Abstract
Colorectal carcinoma (CRC) is the third most common cancer worldwide. Along with many established prognostic factors, tumor budding is emerging as a valuable marker of prognosis. Tumor budding is not yet universally reported but it has recently been suggested in guidelines by ITBCC (International Tumor Budding Consensus Conference). Our aim is to study prognostic implications of tumor budding in CRC. Hundred cases of primary CRC specimens were retrospectively studied from January, 2016, to February, 2017. Tumor bud count and other histopathological parameters were evaluated from hematoxyline and eosin (H & E) stained slides. Survival analysis was done using Cox proportional hazards model. Association of tumor budding and cancer-specific survival was found to be statistically significant (P = 0.018 for average tumor budding and P = 0.035 for highest tumor budding) Tumor budding was found to be significantly associated with other clinicopathological parameters such as T stage, N stage, TNM stage, and lymphovascular invasion with p value < 0.05. Tumor budding is a valuable prognostic indictor for primary CRC and also significantly associated with other prognostic parameters. It should be reported routinely as a guide to prognosis and further management of patients.
Keywords: Tumor budding, Colorectal carcinoma, Survival
Introduction
Colorectal carcinoma (CRC) is the third most common cancer worldwide, and occurs more commonly in men than in women [1]. Although there are many established prognostic factors for CRC, tumor budding is emerging as a novel tool to determine prognosis especially in early stages [2, 3].
Tumor budding has been defined as single tumor cell or cell clusters of four cells or less in number at the invasive front of tumor [4–6]. Tumor budding was first described by Imai [7] and he referred to it as “sprouting.” He also associated it with rapid progression of disease [7]. In 1989, Morodomi et al. [8] coined the term “budding” and showed its association with other histological parameters like lymph node metastasis [8].
Since then, many such studies have been published establishing the relationship between tumor budding and poor prognosis in CRC [5, 6, 9–12]. High tumor bud score in stage I CRC is associated with increased risk of lymph node metastasis [9, 13, 14]. In stage II carcinoma, high tumor budding along with other histologic factors such as tumor differentiation/grade and lymphovascular invasion may suggest need for adjuvant therapy [15–17].
Tumor budding is not reported universally as a part of routine histopathology practice. The International Tumor Budding Consensus Conference (ITBCC) which was held in 2016 proposed guidelines for assessment and inclusion of tumor budding in routine CRC reporting [4]. Royal College of Pathologists and College of American Pathologists (CAP) have incorporated tumor budding in datasets for reporting of CRC as an optional element [18, 19].
Our aim here is to study the prognostic implications of tumor budding in CRC, and its relation with other histological parameters.
Materials and methods
We retrospectively studied 100 cases of resected primary CRC specimens reported in our institute, from January 2016 to February 2017. Cases in which pre-resection treatment was given and cases for which hematoxylin and eosin (H & E) stained slides could not be retrieved were excluded from the study. For each case, demographic details were recorded. In the resected specimen, macroscopic tumor location, appearance, tumor size, and margin status were noted.
From each case, at least three and average five H & E stained slides were studied from tumor with full thickness of wall. All the slides were examined to study histological type of tumor, tumor grade, depth of infiltration, lymphovascular permeation, perineural invasion, tumor budding, and tumor infiltrating lymphocytes (TILs) score. Stage of tumor was determined according to TNM staging (pTNM, AJCC 8th edition) [20].
For assessment of tumor budding, all slides were screened at × 10 objective to identify fields with the highest peritumoral bud count at the interface of tumor and stroma. Ten worst fields were further screened at × 20 objective. Highest bud count in a “hotspot” area (0.785 mm2) was noted according to guidelines by ITBCC [4]. Average of tumor buds in ten worst fields was also noted for each case. Normalization factor used for 18-mm field diameter of eyepiece lens was 0.810. A 3-tier system was used to categorize both average tumor bud count and highest count into low (0–4), intermediate (5–9), and high (10 or more). This assessment of tumor budding was done independently by two pathologists. Wherever needed, deeper sections were examined to avoid falsely high tumor bud counts due to ruptured glands.
For mucinous carcinomas, only tumor cells which met the definition and criteria of tumor budding were considered for counting. Tumor cells and clusters floating in pools of mucin were not counted as tumor buds.
Similarly, for poorly differentiated carcinomas, tumor cell clusters of 5 or more cells were not considered as tumor buds.
TIL score was assessed as percentage of tumor stroma occupied by mononuclear cells, i.e., lymphocytes and plasma cells, and then categorized into weak (0–15%), moderate (16–55%), and strong (56–100%); a similar scoring system is used by Fuchs et al. [21].
All the cases were followed up till April, 2020, for documentation of any recurrence, metastasis, or death.
Statistical analysis
Overall survival (OS) and cancer-specific survival (CSS) for various clinical and histopathological parameters including tumor budding were examined using survival analysis and Cox proportional hazards regression from which P values and hazard ratios (HR) at 95% confidence interval (CI) were obtained.
Correlation between tumor budding and other clinico-pathological factors was established by Log-rank test and Chi square test from which P values were obtained.
P value of < 0.05 was considered significant.
All the data analysis and statistical analysis were performed using software MS Excel and SPSS version 21.
Results
All 100 cases of CRC fell into age range of 25 to 80 years; out of which, 71% cases were aged 65 or less and 29% were aged > 65 years. Sixty-seven percent cases were men and 33% cases were women.
Seventy-four percent cases were adenocarcinoma, 21% cases were mucinous carcinoma, and 5% cases were signet ring carcinoma. Tumor was well differentiated in 2% cases, moderately differentiated in 62% cases, and poorly differentiated in 36% cases. Site of tumor was in colon in 66% cases and rectum in rest 34% cases.
Only single (1%) case was T1 tumor, 36% were T2, 60% were T3 tumors, and 3% cases were T4 tumors. Out of 100, 53% were N0, 28% were N1, and 19% were N2 tumors. Total 21% cases were stage I, 24% were stage II, 45% were stage III, and 10% were stage IV tumors.
When average tumor bud count was considered, low bud score was found in 3% cases, intermediate in 30% cases, and high in 67% cases. When highest tumor bud count was considered, low bud score was found in only 1% case, intermediate in 22% cases, and high in 77% cases. We noticed high inter-observer agreement (interobserver variability-0.8) in the assessment of tumor budding, and hence, a consensus between both was taken for final analysis.
Lymphatic permeation was found in 36% cases, whereas vascular permeation and perineural invasion were found in 20% and 21% cases, respectively.
TIL score was weak in 29% cases, moderate in 66% cases, and strong in 5% cases.
Figures 1 and 2 show microscopic images of hematoxylin and eosin stained slides showing tumor budding at × 20 magnification.
Fig. 1.
Tumor budding at × 20 objective, H & E stain. Black arrows show tumor buds at invasive edge
Fig. 2.
Tumor budding at × 20 objective, H & E stain. Black arrows show tumor buds at invasive edge
When all the patients were followed up, 49 patients survived till the end of the study, 34 had cancer related deaths, and 17 cases were lost to follow-up. Overall survival (OS) of CRC was 65% in our study. Median follow-up was 27 months.
Results of correlation of CSS and various clinical and histopathological parameters are shown in Table 1. T stage (P = 0.014), TNM stage (P = 0.013), vascular permeation (P = 0.006), and tumor budding (P = 0.018 for average tumor budding and P = 0.035 for highest tumor budding) were significantly associated with survival of the patient.
Table 1.
Correlation of survival and various clinical and histopathological parameters
| Variables | Value label | N | N of events | Univariate analysis (CSS) | Sig value – P value | Survival % |
|---|---|---|---|---|---|---|
| Age | < = 65 | 71 | 29 | 1.61 (0.53–4.95) | 0.403 | 59.15 |
| > 65 | 29 | 6 | 79.31 | |||
| Sex | Women | 33 | 11 | 1.03 (0.45–2.38) | 0.94 | 66.66 |
| Men | 67 | 24 | 64.17 | |||
| Histological type | Adeno | 74 | 22 | 1.20 (0.73–1.96) | 0.46 | 70.27 |
| Mucinous | 21 | 9 | 57.14 | |||
| Signet ring | 5 | 4 | 20 | |||
| Differentiation | Well | 2 | 1 | 1.15 (0.41–3.18) | 0.79 | 50 |
| Moderate | 62 | 12 | 80.64 | |||
| Poor | 36 | 23 | 36.11 | |||
| Site | Rectum | 34 | 11 | 1.50 (0.47–4.83) | 0.49 | 67.64 |
| Colon | 66 | 24 | 63.63 | |||
| T stage | T1 | 1 | 0 | 2.02 (0.79–5.18) | 0.014 | 100 |
| T2 | 36 | 7 | 80.55 | |||
| T3 | 60 | 25 | 58.33 | |||
| T4 | 3 | 3 | 0 | |||
| N stage | N0 | 53 | 11 | 1.72 (1.01–2.95) | 0.046 | 79.25 |
| N1 | 28 | 8 | 71.43 | |||
| N2 | 19 | 16 | 15.79 | |||
| TNM stage | Stage 1 | 21 | 1 | 2.35 (1.2–4.6) | 0.013 | 95.24 |
| Stage 2 | 24 | 6 | 75 | |||
| Stage 3 | 45 | 19 | 57.78 | |||
| Stage 4 | 10 | 9 | 10 | |||
| LP | Absent | 64 | 15 | 0.49 (0.19–1.28) | 0.14 | 76.56 |
| Present | 36 | 20 | 44.44 | |||
| VP | Absent | 80 | 22 | 2.65 (0.94–7.48) | 0.006 | 72.5 |
| Present | 20 | 13 | 35 | |||
| PNI | Absent | 79 | 28 | 0.65 (0.25–1.67) | 0.37 | 64.55 |
| Present | 21 | 7 | 66.66 | |||
| TILs Score | Weak | 29 | 16 | 1.31 (0.82–2.09) | 0.25 | 37.93 |
| Moderate | 66 | 18 | 97.48 | |||
| Strong | 5 | 1 | 20 | |||
| Average tumor budding | Low | 3 | 0 | 1.25 (0.14–11.02) | 0.018 | 100 |
| Intermediate | 30 | 2 | 93.33 | |||
| High | 67 | 32 | 52.20 | |||
| Highest tumor budding | Low | 1 | 0 | 2.444 (.0212–28.172) | 0.035 | 100.00 |
| Intermediate | 22 | 2 | 90.9 | |||
| High | 77 | 32 | 58.44 |
CSS cancer-specific survival, Adeno adenocarcinoma, LP lymphatic permeation, VP vascular permeation, PNI perineural invasion, TILs tumor infiltrating lymphocytes
Table 2 shows correlation of tumor budding with T stage, N stage, and TNM stage when average tumor bud counts were considered. Table 3 shows correlation of tumor budding with T stage, N stage, and TNM stage when highest tumor bud counts were considered. These parameters were significantly associated with both average and highest tumor bud counts with p values < 0.05, except T stage and average tumor bud counts (p value-0.05).
Table 2.
Correlation Of tumor budding with clinicopathological parameters using average tumor bud counts
| Variables | Grade_avg_tumor_budding | P value | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Low | Intermediate | High | |||||||||
| Total N | No of events | Survival | Total N | No of events | Survival | Total N | No of events | Survival | |||
| T stage | T1 | - | - | - | 1 | 0 | 100 | - | - | - | 0.05 |
| T2 | 2 | 0 | 100 | 16 | 0 | 100 | 18 | 5 | 72.2 | ||
| T3 | 1 | 0 | 100 | 13 | 2 | 84.6 | 46 | 25 | 45.7 | ||
| T4 | - | - | - | - | - | - | 3 | 2 | 33.3 | ||
| N stage | N0 | 2 | 0 | 100 | 24 | 2 | 91.7 | 27 | 7 | 74.1 | 0.005 |
| N1 | 1 | 0 | 100 | 5 | 0 | 100 | 22 | 8 | 63.6 | ||
| N2 | - | - | - | 1 | 0 | 100 | 18 | 17 | 5.6 | ||
| TNM stage | Stage 1 | - | - | - | 13 | 0 | 100 | 8 | 1 | 87.5 | 0.008 |
| Stage 2 | 1 | 0 | 100 | 8 | 2 | 75 | 15 | 3 | 80 | ||
| Stage 3 | 1 | 0 | 100 | 8 | 0 | 100 | 36 | 20 | 44.4 | ||
| Stage 4 | 1 | 0 | 100 | 1 | 0 | 100 | 8 | 8 | 0 | ||
Adeno adenocarcinoma, LP lymphatic permeation, VP vascular permeation, PNI perineural invasion, TILs tumor infiltrating lymphocytes, W weak, M moderate, S strong
Table 3.
Correlation of tumor budding with clinicopathological parameters using highest tumor bud counts
| Variables | Grade_final_highest_tumor_budding | P value | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Low | Intermediate | High | |||||||||
| Total N | No of events | Survival | Total N | No of events | Survival | Total N | No of events | Survival | |||
| T stage | T1 | - | - | - | 1 | 0 | 100 | - | - | - | 0.0091 |
| T2 | 1 | 0 | 100 | 12 | 0 | 100 | 23 | 5 | 78.3 | ||
| T3 | - | - | - | 9 | 1 | 88.9 | 51 | 26 | 49 | ||
| T4 | - | - | - | - | - | - | 3 | 2 | 33.3 | ||
| N stage | N0 | 1 | 0 | 100 | 17 | 1 | 94.1 | 35 | 8 | 77.1 | 0.0078 |
| N1 | - | - | - | 4 | 0 | 100 | 24 | 8 | 66.7 | ||
| N2 | - | - | - | 1 | 0 | 100 | 18 | 17 | 5.6 | ||
| TNM stage | Stage 1 | - | - | - | 10 | 0 | 100 | 11 | 1 | 90.9 | 0.002 |
| Stage 2 | - | - | - | 6 | 1 | 83.3 | 18 | 4 | 77.8 | ||
| Stage 3 | - | - | - | 5 | 0 | 100 | 40 | 20 | 50 | ||
| Stage 4 | 1 | 0 | 100 | 1 | 0 | 100 | 8 | 8 | 0 | ||
Adeno adenocarcinoma, LP lymphatic permeation, VP vascular permeation, PNI perineural invasion, TILs tumor infiltrating lymphocytes, W weak, M moderate, S strong
Figure 3 shows Kaplan–Meier curves showing relationship of average tumor bud counts and cancer specific survival for T stage, N stage, and TNM stage. Figure 4 shows Kaplan–Meier curves showing relationship of highest tumor bud counts and cancer-specific survival for T stage, N stage, and TNM stage.
Fig. 3.
Kaplan–Meier curves showing relationship of average tumor bud counts and cancer-specific survival for T stage, N stage, and TNM stage
Fig. 4.
Kaplan–Meier curves showing relationship of highest tumor bud counts and cancer-specific survival for T stage, N stage, and TNM stage
On follow-up, out of all cases which showed high tumor bud score (with respect to both average and highest tumor bud counts), five cases showed distant organ metastasis (liver, lung, bones, urinary bladder); two cases showed local recurrence as well as widespread metastasis in lymph nodes, lung, liver, and peritoneal metastasis including ascites; two cases showed only peritoneal metastasis; and single case showed local recurrence with lymph node metastasis. Mean time to recurrence or metastasis in all of these cases was 17 months (range 6–31 months).
Single case showed that lung and peritoneal metastasis on 20 months of follow-up had intermediate tumor bud score based on average bud count and high tumor bud score based on highest bud count.
Cases with intermediate bud score showed predominantly local recurrence on follow-up. Out of 30 cases two showed local recurrences at 15 months and 32 months, respectively, after initial treatment. Another case showed widespread metastasis after 24 months of follow-up.
Single case which showed adrenal metastasis after 12 months of follow-up had low tumor bud score based on average bud count and intermediate tumor bud score based on highest bud count.
Rest of the cases with high and intermediate bud score were survived till the end of follow-up. All the cases with low tumor bud score survived till the end of follow-up. Seventeen cases were lost to follow-up.
Discussion
Results of our study effectively show that tumor budding can be used as a prompt prognostic indicator for CRC. It is significantly associated with cancer-specific survival along with other already established prognostic factors such as T stage, N stage, TNM stage, and lympho-vascular permeation. Hazard ratios (HR) for tumor budding, when average counts are considered and highest bud counts are considered, are 1.25 and 2.444, respectively (CI 95%), which is superseded only by vascular permeation (HR 2.65, CI 95%). These findings are comparable with findings of some other studies [22, 23].
Van Wyk et al. [22] showed tumor budding as an independent marker of cancer-specific survival in CRC with highest hazard ratio compared to other clinico-pathological parameters [22].
Studies have suggested that assessing tumor budding plays an important role in predicting prognosis of patients especially in early (stage I & II) CRC [22, 24, 25]. High tumor bud score in pT1 patients is associated with increased chances of lymph node metastasis [24–26].
In our study, 28 cases (28%) were in N1 stage and 19 cases (19%) were in N2 stage at the time of initial presentation. Out of those, high tumor bud score was found in 22 (79%) cases with N1 stage and 18 (95%) cases with N2 stage. Our findings suggest that high tumor bud score is a significant risk factor for lymph node metastasis and can be used to predict the extent of lymphadenectomy. However, in our study, 27 out of 53 (51%) N0 cases also had high tumor bud score.
A study done by Deb B and Jacob SE suggest that tumor bud score in preoperative biopsies is indicated to be a positive predictor of lymph node metastasis in colorectal carcinomas [27].
In stage II CRC, tumor budding is an independent predictor of survival and guides in further treatment strategy and adjuvant therapy [22, 24, 25].
Rogers AC et al. mention in their meta-analysis that, although, early stage CRCs (stage I & II) are generally treated with surgery only; tumor bud score can be used to stratify these cases into a subset of cases which might benefit from further adjuvant therapy [28]. They also mention various studies which suggest that tumors showing budding might not respond to neoadjuvant chemotherapy [28]. However, we did not include cases with neoadjuvant therapy for our study.
In another study by Zlobec I et al., they conclude that tumor budding in conjunction with K-RAS mutation analysis can be used to predict response to anti-EGFR therapy in metastatic colorectal carcinoma [29].
High tumor bud score is also associated with other parameters of poor prognosis such as higher grade of tumor and lymphovascular permeation [25].
As shown in Table 1, 77% of cases had high (≥ 10) tumor bud score based on highest tumor bud counts, which is comparable to other studies [30, 31]. Sixty-seven percent cases had high tumor bud score based on average bud counts. In the study by Mehta et al. [30], only 28.2% cases had high tumor bud score based on average bud counts. This discordance can be due to variations in selection of cohort as well as subjective variation in counting of buds.
Follow-up results of our cases also indicate that cases with high tumor budding developed early recurrences and more widespread metastasis than other cases.
Our study results also imply that both average bud counts and highest tumor bud counts are significantly associated with survival, although HR was greater when highest bud counts are used for analysis.
Tumor budding also showed significant association with TIL score. Certain other studies have proposed combining tumor budding with TIL score for stratification of prognostic groups in CRC [32, 33].
High tumor bud score was observed in four out of five (4/5) cases with signet ring cell carcinoma in our study. Signet ring cell morphology of tumor has a correlation with poor prognosis and decrease survival rate [34].
Although some studies have applied immunohistochemistry markers to identify tumor budding, our results are solely based on assessment of tumor budding on H & E stained slides [35, 36].
Lastly, our study has certain limitations such as limited cohort size and unavailability of higher molecular investigations to establish correlation of tumor budding with particular mutations. But our results certainly add in the existing knowledge regarding tumor budding as a valuable prognostic indictor in CRC.
Conclusion
We conclude that tumor budding has a significant association with cancer-specific survival and prognosis of patient in CRC. High tumor bud score is associated with higher tumor grade, lymphovascular permeation, lymph node metastasis, and overall higher stage of tumor. We might need additional follow-up protocol for patients with high tumor bud score. Tumor budding should be interpreted in a multidisciplinary setting along with other clinicopathological factors. A 3-tier scoring system for tumor budding should be included in guidelines for routine reporting of CRC specimens and can be considered as a significant indicator of prognosis for staging and further management.
Acknowledgements
We thank technical staff for their help and community oncology department for their guidance in statistical analysis.
Author contribution
Ashini Shah and Neetal Desai performed study concept, design, and development of methodology as well as writing and review of the article. Amisha Gami performed development of methodology, writing, and review of the article. Jahnavi Gandhi and Priti Trivedi performed review and revision of the article. All authors approved the final paper.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Subject ontology
Tumor budding, Colorectal carcinoma, Survival.
Contributor Information
Ashini H. Shah, Email: drashini_shah@yahoo.co.in
Amisha J. Gami, Email: amisha.gami@gcriindia.org
Neetal H. Desai, Email: neetu.khushi13@gmail.com
Jahnavi S. Gandhi, Email: jahnavi.gandhi@gcriindia.org
Priti P. Trivedi, Email: priti.trivedi@gcriindia.org
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.