ISSN: 08526834 | E-ISSN:2337-389X
Berkala PENELITIAN HAYATI Volume 31 | No.
3 | December | 2025 Original article Evaluation of Immunohistochemistry and H&E Staining for Detection of Tumor Budding in Colorectal Cancer Khesar Hussein Khalil1 Department of Anatomy.
Biology and Histology.
College of Medicine.
University of Duhok.
Zakho Way 42001.
Duhok.
Iraq Abstract Tumor budding (TB) is an established prognostic marker in colorectal cancer (CRC) and is defined as single tumor cells or clusters of up to four cells at the invasive front.
While the International Tumor Budding Consensus Conference (ITBCC) recommends hematoxylin and eosin (H&E) as the standard for TB evaluation, immunohistochemistry (IHC) with pan-cytokeratin may improve detection in histologically challenging cases.
The current study aims to compare TB assessment using H&E and pan-cytokeratin IHC and evaluate its association with clinicopathological parameters in CRC.
A retrospective analysis was conducted on 98 CRC cases resected between 2017 and 2022.
TB was graded according to ITBCC 2016 criteria on H&E and cytokeratinstained sections .
lone AE1/AE.
Associations with clinicopathological features were analyzed using Chi-square or FisherAos exact tests, with p O 0.
considered significant.
On H&E.
TB was positive in 61 cases .
2%): Bd1 .
6%).
Bd2 .
3%).
Bd3 .
3%).
On IHC.
TB was positive in 67 cases .
4%): Bd1 .
7%).
Bd2 .
4%).
Bd3 .
3%) .
< 0.
001 for increased detectio.
High TB (Bd2AeBd.
correlated significantly with advanced pathological stage .
< 0.
, higher T stage .
< 0.
, lymph node metastasis .
< 0.
, vascular invasion .
= 0.
, perineural invasion .
= 0.
and desmoplasia .
= 0.
Pan-cytokeratin IHC improves TB detection compared with H&E, especially in morphologically complex areas, and high TB is strongly associated with aggressive tumor features.
Selective IHC use may enhance diagnostic accuracy and prognostic assessment in CRC.
Keywords: tumor budding, colorectal cancer, immunohistochemistry, pan-cytokeratin.
H&E, prognosis Received: August 25, 2025 | Revised: September 2, 2025 | Accepted: October 20, 2025 Introduction Colorectal cancer (CRC) ranks as the third most prevalent cancer and the fourth leading cause of cancerrelated mortality (Marcellinaro et al.
, 2.
It frequently arises from benign neoplastic lesions, including adenomatous and serrated polyps, due to the gradual accumulation of genetic and epigenetic alterations, some of which are implicated in the activation of oncogene signaling pathways (Myrmol et al.
, 2.
Despite advancements in diagnostic and therapeutic strategies, the ability of CRC cells to invade surrounding tissues and metastasize remains a major clinical challenge (Bian et al.
, 2.
The invasion-metastasis cascade is a complex process involving multiple biological mechanisms, including tumor budding (TB), which are increasingly recognized as critical factors in CRC progression (Peng et al.
, 2.
TB is a well-known histopathological hallmark in CRC and has been identified as an independent unfavorable prognostic marker (Graham et al.
, 2.
It is defined as the presence of single tumor cells or small clusters of up to four cells at the invasive front of carcinomas, representing a morphological manifestation of epithelialAemesenchymal transition (EMT) (Lugli et al.
and early metastatic potential (Zlobec et al.
, 2.
Numerous studies have indicated that TB is associated with lymph node metastasis (Dawson et al.
, 2.
, local * Corresponding Author:
Khesar Hussein Khalil Department of Anatomy.
Biology and Histology.
College of Medicine.
University of Duhok.
Zakho Way 42001.
Duhok.
Iraq Phone: 964 750 4425372 E-mail: khesar.
khalil@uod.
recurrence, distant metastases, and decreased overall survival, making it an essential factor in patient risk assessment (Rogers et al.
, 2.
Despite its prognostic significance, the evaluation of tumor budding in routine diagnostic practice has encountered persistent challenges due to the absence of standardized definitions and grading criteria (Mitrovic et al.
This issue was discussed by the International Tumor Budding Consensus Conference (ITBCC) in 2016, which suggested a threeAatier scoring system based on hematoxylin and eosin (H&E) staining: Bd1 .
Ae4 bud.
Bd2 .
Ae9 bud.
, and Bd3 (Ou10 bud.
per AuhotspotAy field (Neppl et al.
, 2.
These recommendations enhanced reproducibility and simplified the incorporation of TB into CRC reporting guidelines (Lugli et al.
, 2.
Nevertheless.
H&E evaluation of TB remains challenging in specific histological situations, including desmoplastic stroma, inflammatory cell infiltration, necrosis, and mucin pools, where tumor buds may be hidden (Pihlmann Kristensen et al.
, 2.
Immunohistochemical (IHC) staining using pan-cytokeratin has been recommended to improve detection accuracy by focusing epithelial tumor cells, thereby differentiating them from stromal or inflammatory components (Fisher et al.
This method could improve sensitivity and interobserver consistency, especially in conditions of borderline or ambiguous budding on H&E.
However, an overdependence on IHC raises the risk of inflating budding grades, which may affect treatment decisions (Takamatsu et al.
, 2.
Taking these factors into consideration, a direct comparison of TB detection employing H&E versus pancytokeratin IHC in CRC can yield significant insights for http://dx.
org/10.
23869/bphjbr.
Published by A PBI East Java.
Open Access Ea w.
Khesar H.
Khalil the improvement of diagnostic techniques.
This study aims to evaluate the agreement between these two methodologies, examine their correlation with clinicopathological parameters, and determine whether IHC provides significant diagnostic superiority over traditional H&E in TB assessment.
Methods Ethical Approval This study was approved by the Ethics Committee of the University of Duhok and the Duhok Directorate General of Health (Approval Number: 31072024-6-44 in July 31, 2.
As this was a retrospective study using archival material, informed consent was waived.
Study Design and Cases This retrospective study involved 98 patients who received surgical resection for histologically confirmed colorectal adenocarcinoma from January 2017 to December 2022.
Formalin-fixed paraffin-embedded (FFPE) tissue specimens were obtained from the archives of the Department of Pathology.
Duhok Central laboratory.
Duhok.
Iraq.
Data regarding clinical and pathological aspects, such as patient demographics, tumor location, histological type, grade, and stage, has been collected from pathology reports and medical records.
Exclusion Criteria Patients who had previously received systemic chemotherapy or radiotherapy for their colorectal cancer were excluded from the study.
This restriction was applied to eliminate potential confounding effects of prior treatments, as both chemotherapy and radiotherapy can alter tumor morphology, induce tissue necrosis or fibrosis, and modify molecular signaling pathways Histopathological Evaluation of Tumor Budding Slides stained with H&E for each case were independently examined by two experienced pathologists who were unaware of the clinical outcomes.
TB was evaluated following the 2016 guidelines of ITBCC, as A The AuhotspotAy area at the invasive front was identified under low magnification.
A Tumor buds, defined as single tumor cells or clusters of up to four tumor cells, were counted in one hotspot field measuring 0.
785 mmA at y20 objective A Bud counts were categorized into three grades: Bd1 .
ow budding, 0Ae4 bud.
Bd2 .
ntermediate budding, 5Ae9 bud.
, and Bd3 .
igh budding.
Ou10 bud.
Cases where inflammation, necrosis, mucin pools, or desmoplastic stroma made it difficult to identify tumor buds were recorded separately for additional IHC analysis.
Immunohistochemistry for PanAaCytokeratin Sections measuring 4 M from representative tumor blocks underwent IHC staining for pan-cytokeratin .
lone AE1/AE3.
Dako.
Denmar.
using the EnVision detection system.
Antigen retrieval was conducted with citrate buffer .
H 6.
in a pressure cooker for a duration of 20 minutes.
The chromogen employed was diaminobenzidine (DAB), with hematoxylin used as the counterstain.
Cytokeratin IHC was applied to distinguish between epithelial tumor cells at the invasive front, facilitating improved visualization of tumor buds in regions where H&E assessment was challenging.
Budding was assessed according to the ITBCC criteria applied to cytokeratin-stained sections.
Statistical Analysis Data analysis was conducted using SPSS software 0 (IBM Corp.
Armonk.
NY.
USA).
The relationship between tumor budding grade .
ssessed via H&E and IHC) and clinicopathological variables was analyzed using the Chi-square test or FisherAos exact test, as applicable.
A p-value of 0.
05 or less was considered statistically significant.
Results Clinicopathological Characteristics of the Patients The clinicopathological features of the 98 colorectal cancer cases are summarized in Table 1.
The median age fell within the older adult category, with approximately two thirds of patients aged 50 years or older.
There was nearly an equal number of males and females.
Just over fifty percent of the tumors were situated in the left colon.
The predominant type was conventional adenocarcinomas, with a minor percentage exhibiting mucinous or signet-ring cell histology.
The majority of tumors exhibited moderate differentiation.
Stage II and stage i diseases were the most prevalent, with over two-thirds of tumors classified as T3 or T4.
More than 40% of patients presented with nodal metastases, while a minority exhibited distant metastases (M.
Perineural and vascular invasion were observed in approximately fifty percent and nearly seventy-five percent of cases, respectively.
Lymphocytic infiltration was noted in more than twothirds of tumors, and desmoplasia was also prevalent.
Table 1.
Demographic and clinicopathological characteristics of the study cohort Variable Category < 50 years Age Ou 50 years Male Sex Female Right colon Tumor location Left colon Adenocarcinoma NOS Histology Mucinous adenocarcinoma Signet-ring cell carcinoma Well differentiated Grade Moderately differentiated Poorly differentiated Stage i T stage Berkala PENELITIAN HAYATI | Volume 31 | Number 3 | December | 2025 Tumor Budding in Colorectal Cancer Variable N stage M stage Positive Perineural invasion Negative Positive Vascular invasion Negative Lymphocytic infil- Positive Negative Positive Desmoplasia Negative Category Tumor Budding on H&E Staining According to ITBCC 2016 criteria, tumor budding was identified in nearly two-thirds of cases on H&Estained sections.
Low-grade budding (Bd.
was the most common pattern among positive cases, followed by intermediate (Bd.
and high-grade (Bd.
Over one-third of tumors showed no detectable budding on H&E.
Representative photomicrographs of the three budding grades are shown in Figure 1, and the detailed distribution is summarized in Table 2.
Table 2.
Tumor budding grades on H&E-stained sections TB Grade (H&E) (%) Bd1 .
Ae4 bud.
Bd2 .
Ae9 bud.
Bd3 (Ou10 bud.
Negative .
Total Fig.
Representative photomicrographs of tumor budding grades on H&E staining according to ITBCC 2016 criteria.
Bd1: lowgrade budding .
Ae4 bud.
Bd2: intermediate-grade budding .
Ae9 bud.
Bd3: high-grade budding (Ou10 bud.
: Negative.
Tumor buds are identified as single cells or small clusters of up to four cells at the invasive front.
Scale bars = 100 AAm.
Berkala PENELITIAN HAYATI | Volume 31 | Number 3 | December | 2025 Khesar H.
Khalil Tumor Budding on Pan-CytokeratinAeStained Sections Pan-cytokeratin (AE1/AE.
immunostaining was positive in over two-thirds of cases.
In positive cases, staining markedly improved the visibility of tumor buds, particularly in areas with dense stromal reaction or prominent inflammatory infiltrates.
Among positive cases, low-grade budding (Bd.
was most frequent, followed by intermediate (Bd.
and high-grade (Bd.
The detailed distribution is shown in Table 3, with representative microscopic images in Figure 2.
Table 3.
Tumor budding grades on pan-cytokeratinAestained sections (%) TB Grade (IHC) .
Bd1 .
Ae4 bud.
Bd2 .
Ae9 bud.
Bd3 (Ou10 bud.
Negative Total .
Fig.
Representative photomicrographs of tumor budding grades on IHC staining according to ITBCC 2016 criteria.
Bd1: low-grade budding .
Ae4 bud.
Bd2: intermediate-grade budding .
Ae9 bud.
Bd3: high-grade budding (Ou10 bud.
: Negative.
Tumor buds are identified as single cells or small clusters of up to four cells at the invasive front.
Scale bars = 100 AAm.
Berkala PENELITIAN HAYATI | Volume 31 | Number 3 | December | 2025 Tumor Budding in Colorectal Cancer Comparison between H&E and pan-cytokeratin detection Pan-cytokeratin immunostaining significantly increased the detection of tumor budding compared with H&E, reducing the proportion of negative cases from 8% to 31.
6% .
= 0.
as shown in Figure 3.
While the relative proportions of Bd1AeBd3 grades remained similar between the two staining methods, pancytokeratin facilitated the identification of buds in cases with dense stromal reaction or inflammatory background.
Fig.
Comparison of tumor budding grades assessed on H&E-stained sections and pan-cytokeratin (AE1/AE.
Aestained sections according to ITBCC 2016 criteria.
The number of cases in each budding category (Bd1.
Bd2.
Bd3, and negativ.
is shown for both staining methods.
Pan-cytokeratin staining significantly increased tumor budding detection compared with H&E .
= 0.
Table 4.
Association between tumor budding grade (H&E) and clinicopathological variables.
Tumor Budding Variants Positive (%) Negative (%) p- value <50 23 .
Age Ou50 38 .
Male 35 .
Sex Female 26 .
Right 26 .
Location of Tumor Left 35 .
Histological type Mucinous 8 .
Signet ring cell Ad 1 .
Well 3 .
Grade Moderate 45 .
Poor 13 .
Stage i 33 .
Tumor invasion 36 .
Lymph node involvement 23 .
Metastasis 6 .
Positive 51 .
Vascular invasion Negative 10 .
Positive 38 .
Perineural invasion Negative 23 .
Positive 41 .
Lymphocytic infiltration Negative 20 .
Positive 45 .
Desmoplasia Negative 16 .
Total Berkala PENELITIAN HAYATI | Volume 31 | Number 3 | December | 2025 Khesar H.
Khalil Correlation between Tumor Budding and Clinicopathological Parameters On H&E-stained sections, high TB (Bd2AeBd.
showed significant associations with advanced pathological stage .
< 0.
, higher T stage .
< 0.
, lymph node metastasis .
< 0.
, vascular invasion .
= .
, perineural invasion .
= 0.
, and desmoplasia .
= 0.
No significant correlations were found with age, sex, tumor location, histological subtype, or lymphocytic infiltration (Table .
On pan-cytokeratinAestained sections, high TB was also significantly associated with advanced pathological stage .
= 0.
, lymph node metastasis .
= 0.
vascular invasion .
= 0.
, perineural invasion .
= .
, and desmoplasia .
= 0.
A significant correlation was also observed with histological grade .
= .
, while no association was detected with age, sex, tumor location, or lymphocytic infiltration (Table .
Overall, both staining methods demonstrated strong correlations between high TB and aggressive tumor features, with pan-cytokeratin slightly enhancing the detection of these associations.
Table 5.
Association between tumor budding grade (IHC) and clinicopathological variables.
Pan-Cytokeratin expression Variants Positive (%) Negative (%) p- value <50 25 .
3%))
Age Ou50 42 .
Male 38 .
Sex Female 29 .
Right 30 .
Location of Tumor Left 37 .
Histological type Mucinous 6 .
Signet ring cell Ad 1 .
Well 3 .
Grade Moderate 24 .
Poor 2 .
14 ( 60.
Stage i 32 .
Tumor invasion 39 .
Lymph node involvement 22 .
Metastasis 5 .
Positive 55 .
Vascular invasion Negative 12 .
Positive 39 .
Perineural invasion Negative 28 .
Positive 45 .
Lymphocytic infiltration Negative 22 .
Positive 49 .
Desmoplasia Negative 18 .
Total 67 .
Discussion In this study.
TB in CRC was evaluated using both H&E staining and pan-cytokeratin IHC, comparing detection rates and assessing correlations with clinicopathological parameters.
The data showed that cytokeratin IHC significantly increased the detection of TB compared with H&E alone, and high-grade TB correlated with multiple adverse clinicopathological features, including advanced stage, higher T and N classification, vascular and perineural invasion, and desmoplasia.
The results are consistent with multiple recent investigations that have demonstrated the prognostic importance of TB in CRC.
High TB has been linked to poor overall survival (Petrelli et al.
, 2.
and disease-free survival (Zhang et al.
, 2.
across all disease stages in previous studies.
However, because survival analysis was not available in our cohort, our findings should be interpreted as pathological associations rather than direct prognostic validation.
In particular, studies have shown that high TB correlates with lymph node metastasis, even in early-stage CRC (Satoh et al.
, 2.
, highlighting its role in guiding surgical and adjuvant therapy decisions (Van Wyk et al.
, 2.
A major strength of the present study was the direct comparison between H&E and pan-cytokeratin IHC for TB assessment.
While ITBCC 2016 recommends H&E as the primary method, several authors have demonstrated that IHC can improve bud detection in morphologically challenging cases.
For example.
EL-Gendi .
(El- Berkala PENELITIAN HAYATI | Volume 31 | Number 3 | December | 2025 Tumor Budding in Colorectal Cancer Gendi and Al-Gendi, 2.
and Caie et al.
(Caie et al.
, 2.
reported that cytokeratin staining increased TB counts, particularly in cases with dense inflammatory infiltrates or desmoplastic stroma, and broken glands, similar to the findings of the current study.
Importantly, our findings suggest that the added value of IHC may be greatest in specific clinical scenarios.
In early-stage disease .
articularly pT1 and stage II CRC), where TB assessment may influence surgical strategy or adjuvant therapy decisions.
IHC can provide greater diagnostic Likewise, in borderline cases near the Bd1/Bd2 threshold.
IHC may clarify grading and reduce ambiguity.
In post-neoadjuvant settings, where fibrosis, necrosis, or mucin pools can obscure buds, cytokeratin IHC offers an advantage in visualizing residual tumor cells (Ichimasa et al.
, 2.
However, increased sensitivity of immunohistochemistry (IHC) in detecting cytokeratins can indeed lead to potential drawbacks, particularly in the context of overestimating tumor grade (Barak et al.
, 2.
While cytokeratins are valuable biomarkers for diagnosing various cancers, their expression can also highlight nonbudding epithelial elements, complicating the interpretation of results (Cserni et al.
, 2.
Therefore, we recommend selective use of IHC only when H&E assessment is equivocal, to maximize accuracy without compromising specificity.
The findings of this study also support the growing body of evidence suggesting that TB should be routinely incorporated into pathology reports as part of CRC risk assessment (Berg and Schaeffer, 2.
, particularly in early-stage disease where therapeutic decisions are nuanced (Cho and Kakar, 2018.
Graham et al.
, 2.
However, definitive prognostic conclusions in our cohort cannot be made without survival data, and future multicenter prospective studies are warranted.
Limitation This study has several limitations.
It was retrospective and single-center in design, which may restrict generalizability.
Survival outcomes were not available, limiting our ability to directly validate the prognostic value of TB in this cohort.
Reliance on IHC carries the potential risk of overestimating bud counts, as cytokeratin can highlight non-budding epithelial Finally, interobserver reproducibility was not formally assessed, which could influence consistency in TB reporting.
Conclusion Pan-cytokeratin IHC enhances the detection of tumor budding compared with H&E staining, particularly in histologically complex areas.
High-grade TB was strongly associated with multiple adverse pathological features in our cohort.
While our findings align with prior literature supporting the prognostic relevance of TB, survival analysis was not available, and conclusions regarding patient outcomes should therefore be drawn Selective use of cytokeratin IHC may nonetheless improve diagnostic accuracy and facilitate more reliable assessment in clinically relevant scenarios such as early-stage CRC, borderline budding cases, and post-neoadjuvant settings.
References