Histopathology 2007, 50, 151–162. DOI: 10.1111/j.1365-2559.2006.02551.x
REVIEW
Tumour budding in colorectal carcinoma
F Prall
Institute of Pathology, University of Rostock, Rostock, Germany
Prall F
(2007) Histopathology 50, 151–162
Tumour budding in colorectal carcinoma
The term tumour budding denotes that at the invasion
front of colorectal adenocarcinomas tumour cells,
singly or in small aggregates, become detached from
the neoplastic glands. This morphological feature is
increasingly being recognized as a strong and robust
adverse prognostic factor. Biologically, tumour budding
is closely related to the epithelial–mesenchymal transition. In this review the morphological features of
tumour budding are discussed, as observed by the
surgical pathologist reporting colorectal carcinoma
resection specimens. The morphological features are
put into context with the rapidly expanding knowledge
of the epithelial–mesenchymal transition in general,
and the molecular pathology of colorectal carcinoma in
particular. Finally, a systematic analysis of the relevant
published clinicopathological studies emphasizes the
potential of tumour budding as a prognostic factor for
routine surgical pathology.
Keywords: colorectal carcinoma, epithelial–mesenchymal transition, prognosis, tumour budding
Abbreviations: APC, adenomatous polyposis coli; BMP, bone morphogenic protein; DMH, 1,2-dimethylhydrazine
dihydrochloride; EGF, epidermal growth factor; ERK, extracellular mitogen-activated kinase; FGF, fibroblast growth
factor; GSK-3, glycogen synthase kinase-3; HGF, hepatocyte growth factor; HNPCC, hereditary non-polyposis
carcinoma syndrome; LEF, lymphocyte enhancing factor; LOH, loss of heterozygosity; MSI-H, high-degree
microsatellite-instable; MSI-L, low-degree microsatellite-instable; MSS, microsatellite-stable; PDGF, platelet-derived
growth factor; PI3-K, phosphoinositide 3-kinase; RTK, receptor tyrosine-kinase; TCF, T-cell factor; TGF-b,
transforming growth factor-beta; TNM, Tumour Node Metastasis; uPA, urokinase plasminogen activator
Introduction
Tumour budding is a peculiar feature observed in many
colorectal adenocarcinomas. The term has been coined
by surgical pathologists1 to denote the presence at the
invasive front of a subset of colorectal adenocarcinomas of tiny cords of neoplastic epithelium that extend
from the neoplastic glands into the adjacent stroma,
and small aggregates of neoplastic epithelium that
appear to have detached and migrated a short distance
into a usually quite desmoplastic stroma (see Figure 1).
Intuitively, this feature appears to represent a distinct
component of tumour invasion. Arbitrarily, tumour
Address for correspondence: PD Dr med. Friedrich Prall, Institute of
Pathology, University of Rostock, Strempelstraße 14, D-18069
Rostock, Germany. e-mail: [email protected]
2007 The Author. Journal compilation 2007 Blackwell Publishing Limited.
buds have been defined as comprising five tumour cells
or less, although in Japanese publications the limit
is often set at four tumour cells. Depending on the
observer’s ⁄ researcher’s background, names other than
tumour budding have been given to this phenomenon
or to closely related findings in in vitro or experimental
systems, viz. focal de-differentiation, tumour cell dissociation or epithelial–mesenchymal transition. As may
be expected with an evolving concept, the nomenclature is not settled. In fact, the role of tumour budding in
the surgical pathology of colorectal carcinoma and its
important link to tumour biology has largely escaped
the attention of histopathologists and the need for
consensus on nomenclature has not yet arisen.
Tumour budding can be appreciated in conventional
slides when it is prominent, but careful observation is
still required. A more complete assessment of tumour
budding is achieved more easily if the neoplastic
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F Prall
interest into tumour budding by academic histopathologists, the links between tumour budding and the
molecular pathology of colorectal carcinoma as well
as the general biology of malignant invasion are
discussed.
A
Phenotypes of invasion in colorectal
carcinoma and tumour budding
B
Figure 1. Example of tumour budding in a colonic carcinoma as seen
on pan-cytokeratin immunostains (monoclonal MNF116). A, Note
small clusters of tumour cells away from the neoplastic glands.
B, Details from the area boxed in A: cytoplasmic pseudo-fragments
are seen as anucleate globules (arrows).
epithelium is highlighted by pan-cytokeratin immunostains. However, since pan-cytokeratin immunostains
are not usually prepared when routinely working up
resection specimens of colorectal carcinomas, a broad
appreciation of tumour budding has not been prompted
by routine surgical pathology practice.
This review serves a dual purpose. First, it aims to
draw attention to and acquaint the reader with the
histomorphological features of tumour budding and its
significance for surgical pathology, which lies in its
potential as a robust and strong adverse prognosticator. Second, in order to promote future research
It is a time-honoured practice to type, grade and stage
colorectal carcinomas by histopathological work-up of
the resection specimens. The discussion of tumour
budding in this review is relevant only to primary
colorectal adenocarcinomas, ordinary or mucinous.
Clearly, assessment of tumour budding is not part of
the staging of colorectal carcinoma. Some morphological features of tumour grading, however, coincide
with tumour budding, and this may give rise to
misconceptions. Grading of colorectal carcinomas as
defined by the World Health Organization (WHO) is
undertaken according to the degree of neoplastic gland
formation, but a poor degree of tumour differentiation
(i.e. high-grade) as defined by the WHO definition and
tumour budding must be clearly discriminated, morphologically and conceptually. Poor differentiation,
implying the absence or near absence of neoplastic
glands, encompasses the totality of a colorectal carcinoma or a distinct subclone, whereas tumour buds are
seen mainly at the invasive border. In fact, most
colorectal adenocarcinomas with a high degree of
tumour budding by WHO criteria are well or moderately differentiated. On the other hand, the tumour cell
aggregates in many high-grade colorectal carcinomas
are much larger than five cells. Thus, by definition,
such high-grade neoplastic glands are not tumour
buds. Of note, there is a significant proportion of highgrade colorectal carcinomas that show very limited
tumour budding. The morphological distinction becomes blurred, however, if a colorectal carcinoma is
high grade and has a diffuse growth pattern. In this
instance—although quite rare for colorectal carcinomas—a large proportion of the tumour cells are
dissociated and arranged singly or in small clusters.
By definition, these colorectal carcinomas have to be
classified as high in tumour budding, even though a
detailed morphological analysis will reveal that in these
cases the characteristic spindled epithelial cells projecting from the main mass of the neoplastic glands are
frequently absent.
A detailed description of tumour budding at the light
microscopic and ultrastructural level was published by
Gabbert et al.2 more than 20 years ago. In their study
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
Tumour budding in colorectal carcinoma
of experimental colonic adenocarcinomas induced in
Wistar rats by 1,2-dimethylhydrazine dihydrochloride
(DMH), these authors examined a total of 266 tumours.
Most (79%) were classified as differentiated adenocarcinoma with fully formed neoplastic glands that by
light microscopy were very similar to human colorectal
carcinomas. Ultrastructurally, the tumour cells were
observed to have an at least rudimentary brush border
lining the lumens of the neoplastic glands, fully
developed desmosomes and junctional complexes, and
the neoplastic glands were invested by a complete
basement membrane. The remaining DMH-incuded
tumours in their series were undifferentiated adenocarcinomas (21%). These tumours were observed to
grow in a trabecular or solid pattern without lumens,
and with a tendency to lose coherence and to detach as
single cells. Ultrastructurally, undifferentiated adenocarcinomas failed to elaborate junctional complexes
and desmosomes were often immature. Furthermore, a
polygonal rim of cytoplasm surrounded the nucleus
without polarity, the cytoplasmic border was smooth
and a brush border was not seen. Whereas the
differentiated adenocarcinomas in the study by Gabbert
et al. very much resembled low- or moderate-grade
colorectal adenocarcinomas of the standard type, the
undifferentiated adenocarcinomas of their study can be
likened morphologically to the much more unusual
high-grade, diffusely infiltrating colorectal carcinomas
of humans.
The very pertinent observation in the study by
Gabbert et al. derives from the description of the
invasive border of the differentiated adenocarcinomas.
On close inspection of these tumour areas by both light
and electron microscopy, the authors observed that the
neoplastic glands were not arranged regularly. Instead,
there were small strands and cords of tumour cells
projecting from the neoplastic glands and at the deepest border discontinuous small aggregates or single
tumour cells were found. Ultrastructurally, these
tumours cell aggregates or single tumour cells did not
elaborate junctional complexes, often had incomplete
desmosomes, a basement membrane was missing or
rudimentary, and a brush border was absent or, at
most, incomplete. The conclusion drawn by the
authors was that at the invasive border differentiated
adenocarcinomas focally acquire the phenotype of
undifferentiated adenocarcinomas. Hence, the concept
of de-differentiation at the invasive margin was established by these authors, and gave rise to the term focal
de-differentiation as an alternative designation of
tumour budding.
Based on these morphological observations, Gabbert
et al. hypothesized that tumour cell migration occurred
153
as a component of the de-differentiation observed at the
invasive margin. Interestingly, these authors also noted
in tumour buds (the areas of de-differentiation in
otherwise differentiated adenocarcinomas in their
terms) the ultrastructural presence of ‘pseudopodialike cytoplasmic protrusions which are in direct contact
with the adjacent interstitial tissue’ (Figure 6 of their
study).
Since the recognition of tumour budding in the
surgical pathology of colorectal carcinoma has occurred relatively recently, whereas the heyday of
electron microscopy is in the past, systematic ultrastructural studies of tumour budding in colorectal
carcinomas are lacking. In particular, ultrastructural
studies investigating cytoplasmic pseudopodia in relation to tumour buds in colorectal carcinomas have not
yet been published (see Figure 2 for author’s electron
microscopic image). However, the morphological link
between tumour budding and tumour cell migration at
the invasive margin by means of pseudopod formation
has recently been proposed in an immunohistochemical study by Shinto et al.3 These authors pointed out
that on high magnification in the immediate vicinity of
tumour buds, pan-cytokeratin immunostains revealed
non-nucleated cytoplasmic droplets of 1 lm diameter
Figure 2. Electron microscopic picture of budding tumour cells in a
colorectal carcinoma. Note absence of desmosomes and basement
membranes; ruffled cytoplasmic membrane protruding at the leading
edge (arrowed, higher magnification in the inset). In this tumour bud,
an abortive intracytoplasmic lumen has formed (arrowheads).
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
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F Prall
and less (see Figure 1). By submitting step sections
to pan-cytokeratin immunohistochemistry, they demonstrated that these structures are continuous with
the cytoplasm of budding tumour cells. The term
‘cytoplasmic pseudo-fragments’ was applied, and the
cytoplasmic pseudo-fragments were interpreted as
podia-like cytoplasmic extensions. Conceivably, these
are formed during tumour cell migration.
Tumour budding must be clearly discriminated from
colorectal carcinomas with an infiltrative growth
pattern. This aggressive feature was originally described by Jass et al.4 and is observed in about 25% of
colorectal carcinomas; most of the remaining larger
fraction comprises colorectal carcinomas with an
expansive growth pattern. The classification of a
colorectal carcinoma as expansive versus infiltrative
is a feature observed at low magnification. At this level,
colorectal carcinomas growing with an infiltrative
pattern dissect the tunica muscularis with longstretched but fully formed neoplastic glands spaced
fairly evenly from each other, usually with little
intervening desmoplastic stroma; this has been named
‘streaming dissection’. Having reached the extramural
fat, as is the case for most colorectal carcinomas with
an infiltrative growth pattern, the adipose tissue is
dissected by microglandular structures with, again, a
desmoplastic stroma being characteristically largely
absent at the interface. Histopathologists must be
aware that, not infrequently, the infiltrative pattern is
found only in part of a tumour. After scanning and
coming to high magnification, tumour budding and
cytoplasmic pseudo-fragmentation will be observed as a
superimposed pattern. Thus, independent of growth
pattern, tumour budding can be found in both types
of colorectal carcinoma, in those growing with an
expansive as well as in those growing with an
infiltrative growth pattern.
Provided that there is sufficient attention to detail
(and pan-cytokeratin immunostains are available),
grading, typing of the invasive margin and determination of the degree of tumour budding and cytoplasmic
pseudo-fragmentation can be performed with confidence. It is only for the very small fraction of high-grade,
diffusely infiltrating colorectal carcinomas that distinctions between grades, tumour growth pattern and
tumour budding become blurred.
Invasive phenotypes have been discussed in some
detail in this section. The rationale for this is, first, to
enable surgical pathologists previously not acquainted
with these features to appreciate them in their daily
practice, and second, morphological detail sets the
stage for the following discussion, which links tumour
budding to the large body of information gathered by
cellular biologists on the mechanisms of malignant
invasion.
Epithelial–mesenchymal transition: linking
tumour budding in colorectal carcinoma
to cellular biology
By becoming aware of tumour budding in colorectal
carcinomas, surgical pathologists have chanced upon a
field of research that is currently under intensive
investigation by basic scientists. Under the heading of
epithelial–mesenchymal transition, a process with very
similar morphological features is known to occur
during tumour cell invasion, and during gastrulation
in embryonic development (reviewed in 5,6).
When undergoing an epithelial–mesenchymal transition, in the early phase, epithelial cells reduce intercellular contacts and cell–matrix contacts and
reorganize the cytoskeleton to form cell membrane
ruffles (lamellipodia) or cytoplasmic protrusions (filopodia). Later, migration follows when new cell–matrix
contacts are formed at the leading edge; these provide
anchorage for the contraction of the cell body which
draws behind the backward pole. Furthermore, pericellular matrix degradation is initiated. For colorectal
carcinoma, in vitro, the most important molecular
components are:7,8 E-cadherin (for intercellular contacts, i.e. adherens junctions), integrins (for cell–matrix
contacts, i.e. desmosomes), actin (forming podia and
joining the backward part of the cytoskeleton to the
cell membrane at the leading edge) and myosin (for
contraction of the backward part of the cell body).
Membrane-type metalloproteinases and the urokinase
plasminogen activator (uPA) ⁄ uPA receptor system are
important in pericellular proteolysis.9
Adapting the terminology of the epithelial–mesenchymal transition to a colorectal carcinoma with a
budding invasion phenotype, the scenario is: adhesion
junctions mediated by E-cadherin are broken up and
tumour cell complexes dissociate, reorganization of
the actin cytoskeleton allows budding tumour cells to
extend podia into the migration direction, integrins
(a3b1 integrin in particular) localize to the podia
forming new attachments to the extracellular matrix,
and the uPA receptor localizes to the podia also, as an
important factor to initiate the matrix degradation.
Tumour budding in this scenario is a highly dynamic
process giving temporal heterogeneity to a tumour.
Furthermore, matrix degradation at the leading edge is
followed by synthesis of new extracellular matrix of a
different composition, and this gives spatial heterogeneity. It is important to note that the budding phenotype
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
Tumour budding in colorectal carcinoma
of colorectal carcinoma invasion (and, putatively, the
same cellular mechanisms) is recapitulated when
metastases are formed at distant sites.7 It follows
that the microenvironment plays an important role
in inducing an epithelial–mesenchymal transition in
colorectal carcinomas, and in this, cell–cell as well as
matrix–cell signal transduction is relevant.
Signal transduction in colorectal carcinoma
and tumour budding
There are three major signal transduction pathways
known to be important for colorectal carcinomas (see
Figure 3).
155
3-kinase (PI3-K) to cytosolic Rho family GTPases
(e.g. Rac, Rho and Cdc42).10 These are key regulators
of actin assembly and they control the formation of
filopodia and lamellipodia, thus defining polarity and
the leading edge of migrating cancer cells. Furthermore, PI3-K activates the cytoplasmic serine-threonine
kinase Akt (also known as protein kinase B) and this
interferes with the caspase pathway to apoptosis.11
Tyrosine-kinase receptors EGF, FGF, PDGF and HGF
are expressed by many colorectal carcinomas and a
basic supply of these growth factors is secreted by
tumour cells and stromal cells.12–14 This, however, is
topped by activating codon 12 or 13 point-mutations
of the K-ras gene that are found in about 35% of
colorectal carcinomas,15 leading to a strong, intrinsic
activation of this pathway.
tyrosine-kin ase r ec ep tor pa thwa y s
(ras pathways)
In this signalling cascade, membranous receptor tyrosine-kinases (RTK) bind ligands, preferentially growth
factors [e.g. epidermal growth factor (EGF), hepatocyte
growth factor (HGF), platelet-derived growth factor
(PDGF) and fibroblast growth factor (FGF)]. Activated
receptors dimerize and autocatalytically phosphorylate
tyrosine residues residing in the cytoplasmic domains.
The signal is relayed by Ras proteins located at the
inner cytoplasmic membrane to Raf proteins, and a
cascade of phosphorylations of cytoplasmic kinases is
initiated which, finally, leads to phosphorylation of the
extracellular mitogen-activated kinases (ERKs). Phosphorylated ERKs translocate to the nucleus and activate transcription factors (e.g. c-Jun, c-Myc and c-Fos)
that act on cell proliferation control.
Besides this signalling cascade to the nucleus, Ras
proteins can relay the signal through phosphoinositide
Extracellular:
FGF, EGF, PDGF, HGF
TGF-βRII
Ras
Cytoplasmic:
SMAD 2/3
SMAD4
Raf
AKT
frz
β-CAT
PI3-K
Rac
GSK-3/APC/Axin
Rho
β-CAT
Cdc42
BAD
WNTs
E-CAD
RTK
actin
repression
TGF-β, BMP, activin
ERK1/2
Nuclear:
SMAD 2/3
ERK1/2
Slug, snail
β-CAT
SMAD4
Cellular effects: Proliferation and apotosis dysregulation,
matrix degradation/synthesis, podia formation, migratory reaction.
Figure 3. Schematic overview of three main signal-transduction
pathways that by dysregulation ⁄ activation are implicated in tumour
budding. Abbreviations in the text.
w n t -signa lling p ath way
In fully formed neoplastic glands (as well as in normal colonic crypt epithelium) b-catenin together with
E-cadherin resides at the cell membrane to form
junctional complexes, but b-catenin released to the
cytoplasm is taken up by the so-called destruction
complex that consists of the adenomatous polyposis coli
(APC) protein and glycogen synthase kinase-3 (GSK-3).
By this destruction complex, b-catenin is ubiquitinated
and thus directed to degradation. In Wnt-signalling
(e.g. in embryonic gastrulation), the binding of the
evolutionarily conserved Wnt-growth factors (Wnts) to
the frizzled receptor induces a release of b-catenin from
the destruction complex. Cytosolic b-catenin then
translocates to the nucleus to activate transcription
factors of the T-cell factor ⁄ lymphocyte enhancing
factor (TCF ⁄ LEF-1) family. Among others, there are
induced slug and snail (transcription factor repressing the E-cadherin gene),16,17 survivin (inhibitor of
apoptosis),18 cyclin D1 and c-myc (both promoting
proliferation),19,20 laminin c2 (extracellular matrix
component positively influencing cell migration)21
and matrix metalloproteinase 7 (inducing and sustaining matrix breakdown during desmoplasia).22 All these
factors, in vitro, cooperate to induce epithelial–mesenchymal transition.
As APC gene mutation in most cases causes truncation of the APC protein, and APC gene mutations are
frequent in colorectal carcinoma (up to 70%, depending on the composition of the series),23,24 Wnt disruption may be expected to be common. Besides gene
mutation, APC can also be compromised by loss of
heterozygosity (LOH) and ⁄ or promoter methylation.24,25 As putative direct evidence of Wnt dysregulation, b-catenin immunohistochemistry in colorectal
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
156
F Prall
carcinomas reveals a striking pattern:26 towards the
centre of a colorectal carcinoma the fully formed
neoplastic glands show membranous immunostaining,
but in tumour buds this membranous immunostaining
is lost. Instead, immunostaining of the tumour cell
nuclei is very strong. In a systematic study relating
APC gene aberrations to b-catenin translocation to
the nucleus,27 a significant association was indeed
observed. However, there remain a number of cases
showing b-catenin nuclear translocation without any
APC gene aberrations. In another fraction, APC gene
aberrations were observed without b-catenin nuclear
translocation. In vivo, apparently, Wnt dysregulation
is neither necessary nor sufficient, of itself, to cause
tumour budding and several factors have to coincide.
tran sformin g g rowth f ac t or - b et a s ig n a l l in g
an d o th er serine-threonine kin ases
Transforming growth factor-beta (TGF-b) and related
ligands such as bone morphogenic protein (BMP) and
activin bind to cell-surface receptor serine-threonine
kinases, TGFbRII in the case of TGF-b. Ligand binding
stabilizes receptor dimers and activates their serinethreonine kinase activity to phosphorylate SMADs 1, 2
or 3. These form heterodimers with SMAD4 (syn.
DPC4) and this heterodimer translocates to the nucleus
to act as a transcription factor. Various different effects
of TGF-b signalling have been observed in vitro,
including arrest of cell proliferation,28 increased synthesis of extracellular matrix (fibrosis in inflammation
and desmoplasia in tumour invasion)29 and changes
of epithelial cells paralleling epithelial–mesenchymal
transition.30 Besides signal transduction by SMAD4,
apparently there exists an alternative TGF-b signalling
pathway which by gene chip expression analysis has
been shown to activate genes preferentially involved in
the epithelial–mesenchymal transition.31
TGF-b and the TGF-b receptor are expressed in the
majority of colorectal carcinomas32 and it is particulary frequent in colorectal carcinomas with prominent
tumour budding.33 Putatively, the growth arrest
observed in tumour buds34 in most colorectal carcinomas would depend on TGF-b signalling. Apparently,
activation rather than disruption of TGF-b signalling
operates in most colorectal carcinomas. Nevertheless,
there is a smaller fraction known to be compromised in
TGF-b signalling, either by mutations in the TGFbRII
gene (particularly in highly microsatellite-instable
tumours, see below)35 or, infrequently, by mutations
of the TGFb gene itself32 or the SMAD4 gene.36 SMAD4
is also a target of LOH 18q21 in about 30% of
colorectal carcinomas, but this does not necessarily
abrogate TGF-b expression completely and, accordingly, LOH 18q21 does not correlate with the degree of
tumour budding.27 Mutations of other SMADs are
uncommon in colorectal carcinomas.36
Taking all data together, the disruption or activation
of the signal transduction pathways discussed above
are very important for understanding the cellular
biology of tumour budding in colorectal carcinoma.
Using colorectal carcinoma cell cultures, experimental
manipulation of either one of these has been shown
to produce full epithelial–mesenchymal transition, or
at least cellular changes that precede this transition
(e.g. breakdown of adherens junctions or formation of
podia).8,37 Interactions between these signalling pathways have been observed and evidence points to
cooperation between these rather than dysregulation ⁄ activation of a single pathway as a key factor in
tumour budding37 (see Figure 3). Within the budding
part of a colorectal carcinoma, tumour cells and their
microenvironment are probably finely tuned to fit this
process precisely.
In various experimental systems, these pathways
converge by each being capable of inducing the zincfinger transcription factors snail and ⁄ or slug.38,39 Snail
and slug, in their turn, in colorectal carcinoma cell
cultures have been shown to repress E-cadherin16,17
and the loss of E-cadherin by colorectal carcinoma cells
occurs as an early event in epithelial–mesenchymal
transition.8,37 In keeping with this, in clinical specimens of colorectal carcinoma, loss or reduction of
E-cadherin is a regular finding in budding carcinoma
cells.7,27 The transient repression of E-cadherin seems
to be a key event when tumour budding is initiated,
and E-cadherin is re-expressed upon re-differentiation.
Thus, induction of snail and slug and their effect
on E-cadherin link the outside–in signals set by the
microenvironment to the invasion process of tumour
budding.
In clinical specimens complexity is increased further
by the fact that some colorectal carcinomas invade
deeply, but dissociation of tumour cells is minimal or
even absent. This also has been recapitulated by in vitro
studies with colorectal carcinoma cells, and the concept
of cohort migration has been introduced.40 In these
studies, large aggregates of colorectal carcinoma cells
(much larger than tumour buds) at their circumference
induced matrix degradation and moved as large,
coherent clusters. The concept of cohort migration,
therefore, signifies that colon carcinomas during invasion are in a state of active locomotion. They initiate
and sustain remodelling of the adjacent extracellular
matrix41 but, in contrast to tumour budding, they
retain cell–cell contacts to remain in large aggregates.
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
Tumour budding in colorectal carcinoma
Tumour budding related to types of
colorectal carcinoma
Since we have entered an ‘era of more than one type of
colorectal cancer’,42 an attempt is warranted to put
into context clinicopathological tumour types with
tumour budding.
s p o r a d i c co l o r e c t a l ca rc i n o ma ,
mi c r o sa t e ll i t e - st a b l e/ l o w- de g r ee
microsatellite-instable
These make up the majority of colorectal carcinomas.
Tumour budding and cytoplasmic pseudo-fragmentation are frequent among these carcinomas and the
highest values are observed in this group of tumours.27
Furthermore, nuclear translocation of b-catenin is
commonly observed in this type of colorectal carcinoma, indicating Wnt-dysregulation. However, there
remains a 30% fraction of sporadic microsatellitestable (MSS) ⁄ low-degree microsatellite-instable (MSI-L)
colorectal carcinomas that do not show any nuclear b-catenin translocation, some even with a high
degree of tumour budding and cytoplasmic pseudofragmentation.27
s p o r a d i c co l o r e c t a l ca rc i n o ma , hi g h - de g r e e
microsatellite-instable
These carcinomas make up about 10% of all colorectal
carcinomas (as most are right-sided, approximately
20% of colonic carcinomas). Tumour budding is
virtually absent from these carcinomas.27,43 Of interest,
as the TGFbRII gene contains a polyA repeat within the
coding region, frequencies of TGFbRII gene mutations
approach 90% in high-degree microsatellite-instable
(MSI-H) tumours.35 Therefore, disruption of TGF-b
signalling might be an explanation for the low degree
of tumour budding. Furthermore, pro-budding Wntaberrations and ras gene mutations are infrequent in
these tumours.35
colorectal carcinoma i n the hereditary
non-p olyposis c arcinoma syn drome
In this relatively rare type of colorectal carcinoma,
germ-line mutations of one of the DNA mismatchrepair genes usually cause MSI-H. Significant tumour
budding does occur in many of these carcinomas.43 As
an explanation, even though somatic APC gene mutations are infrequent (only around 25%), in the absence
of APC gene mutations, b-catenin gene mutations
157
have been reported in many cases (44%).44 Thus, the
majority of hereditary non-polyposis carcinoma syndrome (HNPCC) carcinomas harbour genetic Wnt
aberrations, and this could (partly) explain why, in
contrast to the sporadic MSI-H tumours, tumour
budding occurs fairly frequently.
c o l or e c t a l ca rc in o ma in f a m i l ia l
adenomatou s p ol yposis
Systematic studies on tumour budding in colorectal
carcinomas arising in familial adenomatous polyposis
are lacking thus far. Since APC mutation is a rule in
these carcinomas, and APC LOH is frequent, tumour
budding may be expected to be frequent among these
colorectal cancers.
colorectal carc inoma in u l c e r a t i v e c o l i t i s
Systematic studies on tumour budding in colorectal
carcinoma arising in long-standing ulcerative colitis
are lacking. These tumours are traditionally separated
from the rest on clinical grounds. Though APC gene
mutations and K-ras gene mutations seem to be less
frequent, the molecular pathology as unravelled so far
does not allow a clear distinction of these from sporadic
colorectal carcinoma.45,46 If indeed similar pathways
are operative, tumour budding in ulcerative colitisrelated colorectal carcinomas may be expected to be
similar to sporadic colorectal carcinomas. Accordingly,
evidence of Wnt dysregulation has been reported for
ulcerative colitis-related colorectal carcinoma.47
Taking all data together, tumour budding is intimately
bound up with the molecular pathology and cellular
biology of colorectal carcinoma and there is still much to
be learned. Importantly, tumour budding is not only a
stimulating theoretical issue. The immediate relevance
of tumour budding to surgical pathology stems from the
fact that it is increasingly being recognized as a strong
and robust adverse prognostic factor.
Tumour budding as a prognostic factor in the
surgical pathology of colorectal carcinoma
Diagnosis and treatment of colorectal carcinoma have
evolved considerably during recent years and this has
had an immediate impact on the surgical pathology of
biopsies and resection specimens. Specifically, decisions
for or against limited or full resections, and for or
against adjuvant therapy have to be made, and in both
instances these decisions are made largely on the basis
of the histopathological findings.
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F Prall
With a frequency increasing with the size of the
polyp,48 colorectal carcinomas with limited invasion
are found in colorectal adenomas removed by polypectomy or mucosectomy. For carcinomas of the rectum,
the depth of invasion can be assessed preoperatively
with some precision by endoscopy and imaging techniques such as transrectal endosonography or magnetic resonance imaging. In carcinomas with limited
invasion (pT1), a decision for a local resection is often
made. In the case of both polypectomy and local
excision of early rectal cancer, the possibilty arises
of unrecognized regional lymph node metastases left
in situ.
Alternatively, if a surgical resection of a colorectal
carcinoma with formal lymphadenectomy has been
performed, there arises the question of adjuvant
chemotherapy (for colonic carcinoma) or chemoradiation (for carcinoma of the rectum).49 Currently, the
decision for or against adjuvant treatment is made
by clinicopathological staging [International Union
Against Cancer-Tumour Node Metastasis (TNM), or
Dukes’ staging]. For patients with node-positive
colonic carcinoma, adjuvant 5-fluorouracil-based regimens are standard care now, but additional (and
more toxic and costly) regimens have been proposed50 and further therapeutic advances may be
expected. Furthermore, a small proportion of patients
with node-negative colorectal carcinoma follow an
adverse clinical course, and for these patients adjuvant treatment could be beneficial. Therefore, in this
era of treatment diversification for colorectal carcinoma, patient stratification for risk of progression beyond TNM has become important. Tumour
budding is now showing increasing promise in
clinicopathological studies as a prognostic factor
in colorectal cancer that is independent of TNM
staging.51–54
t u m o ur b u d di n g as pr o gn o s t i c f a c t or :
t h e su r v i va l a n a l y s e s
A detailed summary of currently published clinicopathological studies addressing the prognostic impact
of tumour budding is given in Table 1. Overall, data on
more than 2100 patients are published. All patients are
reported to have undergone a potentially curative
(cM0 ⁄ pM0, R0) resection of a single, non-metachronous colorectal carcinoma including lymphadenectomy, and all patients were selected according to
stringent inclusion criteria to exclude bias. Depending
on the studies, end-points were metachronous distant
metastases, tumour-specific survival, or both. Although
the studies are retrospective, the careful selection of
patients as well as long and complete follow-up gives
them authority.
By univariate analyses, in each of these studies a
strong adverse prognostic impact of tumour budding
was observed. Relative risks can be calculated from the
published data, and for patients classified as high in
tumour budding (BUDhigh) the relative risk of succumbing to their disease or developing metachronous
metastases is two- to threefold. Furthermore, multivariate regressions were performed in some of these
studies and tumour budding was found to add prognostic information to the TNM criteria.
To be a prognostic factor in surgical pathology,
reproducibility is a major issue and this has been
addressed in two of the studies.51,53 A very important
advantage of tumour budding is the potential for
objective assessment by counting. Setting a threshold of
10 tumour buds per field of vision (0.375 mm2), Ueno
et al. report a j-value of 0.84 for their intra-observer
study and a j-value of 0.874 was obtained in our
own series.51,53 Furthermore, using a pan-cytokeratin
immunostain and a preset cut-off (25 tumour buds ⁄
0.785 mm2, 20· objective), the classification of a given
case is very rapid.
In a recent study, high-degree podia formation,
assessed as cytoplasmic pseudo-fragmentation on pancytokeratin immunostains, was observed to have an
adverse prognostic impact independent of tumour
budding.55 Although of uncertain practical significance
for surgical pathology at present, this finding has an
interesting tumour biological implication. Specifically,
it could signify that during tumour cell migration with
prominent podia formation, amoeboid movement56
dominates over cellular discohesion.
t u m o u r b u d d i n g t o p r e di c t s y n c h r on o u s
l y mp h no d e m et a s t a se s
Involvement of the resection margins by colorectal
carcinoma after polypectomy or endoscopic mucosectomy provides a clear case for a completion operation. However, independent of involvement of
resection margins, approximately 10% of patients
with early invasive colorectal carcinoma are observed
to have synchronous metastases to regional lymph
nodes. Therefore, after polypectomy or endoscopic
mucosectomy, patients with early invasive colorectal
carcinoma have to be stratified to either a low-risk
group (with a risk of synchronous lymph node
metastases approaching 0%) or a high-risk group of
patients for whom additional surgery with lymphadenectomy is mandatory. It has been shown by a
number of clinicopathological studies that to minimize
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
Counting H&E
Colorectal ND–
43.4
30.1
25.6
Scoring
40.1
49.8 ⁄ 32.7
27.5 ⁄ 4.3
50.6 ⁄ 8.1
ND
71.1 ⁄ 20.0
Cox
regression***
ND
ND
58.0 ⁄ 95.0––
40.7 ⁄ 84.0
ND
Venous invasion (V)
ND
pN, pT
II: 29.1 ⁄ 68.3
ND
III: 19.0 ⁄ 66.2§§
BUDhigh Metastatic†† Survival‡‡
(%)
(%)
(%)
Counting IHC** 33.3
5-FU, N ¼ 41‡ Scoring
None
Colorectal 5-FU, N ¼ 18
RCT, N ¼ 35§
Colon
Rectal
Scoring
Method of
assessment
*Patients with complete follow-up.
†Patients with complete follow-up.
‡5-fluororacil-based chemotherapy for patients in UICC stage III.
§Postoperative (adjuvant) radiochemotherapy for patients with carcinoma of the rectum; 5-FU-based chemotherapy for patients with colon carcinoma.
–No data given.
**Counting on pan-cytokeratin immunostains.
††Percentage of patients with metachronous metastases during follow-up: BUDhigh ⁄ BUDlow.
‡‡Percentage of patients surviving follow-up: BUDhigh ⁄ BUDlow.
§§Patients in UICC stages II or III, respectively.
––Actuarial survival extrapolated from Kaplan–Meier plots.
***Additional independent factors (Cox regression analysis).
1986–2000 I–III
182† 1994–1999 I ⁄ II
Nakamura et al.54 491
Prall et al.53
1960–1969 I–III
Adjuvant
therapy
Colorectal None
Stages
(UICC) Location
1970–1985 I–III
Years of
operation
179* 1985–1997 II ⁄ III
638
Ueno et al.51
Okuyama et al.
663
Hase et al.1
52
No.
Author
Table 1. Clinicopathological studies addressing tumour budding as prognostic factor
Tumour budding in colorectal carcinoma
2007 The Author. Journal compilation 2007 Blackwell Publishing Ltd, Histopathology, 50, 151–162.
159
160
F Prall
Table 2. Clinicopathological studies addressing tumour budding as a risk factor for synchronous regional lymph node
metastases in early invasive colorectal carcinoma (pT1)
Author
No.
Years of
operation
Node positive Method of
of all (%)
assessment
Hase et al.58
79 1970–1985 13.9
Ueno et al.59
251 1980–2005 13.1
Wang et al.60
159 1969–2002 10.1
Kazama et al.61
56 1990–2001 14.2*
BUDhigh Node
(%)
positive (%)‡
Other risk factors by
multivariate regression
25.0 ⁄ 0
Depth of infiltration (SM),
grading, lymphatic invasion
Counting H&E 15.1
42.1 ⁄ 7.9
Depth of infiltration
(> 500 lm), grading,
lymphatic invasion
Scoring
15.1
45.8 ⁄ 3.7
Depth of infiltration (SM),
grading, lymphatic invasion
Scoring IHC†
75.0
38 ⁄ 0
Scoring
55.7
Lymphatic invasion
*Eight of 56 cases. Using pan-cytokeratin immunostains, an additional eight cases were observed to have micrometastases or
disseminated tumour cells in regional lymph nodes.
†Pan-cytokeratin immunostains used for scoring.
‡Percentage of cases with synchronous regional lymph node metastases: BUDhigh ⁄ BUDlow.
the risk of unrecognized nodal metastases for a
patient, a completion operation should be undertaken
if the carcinoma invades deeply into the submucosa
(depth measured in lm, or SM classification), if the
carcinoma is poorly differentiated (G3), or lymphatic
permeation is observed (L1).57
Tumour budding is an additional factor that can be
entered into this risk assessment and clinicopathological studies addressing this issue are summarized
in Table 2. Overall, there are published data on more
than 500 patients with early invasive colorectal
carcinoma for whom tumour budding was related
to nodal status as assessed by histopathological
examination of surgical resection specimens, either
from lymphadenectomies concurrent with tumour
resection or from completion resections. In all these
studies, the groups of patients with high-degree
tumour budding were observed to have rates of
lymph node involvement around 30%, whereas the
rate was much lower in groups with little tumour
budding. Importantly, in these studies depth of
invasion, tumour grade and lymphatic permeation
were also assessed, and by multivariate analysis
tumour budding was observed to be an independent
factor. These results are borne out by the unpublished
series of more than 900 early invasive colorectal
carcinomas that has been investigated by M. Stolte’s
group (M. Vieth, Bayreuth, Germany, personal communication; manuscript in preparation). Therefore, by
integrating tumour budding, a group of patients can
be delineated for whom the risk of synchronous
regional lymph node metastases can be confidently
stated to approach zero.
Conclusion
In this review, tumour budding has been strongly
advocated as an important topic of research and also as
a promising prognostic factor beyond TNM. However, it
must be borne in mind that much more research is
needed. Presently, we are only beginning to understand
the cellular biology of tumour budding. Considering the
diversity of the molecular pathology at the base of
colorectal carcinoma, the cellular biology must be
extremely complex. Furthermore, several issues have to
be resolved if tumour budding is to be incorporated into
the routine practice of surgical pathology. Tables 1
and 2 indicate that the methods to assess tumour
budding vary between investigators, and cut-offs are
not defined uniformly since rates of high versus low
budding differ considerably between studies. It is hoped
that a broader interest in tumour budding will stimulate continued efforts in this respect.
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