Table 1.
Summary of studies examining tumor buds by immunohistochemistry.
| Biological role | Reference | Markers and methods | Cohort | Budding systematically assessed? Scoring method (reference) | Results/relevance |
|---|---|---|---|---|---|
| Wnt signaling | Gosens (25) | EpCAM: three different antibodies (Ber-EP4, 311-1K1 and a polyclonal antibody), double staining for β-catenin and Ep-CAM. mRNA in situ hybridization of Ep-CAM, WTS | 133 rectal cancers (Dutch RT + TME trial), Stage II–IV | Yes (Ueno) (9) | Tumor buds showed lack of membranous and increased cytoplasmic Ep-CAM staining and nuclear expression of β-catenin. Reduced Ep-CAM staining at the invasive margins correlated with tumor-budding, grade, and increased risk of LR |
| Wnt signaling | Brabletz (27) | β-catenin, WTS | 44 Stage I–III CRC | No | Expression of nuclear β-catenin in 54% of all cases. Strong nuclear staining predominantly at tumor front (80–100%) with strongest staining in tumor buds. Tumor center often without nuclear staining but with retained membranous staining |
| Wnt signaling | El-Bahrawy (28) | E-Cadherin, α-, β-, and γ-catenin (each immunohistochemistry and mRNA), WTS | 30 Dukes A-C CRC | No | Cytoplasmic accumulation of E-cadherin and catenins in over 80% of cases. Increased staining of β-catenin toward tumor front |
| Wnt signaling | Lauscher (29) | Pontin, β-catenin, WTS. Pontin western blot on six cases | 34 CRC Stage I–IV | No | Cytoplasmic pontin expression in all cases, additional nuclear positivity in 50% of cases. Nuclear pontin correlated with nuclear β-catenin in all cases. Nuclear pontin staining stronger at invasive margin and tumor buds in comparison to tumor center (41.2 and 37.9% of cases). Sample size insufficient for significant correlation to stage |
| Wnt signaling | Garcia-Solano (22) | β-catenin, e-cadherin, p-cadherin, laminin5γ2, SMAD4, WTS | 20 SAC (defined by histomorphologic criteria, no features of MSI-high tumors) with stage matched 20 CAC | Yes (Ueno) | Increased expression of laminin5γ2, decreased expression of nuclear β-catenin and membranous e-cadherin in tumor buds of SAC in comparison to CAC |
| Wnt signaling | Shinto (21) | laminin5γ2, β-catenin (assessed in tumor buds), MUC2, MUC5AC (assessed on entire tumor), WTS. Laminin5γ2 promoter methylation | 80 CRC with high-grade budding: 9 sporadic MMR-deficient, 7 Lynch MMR-deficient and 64 sporadic MMR-proficient, Stage n/a | Yes (Ueno) | 3/9 sporadic MMRd laminin5γ2 compared to 46/64 sporadic MMRp (p 0.05) and 2/7 Lynch (p = 0.03). Nuclear β-catenin more frequent in MMRp than MMRd cancers (p 0.01). No difference in methylation among subsets but correlation between methylation and negative laminin5γ2 |
| Cell differentiation cell cycle | Harbaum (30) | CK7, CK20, E-cadherin, MUC2, and MIB1. CK7: 370 cancers on multi-punch TMA, CK7 positive cases re-evaluated on WTS with all markers | 370 CRC Stage I–IV | Yes (Ueno) | 32 cases positive for CK7. CK7 positivity prevailed in tumor buds, these cells were positive for CK20 and negative for E-Cadherin, MUC2 and MIB1 on serial sections. Raises the notion of “EET” (epithelial–epithelial transition) |
| Wnt signaling | Brabletz (31) | CK18, β-catenin, e-cadherin, Ki-67, WTS | 72 CRC Stage n/a | No | Nuclear β catenin in tumor buds accompanied by reduced E-cadherin and Ki-67 reactivity, inverse immunoprofile in main tumor and metastases |
| Wnt signaling | Horkko (32) | Tumor-budding margin on all cases, β-catenin (108 cases), MNF116 (53 cases to assess separately for budding), WTS | 466 CRC Dukes A-D | Yes (Ueno) | Nuclear β catenin increased at invasive front and in tumor buds, but no correlation between expression presence/absence of budding |
| Wnt signaling | Guzinska-Ustymowicz (38) | MMP-9 and cathepsin B, WTS | 55 pT3 G2 CRC | Yes (Morodomi) (37) | Expression of MMP-9 and Cathepsin B associated with lymph node involvement (p < 0.01) |
| Wnt signaling | Rubio (39) | MNF116, Ki-67, laminin5 | 6 CRC (preliminary report), Stage n/a | Hotspot on HE | Mean positivity of buds in comparative fields: MNF 116: 86.2, Ki-67: 9.7, laminin5: 9.3 |
| Wnt signaling | Gavert (42) | β-catenin, L1, ADAM10, WTS | 25 CRC, Stage n/a | No | L1 not detected in main tumor body, but at invasive front and tumor buds, co-localization with ADAM10, and nuclear β-catenin |
| Wnt signaling | Gavert (52) | NFκB, L1, ezrin, WTS | 25 CRC, Stage n/a | No | Tumor buds co-express ezrin, nuclear NfKb and L1, central tumor regions with relative lack immunoreactivity. Together with functional data supports hypothesis that L1-mediated activation of NFkB signaling is a major route of CRC tumor progression |
| CSC | Hostettler (49) | CK22, CD133, CD166, CD24, CD44s, CD90, EpCAM, ALDH1, ABCG5, evaluation within tumor buds on WTS | 101 cases with densest budding out of cohort with 300 CRC patients, Stage n/a | Yes (Ueno) | CD90, CD44s, and CD133 infrequent in buds (<5%). ALDH1, CD24 and CD166 in 16.5, 16.2, and 34%. ABCG5 and EpCAM in 35 and 69% of cases. EpCAM and ABCG5 in buds significantly associated with worse prognosis, especially in node-negative patients with ABCG5 positive buds |
| CSC | Kleist (56) | Lgr5, WTS | 89 cases Stage I–IV, additional distant metastases from 31 patients | Yes (Prall) (36) | 12.9% of cases had Lgr5 positive buds, distant metastases from these cases had 6- to 11.5-fold higher expression rates |
| Cell cycle | Dawson (59) | Ki-67 (WTS), Caspase3, M30Cytodeath (multi-punch TMA) | 188 Stage I–IV CRC | Yes (Karamitopoulou) (35) | Ki-67 expression in 0.3% of buds, in 35% tumor center (p 0.0001). Caspase-3 comparatively lower in tumor buds than other compartments (p 0.0001). Rare cases with Ki-67 and caspase3 immunoreactivity associated with poorer prognosis |
| RAS/RAF | Koelzer (67) | RKIP, NFkB, E-Cadherin WTS RKIP, matched NFκB, and E-Cadherin on multi-punch TMA | 178 Stage I–IV CRC | Yes (Karamitopoulou) | 0.9% of tumor buds positive for RKIP, but expression in main tumor body rather than buds predictive for metastatic disease, vascular invasion, budding, and invasive tumor border configuration. RKIP expression correlated with NFkB expression |
| RAS/MAPK | Dawson (68) | TrkB, multi-punch TMA | 211 Stage I–IV CRC | Yes (Karamitopoulou) | Trkb(m) overexpressed in buds in comparison to main tumor body (p < 0.0001) and associated with KRAS mutation. High expression of membranous Trkb-independent adverse prognostic factor. Inverse correlations between expression profile of Trkb(m) and Ki-67 as well as Caspase-3 (53) |
| Cytokine signaling | Akishima-Fukusawa (71) | CXCL12, WTS | 165 Stage II–III CRC | Yes (Ueno) | CXCL12-positive budding divided into high- and low-grade, staining in the tumor divided into high and low expression. Patients with high-grade CXCL12 budding and high CXCL12 expression had shorter survival than patients with low-grade CXCL12 budding and low CXCL12 expression. CXCL12 expression in buds independent adverse prognostic factor in multivariate analysis irrespective of budding grade |
| Wnt signaling, cell differentiation | Brabletz (76) | β.catenin, Cdx2, laminin5γ2 WTS, additional to cell culture experiments and immunofluorescence | 45 CRC cases, Stage n/a | No | Cdx2 expression was lost in tumor buds but re-expressed in metastases, cell culture experiments demonstrate transient transcriptional down-regulation of Cdx2 triggered by collagen type I |
| Stromal cell interaction | Galvan (79) | TWIST1 and TWIST2 immunohistochemistry on 2 cohorts: cohort 1 (multi-punch TMA) + promoter methylation. Cohort 2: TMA from pre-operative biopsies (prognostic effects). Immunohistochemistry for both markers and promoter methylation in six cell lines. LCM in one tumor-budding high and one tumor-budding low case | Cohort 1: 185 Stage I–IV CRC, Cohort 2: 112 Stage I–IV CRC | Yes [cohort 1: Karamitopoulou, cohort 2: Zlobec (3)] | TWIST 1 and 2 expression restricted to stromal cells. Inverse correlation between TWIST1 protein expression and methylation (Cohort 1) suggests hypermethylation as a mechanism of TWIST1 regulation. TWIST 1 and 2 protein expression significantly correlated with low- and high-grade budding phenotype. LCM of high-grade tumor-budding case with positive TWIST1/2 stroma and no methylation, inverse pattern in low-grade tumor-budding case. TWIST1 (Cohort 2) associated with adverse tumor features and independent prognostic factor. |
| Stromal cell interaction | Karagiannis (81) | Bone morphogenic protein antagonists HTRA3, FST and GREM1, markers assessed in tumors and cancer-associated fibroblasts, WTS | 2 cohorts: 1:30 patients with 10 each no, low and high-grade budding. 2: 219 Stage II CRC | Yes (Ueno) | HTRA3 staining in the epithelial tumor component was differentially regulated between areas with and without tumor-budding, correlation between HETRA3 staining and the presence of budding and with significantly increased expression in tumor-budding cells themselves. Epithelial HTRA3 expression-independent adverse prognostic factor |
WTS, whole tissue sections; LR, local recurrence; SAC, serrated adenocarcinomas; CAC, conventional adenocarcinomas; MMRd, mismatch repair deficient; MMRp, mismatch repair proficient; LCM, laser capture microdissection.