Tumor Budding In Colorectal Cancer Research Articles

Standardization of whole slide image morphologic assessment with definition of a new application: Digital slide dynamic morphometry

Background: In histopathology, the quantitative assessment of various morphologic features is based on methods originally conceived on specific areas observed through the microscope used. Failure to reproduce the same reference field of view using a different microscope will change the score assessed. Visualization of a digital slide on a screen through a dedicated viewer allows selection of the magnification. However, the field of view is rectangular, unlike the circular field of optical microscopy. In addition, the size of the selected area is not evident, and must be calculated. Materials and Methods: A digital slide morphometric system was conceived to reproduce the various methods published for assessing tumor budding in colorectal cancer. Eighteen international experts in colorectal cancer were invited to participate in a web-based study by assessing tumor budding with five different methods in 100 digital slides. Results: The specific areas to be tested by each method were marked by colored circles. The areas were grouped in a target-like pattern and then saved as an .xml file. When a digital slide was opened, the .xml file was imported in order to perform the measurements. Since the morphometric tool is composed of layers that can be freely moved on top of the digital slide, the technique was named digital slide dynamic morphometry. Twelve investigators completed the task, the majority of them performing the multiple evaluations of each of the cases in less than 12 minutes. Conclusions: Digital slide dynamic morphometry has various potential applications and might be a useful tool for the assessment of histologic parameters originally conceived for optical microscopy that need to be quantified.

Use of tumor budding with K-RAS gene analysis to identify potentially responsive metastatic colorectal cancer patients treated with anti-EGFR therapies.

e14042 Background: Tumor budding in colorectal cancer is recognized by the International Union against Cancer as a highly relevant, additional prognostic factor and defined as single cells or clusters of up to 5 cells at the invasive tumor front. In metastatic colorectal cancer (mCRC), K-RAS gene analysis is now routinely performed to identify patients who are more likely to respond to anti-EGFR therapies. The aim of this study was to assess the predictive value of tumor budding and to determine whether this feature can complement K-RAS gene analysis to help identify mCRC patients who may benefit from anti-EGFR therapies. Methods: 43 patients with mCRC treated with cetuximab or panitumumab were entered into this study. Objective tumor response was evaluated using the RECIST guidelines. 30 patients had progressive or stable disease and classified as non-responsive (NR), while 13 patients had complete or partial response. Mutation in K-RAS occurred in 11 (35.6) cases. Tumor budding was evaluated from whole tissue sections stained for pan-cytokeratin. Tumor buds in the densest region of tumor budding were counted using a 40x objective. Based on receiver-operating characteristics (ROC) curve analysis, the cut-off score for “low” and “high” tumor budding (LTB and HTB) was 15 buds/high power field. Results: Tumor budding and K-RAS mutation both correctly classified patients into response groups with 68% accuracy. All patients with K-RAS mutations were NR to therapy. Of the remaining 32 patients with wild-type (WT) K-RAS, 7 (21.9%) had HTB and all were NR to therapy. All patients with PR were K-RAS WT and had LTB. The combination of K-RAS and tumor budding improved the overall accuracy of K-RAS mutation alone to 80%. Conclusions: This appears to be the first evidence suggesting that a histomorphological parameter, namely tumor budding, could have potential predictive value in patients with mCRC treated with anti-EGFR agents. The analysis of tumor budding in addition to K-RAS gene status may represent an effective approach to further optimize the management of mCRC patients treated with anti-EGFR therapies. No significant financial relationships to disclose.

Immune response to tumor budding ratio: A new potential prognostic score to improve risk stratification for patients with colorectal cancer.

e14033 Background: Tumor budding in colorectal cancer is the histologic hallmark of epithelial mesencyhmal transition and is considered an additional prognostic factor by the International Union against Cancer. Abundant tumor budding is associated with lymph node metastasis, vascular invasion and distant metastasis and inversely related to peritumoral lymphocytic inflammation implicating tumor immunity in events occurring at the invasive front. Recently a high CD8+/budding ratio was identified as an independent prognostic factor. The aim of this study was to assess the ratio of various immune cell types to tumor budding ratio in order to define a potential new prognostic score for patients with this disease. Methods: Whole tissue sections from 297 patients with colorectal cancer were cut in series and double immunostaining was performed for CK22 with antibodies directed against CD3, CD8, CD16, CD20, CD21, CD56, CD68, CD138, Foxp3, Granzyme B, and mast cell tryptase. The densest region of tumor budding at the invasive front was identified. Using a 40x objective, the number of tumor buds and positively stained cells for each marker were counted. Results: All protein markers where inversely correlated with the number of tumor buds. After multivariable analysis with pT category, pN category and vascular invasion, a hierarchy of marker/budding ratios identified high Granzyme B+/budding (HR: 0.5 [0.31-0.82; p = 0.006]) as having the strongest positive independent effect on outcome, followed by high CD68+/budding (HR: 0.52 [0.33- 0.84; p = 0.007]), CD8+/budding (HR: 0.54 [0.34-0.84; p = 0.007]), CD16+/budding (HR: 0.56 [0.35-0.9; p = 0.015]), FoxP3+/budding (HR: 0.57 [0.35-0.91; p = 0.019]) and CD3+/budding (HR: 0.58 [0.37-0.9; p = 0.017]). Conclusions: High ratios of CD8+ cytotoxic T- lymphocytes, granzyme B+ or CD68+ cells to tumor budding represent not only a possible mechanism by which budding cells are prevented from disseminating into vessels, but also may lead to an additional prognostic score to improve risk stratification for patients with colorectal cancer. No significant financial relationships to disclose.

Role of APAF‐1, E‐cadherin and peritumoural lymphocytic infiltration in tumour budding in colorectal cancer

Tumour budding or dedifferentiation at the invasive margin of colorectal cancer (CRC) is an important prognostic marker and linked mechanistically to dysregulation of Wnt pathway signalling. Since budding is observed in only 40% of CRCs, we hypothesized that Wnt pathway dysregulation may be a necessary but insufficient explanation for budding and that buds may be destroyed selectively by tumour immune mechanisms. Twenty potential markers of tumour budding were evaluated in tissue microarrays (TMAs) obtained from the main tumour body of 1164 DNA mismatch repair-proficient CRCs and the findings were correlated with tumour budding, lymphocytic infiltration and survival. Loss of expression of E-cadherin and APAF-1 were independent predictors of budding (sensitivity 70.3% and specificity 48.2% when one or the other was lost). Peritumoral lymphocytes (PTLs) were observed more frequently in CRCs with loss of either E-cadherin or APAF-1 that were budding-negative. PTLs and tumour-infiltrating lymphocytes (TILs) were strongly correlated. The absence of TILs increased the adverse prognostic impact of E-cadherin and APAF-1 loss. Co-occurrence of E-cadherin loss, APAF-1 loss and low TIL counts in CRCs was an independent prognostic factor. The findings were verified in whole tissue sections from 88 CRCs with known KRAS mutation status (which was not associated with budding). Loss of E-cadherin and APAF-1 within the main body of CRCs are independent predictors of tumour budding. The prognostic benefit of lymphocytic infiltration may be explained by the immune destruction of budding cells.

A novel classification of tumour budding in colorectal cancer based on the presence of cytoplasmic pseudo‐fragments around budding foci

Tumour budding is an adverse prognostic factor in colorectal cancer (CRC). We have investigated the significance of cytoplasmic fragments occurring in the immediate vicinity of tumour budding foci. Seventy-three CRCs with high-grade budding (> 10 budding foci in a x 20 objective field) were classified according to extent of budding (10-19 versus 20+ foci) and by the presence or absence of cytoplasmic fragments identified by immunostaining for cytokeratin. In serial sections, cytoplasmic fragments were shown to be dendritic cell processes in continuity with budding tumour cells and were renamed pseudo-fragments. Cytoplasmic pseudo-fragments, but not extent of budding, were associated with aberrant expression of beta-catenin (P = 0.045) and laminin-5 gamma2 (P < 0.0001), and with absent peritumoral lymphocytic infiltration (P = 0.0077). Cytoplasmic pseudo-fragments had a stronger association with infiltrating growth pattern (P = 0.0014) than extent of tumour budding (P = 0.014). There was no association between extent of budding and cytoplasmic pseudo-fragments (P = 0.12). Cytoplasmic pseudo-fragments may be a marker for an activated budding phenotype that is associated with cell motility and increased invasiveness in CRC and is independent of the extent of budding.

APC mutation and tumour budding in colorectal cancer

Aim: To determine the frequency of tumour budding and somatic APC mutation in a series of colorectal cancers stratified according to DNA microsatellite instability (MSI) status. Material/Methods: Ninety five colorectal...