Evaluation Of Tumor Budding Research Articles

Tumor budding outperforms ypT and ypN classification in predicting outcome of rectal cancer after neoadjuvant chemoradiotherapy

BackgroundBudding is a complementary prognostic factor for colorectal cancer. In this study, we aimed to clarify the role of tumor budding in rectal cancer patients after preoperative chemoradiotherapy.MethodsA total of 124 patients with rectal cancer treated with neoadjuvant chemoradiotherapy and consecutive surgery were included. Surgical specimens were evaluated for budding and routine clinicopathological features. Budding was evaluated on hematoxylin and eosin (H&E)-stained slides and by cytokeratin immunohistochemical (IHC) staining.ResultsA budding rate of 36.9% (n = 38) by H&E and 55.6% (n = 55) by IHC was observed. Budding was significantly associated with a high ypT and ypN status, poor differentiation, and low degrees of tumor regression. Moreover, budding was strongly predictive of a worse patient outcome, as measured by tumor recurrence or death. In multivariate analyses, budding remained the only significant parameter for overall survival and was even superior to the ypT and ypN status (budding in H&E: hazard ratio (HR) 2.72, 95% confidence interval (95% CI) 1.15–6.44, p = 0.023; budding in IHC: HR 5.19, 95% CI 1.62–16.61, p = 0.006).ConclusionBudding is a strong prognostic predictor of survival in rectal cancer patients after neoadjuvant therapy. A standardized evaluation of tumor budding after neoadjuvant therapy may thus aid in risk stratification and guide the clinical management of patients with rectal cancer. Immunostaining can help to enhance the diagnostic accuracy and prognostic significance.

Evaluation of Tumor Budding in Primary Colorectal Cancer and Corresponding Liver Metastases Based on H&E and Pancytokeratin Staining.

In colorectal cancer, tumor budding is associated with tumor progression and represents an additional prognostic factor in the TNM classification. Tumor buds can be found at the invasive front (peritumoral budding; PTB) and in the tumor center (intratumoral budding; ITB) of primary tumors. Previous studies have shown that tumor buds are also present in colorectal liver metastases (CRLM). Data on the prognostic and predictive role in this clinical context are still sparse and no standardized approach to evaluate budding in CRLM has been published so far. This study aimed to analyze and correlate perimetastatic (PMB) and intrametastatic budding (IMB) on H&E and pancytokeratin staining, compare it to budding results in corresponding primary tumors and to propose a standardized scoring system in CRLM as the basis for future studies. Tumor tissue of 81 primary tumors and 139 corresponding CRLM was used for ngTMA construction. For each primary tumor and metastasis, two punches from the center and two punches from the periphery from areas with highest tumor budding density were included. TMA slides were stained for H&E and pancytokeratin (Pan-CK). PTB, ITB, PMB, and IMB were analyzed and classified as bd1, bd2, and bd3 according to ITBCC guidelines. ITB and PTB as well as IMB and PMB showed significant correlation on H&E and Pan-CK staining. No correlation was found for tumor bud counts in primary tumors and corresponding metastases. The agreement for categorized tumor bud counts showed fair to good agreement for metastases and poor agreement for primary tumors between different classes on H&E and Pan-CK staining. Based on our results, tumor budding in primary tumors and CRLM seems to be different processes which might be the results of differing surrounding microenvironments. The evaluation of tumor budding in CRLM is challenging in cases without desmoplastic stroma reaction or intense perimetastatic ductular reaction. We therefore propose to evaluate tumor budding only in metastases with desmoplastic stroma reaction based on H&E staining since important morphological features are obscured on Pan-CK staining.

EXPRESSION OF THE CANCER STEM CELL MARKER ALDH1 IS INCREASED IN THE BUDDING AREA OF ORAL SQUAMOUS CELL CARCINOMA

Objectives This study aimed to evaluate the expression of the cancer stem cell marker ALDH1 and its association with tumor budding, a morphological marker of cancer invasion, in oral squamous cell carcinoma (OSCC). Findings 163 OSCC samples were obtained by incisional biopsies. Immunohistochemistry was performed to detect positive cells for ALDH1 (cancer stem cell marker) and for AE1/AE3 (multi-cytokeratin to identify OSCC cells in tumor budding evaluation). A positive expression of ALDH1 was observed in 47.24% of the samples. In the tumor budding evaluation, samples were classified as low-or high-intensity tumor budding. Association between the ALDH1 expression and tumor budding was assessed using the chi-square test. However, no association was observed (p>0.05). In samples with high-intensity tumor budding, differences in the ALDH1 expression between the budding area and the area outside the budding were evaluated using the McNemar test. The ALDH1 expression was higher in the budding area than in the area outside the budding (p Conclusion The findings reinforce the idea that cells at the tumor budding area show phenotypic characteristics of cancer stem cells. The debate concerning the model of oral carcinogenesis by cancer stem cells was also strengthen. Supported by FAPEMIG (APQ-01806/14 and PPM-00653-16).

Immunohistochemical evaluation of tumor budding for stratifying T1 colorectal cancer: optimal cut-off value and a novel computer-assisted semiautomatic method

High-grade tumor budding is an adverse prognostic factor for submucosal invasive (T1) colorectal cancer used to predict the risk for lymph node metastasis in endoscopically resected specimens. Cytokeratin immunohistochemistry is a potential option for evaluating tumor budding. The optimal cut-off value between low- and high-grade budding has not yet been determined, however, and the high inter-observer variability in selecting budding foci remains problematic. We explored the optimal cut-off value for predicting lymph node metastasis using cytokeratin immunohistochemistry, and developed a novel computer-assisted semiautomatic quantification method to reduce inter-observer variability. A retrospective single-institution study of 463 T1 colorectal cancer cases was conducted. Cases were split into derivation and validation datasets. Tumor budding foci were counted manually and semiautomatically using Image J software on cytokeratin immunohistochemistry-stained specimens. We determined the cut-off values and compared inter-observer variability among pathologists between the two methods. Univariate and multivariate analyses of the derivation dataset were performed to select the risk factors for lymph node metastasis. Predictive simulation for the validation dataset was conducted. The optimal cut-off values for the manual and semiautomatic methods were ≥10 and ≥12, respectively. For both methods, multivariate analyses revealed that venous invasion, lymphatic invasion, and high-grade tumor budding were independent risk factors for lymph node metastasis. The semiautomatic method provided significantly better inter-observer agreement. The predictive and observed lymph node metastasis frequencies were highly correlated in the validation dataset.

Reproducibility, Repeatability, and Level of Difficulty of Two Methods for Tumor Budding Evaluation in Oral Carcinoma

Objective: To analyze the reproducibility, repeatability, and difficulty level of 2 tumor budding evaluation methods, staining with hematoxylin and eosin (HE) and immunostaining for multicytokeratin, in oral squamous cell carcinoma (OSCC).

Automatic evaluation of tumor budding in immunohistochemically stained colorectal carcinomas and correlation to clinical outcome

BackgroundTumor budding, meaning a detachment of tumor cells at the invasion front of colorectal carcinoma (CRC) into single cells or clusters (<=5 tumor cells), has been shown to correlate to an inferior clinical outcome by several independent studies. Therefore, it has been discussed as a complementary prognostic factor to the TNM staging system, and it is already included in national guidelines as an additional prognostic parameter. However, its application by manual evaluation in routine pathology is hampered due to the use of several slightly different assessment systems, a time-consuming manual counting process and a high inter-observer variability. Hence, we established and validated an automatic image processing approach to reliably quantify tumor budding in immunohistochemically (IHC) stained sections of CRC samples.MethodsThis approach combines classical segmentation methods (like morphological operations) and machine learning techniques (k-means and hierarchical clustering, convolutional neural networks) to reliably detect tumor buds in colorectal carcinoma samples immunohistochemically stained for pan-cytokeratin. As a possible application, we tested it on whole-slide images as well as on tissue microarrays (TMA) from a clinically well-annotated CRC cohort.ResultsOur automatic tumor budding evaluation tool detected the absolute number of tumor buds per image with a very good correlation to the manually segmented ground truth (R2 value of 0.86).Furthermore the automatic evaluation of whole-slide images from 20 CRC-patients, we found that neither the detected number of tumor buds at the invasion front nor the number in hotspots was associated with the nodal status. However, the number of spatial clusters of tumor buds (budding hotspots) significantly correlated to the nodal status (p-value = 0.003 for N0 vs. N1/N2). TMAs were not feasible for tumor budding evaluation, as the spatial relationship of tumor buds (especially hotspots) was not preserved.ConclusionsAutomatic image processing is a feasible and valid assessment tool for tumor budding in CRC on whole-slide images. Interestingly, only the spatial clustering of the tumor buds in hotspots (and especially the number of hotspots) and not the absolute number of tumor buds showed a clinically relevant correlation with patient outcome in our data.

Digital image analysis of pan-cytokeratin stained tumor slides for evaluation of tumor budding in pT1/pT2 colorectal cancer: Results of a feasibility study

Tumor budding is an independent prognostic factor in colorectal cancer. However, varying degrees of interobserver agreement and reproducibility challenges the use of tumor budding in diagnostics. Immunohistochemical staining of tumor slides with pan-cytokeratin visualizes the budding tumor cells and has been suggested to improve reproducibility. Here we demonstrate the methodology of tumor budding assessment using digital image analysis based on tumor slides stained for pan-cytokeratin, and investigate interobserver agreement, agreement between manual and digital assessment methods and digital reproducibility between users.Tumor slides from 126 patients with pT1/pT2 colorectal cancer were stained with pan-cytokeratin and tumor budding at the invasive tumor front was assessed by conventional manual microscopy. A digital image analysis algorithm for identification and quantification of budding tumor cells was developed and tested on the pan-cytokeratin stained slides.Manual assessment of tumor budding using pan-cytokeratin stained tumor slides exhibited high correlations (Spearman Rank 0.84-0.89, p < 0.001),excellent agreement between observers (Intra-class correlation coefficient (ICC): 0.86 -0.87) and 2.20 higher odds for regional metastases with increasing budding counts (p = 0.017). Digital image analysis correlated well to manual assessment (Spearman Rank 0.71-0.88) and agreement between the two methods was good (ICC 0.62-0.82). However, only a trend towards increased odds for metastatic progression was found for the adjusted digital estimates (p = 0.076). Digital estimates were higher than manual estimates, demonstrated by a systematic median difference of 3–4.5 buds. Image analysis was highly reproducible between users of the algorithm (ICC 0.98). In conclusion, assessment of tumor budding using pan-cytokeratin stained tumor slides is a method with high correlation and agreement between observers. Digital image analysis quantifies budding tumor cells in high agreement with manual estimates, but approval of the digital slides by a pathologist is mandatory. The method qualifies for further investigation.

Interobserver variability in the H&E-based assessment of tumor budding in pT3/4 colon cancer: does it affect the prognostic relevance?

Tumor budding is a mostly accepted adverse prognostic factor in colorectal carcinoma. It is on the cusp of a widespread use after agreement was reached recently on uniform assessment criteria. We investigated whether the interobserver variability has a direct influence on the prognostic relevance in pT3/4 colon cancer in the background of different levels of experience of the investigators. In total, six investigators with different levels of experience evaluated tumor budding on H&E slides in 244 cases with primary diagnosed (2002-2011) colon carcinoma (pT3/4, N+/-, M0). High-grade tumor budding/budding grade 3 (defined as majority assessment among the investigators) was significantly associated with an adverse outcome (overall survival p = 0.03, cancer-specific survival p = 0.08) and the occurrence of distant metastasis (p = 0.009). However, a detailed analysis of the rating results of the individual investigators revealed that only ratings of one investigator (advanced resident) were associated with an adverse outcome (p = 0.01 cancer-specific survival, overall survival p = 0.09, distant metastasis p = 0.002). The results of another investigator (consultant) were significantly associated with distant metastasis (p = 0.007). The kappa values among the investigators have a range between 0.077 and 0.357 (median 0.166). Total agreement of all investigators existed in 109 cases (44.7%). Our results demonstrate that the evaluation of tumor budding on H&E slides in pT3/4 colon cancer goes along with a considerable interobserver variability among investigators of different levels of experience. Furthermore, our results reveal that these findings directly influence the prognostic value.

Pathologic evaluation of tumor budding as a prognostic factor for Stage I–II tongue squamous cell carcinoma

ObjectiveTongue squamous cell carcinoma (TSCC) has the tendency of rapid locoregional invasion and high cervical lymph node metastasis compared with other squamous cell carcinomas. Tumor budding has been reported as a useful prognostic factor for various cancers. In this study, we reported the usefulness of tumor budding evaluation as a prognostic factor for Stage I–II TSCC, in comparison with the Yamamoto-Kohama mode of invasion (YK classification). Materials and methodsA total of 23 patients with Stage I–II TSCC were evaluated for tumor budding and YK classification. Tumor budding was classified as low or high-grade based on presence of <5 or ≥5 tumor buds, respectively, in hematoxylin and eosin-stained specimens (BD classification). YK classification was similarly designated as either low (YK-1/-2/-3) or high-grade (YK-4C/-4D). Statistical analysis was carried out to evaluate the correlation of BD and YK classifications with disease free survival (DFS). ResultsLow-grade BD and YK classifications were confirmed in 15 and 11 patients (65.2% and 47.8%), respectively, while high-grade BD and YK classifications were confirmed in 8 and 12 patients (34.8% and 52.2%), respectively. DFS of patients with low-grade BD and YK classifications were significantly longer than those with high-grades (p = 0.047 and p = 0.017, respectively). ConclusionHigh-grade BD and YK classifications were associated with poor prognosis in patients with Stage I–II TSCC. Strict follow-up observation or postoperative adjuvant therapy should be considered when high-grade BD and YK classifications are confirmed.

Tumor budding as a novel predictor of occult metastasis in cT2N0 tongue squamous cell carcinoma

Occult neck metastasis is an important prognostic factor in patients with tongue squamous cell carcinoma (TSCC) who are deemed clinically negative for neck metastasis. The purpose of this study was to identify predictive factors for occult neck metastasis arising from TSCC and to determine patient prognosis. Ninety-seven patients with cT2N0 TSCC who underwent surgical resection of their primary lesion as initial therapy were enrolled in this retrospective study. Cutoff values for depth of invasion (≥3.3 mm) and the tumor budding score (≥4) were determined using receiver operator characteristic analyses. Univariate and multivariate analyses revealed that a tumor budding score ≥4 is a significant independent predictive factor for the occurrence of occult neck metastasis, which in turn is a significant independent prognostic factor. When evaluating tumor budding, we demonstrated greater interobserver and intraobserver agreement when using immunohistochemical staining for cytokeratin AE1/AE3 than with hematoxylin and eosin staining (HE). We conclude that the evaluation of tumor budding is effective for identifying populations at high risk of occult neck metastasis, which will enable the planning of appropriate therapeutic strategies for patients with cT2N0 TSCC. Furthermore, cytokeratin staining is recommended over HE staining for simpler and more accurate evaluation of tumor budding.

Cell proliferation is associated with intensity of tumor budding in oral squamous cell carcinoma

Tumor budding is a morphological marker of cancer invasion, defined as the presence of isolated or small clusters of neoplastic cells at the tumor invasive front. This study aimed to evaluate the association between intensity of tumor budding and cell proliferation in oral squamous cell carcinoma (OSCC). Immunohistochemistry was employed in 163 OSCC samples to detect the cell proliferation marker Ki-67 and multicytokeratin (to identify OSCC cells in tumor budding evaluation). The Mann-Whitney test was used to evaluate differences in the cell proliferation index between samples with high-intensity tumor budding and samples with low-intensity or no tumor budding. In samples with high-intensity tumor budding, the Wilcoxon test was used to evaluate differences in the cell proliferation index between the budding area and the area outside the budding. The chi-square test assessed the association between cell proliferation index and intensity of tumor budding. The cell proliferation index was higher in samples with high-intensity tumor budding than in samples with low-intensity or no tumor budding (P < .05). Tumors with high-intensity tumor budding showed a higher cell proliferation index in the budding area than in the area outside the budding (P < .05). Finally, samples showing high-intensity tumor budding were associated with high cell proliferation index (P < .05). Cell proliferation is positively associated with intensity of tumor budding in OSCC. Moreover, in tumors showing high-intensity tumor budding, the budding area is the location of higher cell proliferation. These findings reinforce the hypothesis that tumor budding is associated with the biological behavior of OSCC.

Cytokeratin immunohistochemistry improves interobserver variability between unskilled pathologists in the evaluation of tumor budding in T1 colorectal cancer.

Tumor budding is a major risk factor for T1 colorectal cancer. Quality control of the pathological diagnosis of budding is crucial, irrespective of the pathologist's experience. This study examines the interobserver variability according to pathologists' experience and evaluates the influence of cytokeratin (CK) immunostaining in the assessment of budding. Hematoxylin-eosin (HE) and CK-immunostained slides of 40 cases with T1 primary colorectal cancer were examined. Budding grades were individually evaluated by 12 pathologists who we categorized into three groups by their experience (expert, with >10 years of experience (n = 4), senior, with 5-10 years (n = 4), and junior, < 5 years (n = 4)). The results revealed a tendency for the more experienced pathologists to assign higher budding grades compared to the less-experienced pathologists. In the junior group, the interobserver variability obtained with HE slides was poor, but it was markedly improved in the evaluation using CK-immunostained slides. The benefit of CK immunostaining was only slight in the expert group. CK immunostaining would be useful when a pathologist is not experienced enough or does not have enough confidence in the assessment of budding.

Laminin‐5 gamma 2 chain expression is associated with intensity of tumor budding and density of stromal myofibroblasts in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide. Laminin-5 gamma 2 chain (laminin-5 γ2) is a protein associated to a migratory phenotype in epithelial neoplastic cells. Stromal myofibroblasts also play a significant role in tumor invasion, due to its ability to modify the extracellular matrix. Tumor budding is a morphologic marker of tumor invasion. The aim of this study was to evaluate the expression of laminin-5 γ2 in OSCC and its association with intensity of tumor budding and density of stromal myofibroblasts. Paraffin-embedded archival samples of 57 OSCC patients were evaluated. Immunohistochemistry was employed to detect laminin-5 γ2, alpha smooth muscle actin (marker of stromal myofibroblasts), and multicytokeratin (to identify OSCC cells in tumor budding evaluation). Laminin-5 γ2 expression and its association with intensity of tumor budding and density of stromal myofibroblasts were analyzed. Association among intensity of tumor budding and density of stromal myofibroblasts was also evaluated. Higher laminin-5 γ2 expression was associated with high-intensity tumor budding (P < 0.05) and with higher density of stromal myofibroblasts (P < 0.05). Moreover, high-intensity tumor budding was associated with higher density of stromal myofibroblasts (P < 0.05). In OSCC, higher laminin-5 γ2 expression is associated with high-intensity tumor budding and with higher density of stromal myofibroblasts, suggesting that this expression is related to the establishment of an invasive phenotype of neoplastic cells and a permissive environment for tumor invasion in this neoplasia.

Tumor budding predicts response to anti-EGFR therapies in metastatic colorectal cancer patients

To investigate whether the evaluation of tumor budding can complement K-RAS analysis to improve the individualized prediction of response to anti-epidermal growth factor receptor based therapies in metastatic colorectal cancer (mCRC) patients. Forty-three patients with mCRC treated with cetuximab or panitumumab were entered into this study. According to the Response Evaluation Criteria in Solid Tumors criteria, 30 patients had stable or progressive disease (non-responsive), while 13 patients had a partial response. Tumor buds were evaluated from whole tissue sections stained for pan-cytokeratin, evaluated in the densest region using a 40 × objective and "high-grade" tumor budding was defined as 15 buds/high-power field. Tumor buds and K-RAS mutation both correctly classified 68% of patients. All patients with K-RAS mutation (n = 7) or high-grade tumor budding (n = 11) were non-responsive, of which 4 patients had both features. All 13 partial responders were K-RAS wild-type with low-grade tumor budding. Combined, the predictive value of K-RAS and tumor budding was 80%. Additionally, high-grade tumor budding was significantly related to worse progression-free survival [HR (95% CI): 2.8 (1.3-6.0, P = 0.008)]. If confirmed in larger cohorts, the addition of tumor budding to K-RAS analysis may represent an effective approach for individualized patient management in the metastatic setting.

Actual number of tumor budding as a new tool for the individualization of treatment of T1 colorectal carcinomas

Treatment of T1 colorectal carcinomas, either local excision including endoscopic polypectomy or radical surgery, has always been problematic in everyday practice. Although previous studies have revealed that tumor budding at the invasive margin can be a marker for the malignant potential of T1 colorectal carcinomas, the evaluation of tumor budding has not been standardized as yet. In the present study, we attempted to apply the actual number of tumor budding units for the individualization of treatment in T1 colorectal carcinomas. In 76 T1 colorectal carcinomas, associations between lymph node metastasis and clinicopathological parameters were examined statistically. A mathematical formula for predicting the risk of lymph node metastasis was constructed and decision analysis was attempted to determine individually the indication for additional surgery after endoscopic mucosal resection of T1 colorectal carcinomas. Of the clinicopathological parameters examined, multivariate analysis showed that the actual number of tumor budding units alone was significantly associated with lymph node metastasis. The probability of lymph node metastasis was calculated as Z = 0.07 x (budding counts) - 3.726, probability = 1/1 + e(-Z). The more the budding counts, the higher the probability of lymph node metastasis. This formula was able to accurately predict lymph node metastasis in successive cases. The actual number of tumor budding units can be applied to decision analysis in determining an indication for additional surgery after endoscopic mucosal resection of T1 colorectal carcinomas. The actual number of tumor budding units may be useful in the decision making for patient-oriented treatment of T1 colorectal carcinomas.