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Contains fulltext : 235824.pdf (Publisher’s version ) (Closed access) AIM: Tumour budding ('attacker') and CD8(+) T cells ('defender') are recognised as important parameters for risk stratification in colon cancers and, combined, may have an even stronger clinical impact. Here, we determine the value of tumour budding and CD8(+) in rectal cancer patients treated with/without neoadjuvant therapy. METHODS AND RESULTS: Using digital scans of all tumour slides/case, we analysed CD8(+) T cell counts in two patient cohorts: 45 neoadjuvantly treated and 47 primarily surgically treated (totalling n = 543 slides) after double-staining of the surgical resection specimen for pan-cytokeratin and CD8(+) . Tumour buds in hot-spots were manually counted (area = 0.785 mm(2) ) and CD8(+) T cell counts were analysed separately both in tumour budding hot-spots and the densest CD8(+) regions throughout the tumour. In neoadjuvantly treated patients, only tumour budding and not CD8(+) T cells was associated with tumour features, including more advanced ypT (P = 0.0062), venous invasion (P = 0.002), lymphatic invasion (P = 0.0003) and perineural invasion (P = 0.0017), as well as higher American Joint Committee on Cancer (AJCC) tumour regression score (P = 0.0035), indicating less tumour response. Overall survival was also worse in patients with high-grade budding in univariate analysis only. In contrast, all three variables, namely tumour budding (P = 0.0347), CD8(+) T cells in budding hot-spots (P = 0.0382) and CD8(+) T cells in the densest areas (P = 0.0117) were also associated with worse (budding) and better (CD8) survival time in the multivariate setting. CONCLUSION: In rectal cancer, tumour budding has clinical relevance in both primarily surgically treated patients and in those with neoadjuvantly treated patients, where it characterises highly aggressive residual disease. CD8(+) T cell counts appear not to have prognostic relevance in the neoadjuvant context.

AIMS The tumour-node-metastasis classification system is used for prognostication purposes and to guide patient management. However, in colorectal cancer (CRC), additional markers are needed to stratify prognostic subgroups. Two promising markers have emerged from large bodies of research: tumour budding and T cell host response (CD3, CD8 and CD45RO infiltrates). However, attempts to combine these two parameters have been sparse. The aim of this study was to perform an assessment of potential protagonists that could be used in a combined score (budding/T cell score, BTS). METHODS AND RESULTS This descriptive, retrospective study was performed on a multipunch tissue microarray containing material from 345 patients with stages I-IV CRC. Areas from tumour centre, front and microenvironment were stained for pancytokeratin/CD3, pancytokeratin/CD8 and pancytokeratin/CD45RO. Tumour buds were scored manually and T cell infiltrates digitally using open-source software. Tumour buds, T cell counts and combined BTS were associated with clinicopathological features and overall survival (OS). A higher combined BTS score (buds/CD8, tumour centre) performed better than budding or CD8/CD3 alone in predicting nodal metastases (P

In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high-level microsatellite instability (MSI-H). Cases with MSI-H or high-level CpG island methylator phenotype (CIMP-H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference.

Puppa G, Senore C, Sheahan K, Vieth M, Lugli A, Zlobec I, Pecori S, Wang L M, Langner C, Mitomi H, Nakamura T, Watanabe M, Ueno H, Chasle J, Conley S A, Herlin P, Lauwers G Y & Risio M (2012) Histopathology Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using virtual microscopy Aims: Despite the established prognostic relevance of tumour budding in colorectal cancer, the reproducibility of the methods reported for its assessment has not yet been determined, limiting its use and reporting in routine pathology practice. Methods and results: A morphometric system within telepathology was devised to evaluate the reproducibility of the various methods published for the assessment of tumour budding in colorectal cancer. Five methods were selected to evaluate the diagnostic reproducibility among 10 investigators, using haematoxylin and eosin (H&E) and AE1-3 cytokeratin-immunostained, whole-slide digital scans from 50 pT1-pT4 colorectal cancers. The overall interobserver agreement was fair for all methods, and increased to moderate for pT1 cancers. The intraobserver agreement was also fair for all methods and moderate for pT1 cancers. Agreement was dependent on the participants' experience with tumour budding reporting and performance time. Cytokeratin immunohistochemistry detected a higher percentage of tumour budding-positive cases with all methods compared to H&E-stained slides, but did not influence agreement levels. Conclusions: An overall fair level of diagnostic agreement for tumour budding in colorectal cancer was demonstrated, which was significantly higher in early cancer and among experienced gastrointestinal pathologists. Cytokeratin immunostaining facilitated detection of budding cancer cells, but did not result in improved interobserver agreement.

A tumour bud is defined as a single tumour cell or tumour cell cluster of up to five cells at the invasive tumour front. Significant differences in survival have been detected in colorectal cancer patients with low- compared to high-grade budding. The aim of this study was to identify potential multi-marker phenotypes characterizing low- and high-grade budding in mismatch repair (MMR)-proficient colorectal cancer.

In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high-level microsatellite instability (MSI-H). Cases with MSI-H or high-level CpG island methylator phenotype (CIMP-H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference.MSI, KRAS, BRAF, CIMP and 0(6)-methylguanine-DNA methyltransferase (MGMT) were investigated in tissues from 127 colorectal cancer patients. Tumour budding was scored using pan-cytokeratin-stained whole tissue sections within the densest area of buds (×40). Tumour budding was not associated with KRAS, BRAF, MGMT or CIMP, but was correlated inversely with MSI-H (P = 0.0049). Multivariate survival time analysis revealed that tumour budding was independent of all five molecular features and was predicted by MSI status [odds ratio (OR): 4.29, 95% confidence interval (CI) 1.5-12.1; P = 0.006)], but not CIMP (OR: 0.81, 95% CI 0.3-2.5; P = 0.714).These findings underline that MSI, rather than CIMP, plays a role in conferring a tumour budding phenotype. Budding retains its unfavourable prognostic effect independently of these five molecular features. Continued efforts to standardize the assessment of tumour budding are necessary to integrate this feature into daily diagnostic routine.

Tumour budding in colorectal cancer is established as a poor prognostic factor. The inverse correlation of tumour buds with peritumoural lymphocytic inflammation suggests an interaction with specific immune responses. The aims of this study were to characterize the immunological microenvironment of tumour buds and its impact on prognosis in mismatch repair (MMR)-proficient and -deficient colorectal cancers.A total of 297 colorectal cancers were double-immunostained for CK22 plus one of the following: CD138, CD16, CD20, CD21, CD56, CD68, CD8, forkhead box P2 (FoxP3), granzyme B, mast cell tryptase, CD3 or T cell intracellular antigen-1 (TIA)-1. Tumour buds and immune cells within the region of densest budding were evaluated [×40 high-power field (HPF)] simultaneously. In both MMR-proficient and -deficient cancers, CD8(+), FoxP3(+) and CD68(+) cells were observed most frequently (40 cells/HPF) and were independent prognostic factors. A combined prognostic score of tumour budding and CD8(+), FoxP3(+) and CD68(+) distinctly identified patients with low-, moderate- or high-risk colorectal cancers with 5-year survival rates of 75.2% [confidence interval 95% (CI): 66-83], 56.3% (95% CI: 43-68) and 25.2% (95% CI: 14-38), respectively, in MMR-proficient and -deficient cancers.The combined assessment of tumour budding with CD8, FoxP3 and CD68 lymphocytes could represent a basis for a prognostic score similar to the Bloom Richardson grade (BRE) and Gleason scores for breast and prostatic cancers.

A tumour bud is defined as a single tumour cell or tumour cell cluster of up to five cells at the invasive tumour front. Significant differences in survival have been detected in colorectal cancer patients with low- compared to high-grade budding. The aim of this study was to identify potential multi-marker phenotypes characterizing low- and high-grade budding in mismatch repair (MMR)-proficient colorectal cancer.Established and promising prognostic proteins such as epidermal growth factor receptor (EGFR), pERK, RHAMM, RKIP, beta-catenin, E-cadherin, pAKT, p16, p21, Ki67, Bcl-2, vascular endothelial growth factor (VEGF), apoptotic protease activating factor-1 (APAF-1), MUC1, EphB2, matrix metalloproteinase 7, pSMAD2, CDX2, laminin5gamma2 and MST1 were analysed on 208 MMR-proficient colorectal cancers with complete clinicopathological data. The most accurate markers for predicting high-grade budding (more than six tumour buds) were EphB2 (P0.001), Bcl-2 (P0.001), RKIP (P0.001), E-cadherin (P = 0.004), laminin5gamma2 (P = 0.004) and APAF-1 (P = 0.005). On multivariable analysis, only loss of Bcl-2 (P0.001) and EphB2 (P0.001) were independent predictors of high-grade budding. Bcl-2-/EphB2- tumours were more frequently poorly differentiated (P0.001), of advanced pT stage (P = 0.002), lymph node positive (P = 0.023), presented vascular (P = 0.053) and lymphatic invasion (P = 0.005) and had a negative impact on patient survival (P = 0.012).The multi-marker phenotype EphB2-/Bcl-2- is an independent predictor of high-grade budding and implies increased aggressive behaviour in MMR-proficient colorectal cancer.

Aims: To investigate dysregulation of the wnt signal-ling pathway by assessing b-catenin expression ⁄increasing expression and loss of cytoplasmic ade-nomatous polyposis coli (APC) and membranousE-cadherin in colorectal cancer (CRC) and determiningthe prognostic significance of these variables.Methods and results: Unselected, non-consecutive CRCresections (n ¼ 1420) were subdivided into threegroups: mismatch repair (MMR)-proficient, MLH1– andpresumed hereditary non-polyposis colonic cancer(HNPCC). Immunohistochemical analysis of b-cateninexpression (0% versus > 0%) and increasing expression(increasing percentage-positivity) and loss of APC andE-cadherin was performed using the tissue microarraytechnique. In MMR-proficient CRC, increased nuclearb-catenin expression and loss of membranous E-cadherinwere independently associated with higher N stage (P ¼0.03 and < 0.0001), vascular invasion (P