pushing finger-like projection. Pattern 3 represents invasive tumor islands with >15 cells per island. Pattern 4 represents invasive tumor islands with less than 15 cells per island. Pattern 5 was defined by the presence of a tumor island outside the main tumor at a distance of >1 mm. In a given case showing multiple patterns of invasion, the score was determined by the highest pattern present, even if present focally. Among these five patterns, POI 4 and 5 were classified as WPOI.
The above histomorphological prognostic factors were compared with TB intensity and statistically analyzed as shown in [Table 3].
|Table 3: Comparison of Tumor Budding Intensity with histomorphological prognostic markers|
Click here to view
Evaluation of EMT by IHC for E-cadherin and vimentin
E-cadherin and Vimentin play an important role in epithelial cell adhesion and tissue architecture maintenance and are important biomarkers of EMT. Primary antibody used for Vimentin: Monoclonal Mouse Anti Vimentin clone V-9 (BSB6022: Bio SB, USA). Primary antibody used for E Cadherin: Monoclonal Rabbit Anti-E cadherin clone EP6 (BSB5463; Bio SB, USA). Prediluted antibodies were used. Tumor cells showing brown cytoplasmic staining were taken as positive for Vimentin and brown membranous was taken as positive for E-Cadherin.
When the positivity of E-cadherin was more than 90%, the case was designated as having preserved expression; otherwise, the case was designated as having reduced expression. For the Vimentin expression, no detectable or <10% positive staining of tumor cells was served as negative, while ≥10% positive staining of tumor cells was defined as positive.,, Results are shown in [Table 4].
|Table 4: Association of the tumor budding intensity with E-cadherin and Vimentin expression|
Click here to view
Approval by the ethics committee was taken.
The data were tabulated using a Microsoft excel sheet and parameters in different groups were compared. The Chi-square test was applied and P value of <0.05 was considered significant.
| Results|| |
The mean age of the patients was 49 years with a majority of cases in the fourth and fifth decade of life. The male to female ratio was 4.4:1. The majority, i.e. 118/200 (59%) cases had a positive history of oral tobacco intake. Most of the tumors 92/200 (46%) cases were 2-4 cm in size. Buccal mucosa was the most common site of tumor involvement with 100/200 (50%) cases. Most of the tumors, i.e., 96/200 (48%) cases were moderately differentiated squamous cell carcinoma (Grade 2).
TB grading was compared on H and E and IHC as shown in [Table 2]. Out of 200 cases evaluated, 154 (77%) cases showed TB. High intensity budding (>5 TB/hpf) was observed in 88 cases (57.14%) on H and E. The number of cases showing high TB intensity increased to 100 (64.9%) cases on IHC. This difference was found to be statistically significant (P value = 0.00432).
TB intensity was further compared with histomorphological prognostic parameters as detailed in [Table 3]. A significant association was seen with tumor grade and DOI.
Assessment of EMT showed upregulation of Vimentin in tumor buds in 65/154 cases (42.2%). Out of these 57/65 (87.6%) showed high-budding intensity. Membranous E-cadherin was generally reduced in tumor buds. Only 24/62 (38.7%) high-budding tumors showed preservation of membranous E-cadherin expression [Table 4]
| Discussion|| |
Our study aimed to investigate the role of TB in OSCC and its correlation with newer prognostic histomorphological markers along with its association with EMT.
The invasive front of the tumor shows striking disorganization at the tumor architecture level along with dedifferentiation at the cellular level. These changes ultimately resulted in isolated tumor cells and small clusters showing a uniform phenotype i.e., tumor buds.
Tumor buds have features compatible with cell mobility (loss of cell junctions and basement membrane, presence of cytoplasmic microfilaments and pseudopodia, versatile cell shape), which help mobilize cancer cells from the main tumor mass, followed by the invasion of host tissues through locomotion. Thus, it may be utilized as a grading and prognostic marker. TB was first mentioned in colorectal cancers for tumor staging along with stromal reaction and lympho-vascular invasion. Since then numerous studies have demonstrated its vital role in gastric, breast, and lung cancers.,,,,,, However, the role of TB in OSCC has not been validated yet.
Considerable heterogeneity exists in the methodology used for assessing TB. We compared both methods for TB counting i.e., H&E staining and IHC for Cytokeratin AE1/AE3 as shown in [Table 2]. A statistically significant increase in the number of high TB cases was noted on IHC in comparison to H and E analysis in the present study (P = 0.004). Our study supports the findings of earlier studies which suggest that the IHC technique has played a significant role in overcoming the shortcomings of H and E for example abundant inflammatory infiltrate and stromal cells can obscure or mimic TB.
Moreover, one recent study reported that among relatively inexperienced pathologists (<5 years of practice), pan-cytokeratin immunostaining markedly increased the inter-observer reproducibility as compared to the H and E staining.
TB intensity was divided into low grade and high grade. There is still a great deal of debate around choosing the cutoff value for stratifying patients which best separates them into prognostic categories followed by appropriate therapeutic decisions. A cut off value of ≤5 TB/hpf and >5 TB/hpf was taken in our study which correlated with survival.,,,, We compared TB intensity with prognostic histomorphological features.
One of the established survival predictors in any carcinoma is tumor grade. The correlation of TB with grade is well established in colorectal and esophageal cancers.,, However in the present study, a highly statistically significant correlation has been noted in TB with tumor grade in OSCC.
Penetration of tumor cells into lymphovascular spaces through the endothelial cell layer is considered to be a significant step in the process of tumor metastases, which is one of the most consistent prognostic markers in carcinomas and indicates a worse prognosis. The present study was statistically significant with the fact that high TB intensity cases had more incidence of LVI and therefore a higher chance of lymph node metastasis.,,,,,
TB and DOI have been documented as poor prognostic factors in OSCC in previous studies. Almangush et al. found that higher TB intensity and DOI ≥4 mm were associated with poor prognosis in early-stage oral tongue SCC. In our study, the TB intensity was seen to increase along with the increasing depth of tumor invasion showing a statistically significant association.
Several different studies have reported the relationships among regional metastasis and pattern of invasion but little is known about the clinical and prognostic value of the combination of WPOI and TB at the invasive tumor front. With increasing aggressiveness of the tumor, there is loss of cohesion in the tumor cells leading to change in POI starting from broad pushing margins to the collection of 15 or more tumor cells. The present study revealed that the WPOI was well correlated and showed a statistically significant correlation with TB intensity.,
Tumor cells in tumor buds exhibit distinct morphological features, including de-differentiation and loss of cell–cell adhesion. This fibroblast-like morphological appearance is characteristic of cells undergoing EMT which allows for greater mobility to the tumor cells leading to a more aggressive and invasive ability, thereby leading to a poor prognosis and survival. It is characterized at the molecular level by the loss of E-cadherin (a key component of the adherens junctions) and the increase in expression of Vimentin (a ubiquitous mesenchymal intermediate filament).,, Our results confirmed that the expression of E-cadherin is significantly reduced in cells located in the invasive tumor front and cells located in tumor buds when compared with those located in the central/superficial portions of the tumor samples. The observed reduction in E-cadherin in Invasive tumor front (ITF) and budding cells is accompanied by an increase in Vimentin expression. (P value < 0.001). This is similar to the results mentioned in previous studies.
| Conclusion|| |
To conclude, TB is frequently noted in oral cancers and a highly significant correlation has been noted between the TB and conventional prognostic markers including tumor grade, lymph node metastasis, LVI, pattern of invasion, and depth of invasion. Evaluation of TB by IHC CK AE1/AE3 shows a higher reproducibility and replicability compared to H and E sections alone. It can be used as an independent, significant, and poor prognostic marker wherein management and prognosis can be accurately ascertained. Moreover, tumor buds showing epithelial to mesenchymal transformation, further add to the aggressiveness of the tumor leading to a much worse prognosis. Since EMT has also been shown to cause resistance to therapy in cancers, additional studies may be required to assess the molecular events associated with it for targeted therapy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Boxberg M, Kuhn PH, Reiser M, Erb A, Steiger K, Pickhard A, et al
. Tumor Budding and Cell Nest Size Are Highly Prognostic in Laryngeal and Hypopharyngeal Squamous Cell Carcinoma. The American Journal of Surgical Pathology 2019;43:303-13.
Zhang S, Wang X, Gupta A, Fang X, Wang L, Zhang C. Expression of IL-17 with tumor budding as a prognostic marker in oral squamous cell carcinoma. American Journal of Translational Research 2019;11:1876.
Teramoto H, Koike M, Tanaka C, et al.
Tumor budding as a useful prognostic marker in T1- stage squamous cell carcinoma of esophagus. J Surg Oncol 2013;108:42-6.
Attramadal CG, Kumar S, Boysen ME, Dhakal HP, Nesland JM, Bryne M. Tumor budding, EMT and cancer stem cells in T1-2/N0 oral squamous cell carcinomas. Anticancer Research 2015;35:6111-20.
Hong KO, Oh KY, Shin WJ, Yoon HJ, Lee JI, Hong SD. Tumor budding is associated with poor prognosis of oral squamous cell carcinoma and histologically represents an epithelial-mesenchymal transition process. Human Pathology 2018;80:123-9.
Chatterjee D, Bansal V, Malik V, Bhagat R, Punia RS, Handa U, et al
. Tumor Budding and Worse Pattern of Invasion Can Predict Nodal Metastasis in Oral Cancers and Associated With Poor Survival in Early- Stage Tumor. Ear Nose Throat J 2019;98:E112-9.
Wang C, Huang H, Huang Z, Wang A, Chen X, Huang L, et al
. Tumor budding correlates with poor prognosis and epithelial-mesenchymal transition in tongue squamous cell carcinoma. Journal of Oral Pathology & Medicine 2011;40:545-51.
Masugi Y, Yamazaki K, Hibi T, Aiura K, Kitagawa Y, Sakamoto M. Solitary cell infiltration is a novel indicator of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer. Hum Pathol 2010;41:1061-8.
Uchikado Y, Natsugoe S, Okumura H, et al
. Slug expression in the E-cadherin preserved tumors is related to prognosis in patients with esophageal squamous cell carcinoma. Clin Cancer Res 2005;11:1174-80.
Masuda R, Kijima H, Imamura N, et al.
Tumor budding is a significant indicator of a poor prognosis in lung squamous cell carcinoma patients. Mol Med Rep 2012;6:937-43.
Sadek SA, 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;63:32-7.
] [Full text]
Nakanishi Y, Ohara M, Doumen H, Kimura N, Ishidate T, Kondo S. Correlation between tumor budding and post-resection prognosis in patients with invasive squamous cell carcinoma of the thoracic esophagus. World Journal of Surgery 2011;35:349-56.
Kumarguru BN, Ramaswamy AS, Shaik S, Karri A, Srinivas VS, Prashant BM. Tumor budding in invasive breast cancer - An indispensable budding touchstone. Indian J Pathol Microbiol 2020;63(Special):S117-22.
Liang F, Cao W, Wang Y, Li L, Zhang G, Wang Z. The prognostic value of tumor budding in invasive breast cancer. Pathol Res Pract 2013;209:269-75.
Grigore AD, Jolly MK, Jia D, Farach-Carson MC, Levine H. Tumor budding: The name is EMT. Partial EMT. Journal of Clinical Medicine 2016;5:51.
Guil-Luna S, Mena R, Navarrete-Sirvent C, López-Sánchez LM, Khouadri K, Toledano-Fonseca M, et al
. Association of Tumor Budding With Immune Evasion Pathways in Primary Colorectal Cancer and Patient-Derived Xenografts. Frontiers in Medicine 2020:264.
Brandwein- Gensler M, Smith RV. Prognostic indicators in head and neck oncology including the new 7th
edition of the AJCC staging system. Head and Neck Pathol 2010:53-61.
Shimizu S, Miyazaki A, Sonoda T, Koike K, Ogi K, Kobayashi JI, et al
. Tumor budding is an independent prognostic marker in early stage oral squamous cell carcinoma: With special reference to the mode of invasion and worst pattern of invasion. PloS one 2018;13:0195451.
Liu LK, Jiang XY, Zhou XX, Wang DM, Song XL, Jiang HB. Upregulation of vimentin and aberrant expression of E-cadherin/β-catenin complex in oral squamous cell carcinomas: correlation with the clinicopathological features and patient outcome. Modern Pathology. 2010;23:213-24.
Niwa Y, Yamada S, Koike M, Kanda M, Fujii T, Nakayama G, et al
. Epithelial to mesenchymal transition correlates with tumor budding and predicts prognosis in esophageal squamous cell carcinoma. Journal of Surgical Oncology 2014;110:764-9.
Angadi PV, Patil PV, Hallikeri K, Mallapur MD, Hallikerimath S, Kale AD. Tumor budding is an independent prognostic factor for prediction of lymph node metastasis in oral squamous cell carcinoma. International Journal of Surgical Pathology 2015;23:102-10.
Xu B, Salama AM, Valero C, Yuan A, Khimraj A, Saliba M, et al
. The prognostic role of histologic grade, worst pattern of invasion, and tumor budding in early oral tongue squamous cell carcinoma: a comparative study. Virchows Archiv. 2021;479:597-606.
Jensen DH, Dabelsteen E, Specht L, Fiehn AM, Therkildsen MH, Jønson L, et al
. Molecular profiling of tumour budding implicates TGFβ-mediated epithelial–mesenchymal transition as a therapeutic target in oral squamous cell carcinoma. The Journal of pathology. 2015;236:505-16.
Almangush A, Bello IO, Keski-Santti H, et al
. Depth of invasion, tumor budding, and worst pattern of invasion: prognostic indicators in early-stage oral tongue cancer. Head Neck 2014;36:811-8.
Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S, et al
. Epithelial –mesenchymal transition in cancer development and its clinical significance. Cancer Sci 2010;101:293-99.
Maeda M, Johnson KR, Wheelock MJ. Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition. Journal of Cell Science 2005;118:873-87.
Thiery JP. Epithelial–mesenchymal transitions in tumour progression. Nature Reviews Cancer 2002;2:442-54.
Wells A, Yates C, Shepard CR. E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas. Clinical & Experimental Metastasis 2008;25:621-8.
Wang C, Huang H, Huang Z, Wang A, Chen X, Huang L, et al
. Tumor budding correlates with poor prognosis and epithelial-mesenchymal transition in tongue squamous cell carcinoma. J Oral Pathology Med 2011;40:545-51.
Department of Pathology, M.L.N. Medical College, Prayagraj, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]