Human Pathology (2015) xx, xxx–xxx

www.elsevier.com/locate/humpath

Progress in pathology

Tumor budding in colorectal cancer—ready for

diagnostic practice?☆,☆☆
Viktor H. Koelzer MD a,b,⁎, Inti Zlobec PhD b , Alessandro Lugli MD a,b
a
Clinical Pathology Division, Institute of Pathology, University of Bern, CH-3010 Bern, Switzerland
b
Translational Research Unit, Institute of Pathology, University of Bern, CH-3010 Bern, Switzerland

Received 30 April 2015; revised 30 July 2015; accepted 13 August 2015

Keywords:
Summary Tumor budding is an important additional prognostic factor for patients with colorectal cancer
Colorectal cancer;
(CRC). Defined as the presence of single tumor cells or small clusters of up to 5 cells in the tumor stroma, tumor
Tumor budding;
budding has been linked to epithelial-mesenchymal transition. Based on well-designed retrospective studies,
Prognostic factor;
tumor budding has been linked to adverse outcome of CRC patients in 3 clinical scenarios: (1) in malignant
Epithelial-mesenchymal
polyps, detection of tumor buds is a risk factor for lymph node metastasis indicating the need for colorectal
transition;
surgery; (2) tumor budding in stage II CRC is a highly adverse prognostic indicator and may aid patient
Metastasis
selection for adjuvant therapy; (3) in the preoperative setting, presence of tumor budding in biopsy material may
help to identify high-risk rectal cancer patients for neoadjuvant therapy. However, lack of consensus guidelines
for standardized assessment still limits reporting in daily diagnostic practice. This article provides a practical and
comprehensive overview on tumor budding aimed at the practicing pathologist. First, we review the prognostic
value of tumor budding for the management of colon and rectal cancer patients. Second, we outline a practical,
evidence-based proposal for the assessment of tumor budding in the daily sign-out. Last, we summarize the
current knowledge of the molecular characteristics of high-grade budding tumors in the context of personalized
treatment approaches and biomarker discovery.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
The presence of de-differentiated single cells or small
clusters of up to 5 cells at the invasive front of colorectal cancer
☆ tumor budding has proceeded farthest in colon and rectal
Competing interest: The coauthors have no relevant affiliations or financial
involvement with any organization or entity with a financial interest in or
financial conflict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership or options,
expert testimony, royalties, or patents received or pending.
☆☆
Funding/Support: This project received no grant from any funding
agency in the public, commercial, or not-for-profit sectors.
⁎ Corresponding author at: Institute of Pathology, University of Bern,
CH-3010 Bern, Switzerland.
E-mail address: viktor.koelzer@pathology.unibe.ch (V. H. Koelzer).
(CRC) has been termed tumor budding [1] (Fig. 1). Tumor
budding has received increasing attention by gastrointestinal
pathologists as a valuable prognostic factor. It is an indicator of
aggressive tumor biology that may be driven by an
epithelial-mesenchymal transition (EMT)–like process. The
characterization of the clinical and biological implications of
cancers and is rapidly advancing in carcinomas of the upper
gastrointestinal tract [2], pancreas [3,4], breast [5,6], head and
neck [7], and lung [8–10]. Substantial evidence from
retrospective studies suggests that tumor budding is ready to
take center stage as an additional prognostic factor for CRC
patients in distinct clinical settings [11]. Specifically, detection
of tumor budding in malignant polyps may allow the
identification of patients at high risk for nodal metastasis as

http://dx.doi.org/10.1016/j.humpath.2015.08.007
0046-8177/© 2015 Elsevier Inc. All rights reserved.
2 V. H. Koelzer et al.

TUMOR BUDDING (H&E)
* link between tumor progression, vascular invasion, the spread
100µm
Fig. 1 Tumor budding. High-power image (×40) of tumor budding
cells (→) at the invasive front of CRC in a standard H&E stain. Tumor
budding is defined as the presence of single cells or small clusters of
cancer cells in the tumor stroma and is frequently found in tumors with
an invasive growth pattern and desmoplastic stromal response.
Detection of tumor budding is frequently associated with lympho-
vascular invasion (*) in the tumor microenvironment.
candidates for surgical resection [12]. In stage II CRC tumor
budding may indicate a high chance for metastatic relapse and
may complement clinical decision making on adjuvant therapy
and follow-up [13]. Last, detection of tumor budding in
preoperative biopsies may allow the identification of rectal
cancer patients for for neoadjuvant treatment and risk-adapted
surgery to reduce the risk of local recurrence [14,15] (Fig. 2).
The importance of tumor budding additional prognostic factor is
reflected in publications by the Union for International Cancer
Control (UICC) [16], the Association of Directors of Anatomic
and Surgical Pathology [17], and inclusion in the guidelines for
CRC screening, diagnosis, and treatment in Europe and Japan
[18–21].In the UpToDate information resource, tumor budding
is currently listed as a category IIB prognostic factor [22].
Convincing evidence suggests that tumor budding may be
the visible correlate of an EMT-related process in the tumor
microenvironment of CRC [23]. The detection of tumor buds
at the invasive front may therefore represent a morphologic
of circulating tumor cells, and the establishment of metastatic
disease. Because metastasis is the leading cause of death for
CRC patients, the characterization of signaling aberrations
leading to tumor budding may represent an important area of
research to discover new options for precision therapy.
Tumor budding is frequently observed in daily diagnostic
practice but infrequently reported. In particular, standardized
reporting is currently held back by disagreement concerning
the most reproducible scoring method, the selection of the best
block for budding assessment, and the use of immunohisto-
chemistry. The purpose of this review is therefore to present a
practical and comprehensive overview on tumor budding
aimed at the practicing pathologist.
2. Materials and methods
For review of the primary and secondary literature
concerning the prognostic impact of tumor budding in CRC
(stage I, stage II, preoperative biopsies), electronic keyword

TUMOR BUDDING
Potential clinical scenarios
Malignant polyps

Tumor budding as a predictor of lymph node metastases

Clinical implication: surgical resection

Stage II CRC

Tumor budding as an adverse prognostic factor

Clinical implication: risk adapted follow up and adjuvant therapy

Pre-operative biopsies of colon and rectal cancer

Tumor budding as an adverse prognostic factor and predictor of lymph node and distant metastasis

Clinical implication: neo-adjuvant therapy and risk adapted surgery

Fig. 2 Potential clinical scenarios to report tumor budding in CRC. Assessment of tumor budding can provide valuable prognostic information in
distinct clinical settings. In malignant polyps, presence of tumor budding as reported by the gastrointestinal pathologist indicates a significantly
elevated risk for nodal metastasis. Surgical resection may be considered. In stage II disease, tumor budding is an independent adverse prognostic
factor that can contribute to the identification of high-risk patients for neoadjuvant therapy and follow-up. Detected in preoperative biopsy material
of colon and rectal cancer patients, tumor budding is an adverse prognostic indicator and correlates with an elevated risk for metastatic spread to
lymph nodes and distant organs. Risk-adapted surgery and neoadjuvant treatment may be a valid option for these patients.
Tumor budding in colorectal cancer
searches using Boolean operators were performed in MED-
LINE, MEDLINE In Process, Scopus, Web of Science,
EMBASE, Google Scholar, and ISI Proceedings. Reference
lists were searched manually to locate additional items. Search
queries were not limited by date and include all literature
published up to the 30th of April 2015 (date of submission).
Case reports or notes were excluded. All items that met the
inclusion criteria were reviewed and assessed for relevance.
Relevant studies were retrieved, catalogued, and categorized
according to the clinical setting of interest. Data tables were
reviewed by all authors of this article.
3. Tumor budding as a histomorphologic
prognostic factor in colon and rectal cancers
3.1. Early stage CRC
Early stage CRC (American Joint Commitee on Cancer
(AJCC) and UICC stage I; pT1/2, pN0, M0) is commonly
identified in the histopathology laboratory after endoscopic
polyp removal. Although most patients fare well after the
complete endoscopic removal of a malignant polyp, up to 17%
already have clinically unapparent micrometastasis in
locoregional lymph nodes at the time of resection [24,25].
Additional histomorphologic factors are therefore needed to
identify patients that may be candidates for colorectal surgery.
Based on retrospective cohort studies, the assessment of tumor
budding can provide important additional information on the
risk of nodal metastasis in this setting (Table 1).
These results have been well confirmed by recent
meta-analyses using robust statistical methods: in a system-
atic review of 23 cohort studies including 4510 pT1 patients,
Beaton and colleagues [24] report high-grade tumor budding
to be significantly associated with nodal metastasis (odds
ratio [OR], 7.74; 95% confidence interval [CI], 4.47-13.39;
P b .001) exceeding the relative risk (RR) of other
established adverse prognostic indicators including greater
than 1-mm depth of submucosal invasion (OR, 3.87; 95% CI,
1.50-10.00; P = .005), lymphatic invasion (OR, 4.81; 95%
CI, 3.14-7.37; P b .00001), and poor tumor differentiation
(OR, 5.60; 95% CI, 2.90-10.82; P b .00001). These findings
are underlined by separate studies from Japan [26] and the
Netherlands [27]: in a systematic review of 3556 pT1
carcinomas from 30 different Japanese institutions, Ueno and
colleagues [26] report the presence of tumor budding, poorly
differentiated clusters, poor tumor differentiation, vascular
invasion, and submucosal invasion depth as significant risk
factors for lymph node metastasis (all, P b .0001). Based on
a meta-analysis of 17 published studies including 3621
patients, Bosch and associates [27] confirm lymphatic
invasion (RR, 5.2; 95% CI, 4.0-6.8), submucosal invasion
at least 1 mm (RR, 5.2; 95% CI, 1.8-15.4), budding (RR, 5.1;
95% CI, 3.6-7.3), and poor histologic differentiation (RR,
4.8; 95% CI, 3.3-6.9) as significant pathological risk factors
for nodal metastasis in pT1 CRC.
3
Importantly, it is already considered good practice to
report tumor budding in early stage CRC according to the
European guidelines for quality assurance in CRC screening
and diagnosis [18]. The importance of including a comment
on tumor budding in early stage lesions is further highlighted
by the European Society for Medical Oncology recommen-
dations for clinical management of patients with colon and
rectal cancers: the 2012 guidelines advocate standard
surgical resection for early stage CRC if high-risk features
including grading greater than 2, invasion of submucosa,
lymphatic or venous invasion, resection margins less than 1
mm, tumor budding, or invasive carcinoma in a sessile polyp
are detected, even after definite R0 removal [19].
3.2. Stage II CRC
AJCC/UICC stage II CRC (pT3/4, pN0, M0) encompass a
heterogeneous group of patients with an observed 5-year
survival ranging from 66.5% (stage IIA) to 37.3% (stage IIC)
[28]. This contrasts with an observed 5-year survival of 73.1%
(stage IIIA) and 46.3% (stage IIIC) for node-positive patients
[28]. Clearly, some node-negative, stage II patients will face
early relapse of disease after resection of the primary tumor due
to the outgrowth of clinically unapparent micrometastasis. The
identification of these stage II patients with an elevated risk of
recurrence through the use of additional pathological risk
factors is therefore important to support clinical decision
making on follow-up and adjuvant therapy. For high-risk
patients, adjuvant therapy can be considered based on a careful
assessment of potential risks and benefits including prognosis
[29–31]. According to the 2015 NCCN guidelines, high-risk
features include “poorly differentiated histology (exclusive of
those cancers that are high microsatellite instability), lymphat-
ic/vascular invasion, bowel obstruction, b12 lymph nodes
examined, perineural invasion, localized perforation, or close,
indeterminate, or positive margins” [31].
Importantly, the presence of tumor budding is increas-
ingly recognized as an additional high-risk feature for
AJCC/UICC stage II CRC patients. Based on a series of
well-designed retrospective cohort studies, high-grade
tumor budding is significantly linked to poor overall and
disease-free survival of curatively resected stage II CRC
[13,32–37] (Table 2). In fact, survival rates of stage II
patients with high-grade tumor budding were found not to
differ significantly in a direct comparison with a matched
cohort of stage III patients in a retrospective analysis
including 446 CRC patients [13]. Furthermore, presence of
tumor budding was consistently related to aggressive
clinicopathological features including lymphatic and ve-
nous invasion, higher tumor grade, and an infiltrative tumor
margin [13,32–37]. Taken together, tumor budding is an
important additional prognostic indicator in stage II disease
and may aid identification of high-risk patients for adjuvant
therapy. Tumor budding is therefore a promising candidate
for inclusion into reporting guidelines of stage II disease and
validation in prospective clinical trials.
 4
Table 1 Tumor budding as a risk factor for lymph node metastasis in stage I CRC
Study Year n (no. of EMR) Assessment method High-grade TB % Clinicopathological features Clinicopathological features significantly
analyzed for correlation with associated with lymph node metastasis (P b .05)
lymph node metastasis
Okuyama et al [80] 2002 101 (0) Morodomi et al [81] Any TB found Age, sex, SMd, L, V, TB, location, size TB, L1, V1, location in rectum
H&E in 42%
Egashira et al [82] 2004 140 (0) Morodomi et al [81] Any TB found SMd, SMw, L, V, TB, LInf, differentiation L1, V1, depth of invasion, cribriform-type
H&E in 60% of invasive component, structural atypia structural atypia, low LInf
of invasive component, border
Ueno et al [63] 2004 285 (80) Ueno et al [63] H&E 15.1% Age, sex, G, SMd, SMw, L, TB, G, LVI, cribriform growth pattern
V, TB, location, size, TuConf
Kitajima et al [83] 2004 865 Hase et al [52] H&E 42.3% G, SMd, L, V, TB, status of TB, SMd, L1, V1, status of muscularis mucosae,
muscularis mucosae, Histological histological type at deepest portion
type at deepest portion
Wang et al [84] 2005 159 (0) Morodomi et al [81] 15.1% Age, G, SMd, L, V, Pn, TB, TB, G, L1, low LInf
H&E border, location, CEA level,
Borrmann type, mucinous
component, no. of lymph nodes
sampled, LInf, MSI
Kazama et al [85] 2006 56 (0) Morodomi et al [81] 75.0% TB TB
PanCK
Masaki et al [86] 2006 76 (21) Ono et al [87] H&E Continuous score G, SMd, SMw, L, V, TB, size, TuConf TB, G
Kawaura et al [88] 2007 122 Ueno et al [63] H&E 18.8% G, SMd, L, V, TB, size, location, TB, SMd, L1, V1, G (univariate)
LInf, TuConf, histologic type at L1, V1 (multivariate)
deepest portion, lymphatic density,
adenomatous component
Kaneko et al [89] 2007 214 (N.S.) Ueno et al [63] H&E 18.2% G, SMd, L, V, TB, size, location, TB, TuConf, G, L1
TuConf, no. of lymph nodes sampled
Sohn et al [90] 2007 48 (0) Ueno et al [63] H&E 20.8% G, SMd, LVI, TB, size TB, LVI
Yasuda et al [91] 2007 86 (0) Morodomi et al [81] 46.5% Age, sex, G, SMd, LVI, TB, location, TB, LVI
H&E size, TuConf
Ishikawa et al [92] 2008 71 (0) Morodomi et al [81] 50.7% (cutoff=4) Age, sex, G, SMd, L, V, TB, location, TB (multivariate), L1
H&E and PanCK size, TuConf,
Yamauchi et al [93] 2008 164 (62) Ueno et al [63] H&E 14.6% G, SMd, L, V, TB TB, G
Choi et al [94] 2009 168 (0) Ueno et al [46] H&E 18.4% G, SMd, TB, LVI, V, resection status TB (multivariate)
Ogawa et al [95] 2009 98 (15) Ueno et al [46] H&E 48.9% Age, sex, G, SMd, L, V, TB, location, TB, G (multivariate)
size, TuConf

V. H. Koelzer et al.
Suzuki et al [47] 2009 124 (0) Ueno et al [46] H&E, 7.3% (H&E) L, V, TB TB, V1 (using elastic stain)
PanCK 17.7% (PanCK)
Kajiwara et al [96] 2010 244 (0) Ueno et al [46] H&E 44.7% Age, sex, G, SMd, L, V, TB, location, TB, L1, poorly differentiated component, myxoid
size, border TuConf, annularity, poorly cancer stroma, border (univariate)
differentiated component, myxoid Poorly differentiated component, L1, myxoid
cancer stroma, infiltration level in cancer stroma (multivariate)
muscularis propria,
 Tumor budding in colorectal cancer
Komori et al [97] 2010 111 (0) Ueno et al [46] H&E SMd, SMw, L, V, TB, area of SM TB, Histological type
invasion, histologic type at deepest
invasion
Tateishi et al [98] 2010 322 (0) Ueno et al [46] H&E 32.9% G, SMd, L, V, TB, disruption of TB (multivariate), L1, G
muscularis mucosae
Ueno et al [99] 2010 296 (67) Ueno et al [63] H&E 11.8% G, SMd, SMw, L, V, TB, size, TB, SMd, SMw, LVI, cribriform pattern, extent
TuConf, growth pattern, adenoma of poorly differentiated component, extent of
component, extent of poorly mucin-producing component, grade of least-
differentiated component, cribriform differentiated component
pattern, extent of mucin-producing
component
Akishima-Fukasawa 2011 111 (0) Ueno et al [63] H&E 50.5% G, SMd, SMw, L, V, TB, TuConf, TB, SMd, G, L1, border, disruption of muscularis
et al [100] border, disruption of muscularis mucosa, neutrophil infiltration in cancer cells,
mucosae, neutrophil infiltration in fibrotic cancer-type stroma, Crohn-like reaction,
cancer cells, fibrotic cancer-type microscopic abscess formation
stroma, LInf, Crohn-like reaction,
microscopic abscess formation
Keum et al [101] 2012 434 (0) Ueno et al [46] H&E 40% in patients Age, sex, G, L, V, TB, location, TB found in all 20 patients with recurrence
with recurrence size, border, TuConf, annularity,
poorly differentiated component,
myxoid cancer stroma, infiltration
level in muscularis propria
Kye et al [102] 2012 55 (0) Ueno et al [46] H&E 36% Age, sex, SMd, LVI, TB, location, TB (multivariate)
size, TuConf, circumference ratio,
presence of microacinar structures
Nakadoi et al [103] 2012 499 (150) Ueno et al [63] H&E Colon 9.2%; G, SMd, LVI, TB TB (multivariate), G, SMd, LVI
rectum 15.3%
Suh et al [104] 2012 435 (111) Ueno et al [46] H&E 17.6% G, SMd, LVI, TB, background TB, G, LVI, No background adenoma
adenoma
Oka et al [105] 2013 118 Modified Ueno et al 15.3% G, SMd, LVI, TB TB
[63] H&E
Umemura et al [106] 2013 142 Ueno et al [63] H&E 11.2% H&E, Age, sex, SMd, L, V, TB, location, TB (as assessed by IHC only), SMd (multivariate)
and β-catenin 9.8% β-catenin TuConf, size, β-catenin expression,
operation method
Beaton et al [24] 2013 Meta-analysis N.A. N.A. G, SMd, LVI, TB TB (OR, 7.74; 95% CI, 4.47-13.39; P b .001),
of 23 studies G (OR, 5.60; 95% CI, 2.90-10.82; P b .00001),
(n = 4510) LVI (OR, 4.81; 95% CI, 3.14-7.37; P b .00001),
SMd ≥1 mm (OR, 3.87; 95% CI, 1.50-10.00,
P = .005)
Ueno et al [107] 2014 3556 patients from Ueno et al [63] H&E 19.4% G, SMd, LVI, TB, PDC G (OR, 7.2; 95% CI, 4.4-11.8; P b .0001),
30 institutions LVI (OR, 4.8; 95% CI, 3.8-6.0; P b .0001),
SMd ≥1mm (OR, 4.0; 95% CI, 2.5-6.5;
P b .0001)
TB (OR, 3.8; 95% CI, 3.0-4.7; P b .0001)
PDC (OR, 3.3; 95% CI, 2.6-4.1; P b .0001)

5
 6
Table 1 (continued)
Study Year n (no. of EMR) Assessment method High-grade TB % Clinicopathological features Clinicopathological features significantly
analyzed for correlation with associated with lymph node metastasis (P b .05)
lymph node metastasis
Bosch et al [27] 2013 Meta-analysis N.A. N.A. G, SMd (based on SM levels, cutoff LVI (RR, 5.2; 95% CI, 4.0-6.8),
of 17 studies value 1 mm and cutoff value 2 mm), SMd ≥1mm (RR, 5.2; 95% CI, 1.8-15.4),
(n = 3621) SMw, L, V, LVI, TB, Linf, border, TB (RR, 5.1; 95% CI, 3.6-7.3),
Tu Conf, poor differentiation at G (RR, 4.8; 95% CI, 3.3-6.9)
invasion front, size, histologic type,
cribriform subtype, microvessel density
Suh et al [108] 2013 150 (75) Ueno et al [46] H&E 29.3% in surgically G, SMd, LVI, TB, tumor size, TB, SMd, LVI (univariate)
treated patients TuConf, margin status TB, SMd (multivariate)
Ryu et al [109] 2014 179 Ueno et al [46] H&E 18.9% Age, sex, G, SMd, LVI, TB, TuConf, TB, G, L1, SMd, border, tumor depth, presence of
Pn, border, tumor depth, preexisting cribriform structures, desmoplasia, micropapillary
adenoma, presence of cribriform structures, mucinous differentiation, absence of
structures, desmoplasia, micropapillary Crohn-like reaction
structures, mucinous differentiation,
Crohn-like reaction, intraglandular
necrosis, clear cell change, signet ring
differentiation, solid differentiation
Caputo et al [110] 2014 48 Ueno et al [46] H&E 12.5% G, SMd, LVI, TB G
Macias-Garcia 2015 97 Ueno et al [63] H&E 13.4% Age, sex, G, SMd, L, V, TB, LInf, TB, G, L1, absence of LInf, border, sessile
et al [111] border, TuConf, size, location, stalk morphology (univariate)
invasion, presence of background G, border, absence of LInf, sessile morphology
adenoma (multivariate)
Kawachi et al [112] 2015 806 Ueno et al [63] H&E 28.9% Age, sex, G, SMd, L, V, TB, size, TB, SMd, G, L1, V1, nonpedunculated TuConf
location, TuConf (univariate)
TB, SMd (multivariate)
Wada et al [113] 2015 Meta-analysis All Ueno et al [63] N.A. G, SMd, LVI, TB, LInf, differential TB (OR, 7.45; 95 % CI, 4.27-13.02; P = .0077),
of 5 studies H&E/IHC subtype at the deepest portion, L1 (OR, 5.19; 95 % CI, 3.31-8.15; P = .01)
(n = 770) lymphatic vessel density,
adenomatous component
Abbreviations: Border, tumor border configuration; CEA, carcinoembryonic antigen; CI, confidence interval; CRC, colorectal carcinoma; EMR: endoscopic mucosal resection; G, tumor grade; H&E,
hematoxylin and eosin; IHC, immunohistochemistry; L1, lymphatic invasion; LInf, lymphoid cell infiltrates; LVI, lymphovascular invasion; MSI, microsatellite instability; N.A., not applicable; N.S., not
significant; OR, odds ratio; PanCK, pan-cytokeratin; PDC, poorly differentiated clusters; Pn, perineural invasion; SMd, depth of submucosal invasion; SMw, width of submucosal invasion; TB, tumor budding;
TuConf, macroscopic tumor configuration; V1, venous invasion.

V. H. Koelzer et al.
 Tumor budding in colorectal cancer
Table 2 Tumor budding as a histopathologic prognostic factor in stage II CRC
Study Year n Method High-grade TB % Clinicopathological End point Outcome
features correlated
with TB (P b .05)
Tanaka et al 2003 138 Hase et al [52] H&E 19.6% Disease recurrence Disease-specific 5-year 5-y survival rates with low-grade TB: 98%;
[33] pT3 survival rate high-grade TB: 74% (P b .0001; univariate)
Okuyama et al 2003 83 Morodomi et al [81] H&E 57.9% L1, pN1, local recurrence, Cumulative disease-specific 5-y survival rates with low-grade TB: 85.0%;
[114] (rectal) liver metastasis 5-y survival rate high-grade TB: 51.8% (P b .002)
pT3
Masaki et al 2005 72 Raw number of buds; H&E N.A. pN1 pN stage N.A.
[115] (rectal)
pT2
Nakamura et al 2008 200 Nakamura et al [13] 34.5% Disease recurrence Cumulative 5-y and 5-y survival rates with low-grade TB: 93.9%;
[13] pT3-4 H&E (peritoneal and 10-y survival rates high-grade TB: 73.9% (P b .001).
liver metastasis) 10-y survival rates with low-grade TB: 90.6%;
high-grade TB: 67.8% (P b .001).
Wang et al [34] 2009 128 Wang et al [34] H&E 45% LVI, infiltrative tumor Disease-specific 5-y 5-y survival rates with low-grade TB: 91%;
pT3 margin survival rate high-grade TB: 63% (P b .0001).
Kevans et al 2011 258 Wang et al [34] H&E 44% TB more frequent in 5-y survival rate TB independently associated with survival
[116] (122 cases microsatellite stable (HR, 7.9; 95% CI, 3-21; P b .001)
analyzed) tumors
Betge et al [35] 2012 120 Modified Ueno et al [46] H&E; 30% LVI, G 5-y progression-free 5-y progression-free survival rates with
pT3-4 area 0.95 mm2; high-grade TB and disease-specific low-grade TB: 91%; high-grade TB:
defined as ≥10 buds/HPF survival rates 65% (P = .009).
5-y disease-specific survival rates with
low-grade TB: 94%; high-grade TB:
72% (P b .001)
Canney et al 2012 205 Wang et al [34] H&E pT3 44%, pT4a TB most frequent in 5-y survival rate Not significant
[36] pT3-4b 73%, pT4b 25% pT4a disease
Horcic et al 2013 105 Interobserver study (PanCK): Hase et al: 60%, Infiltrating tumor Disease-specific 1-HPF and 10-HPF methods (1 HPF,
[37] pT3-4 Hase et al [52], Nakamura Nakamura et al 59.1%, border configuration 5-y survival rate P = .0007; 10 HPF, P = .0067) and
et al [13], Wang et al [34], Ueno et al 83% (except Wang et al) Wang (P = .0459) significantly linked
1 HPF [57], 10 HPFs [50] Wang et al: 41.9% to patient outcome based on a single or
multiple observers (univariate).
1 HPF and 10 HPFs significantly linked
to survival outcome in multivariate analysis.
Lai et al [32] 2014 135 Ueno et al [46] H&E 27 N.A. Progression-free survival 5-y progression-free survival rates with
pT3-4 disease-specific survival low-grade TB: 89%; high-grade TB:
57.6% (P b .001).
5-y disease-specific survival rates with
low-grade TB: 92%; high-grade TB:
66.7% (P b .001)
Abbreviations: Border, tumor border configuration; HR, hazard ratio; L1, lymphatic invasion; LVI, lymphovascular invasion; N.A., not applicable; PanCK, pan-cytokeratin; pN, pathological nodal stage; pT,
pathological tumor stage; TB, tumor budding; 1 HPF, 1-high-power field method; 10 HPF, 10-high-power field method.

7
8 V. H. Koelzer et al.
3.3. Tumor budding in preoperative biopsies of
colon and rectal cancers
The preoperative biopsy of primary CRC is an essential step
for confirmation of diagnosis but currently fails to provide
prognostic information to the clinician beyond a rough
estimation of tumor grade, assessment of mismatch-repair
protein expression, and molecular pathology studies. Recent
studies indicate that preoperative biopsy material may contain
significant prognostic information based on the assessment of
tumor budding and immune cell infiltrates [14,15,38,39]
(Table 3). In particular, presence of tumor budding in colon
and rectal cancers biopsies may be a strong indicator of nodal
and distant metastasis at the time of resection [15,40] and has
been associated with nonresponse to neoadjuvant chemora-
diotherapy and poor survival outcome in rectal cancer patients
[14]. As the infiltrative margin is rarely sampled on biopsy
specimens of CRC, intratumoral budding is assessed for
prognostication [40]. A cutoff of 6 tumor buds in 1 high-power
field (HPF; ×40) has been proposed to identify high-risk
patients, but assessment of tumor budding on a continuous
scale may allow for a more precise risk estimate to complement
clinical decision making in the preoperative setting [15,41]. In
addition to radiology, endoscopic imaging, and ultrasound for
local staging, risk factors present in the preoperative biopsy
may thereby contribute important prognostic and possibly
predictive information for the multidisciplinary management
of colon and rectal cancers patients.
4. Tumor budding: scoring systems and
interobserver variability
Ease of assessment and interobserver reproducibility are
central prerequisites for the establishment of any histomorpho-
logic prognostic indicator in daily diagnostic practice. In
retrospective analyses, commonly used additional prognostic
factors such as tumor grade [42,43] and vascular invasion [44]
have been shown to be only moderately reproducible between
observers. It is therefore essential to identify the most effective
and reproducible method of assessment for tumor budding based
on well-designed interobserver studies and to agree on
transparent guidelines for routine application. This includes
testing the methodological approach in clearly defined clinical
scenarios (eg, scoring of buds in biopsy material, malignant
polyps, and resection specimens [Fig. 2]) to provide reproduc-
ible prognostic data for the interdisciplinary management of
CRC patients.
4.1. Diagnostic reproducibility of tumor budding
Several investigators have included an assessment of
interobserver variation in prognostic studies. Based on
scores from 2 or more observers, reported κ values for tumor
budding scores range from 0.41 (moderate) to 0.938 (very
good) [32,34,45–51]. However, interobserver concordance
was frequently assessed using a limited subset of cases and
scores from a single academic center, which may artificially
reduce statistical divergence. Systematic interobserver
studies comparing different scoring methods with observers
from different institutions are rare: In a virtual microscopy
study including 10 investigators from different centers, the
scoring methods by Hase et al [52], Nakamura et al [53],
Ueno et al [54,55], and Wang et al [34] were systematically
compared using both hematoxylin and eosin (H&E)– and
cytokeratin-stained sections. For these methods, overall
agreement was fair and primarily dependent on the
participants' experience with the assessment of tumor
budding and training in gastrointestinal pathology [56].
Use of immunohistochemistry led to an increase in the
reported tumor budding counts but did not improve
interobserver reproducibility.
In a subsequent study using cytokeratin-stained sections
scored by 3 observers, tumor budding as assessed according
to Hase, Nakamura, Ueno, Wang (conventional and rapid
method), the densest HPF (1-HPF method [57]) and the 10
densest HPFs (10-HPF method [50]) were systematically
compared in respect to prognostic impact and interobserver
reproducibility [37]. In this analysis, the 1-HPF and 10-HPF
methods (intraclass correlation coefficient [ICC] = 0.83 and
0.91, respectively) significantly outperformed the scoring
methods according to Hase (κ = 0.29-0.51), Nakamura (κ =
0.41-0.52), Ueno (κ = 0.39-0.56), and Wang (conventional
method, κ = 0.46-0.62, and rapid method, κ = 0.46-0.58
respectively).
Notably, only tumor budding as evaluated in 1-HPF and
10-HPF methods had significant prognostic effects on
patient survival in a cohort of 105 well-characterized stage
II CRC patients [37]. The 1-HPF and 10-HPF methods were
subsequently tested in a multicenter interobserver study
involving 6 independent community and academic centers
using both H&E- and cytokeratin-stained slides [41]. In this
analysis, 3 to 4 times more tumor buds were detected in
cytokeratin as compared with H&E-stained slides. Importantly,
excellent interobserver correlations for tumor budding scores
(10 HPFs: ICC= 0.83 and 1 HPF: ICC= 0.85) were reached
using cytokeratin stains, whereas interobserver agreement
for H&E slides was only moderate (10 HPFs: ICC= 0.58 and
1 HPF: ICC= 0.49) [41].
5. Practical, evidence-based proposal for the
assessment of tumor budding in daily
diagnostic practice
Agreement on the optimal method for the assessment of
tumor budding is of key importance to transfer the available
evidence regarding interobserver reproducibility, practica-
bility, and prognostic impact into diagnostic practice. Central
aims are as follows: definition of clear parameters for
selection of the tissue block, optimal visualization of tumor
 Tumor budding in colorectal cancer
Table 3 Tumor budding in preoperative biopsies as an indicator of adverse prognosis and elevated risk of metastasis
Study Year n Method High-grade TB % Clinicopathological features End point Outcome
correlated with TB (P b .05)
Morodomi 1989 112 rectal cancer Morodomi et al 46% (ITB, biopsy) ITB in biopsy associated with N.A. N.A.
et al [81] patients (all with [81] H&E presence of pN1 and L1 in the
preoperative biopsies, resection specimen
40 with matched
resection specimens)
Giger et al 2012 135 colon cancer Modified Nakamura 17% (ITB, biopsy), ITB in biopsy associated with N.A. N.A.
[40] patients (72 with et al [13] H&E 57% (PTB, resection presence of pN1 and higher
preoperative biopsies) specimen) tumor grade in the resection
specimen. More ITB in biopsy
in patients with distant metastasis
Rogers et al 2013 185 rectal cancer Modified Nakamura 20% (ITB, biopsy) ITB in biopsy associated with Disease-Free survival, High-grade ITB in the preoperative
[14] patients (89 with et al [13] H&E more-advanced ypT stage, ypN1, cancer-specific death, biopsy was associated with a lower
preoperative biopsies, residual poorly differentiated chemotherapy response disease-free 5-y survival rate (33% vs
all received neoadjuvant tumors and LVI in resection 78%, P = .001), lower cancer-specific
treatment) specimen 5-y survival rate (61% vs 87%,
P = .021), and predicted cancer-
specific death (HR, 3.51; 95% CI,
1.03-11.93; P = .040) and
nonresponse to neoadjuvant
chemoradiotherapy
Zlobec et al 2014 346 CRC patients 1-HPF method 0-10 buds (n = 97), ITB in biopsy associated with more N.A. N.A.
[15] (185 with preoperative [57] PanCK 11-20 buds (n = 19), advanced pT stage, L1, and V1 in
biopsies, patients who 21-30 buds (n = 7), resection specimen. More ITB in
received neoadjuvant or 31-50 buds (n = 6), biopsy in patients with distant
preoperative treatment N50 buds (n = 4) metastasis
excluded)
Abbreviations: CI, confidence interval; H&E, hematoxylin and eosin; HPF, high-power field; HR, hazard ratio; ITB, intratumoral budding; L1, lymphatic invasion; LVI, lymphovascular invasion; N.A., not
applicable; PanCK, pan-cytokeratin; PTB, peritumoral budding; pN, pathological nodal stage; pT, pathological tumor stage; TB, tumor budding; V1, venous invasion.

9
10 V. H. Koelzer et al.

budding cells, and standardized scoring criteria for the STANDARD HISTOLOGY (H&E)
relevant clinical scenarios (stage II CRC, preoperative
biopsies, malignant polyps).
A

• Selecting the optimal tissue block: the assessment of

resection specimens needs to account for tumor
heterogeneity. This is reached by selecting the block
with the highest grade of tumor budding for further
analysis during sign-out of the H&E slides. Because
tumor budding is most often found in association with
an infiltrative tumor margin [1,58], this can be used as a
surrogate criterion to select the optimal tissue block.

• Optimal visualization of tumor budding cells: although IMMUNOHISTOCHEMISTRY (AE1-AE3/CD8)

tumor budding can be assessed in H&E in unproblematic
cases, tumor buds can be obscured by peritumoral
inflammation and reactive stromal fibroblasts and may
be confused with ruptured glands (Fig. 3). This
contributes to suboptimal interobserver reproducibility
and the failure of standard histology to reflect the true
amount of tumor budding cells in an individual
case [41,56,59,60]. Immunohistochemistry facilitates the
detection of tumor budding cells and may significantly
improve interobserver reproducibility [12,41]. Many
studies have so far failed to provide the exact features
used for the identification of tumor budding cells in
cytokeratin stains. To address this problem, we recently
implemented standardized features for the identification
of tumor buds in CRC resection specimens by cytokeratin
immunohistochemistry [41]:
- Tumor buds are single cells or clusters of up to
5 cells (≤5 cells) present in the peritumoral stroma
- Tumor buds show cytoplasmic reactivity to cytoker-
atin stains and a clearly identifiable nucleus.
- Cytoplasmic pseudofragments, ruptured glands,
mucin pools, and necrosis are excluded.
• Standardized scoring criteria: importantly, tumor budding
cells can be manually quantified under the microscope and
by using digital analysis methods [61,62]. Just as mitotic
counts in breast cancer, tumor budding is a continuous
feature and can be assessed as such. Quantitative
assessment of tumor buds allows for the combination of
stringently defined histomorphologic criteria with a defined
visual field size. Importantly, numeric tumor budding
counts are highly reproducible based on interobserver
studies [37,41,50], whereas qualitative assessment may
suffer from poor interobserver reproducibility [37].
Two central scenarios are met in daily diagnostic practice
when signing out CRC specimens. Most commonly, tumor
budding will be assessed in CRC resection specimens. In this
setting, tumor budding as assessed in the 10 HPPs of highest
density along the tumor invasion front (10-HPF method) has
been shown to reliably capture associations with adverse
clinicopathological features [37,50] with superior interobserver
B
50µm
Fig. 3 Optimal visualization of tumor buds. Comparative
high-power image (×40) of tumor budding in a CRC with strong
peritumoral inflammation, stromal activation, and glandular
destruction. Serial sections were stained with standard H&E and
an immunohistochemical double stain (pan-cytokeratin AE1/AE3,
brown; CD8, red) to visualize tumor budding cells and peritumoral
CD8+ lymphocytes. In cases with strong inflammation, the
differentiation of tumor budding cells from reactive stromal
fibroblasts and activated histiocytes can be challenging using
standard H&E stains. Cytokeratin facilitates detection of tumor
budding cells and allows for the scoring of tumor buds with strong
reproducibility and specificity.
reproducibility [37,41,50]. To assess tumor budding in the
10-HPF method, the invasive front is first scanned at low
magnification (×4-×10) to identify areas of highest budding
density [37]. Tumor buds are then counted under high
magnification (×40) and the tumor budding count is reported
(Fig. 4A). Based on previous studies, patients with an
average of more than 10 buds per HPF (100 buds is total)
have a highly adverse prognosis [50]. However, raw tumor
budding counts can be used to directly calculate the probability
of clinically relevant outcomes [37,41]. We therefore advise to
additionally report the actual tumor budding count to aid
clinical decision making.
In contrast to resection specimens, limited material will be
available for the assessment of tumor budding in preoperative
biopsies and malignant polyps. Importantly, tumor budding
counts as assessed in a single hotspot strongly correlate with
prognosis as shown by using the 1-HPF [15,37,57], Giger
Tumor budding in colorectal cancer 11
RESECTION SPECIMENS – 10HPF METHOD MALIGNANT POLYPS – 1HPF METHOD
Field selection on low power Field selection on low power

A B

4x-10x 4x-10x

Counting buds in 10 high power fields (10HPF) Counting buds in 1 high power field (1HPF)

40x 40x

BIOPSY FRAGMENTS – 1HPF METHOD

Field selection on low power

4x-10x

Counting buds in 1 high power field (1HPF)

40x

Fig. 4 A, For assessment of tumor budding in CRC resection specimens using the 10-HPF method, the invasive front is first scanned at low power
(×5-×10) to identify areas of the highest budding density (top panel). Tumor buds are then counted in a total of 10 HPFs (×40, field area 0.49 mm2)
along the invasion front (bottom panel). B and C, For assessment of tumor budding in malignant polyps or biopsy material, the specimens are first
scanned at low power (×5-×10) to identify the hotspot with the most tumor buds (top panel). Tumor buds are then counted in 1 HPF (×40, field area 0.49
mm2) in the area of highest density (bottom panel).
12
[14,40], and Ueno methods [46,63]. Based on the proven
reproducibility of using pan-cytokeratin stains for the 1-HPF
method in CRC resection specimens [37,41,56], we currently
favor this approach when assessing tumor budding in biopsy
material and malignant polyps (Fig. 4B and C). However,
interobserver reproducibility of the 1-HPF method still needs
to be independently confirmed in this setting.
5.1. Relation of budding to grade, tumor border
configuration, and special morphologic variants
of CRC
Tumor budding is a conceptually independent histomor-
phologic feature of CRC and does not count toward tumor
grade [17]. In fact, tumor budding is also frequently observed
at the invasive front of tumors with a predominantly glandular
growth pattern that are graded as well or moderately
differentiated according to World Health Organization
recommendations [64]. Furthermore, the presence of tumor
budding does not influence the assessment of tumor border
configuration [1,58]. Although tumor budding is most
common in cancers with an invasive-type margin as assessed
under low power, tumor budding is an independent, super-
imposed feature observed at high magnification and should be
separately reported [1].
Two specific, but infrequent histologic subtypes of CRC
can pose a challenge when assessing tumor budding cells at the
invasive front. First, carcinomas with a primary dissociative
growth pattern or signetring cell differentiation do not allow
the differentiation of “tumor buds” from the main tumor body.
It has been previously suggested that primary CRCs with a
diffuse growth pattern should be classified as high-grade tumor
budding by definition [1]. Second, mucinous carcinomas
frequently form pools containing single cancer cells or small
tumor cell clusters. Because these cell clusters frequently lie in
mucin pools and are not surrounded by tumor stroma, they do
not qualify as bona fide tumor buds.
6. Molecular characteristics of tumor budding
in CRC
Molecular features of CRC impact the morphologic
appearance on the histologic slide. Specific genomic and
proteomic changes in the mutational landscape of CRC may
therefore cause the formation of tumor budding cells. The
identification of pathogenic signaling alterations driving the
tumor budding phenotype may allow not only a better
understanding of the malignant progression of colorectal
tumors but also the development of new precision therapeu-
tics targeting the earliest phase of metastatic dissemination.
CRC with high-grade tumor budding share aggressive
molecular and biological features. Correlative evidence
V. H. Koelzer et al.
suggests that tumor budding cells at the invasive front may
be formed in a process likened to EMT. In support of this
theory, tumor budding is particularly frequent in CRC with
constitutive activation of WNT signaling, characterized by
loss of membranous E-cadherin and nuclear translocation of
β-catenin [65–68]. On a molecular level, APC mutations are
frequently found in high-grade budding cancers, providing
additional evidence for WNT signaling as a driver of
invasive growth [65,69]. TGF-β signaling may additionally
contribute to stromal remodeling and activation of EMT
[70,71]. Furthermore, activation of tyrosine-kinase receptor
pathways including RAS, RAF, and several mitogen-
activated protein kinases may add to the activation of EMT
and the generation of the tumor budding phenotype
(reviewed by Dawson and Lugli [72]). Indeed, presence of
activating BRAF and KRAS mutations has been previously
associated with high-grade tumor budding in CRC [73,74].
Once formed, tumor buds display an armamentarium of
enzymes allowing for efficient migration in the tumor stroma.
In particular, expression of metalloproteinases and cathepsin
allows for the breakdown of stromal collagen and efficient
invasion of host tissue [75]. Tumor budding cells have also
been assigned an increased resistance to apoptotic stimuli,
chemotherapeutics, and immunogenic cell death based on a
reduced expression of caspases [76] and Raf-kinase inhibitory
protein (RKIP), an important checkpoint inhibitor of the
RAF-kinase-signaling pathway [77]. Indeed, residual tumor
budding cells can be observed in fibrotic tumor beds after
neoadjuvant treatment of rectal cancer, adding morphologic
evidence to these correlative analyses [78].
Recent studies suggest that tumor budding cells also
interact with the host immune system. In patients with a
strong peritumoral infiltration by CD8+ cytotoxic T cells,
tumor budding is infrequently encountered [57]. As CD8+
T cells represent an important antitumoral effector mecha-
nism, a destruction of tumor budding cells in patients with a
functional immune response at the tumor-host interface may
be postulated. As further evidence of tumor-host interaction,
tumor budding cells may down-regulate components of the
major histocompatibility complex I under selective pressure
of the antitumoral host response [79]. As a consequence,
tumor budding cells and CD8+ T cells can be seen as 2
opposing sides of an attacker-defender model. This offers an
integrative basis to further refine prognostication of CRC
patients based on specific properties of the tumor microen-
vironment [57].
Based on the present evidence at the molecular and protein
level, tumor budding cells may represent an important stage of
CRC progression—they are the first morphologic evidence of
dissociative tumor growth and echo detrimental changes at the
tumor/host interface. It seems likely that tumor budding cells are
indeed the source for circulating tumor cells in CRC patients and
the seed for nodal and distant metastatic disease. Consequently,
targeting tumor budding cells may inhibit metastatic spread of
CRC at the earliest possible time point. Based on our current
knowledge, WNT, RAS, and TGF-β signaling pathways as well
Tumor budding in colorectal cancer
as proteins involved in stromal degradation and the antitumoral
immune response may provide valid options for the develop-
ment of these therapeutic strategies.
7. Conclusions and future perspectives
Tumor budding is a valuable prognostic indicator for CRC
patients. Detection of tumor budding in malignant polyps
indicates a significantly elevated probability of nodal
metastasis. After an endoscopic polyp removal, colorectal
surgery may therefore be indicated in a risk-adapted approach.
In stage II disease, presence of tumor budding is an
independent adverse prognostic indicator. For these patients,
surgery may not be curative and adjuvant therapy considered.
Detection of tumor budding in preoperative biopsies of colon
and rectal cancers may indicate an increased probability for
metastatic dissemination to lymph nodes and distant organs.
Neoadjuvant treatment and risk-adapted tumor surgery can be
considered in this patient population.
To facilitate the application of tumor budding in daily
diagnostic practice, standardized guidelines for reporting and
assessment are essential. Much progress has been made in this
respect over recent years. Based on meaningful retrospective
data and a strong evidence base, tumor budding has been
included in several reporting recommendations in the United
States, Europe, and Asia. Systematic interobserver studies
have contributed to the identification of the most reproducible
assessment methods for tumor budding. The advantages of
using immunohistochemistry to detect tumor buds of CRC
have been demonstrated but must be weighed against the costs.
Together these data form an important base to reach a working
model. Taking the next step toward a unified approach will be
essential to avoid that one of the potentially most valuable
prognostic factors available to gastrointestinal pathologists and
CRC patients is lost in translation.
To attain this goal, specific questions will need to be
addressed: first and foremost, an international, evidence-based
consensus on standardized reporting of tumor budding in CRC
has to be found. In particular, decisions on the optimal scoring
method and whether cutoffs or continuous budding counts
should be included in histopathology reports have to be made.
Second, the normalization of tumor budding scores to visual
field size needs to be systematically pursued for application in
diagnostic practice. Last, the inclusion of tumor budding into
clinical trials should be pursued to confirm the influence on
prognosis in the prospective setting.
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