Introduction

The tumor invasive front is a significant interface of tumor host interactions which regulates tumor progression. Tumor budding (TB) is a histological hallmark at the tumor invasive front which supposedly initiates invasion and eventually metastasis.1 According to the International Tumor Budding Consensus Conference (ITBCC) criteria, TB is defined as a single tumor cell or a cluster of fewer than five tumor cells detached from the main tumor at the invasive front, while clusters of five or more tumor cells lacking gland formation are termed as poorly differentiated clusters (PDCs).23

TB has been designated as an adverse prognostic factor in colorectal cancers (CRCs) by the International Union against Cancer (UICC). The UICC has included TB under “additional prognostic markers,” besides histologic grade, perineural invasion (PNI), and tumor border.456

Tumor grade based on glandular differentiation is an important predictive factor of CRC aggressiveness. However, this system poses a significant interobserver variability due to the absence of objectivity in assessment of glandular component. A novel histological grading system based on PDCs has been highlighted that is expected to achieve an objective assessment of tumor differentiation.7 Besides, PDC is strongly predictive of unfavorable histological features, namely, infiltrating tumor borders, TB, lymph vascular, and PNI in CRCs.8910

PDCs and tumor buds form a continuum of morphological features with a randomly set cut-point of 5 tumor cells to distinguish between the two.23 PDCs possibly represent the evolution of tumor buds, which acquire proliferative and aggregative potential. Thus, they have common molecular, clinical, and pathological features of CRC cases.3

Very few studies have assessed the development of TB and PDC simultaneously in CRCs. Evaluation of these two histological parameters in the same hotspot field at the tumor invasive front is expected to assume a fundamental role in the definition of cancer aggressiveness and prediction of tumor behavior.

PDC and TB can be evaluated in routine hematoxylin and eosin-stained histopathological sections taken from the advancing edge of the tumor.

The present study aims to evaluate TB and PDC simultaneously in CRC cases by morphological assessment. The study further strives to establish a correlation of these two parameters with other known prognostic factors of CRCs.

The goal of our study was to establish a correlation between these two pertinent histological features and to reinforce the importance of their incorporation in routine histopathological reporting of CRC cases as a means to predict clinical outcome.

Materials and Methods

The study was conducted in the department of pathology over a period of 1 year (May 2020 to May 2021). Resection specimens of colorectal carcinoma received in the department of pathology were included in the study. Patients who received presurgical therapy, or refused consent were excluded from the study. After applying the exclusion criteria, 54 cases were included in the study. The current study was a single institutional, noninterventional, observational, prospective study. Clinical parameters were analyzed followed by histomorphological assessment of colonic resection specimens. Pertinent macroscopic findings of each specimen were tabulated after diligent assessment. Formalin-fixed paraffin-embedded tissue blocks were cut at 4 µm thickness and stained with hematoxylin and eosin. Histopathological reporting was done according to the College of American Pathologists protocol for the Examination of Resection Specimens from Patients with Primary Carcinoma of the Colon and Rectum (Version: Colon and Rectum Resection 4.1.0.0; Protocol Posting Date: February 2020). TB was reported by selecting a “hotspot” chosen after review of all available slides with invasive tumor. The total number of buds were reported in an area measuring 0.785 sq.mm, which corresponded to 20× field in LM-52-6000 Mega, LYNX Penta Head Microscope (based on the ITBCC, 2016 recommendation).23 Both total number of buds and a three-tier score (based on 0.785 sq.mm field area) were reported: low (0–4 buds), intermediate (5–9 buds), and high (10 or more buds). PDC was evaluated in the advancing edge of the tumor in the same hotspot field as TB. The cases were categorized into three grades based on the highest PDC count: grade 1 (G1) count less than 5, grade 2 (G2) range between 5 and 9, and grade 3 (G3) 10 or more. TB and PDC data of each CRC case was assessed simultaneously. It was then followed by their correlation with other known prognostic factors, namely, tumor border, radial margin involvement, lymphovascular (LVI) and PNI, dirty tumor necrosis, histological grade, and pathological stage. All cases were initially assessed by a single pathologist blinded to other data followed by review of the cases by a second pathologist. Discordant findings, if any, were finally assessed by a third pathologist and the consensus opinion was incorporated in the final tabulation. Statistical analysis of the data was performed using the “social science statistics” Web site and the statistical tests employed were one-way analysis of variance (ANOVA) calculator, including Tukey's honest significant difference (HSD) and Spearman's rho calculator (https://www.socscistatistics.com/).

Results and Analysis

A total of 54 CRC cases were studied. The patients belonged to a wide age range spanning from 3rd to the 6th decades, majority being in the range of 50 to 54 years (20.4%). Fifty-six percent patients were females. Sixty-one percent of the cases presented with involvement of the left colon. Rectum was the most common tumor location. Bulk of the cases (57.4%) was of histologic grade 2. Note that 81.5% of the cases belonged to pT3. Resection margins were uninvolved in 70.4% of the cases (Table 1).

TB score was done according to the ITBCC guidelines and the study comprised 37% low TB score, and 31.5% each of intermediate and high scores. PDC was evaluated and graded in the advancing edge of the tumor in the same hotspot field as TB. Low, intermediate, and high PDC scores were noted in 9.3, 66.6, and 24.1% cases, respectively, in the present study (Fig. 1).

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Fig. 1: Morphology of tumor budding (TB) and poorly differentiated cluster (PDC) scores in different histologic grade of tumor (hematoxylin and eosin [H&E], 200×). (A) TB score: Low (04) and PDC: G2 (05). (B) TB score: Intermediate (06) and PDC: G3 (10). (C) TB score: High (13) and PDC: G3 (10). (D) TB score: High (10) and PDC: G2 (08).

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Spearman's rho calculator for strength of association between TB and PDC as well as association of TB and PDC individually with known prognostic factors, namely, lymph node metastasis, LVI, and PNI, revealed statistical significance. The correlation of TB with LVI and PNI was found to be statistically significant (p = 0.008 for TB vs. LVI, p = 0.005 for TB vs. PNI); however, the correlation of PDC with LVI and PNI was not statistically significant. Correlation of TB and PDC with histologic grade, primary tumor (pT), and regional lymph node (pN) stage was done based on one-way ANOVA calculator, including Tukey's HSD, which yielded statistically significant results (Table 2). Also, the correlation of TB and PDC individually with overall TNM stage was statistically significant. TB was found to be better correlated to TNM stage compared to PDC (p = 0.004 for TB vs. TNM, p = 0.008 for PDC vs. TNM). Hence, the present study establishes the prognostic significance of TB and PDC as well as their relation with known prognostic factors in CRCs, thereby reinforcing the importance of their incorporation in routine reporting. Furthermore, these two parameters when used in conjunction provide objectivity in grading the CRC cases and strengthens the histologic grading system for better risk stratification.

Discussion

TB is one of the preliminary steps of cancer progression, as TB cells invade extracellular matrix and lymphovascular spaces, thereby giving rise to metastatic deposits in lymph nodes and distant organs. PDCs were first defined in 2008 and are a predictive and prognostic factor in CRCs. Tumor grading by TB and PDC scoring has been used with a view to achieving greater objectivity compared to histologic grading alone.11121314 Our present study was conducted to reinforce the prognostic value of TB when used simultaneously with PDC in CRCs.

According Lugli et al and Marx et al, in CRC, pTB (peritumoral TB) and iTB (intratumoral TB) are important indicators of higher TNM stage and tumor grade, LVI, or nodal and distant metastases.1516 Resemblances in adverse prognostic roles of pTB and iTB were established in a study by Lugli et al.16 The present study also revealed that the TB positively correlated with histologic grade and pTNM stage (p-value is < 0.00001).

In a study by Zlobec et al, it was found that iTB could also identify the patients who might show aggressive behavior and benefit from adjuvant therapy in a subgroup of node-negative CRC.17 Hence, routine reporting of TB will help in identifying a subgroup of patients who will require adjuvant therapy.

Ueno et al in their study delineated that pPDC (peritumoral PDC) was a more accurate prognostic indicator than pTB.311

Archilla et al, in their study delineated the fact that lymph nodal tumor burden significantly correlates with newer prognostic indicators, namely, higher TB and PDC scores in CRCs.18 The current study has revealed similar findings.

As per the study by Mohan et al, statistically significant association was found between TB and LVI (p < 0.01) as well as TB vs. PNI (p = 0.002). The current study also showed similar statistically significant results.19

Study on PDC in colorectal carcinoma by Maurya et al failed to show any significant association between PDC and depth of invasion (pT) with p-value of 0.136.20 While the study by Shah et al revealed significant correlation between TB with T stage, N stage, and TNM stage.21 This was in concordance with the present study in which the correlation between PDC and pT was found to be statistically significant with a p-value of < 0.05.

The current study reinforced the fact that the correlation of TB and PDC individually with overall TNM stage was statistically significant. However, TB was found to be better correlated to TNM stage compared to PDC (p = 0.004 for TB vs. TNM, p = 0.008 for PDC vs. TNM).

TB and PDC are associated with the epithelial-mesenchymal transition phenomena, which is triggered by the Wnt/beta-catenin signaling pathway.22223 Transition of TB to a mesenchymal phenotype is mediated by nuclear translocation of beta-catenin and simultaneous loss of membranous E-cadherin expression. The development of PDC is associated with the upregulation of L1 cell adhesion molecule (L1CAM), which mediates epithelial cell migration and is one of the target factors of the Wnt signaling pathway.2223

The present study strives to establish the importance of simultaneous assessment of TB and PDC in the prognostication of CRCs since both the parameters have significant positive correlation with histologic grade and pTNM stage. However, one weakness of the study is that the survival data of the study population could not be incorporated.

Conclusion

The study reinforces the fact that even in a resource poor setup, morphological assessment of TB and PDC will help achieve a greater objectivity in prognostication of CRCs than histologic grade alone. Identification of cases with higher TB score will in turn help to select subgroups who have a more aggressive behavior and likely to benefit from adjuvant chemotherapy. This current study also emphasizes the importance of scoring PDC in the same hotspot field that is used in the assessment of TB to strengthen the prognostic and predictive significance of TB in CRCs.