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Year : 2017  |  Volume : 54  |  Issue : 4  |  Page : 640--645

Reporting of tumor budding in colorectal adenocarcinomas using ×40 objective: A practical approach for resource constrained set-ups

Paromita Roy1, Jayati Datta1, Manas Roy2, Indranil Mallick3, Mallath Mohandas4,  
1 Department of Pathology, Tata Medical Center, Kolkata, West Bengal, India
2 Department of GI Surgery, Tata Medical Center, Kolkata, West Bengal, India
3 Department of Radiation Oncology, Tata Medical Center, Kolkata, West Bengal, India
4 Department of Medical Oncology, Tata Medical Center, Kolkata, West Bengal, India

Correspondence Address:
Dr. Paromita Roy
Department of Pathology, Tata Medical Center, Kolkata, West Bengal


CONTEXT: Tumor budding (TBud) is recognized as a poor prognostic marker in colorectal cancer (CRC) with important treatment implications in Stage II cancers and malignant polyps. There are multiple propositions for bud count reporting but without an uniformly accepted system. The International TBud consensus conference (ITBCC) proposed mandatory reporting of budding on the single worst ×20 high power field (0.785 mm2 area) with a 3-tier scoring system (low/intermediate/high for 0–4, 5–9, and ≥10 buds/×20 field). AIMS: Due to the lack of availability of ×20 objective, we aimed to validate a simple ×40 field count (0.236 mm2 area) for wider applicability. METHODS: Bud count was done on hematoxylin and eosin-stained slides of 92 archived cases of colon cancer on the worst ×20 and ×40 fields (0.95 mm2 and 0.236 mm2 area) (hotspot method). Count for 0.785 mm2 area was calculated using ITBCC normalization factor of 1.2. Interobserver variability between two observers was assessed. Score groups for ×20 field and proposed score groups for 40× field (low/intermediate/high for 0–1, 2–4 and ≥5 buds) were compared with disease-free survival. RESULTS: High bud score was seen in 20.6% and 31.5% cases, respectively, using the ×20 and ×40 methods. High interobserver concordance was noted (ICC 0.95). Both the ITBCC bud score and our proposed 40× scoring correlated significantly with prognosis (P = 0.030, log-rank test). CONCLUSIONS: In centers lacking 20× objective, we propose using the worst 40× hotspot method for reporting of budding for all CRCs as a simple, reproducible and prognostically significant scoring system.

How to cite this article:
Roy P, Datta J, Roy M, Mallick I, Mohandas M. Reporting of tumor budding in colorectal adenocarcinomas using ×40 objective: A practical approach for resource constrained set-ups.Indian J Cancer 2017;54:640-645

How to cite this URL:
Roy P, Datta J, Roy M, Mallick I, Mohandas M. Reporting of tumor budding in colorectal adenocarcinomas using ×40 objective: A practical approach for resource constrained set-ups. Indian J Cancer [serial online] 2017 [cited 2021 Sep 21 ];54:640-645
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Tumor budding (TBud) is defined as single cells or small groups of tumor cells (up to 4 cell clusters) within the tumor or at the invasive front. TBud is an independent predictor of lymph node metastasis in pT1 colorectal cancer (CRC) and an independent predictor of survival in stage II CRC being associated with lymphovascular invasion, lymph node, and distant metastasis.[1],[2],[3],[4] However, widespread adoption of reporting of this important prognostic parameter has been limited by a lack of uniform scoring systems. The different methods used by various researchers vary significantly regarding the field size and cutoff values used for scoring [Table 1]. This problem was addressed by the International TBud Consensus Conference (ITBCC; Bern, April 2016), and recommendations for reporting of budding in colon cancer was published in May 2017.[5] The conference proposed counting buds on hematoxylin and eosin (HE)-stained slides on the single worst (hotspot) ×20 field (with a 0.785 mm2 field area) and reporting as a three-tier scoring system [Table 1]. In an updated protocol, the College of American Pathologists (CAP) also advocated routine reporting of TBuds in all CRCs based on the ITBCC recommendations.[6], [23]{Table 1}

However, in low-resource countries such as India, availability of ×20 objective is uncommon in most pathology laboratories. Even in academic centers of excellence, none or only few of the microscopes have a ×20 objective. Most lower end microscope models have a nose piece accommodating four objectives – ×4, ×10, ×40, and ×100. This is a big hurdle in the widespread adoption of the ITBCC recommendations for reporting budding in low-resource countries. The ITBCC also provided a score converter for counting buds on a ×20 objective with varied field sizes.[5] At present, there is no validated protocol for counting buds using a ×40 high power field (hpf) and reporting based on the ITBCC system. To address this problem, we used the ITBCC scoring guidelines to propose a novel hotspot 40× field-based scoring system for easy applicability and widespread reporting of TBud in resource-poor laboratory setups.


The hospital electronic medical record (EMR) was searched for CRC patients who underwent resection at our institute between 2011 and 2015. Out of a total of 332 cases, 92 were available for this retrospective review. Exclusion criteria included neoadjuvant treatment, metastatic disease at presentation, less than 3 tumor blocks, and lack of clinical follow-up.

Formalin-fixed paraffin-embedded (FFPE) tissue blocks and HE-stained slides were retrieved from the archives and all tumor sections were reviewed. Fresh recuts were reviewed in cases where the original stain was faded. First, all slides were screened at ×10 to identify fields with the highest bud count at the peritumoral interface. Intratumoral bud count was not done. The ten worst fields were further screened on high power and the absolute TBud count was recorded on the single worst ×20 and ×40 fields with the highest score (hotspot method).[5] Scoring was done independently by two pathologists (PR and JD). Microscopy was performed using Leica DM 1000 LED with a field area of 0.95 mm2 (using ×20 objective) and 0.236 mm2 (on ×40 objective).

For inferring TBud count for a 0.785 mm2 field, we used the score converter provided by ITBCC,[5] i.e., dividing the count done on ×20 objective with 0.950 mm2 field by the normalization factor 1.210.

We compared the absolute counts done on ×40 and ×20 (field area 0.95 mm2 4 times than that of ×40 field) to determine if a simple area-based conversion method would work adequately, to devise a similar normalization factor, and for inferring ×20 counts from bud counts done on 40×. The calculated count (count on ×40 multiplied by 4) was compared with the actual ×20 count using the Pearson correlation test and assessing the correlation using graphical methods.

TBud score was also recorded as an average of ten ×40 hpfs in 28 cases and grouped into two groups – low, showing <10 buds and high, showing ≥10 buds/10 hpf.

Where budding was obscured by dense peritumoral inflammation and reactive stromal fibroblasts, we used immunohistochemistry (IHC) with pan-cytokeratin (panCK) (clone AE1/AE3, Novacastra; on Leica Bondmax IHC platform) to facilitate the detection of TBud. Subsequently, scoring was done on HE sections. Deeper sections were studied to ensure ruptured glands were not counted as TBud, and areas of mucin pools and necrosis were avoided.

Other histopathological variables documented were tumor location, type, grade (low or high based on ≥ or <50% glands), margin status, presence of lymphovascular, perineural and large vessel invasion, and TNM (tumor nodal metastasis) stage (Union for International Cancer Control; UICC TNM 7th edition). Clinical variables noted from the EMR were the evidence of failure (radiological, clinical, biochemical, or pathological), and recurrence-free survival. Data were recorded using the REDCap® version 5.11.4 software.

We assessed the prognostic significance of TBud score grouping done for the normalized ×20 count (0.785 mm2 field), as per the ITBCC recommendation [Table 2][5] using regression methods. We proposed similar score grouping for the ×40 hotspot field counts (0.236 mm2 field) and compared the recurrence-free survival of these groups using the log-rank test. Statistical analysis was performed using SPSS Version 20. Interobserver variability in counting TBud by two observers was done by estimating the intraclass correlation coefficient (ICC) using two-way analysis of variance test.{Table 2}


The clinicopathological variables captured in this retrospective review of 92 cases are listed in [Table 2]. The most common site in our series was sigmoid colon, with nine cases involving more than one site. Specimen length varied from 10 to 84 cm, with one having distal margin involvement and four cases with circumferential resection margin involvement. Predominant tumor type was adenocarcinoma, not otherwise specified, and low grade was more prevalent. Most tumors were of stage II, pT3, and pN0. The median follow-up for our cohort was 44 months.

Absolute TBud count on HE-stained sections ranged from 0 to 80 on ×20 and 0–50 on the worst ×40 hpf. In two cases, TBud count was difficult on HE staining due to dense inflammation; hence, IHC with PanCK was used to identify the tumor cells, following which count was done using HE sections.

A three-tier scoring system for reporting bud count using ×20 objective (0.95 mm2 field) with cutoffs of 0–5, 6–11, and ≥12 buds is listed in [Table 3]. The calculated count for 0.785 mm2 field and score grouping as per the ITBCC recommendations are also listed [Table 3].{Table 3}

Based on the range of absolute counts on the hotspot ×40 field, we grouped our cases into clinically relevant groups as low (0–1 buds/hpf), intermediate (2–4 buds/hpf), and high score (≥5 buds/hpf) [Table 3].

Low scores were the most prevalent group in our cohort for both the ×20 and ×40 methods. On bud counting on 10 ×40 hpfs, majority (78.5%) of our cases had <10 buds/10 hpf.

On correlating with disease-free survival, both the ITBCC score grouping on the corrected ×20 field count (P = 0.050) and our new proposed score grouping done on ×40 hotspot counts (P = 0.030) proved to be highly significantly correlating with prognosis. The 3-year disease-free survival in the bud score groups done on ×40 count, was 87.6%, 70.3%, and 49.6% in the three groups (P = 0.030, log-rank test), whereas that for the normalized ×20 count (0.785 mm2 area) were 79%, 53.5%, and 51.9% (P = 0.050). The score groups done on the ten ×40 hpf counts did not show statistical significance (P = 0.562). The Kaplan–Meier survival plots for ×40 and 20 bud scoring methods are shown in [Figure 1] and [Figure 2].{Figure 1}{Figure 2}

TBud counts on both the ×20 evaluation and ×40 evaluation showed a high interobserver agreement. For ×20 evaluation, the ICC was 0.955 (95% confidence interval [CI] 0.925–0.973); and for the ×40 evaluation it was 0.951 (95% CI 0.918–0.971). This demonstrates near perfect concordance for TBud scoring, which can be reproduced in the clinical setting.

While we noticed that there was good correlation between the worst field ×40 and ×20 counts (Pearson correlation coefficient = 0.729 P < 0.001), simple multiplication of the ×40 count by four did not match ×20 counts. The scatter-plot [Figure 3] shows the mutual values, which are in most cases shifted to left of the dotted line demonstrating that the actual ×20 values are lower than the ×40 values multiplied by four (based on a simple area based conversion; see discussion).{Figure 3}


TBud has gradually evolved as one of the most important prognostic markers to document in various sites such as head and neck, esophagus and lung squamous carcinomas, and breast and colonic adenocarcinomas.[7],[8] It represents an epithelial–mesenchymal transition like process.[9] Specific management guidelines for colon cancer based on bud count have been recently proposed in three clinicopathological settings – surgical resection for adenocarcinoma arising in a polyp, adjuvant treatment for Stage II CRCs, and neoadjuvant treatment decision on preoperative biopsies of CRC.[10],[11],[12]

Although the clinical significance of TBud is undoubted, there has been little concordance among researchers regarding the best method for routine reporting of TBud. Significantly differing criteria have been used in the literature [Table 1]. The initial system was proposed by Morodomi in 1989,[13] followed by Ueno et al. in 2002[14] using a ×25 objective. Other authors have used a ×20 hpf to count TBud.[3],[15],[16],[17] Lugli et al. proposed a scoring system using counts based on an average of 10 worst hpfs using a ×40 objective.[18] This was the only study to propose a ×40 hpf-based TBud scoring system.

ITBCC addressed this problem, and a team of global experts agreed to a unified system of reporting TBud on the single worst ×20 field measuring 0.785 mm. 2 This method has been shown to have high interobserver concordance and is easy to adopt in routine practice.[5]

However, the availability of ×20 objective is a problem in adopting this method in resource-poor nations. An informal E-mail survey of 25 general laboratories, oncologic institutes, and academic centers of excellence in India and neighboring countries (Sri Lanka, Bangladesh, Bhutan, and Myanmar) confirmed this finding. Approximately 60% (15/25) laboratories lacked ×20 completely, while the remaining had the objective in a single (teaching/photography) scope (24%) or a few microscopes in the laboratory (16%). Although the cost of a ×20 objective is not prohibitive, the microscopes mostly have a nose piece for four objectives. The preferred objectives were ×4, ×10, ×40, and ×100. The use of oil immersion ×100 lens for detection of mycobacteria is a vital requirement in histopathology services in India. Laboratories that can afford more expensive microscopes (Leica DM1000 or equivalent) have a nosepiece accommodating five objectives, which are more commonly ×2.5, ×4, ×10, ×40, and ×100 rather than the ×20 objective.

In this review, we applied the ITBCC scoring system for TBud in our cohort and tried to emulate the same method to devise a clinically reproducible, prognostically significant, simple scoring system using a ×40 hotspot field so that laboratories lacking the ×20 objective could still report this important prognostic marker.

Using the ITBCC scoring guidelines we found the incidence of high budding scores to be lower (20.6%) in our cohort than that reported by other authors using various method.[3],[16] Our high score using ×40 hotspot method with a cutoff of >5 buds/hpf was 31.5%. Ueno et al.[14] and Graham et al.[2] using comparable methodologies as the ITBCC guidelines reported similar rates (30.1% and 32% high-grade budding, respectively). In contrast, Karamitopoulou et al. reported high-grade budding in 48% patients using a cutoff of 10 buds/×40 hpf.[18]

We found highly significant correlation with recurrence-free survival using both the ×20 hotspot method (P = 0.050) similar to other studies[5],[19] and the ×40 hotspot method (P = 0.030), as shown by the Kaplan–Meier curves [Figure 1].{Figure 1}

Similar to the ×20 method, the ×40 hotspot method was easy to apply in daily practice without increasing the turn-around-time of reporting. We observed that buds were easier to appreciate on the higher ×40 magnification (rather than ×20) and easier to count manually due to the smaller field area [Figure 4]. A clue for the presence of TBud on ×10 screening for the worst field was an infiltrative invasive front of tumor. Mitrovic et al.[9] reported blurring of the interface between tumor and stroma to be an indicator of TBud.{Figure 4}

The interobserver variability assessment for our study showed excellent concordance between two pathologists for the ×40 method (ICC, 0.955). Variable rates for interobserver concordance of different methods for TBud counting have been reported in the literature (ranging from 0.41 to 0.938).[9] Most authors have had less impressive variability kappa values than those reported by us. Wang et al. reported a moderate interobserver agreement of κ value of 0.51,[3] whereas Ueno et al. reported a weighted κ coefficient of 0.82.[20] Koelzer et al. performed a large multicenter interobserver study and found Pearson correlation rates ranging from 0.46 to 0.91, which improved on cytokeratin stained slides (r = 0.73–0.95).[21] They also showed better concordance rates using an average of 10 fields rather than the worst field count. They also compared seven different scoring methods (Hase, Nakamura, Wang, Ueno, and Lugli 10 hpf and 1 hpf methods) [Table 1], and found the 10 hpf and 1 hpf methods to be most reproducible (ICC = 0.91 and 0.83, respectively). Karamitopoulou et al. also reported excellent agreement using the 10 hpf method (ICC = 0.97).[18]

We appreciate that wrong choice of the worst field can significantly affect the TBud score, and taking an average of ten ×40 hpfs would minimize this error rate. We validated the Lugli et al. method[18] of counting buds on 10 hpfs by dividing our cases into two score groups (<10 buds/10 hpf and ≥10 buds/10 hpf). The prognostic importance in our cohort was not statistically significant (P = 0.562). The lack of adequate number of cases may have limited this prognostic assessment. However, counting buds on ten ×40 fields is tedious for use in routine reporting, and our high interobserver concordance as well as prognostic significance of the ×40 hotspot method makes it the preferred system. The current consensus guidelines (ITBCC) of reporting on a single worst field also support this theory.

We used IHC (with Pan-cytokeratin; AE1/AE3 clone) in only two cases where dense inflammation and prominent reactive fibroblasts in the tumor-stroma interface limited the assessment of TBud. IHC helped identify the tumor cells and was not used for actual scoring, which was done on HE-stained slides. Although bud counts are higher with IHC and reproducibility is better, the increased cost and turn-around-time does not justify routine use of IHC in TBud reporting.[5],[19] Okamura et al. reviewed 265 cases by both routine HE and Cam5.2 IHC, and did not find the latter to be superior to HE staining for predicting lymph node metastasis in pT1 CRC.[22]

We noted that simply multiplying the ×40 field TBud counts by 4 did not correlate with the corresponding absolute counts recorded on the ×20 field [Figure 3]. Hence, an area-based conversion factor, i.e., a normalization factor (as proposed for the ×20 fields of different diameters, by the ITBCC guidelines) cannot be devised for calculating ×20 field scores from counts recorded using a single ×40 field.

Koelzer et al.[19] also proposed using the worst ×40 hpf method. In contrast to our cutoff of 5 buds/hpf being indicative of a high bud score, they recommended a cutoff of 10 buds/hpf for high-risk budding. However, in their study, they used a ×40 field with an area of 0.49 mm2, which is double that of the field used in our study (which is 0.236 mm2), and hence their proposed cut-off is similar to ours for high-grade bud group scoring.

The limitation of our study is the fact that it is a retrospective review done by two observers. Our findings need to be validated using a prospective larger cohort by multiple observers.


TBud assessed on routine HE sections using both ×20 and ×40 worst field score has high reproducibility and correlates significantly with prognosis. The ITBCC guidelines aim to promote the adoption of a uniform system of reporting this vital prognostic marker. Because of lack of easy availability of ×20 objective in low-resource laboratories, we propose counting on the ×40 field (0.236 mm2 area) using the same methodology and applying our proposed score groups for reporting TBud for all CRCs.


We would like to thank Professor Sanjay Kakar, VAMC, University of California San Francisco, for his guidance in doing this study and Mr. TK Giri, Senior Technologist, Department of Pathology, Tata Medical Center, for technical support.

Financial support and sponsorship

This study was supported by Tata Medical Center, Kolkata, India

Conflicts of interest

There are no conflicts of interest.


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