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Tumor budding in colorectal carcinoma time to take notice .pdf



Nombre del archivo original: Tumor budding in colorectal carcinoma time to take notice.pdf
Título: Tumor budding in colorectal carcinoma: time to take notice
Autor: Bojana Mitrovic

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Modern Pathology (2012) 25, 1315–1325
& 2012 USCAP, Inc. All rights reserved 0893-3952/12 $32.00

Tumor budding in colorectal carcinoma:
time to take notice
Bojana Mitrovic, David F Schaeffer, Robert H Riddell and Richard Kirsch
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada

Tumor ‘budding’, loosely defined by the presence of individual cells and small clusters of tumor cells at the
invasive front of carcinomas, has received much recent attention, particularly in the setting of colorectal
carcinoma. It has been postulated to represent an epithelial–mesenchymal transition. Tumor budding is a wellestablished independent adverse prognostic factor in colorectal carcinoma that may allow for stratification of
patients into risk categories more meaningful than those defined by TNM staging, and also potentially guide
treatment decisions, especially in T1 and T3 N0 (Stage II, Dukes’ B) colorectal carcinoma. Unfortunately, its
universal acceptance as a reportable factor has been held back by a lack of definitional uniformity with respect
to both qualitative and quantitative aspects of tumor budding. The purpose of this review is fourfold: (1) to
describe the morphology of tumor budding and its relationship to other potentially important features of the
invasive front; (2) to summarize current knowledge regarding the prognostic significance and potential clinical
implications of this histomorphological feature; (3) to highlight the challenges posed by a lack of data to allow
standardization with respect to the qualitative and quantitative criteria used to define budding; and (4) to
present a practical approach to the assessment of tumor budding in everyday practice.
Modern Pathology (2012) 25, 1315–1325; doi:10.1038/modpathol.2012.94; published online 13 July 2012
Keywords: colorectal carcinoma; epithelial–mesenchymal transition; prognostic factors; tumor budding

Colorectal carcinoma is one of the commonest
human cancers, and one of the leading causes
of cancer-related death.1 Prognosis and treatment
decisions are based primarily on the extent of the
disease, as codified by the TNM staging system.
Unfortunately, a substantial number of tumors
behave poorly despite being categorized as low risk
based on their TNM stage.2 Thus, the search for
additional prognostic factors in the assessment of
colorectal carcinoma has been a major research
focus. Of the histopathological factors studied to
date, the most promising include extramural venous
invasion, the nature of the advancing front (pushing
vs infiltrative), an inflammatory infiltrate, microsatellite instability, and tumor budding—the presence of small discrete clusters of tumor cells at the
invasive edge (Figure 1). There is now overwhelming evidence that tumor budding is an independent
prognostic factor in colorectal carcinoma, particuCorrespondence: Dr B Mitrovic, MD, Department of Pathology
and Laboratory Medicine, Mount Sinai Hospital, 600 University
Avenue, Toronto, Ontario, Canada M5G 1X5.
E-mail: BMitrovic@mtsinai.on.ca
Received 15 December 2011; revised 30 March 2012; accepted 1
April 2012; published online 13 July 2012
www.modernpathology.org

larly in node-negative disease. Thus, its assessment
has the potential to enhance prognostic accuracy
and influence treatment algorithms. When examined carefully, the majority of colorectal carcinomas
display some degree of budding; hence, attempts
have been made at developing scoring systems
to identify a prognostically significant degree of
budding, commonly termed ‘high-grade’ budding.
Definitions of high-grade budding vary substantially
among different authors and even among different
studies by the same authors.
Although tumor budding only entered the mainstream pathology literature in the past decade, it
was first described in the 1950s by Imai,3 who
postulated that the presence of ‘sprouting’ at the
invasive edge of carcinomas reflected a more rapid
tumor growth rate (or ‘Schub’ phase of growth).
Budding has been described in association with
carcinomas at a number of different sites, but it
has been most extensively studied in colorectal
carcinoma.
The biology of tumor budding is not understood
but the prevailing theory is that at least some types
of budding represent an example of epithelial–
mesenchymal transition, a process thought to occur
physiologically during embryological development,

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Figure 1 Tumor budding in colorectal carcinoma (a) Illustration of a colorectal carcinoma with an infiltrative border but no budding.
Tumor buds, defined as individual cells or small clusters (o5 cells) of tumor cells at the invasive front (arrows), are illustrated in these
examples from (b) a malignant polyp and (c and d) a colorectal cancer resection specimen. Note the blurring of the tumor–stroma
interface (c), which corresponds with tumor budding at higher magnification (d). (Original magnification: a— 100; b— 200; c— 20;
d— 400).

and pathologically during fibrosis and tumor invasion.4,5 The epithelial–mesenchymal transition is
characterized by loss of cell adhesion molecules,
cytoskeletal alterations, increased production of
extracellular matrix components, resistance to apoptosis, and ability to degrade basement membrane,
resulting in a phenotype with increased migratory
capacity and invasiveness.6,7 Current theories
relating to the epithelial–mesenchymal transition
and tumor budding have been comprehensively
reviewed elsewhere, most recently by Zlobec et al,8
and will not be discussed here.
The purpose of this review is to examine
practical issues related to tumor budding that
may be of interest to the practicing pathologist.
We will discuss the morphological features
and prognostic significance of tumor budding
and summarize the various scoring systems for its
assessment in order to highlight problems related
to definitional heterogeneity in tumor-budding
studies.
Modern Pathology (2012) 25, 1315–1325

Finally, we will present a practical approach
to the assessment of tumor budding. Although
performing a tumor bud count is easy in theory,
deciding what is or is not a bud can prove
surprisingly difficult in practice. Buds are often
obscured in the setting of a prominent inflammatory
reaction; fibroblasts and stromal cells may occasionally be mistaken for buds; and single-cell buds are
usually inconspicuous and often missed on H&E
evaluation (Figure 2).

Morphological features
Although tumor budding (‘sprouting’) was originally described by Imai in the 1950s, the first detailed
description of tumor buds in the English language
literature was put forth by Gabbert et al9 who used
light and electron microscopy to characterize what
they termed ‘tumor dedifferentiation’ at the invasive
edge of colorectal carcinomas. They identified a

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B Mitrovic et al

Figure 2 ‘Challenging scenarios’. (a) Peritumoral inflammatory cells, including histiocytes, can be difficult to differentiate from tumor
buds, and may sometimes obscure the underlying budding. (b) Immunohistochemistry for anti-cytokeratin may help to highlight tumor
buds in this setting. (c) Given that tumor budding may represent an epithelial–mesenchymal transformation, their separation from
stromal cells can also be challenging and (d) anti-cytokeratin stains may be very useful in identifying tumor buds. (e) Fragmentation of
neoplastic glandular structures (arrow) can sometimes give the impression of extensive tumor budding on H&E and on
immunohistochemistry with (f) anti-cytokeratin. In this example, the small fragments/individual tumor cells highlighted by anticytokeratin in the region of the fragmented gland (arrow between fragments) may be a consequence of fragmentation rather than true
budding. (Original magnification: a— 40; b— 40; c— 100; d— 100; e— 200; f— 200).

subset of colorectal carcinomas with invasive
fronts that displayed a strikingly different architecture compared with the central area of the tumor,

with well-developed glandular structures giving
way to isolated tumor cells and small cell groups,
as illustrated in Figure 1. At the ultrastructural level,
Modern Pathology (2012) 25, 1315–1325

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B Mitrovic et al

these small clusters and isolated tumor cells
appeared poorly differentiated, with large nuclei
and a loss of microvilli and basement membrane,
and poorly developed or absent junctional complexes and desmosomes.
When seen in a single section, tumor buds appear
as clusters of cells that have broken off from the
main tumor mass. By examining serial sections of
high budding tumors stained with anti-cytokeratin
antibodies, Prall et al10 demonstrated that most buds
that appear to represent discrete clusters of cells are
in fact connected to adjacent larger glands. Indeed,
Morodomi et al11 coined the term ‘budding’ because
the undifferentiated single cells and tubular tumor
nests that they counted as buds both appeared to be
budding from larger neoplastic glands.
Shinto et al12,13 used immunohistochemistry with
anti-cytokeratin antibodies to highlight another
feature of buds, termed cytoplasmic pseudofragmentation, whereby cytoplasmic fragments, visible only
by immunohistochemistry, are seen in the immediate vicinity of tumor buds. When examined on serial
sections, some of the fragments were shown to be
connected to the buds (hence the term ‘pseudofragments’). The presence of pseudofragments is associated with high-grade budding, but was shown to
be an independent prognostic factor on multivariate
analysis, suggesting that its presence signifies an
aggressive budding phenotype.
Although tumor buds are usually most prominent
at the invasive front, intratumoral budding has been
described as well. Only one study has attempted to
assess the significance of this finding: Lugli et al14
reported that the presence of intratumoral budding
was strongly correlated with peritumoral budding,
but was found to be an independent prognostic
factor on multivariate analysis.
There is a strong association between budding and
the presence of lymph node metastases and lymphovascular invasion,11,12,15–32 defined by the presence of tumor cells within an endothelium-lined
space, and it has been suggested that buds represent
the part of the tumor that has gained the ability to
invade lymphatics and vascular channels. This idea
is supported by two intriguing morphological
studies: Morodomi et al33 examined serial sections
of high-budding areas to demonstrate that budding
nests are often found adjacent to areas of lymphovascular space invasion, and, in a more recent
study, Ohtsuki et al31 performed double staining
for anti-cytokeratin antibodies and anti-lymphatic
antibodies, finding that a number of ‘buds’ at the
invasive edge of a tumor are in fact located in small
lymphatic spaces. Similarly, the presence of budding has been associated with increased risk of
distant metastases,34–36 suggesting that budding may
also be associated with vascular invasion. A few
tumor-budding studies have used vascular markers and/or elastic stains to assess vascular invasion,11,12,19,22,26,32,37,38 but only four have analyzed
the relationship between budding and vascular
Modern Pathology (2012) 25, 1315–1325

invasion: Kazama et al22 found no relationship
between budding and vascular invasion, whereas
three other studies have reported a statistically
significant correlation between budding and venous
invasion, though the association was not as pronounced as the relationship with lymphatic invasion and lymph node metastases.11,12,32
The tumor–host interaction at the invasive front
may be of prognostic importance in the setting of
tumor budding. Several studies have shown peritumoral lymphocytic infiltration to be an independent prognostic factor in colorectal carcinoma.
Lugli et al23 have demonstrated that a peritumoral
lymphocytic reaction is associated with improved
prognosis in the setting of tumor budding, suggesting that the immune response might target the tumor
buds. The nature of the stroma at the invasive
margin may provide further prognostic information,
with myxoid stroma being associated with worse
prognosis,20,39 but further studies are needed to
confirm the reproducibility and prognostic importance of these findings.

Clinical significance of tumor budding
Tumor budding is associated with other histopathological factors known to portend a worse prognosis,
namely higher tumor grade, infiltrating tumor border,
the presence of lymphovascular and perineural
invasion, and lymph node metastases.11,12,15–32 On
multivariate analysis, budding has consistently
emerged as an independent adverse prognostic factor,
associated with local tumor recurrence and distant
metastases, and significantly worse overall and
disease-free survival.12,15,16,21,27,28,31,32,34–36,38,40,41
The adverse prognostic impact of high-grade
tumor budding is seen in both early and advanced
colorectal carcinoma, and there are several scenarios
in which this feature might influence clinical
decision making, particularly in early colorectal
carcinoma.
Tumor Budding in Early Colorectal Carcinoma

Submucosally invasive colorectal carcinoma is
associated with excellent outcomes and low rates
of lymph node metastases, and a subset of patients
can be successfully managed with endoscopic
mucosal resection or polypectomy,42 thus avoiding
the risks associated with segmental resection.
Identification of candidates for endoscopic resection
rests mainly on the absence of certain high-risk
histopathological features, namely high tumor
grade, lymphovascular invasion, and tumor budding.26,42 Tumor extending to the cauterized margin
can often be managed by endoscopic follow up.
Several large studies have shown tumor budding
in submucosally invasive colorectal carcinoma to
be an independent prognostic factor associated
with lymph node metastases, local recurrence, and

Tumor budding

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cancer-related death.16,19,22,25,26,29,30,37,43,44 In the
largest of these studies, Ueno et al26 demonstrated
that, in submucosally invasive colorectal carcinoma,
high tumor grade, lymphovascular invasion, and
tumor budding are the three factors independently
associated with lymph node metastases. Patients
without any of these three features showed exceptionally low rates of lymph node metastases (1%,
1/138); in the presence of one risk factor, the rate of
nodal metastases increased substantially to 21%
(12/58), and when two or three factors were present,
the risk was 36% (20/55), suggesting that in the
absence of these factors, polypectomy alone is
sufficient treatment for early colorectal carcinoma
provided the resection margins are clear. In this
study, Ueno et al further showed that in the absence
of extensive submucosal invasion (defined as
r4 mm wide and r2 mm deep), the risk of nodal
metastases (including isolated tumor cells, identifiable only with anti-cytokeratin antibodies) in
tumors lacking the three above-mentioned features
was 0; thus, extent of submucosal invasion may
need to be included in the histopathological assessment of T1 tumors if strict criteria are to be applied
for the identification of patients that can safely be
managed without surgical resection.

Tumor Budding in Stage II (T3–4 N0) Colorectal
Carcinoma

As patients with Stage II colorectal carcinoma
have highly variable outcomes, tumor budding
may be particularly useful in identifying high-risk
subgroups within this population. In one of the
earlier studies of tumor budding, Hase et al18
demonstrated that 5-year survival rates of Dukes B
(stage II, T3–4 N0) patients with high-grade budding
are significantly worse than those of Dukes C (N þ )
patients without budding (29 percent vs 66 percent;
Po0.001). A number of more recent studies have
confirmed that patients with Stage II colorectal
carcinoma do significantly worse when high-grade
budding is present,34–36,38,41,45 and several studies
have shown that survival rates of patients with Stage
II colorectal carcinoma with high-grade budding are
equivalent to survival rates of patients with Stage III
colorectal carcinoma.34–36 In their studies of Stage II
and III pT3 tumors, Okuyama et al34,35 found that
tumor budding was the only factor on multivariate
analysis to be associated with decreased survival
and was more prognostically significant than lymph
node metastases.
Currently, the indications for chemotherapy in
Stage II colorectal carcinoma are unclear, and adjuvant therapy is considered only in subgroups with
adverse prognostic features, eg, high-histological
grade, pT4 disease, and extramural venous invasion.
The QUASAR trial demonstrated that chemotherapy
does result in increased survival in Stage II patients,
but the improvement was small and may not justify

the costs and side effects of chemotherapy in all
patients.46 The significantly worse outcome experienced by Stage II patients with tumor budding
has prompted some authors to suggest that adjuvant chemotherapy should be considered in these
patients,18,34–36,41 but there are no published data
assessing the effectiveness of chemotherapy in Stage
II colorectal carcinoma with tumor budding.
Tumor Budding in Stage III (T1–4 N þ ) Colorectal
Carcinoma

Data regarding budding in Stage III colorectal
carcinoma are limited, and the two studies reporting
on the prognostic significance of budding in this
subgroup of patients have produced conflicting
results. Choi et al15 studied tumor budding in 103
patients with Stage III rectal carcinoma; budding
was an independent poor prognostic factor: when
added to N stage as a prognostic variable, there was
better prognostic stratification with respect to 5-year
disease-free survival compared with the Americal
Joint Comittee on Cancer nodal staging alone. On the
other hand, in a series of 477 Stage III colorectal
carcinomas reported on by Sy et al,47 although there
was a survival difference between high- and lowbudding groups, on multivariate analysis budding
was not found to be an independent prognostic
factor. Although more studies are needed to define
the prognostic significance of tumor budding in this
group, it is not likely that the reporting of this factor
will impact the clinical management of these
patients.
Tumor Budding in Metastatic Colorectal Carcinoma

The significance of tumor budding in metastatic
colorectal carcinoma has received little attention.
However, one study showed that the presence of
tumor budding predicts poor response to anti-EGFR
therapy in patients with metastatic colorectal carcinoma: Zlobec et al48 studied a series of 43 patients
with metastatic colorectal carcinoma treated with
cetuximab or penitumumab, and found all patients
with a KRAS mutation (n ¼ 7) and/or high-grade
tumor budding (n ¼ 11, including 4 with KRAS
mutation) to be nonresponsive to anti-EGFR therapy.
Although this finding needs to be confirmed in
larger cohorts, it raises the interesting possibility
that tumor budding in addition to K-RAS analysis
may more accurately determine eligibility for antiEGFR therapy.
Tumor Budding in Microsatellite Unstable Tumors

The association between tumor budding and poor
prognosis in sporadic microsatellite stable colorectal
carcinoma is well established, but whether this can
be extrapolated to sporadic and Lynch syndromerelated high frequency microsatellite unstable
Modern Pathology (2012) 25, 1315–1325

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colorectal carcinoma is unclear. Many studies of
tumor budding have excluded Lynch syndromerelated cases, while others have not attempted to
analyze the groups separately. Only a few studies
have compared the frequency of tumor budding in
these different types of colorectal carcinomas: in
these studies, the frequency of tumor budding was
approximately 50% in sporadic microsatellite stable
and low-frequency microsatellite unstable tumors;
lower rates of tumor budding were seen in Lynch
syndrome-associated tumors (B20%); whereas
tumor budding was found to be virtually absent
in sporadic high-frequency microsatellite unstable
colorectal carcinoma.23,49,50 It has been postulated
that the relative infrequency of budding in microsatellite unstable tumors may, at least in part,
explain the relatively better prognosis of these
tumors.49 Interestingly, compared with mismatch
repair-proficient tumors, mismatch repair-deficient
tumors have been shown to display less cytoplasmic
pseudofragmentation in the context of high-grade
budding,51 and to be associated with less intratumoral budding,14 both of which are associated with a
worse prognosis. Given the differing biological
pathways of microsatellite stable, sporadic highfrequency microsatellite unstable and Lynch syndrome-related tumors, it seems likely that the
frequency and clinical significance of tumor budding
will vary significantly among these three groups, but
awaits confirmation in controlled studies.

Scoring systems
Although a large number of studies have shown
tumor budding to be an independent adverse
prognostic factor in colorectal carcinoma, study
methodologies have varied widely. Different authors
have used different scoring systems as well as
differing definitions as to what constitutes a bud.
The most widely cited scoring system, developed by
Ueno et al,27 defines ‘high-grade budding’ as 410
buds composed of fewer than 5 cells in a 25 field
(field area ¼ 0.385 mm2). Other authors have defined
buds as being composed of r5 cells,38 while some
have not set an upper cell limit and instead count
both single cells and bundles of five or more cells as
buds, as proposed by Morodomi et al11 and
commonly applied in the Japanese literature. This
heterogeneity in defining budding is compounded
by the use of varying field diameters and cutoffs for
defining ‘high-grade’ budding, especially when a
score is given as #/hpf rather than/mm2. Although
most authors have used Ueno’s definition of ‘highgrade budding’ (410 buds of fewer than 5 cells),
all have used a 20 objective, rather than the
25 objective used by Ueno et al, resulting in
significantly greater field areas than that originally
reported by Ueno. Furthermore, while the majority
of studies have evaluated only areas with the
greatest amount of budding, others have proposed
Modern Pathology (2012) 25, 1315–1325

calculating an average bud count,11 counting the
number of buds along the entire invasive
front19,24,52,53 or calculating the proportion of invasive front with tumor budding.21,36,40 Such methodological heterogeneity is illustrated in Table 1,
which summarizes the bud counting methods used
in a sample of tumor-budding studies.
Establishing a Prognostically Significant Cutoff for
‘High-Grade’ Budding

Although Ueno’s definition of ‘high-grade’ budding
(ie, 10 buds in a 25 field) is the most widely
applied in the literature, there is no evidence that
this is the optimal cutoff for evaluating budding.
Only a handful of studies have attempted to analyze
their data so as to generate a clinically useful
threshold for the assessment of high-grade budding.
The results and methodologies of these studies are
summarized in Table 2. In addition, Masaki et al24,53
have proposed a formula based on the total number
of buds counted along the entire invasive margin to
predict the probability of lymph node metastases
in T1 and T2 colorectal carcinoma: although this
is an interesting idea, it is not practical for use in
everyday practice.
Subjectivity in the Qualitative Assessment of Tumour
Budding

In addition to the quantitative issues of inconsistent
cutoffs and differing field diameters, there are a
number of qualitative considerations contributing to
subjectivity in defining a tumour bud. Although
only a few authors have specifically commented on
this problem, the identification of tumor buds can
prove very difficult in several circumstances, eg, (1)
stromal cells or histiocytes masquerading as buds,
(2) marked inflammation obscuring buds, (3) difficulties in determining whether a small cluster of
cells represents a true bud or mechanical fragmentation of a larger gland, etc (Figure 2). Most studies of
tumor budding have not provided much detail
regarding qualitative criteria used to include or
exclude a potential ‘bud’. In addition, there appear
to be discrepancies in the qualitative assessment of
budding, even among studies ostensibly using the
same objective criteria of clusters of o5 cells: eg,
while Jass et al49 counted only ‘discrete clusters’ of
cells as buds, Ha et al17 defined buds as clusters
‘appearing to bud from a larger gland’, suggesting
that they may have also counted buds that were not
completely separate from adjacent glands. Although
such variability may be reduced to some degree by
establishing consensus criteria, there will likely
remain a subset of cases in which budding will be
difficult to evaluate owing to the resemblance of
buds to surrounding stromal cells and/or the
concealment of buds in the setting of a prominent
peritumoral inflammatory reaction (Figure 2).

Table 1 Summary of scoring systems for tumor budding evaluation in a selected sample of relevant studies
Evaluation
method

Bud definition

Cutoff for ‘high-grade’ budding

Proportion of cases classified as high
grade

Morodomi11

40 advanced rectal
carcinomas

H&E

Isolated cancer cells
and bundles of 5cells

Count performed at four locations
(1.25 mm2 field area), and average taken
Three categories:
0–4, negative;
4–14, mildly positive;
414, strongly positive

Mildly positive, 37.5%;
Strongly positive, 27.5%

Hase18

663 colorectal carcinomas

H&E

Not specified
(subjective evaluation)

N/A: classified according to subjective
impression

26%

Ueno27

638 rectal carcinomas

H&E

o5 cells

10 buds in 25 field (0.385 mm2)

30.1%

Okuyama35

196 pT3 well to moderately H&E
differentiated colorectal
carcinomas

Up to 5 cells

N/A: classified according to subjective
impression

43.3%

Jass49

95 colorectal carcinomas,
stratified according to MSI
status

H&E

Up to 5 cells

5 buds in 40 field (area not specified)

32%

Guzinska-Ustymowicz16 24 T1 rectal carcinomas

H&E

Up to 5 cells

Any budding considered positive

35%

Ha17

90 well to moderately
differentiated rectal
carcinomas

H&E

o5cells

47 buds in 20 field (area not specified)
Cutoff selected based on mean intensity
of budding (7.5)

N/A (mean bud count selected as
cutoff such that 50% of cases would
be classified as ‘high grade’)

Kanazawa22

159 colorectal carcinomas

H&E

‘Small cluster’
of cells

Divided into three groups based on
proportion of invasive front with budding:
0–1/3, mild;
1/3–2/3, moderate;
42/3, marked

29% marked
37% moderate

Losi44

295 Stage I colorectal
carcinomas

Immunohistochemistry

o5 cells

45 buds in 20 field (area not specified)

6%

Ishikawa43

71 submucosally invasive
colorectal carcinomas

Immunohistochemistry

o5 cells

44 buds in 40 field (area not specified)

51%

Yamauchi29

164 T1 colorectal
carcinomas

Immunohistochemistry

o5 cells

5 buds in 20 field (area not specified)

15%

Wang41

128 pT3 N0 colorectal
carcinomas

Immunohistochemistry

o5 cells

Budding was counted in 5 fields
(20 0.95 mm2); a median count
of 1 buds considered positive

45%

Tumor budding

Patient population

B Mitrovic et al

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Modern Pathology (2012) 25, 1315–1325

First author

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B Mitrovic et al

Table 2 Summary of studies proposing evidence-based cutoffs for ‘high-grade’ budding
First
author

Method of
evaluation

Bud
Method for determination of optimal cutoff
definition

Resulting definition of
‘high-grade’ budding

Choi15

H&E

o5 cells

410 buds in 20 field
(area not specified)

Sy47

H&E

o5 cells

Prall38

Immunohisto- Up to
chemistry
5 cells

Lugli23

Immunohisto- Up to
chemistry
5 cells

Zlobec48

Immunohisto- o5 cells
chemistry

Patients were divided semiquantitatively into four budding groups
based on quartiles. The cutoff considered the best discriminator of
disease-free survival was between groups 3 and 4.
Patients were divided arbitrarily into five budding groups.
As there were no statistically significant differences among
the first four groups, while the highest budding group showed
significantly worse survival, the cutoff between groups 4 and 5
was deemed the most prognostically significant.
ROC curves were constructed to define the budding cutoff with
the greatest sensitivity and specificity for predicting the
development of metastases.
ROC curves were constructed to define the budding cutoff with
the greatest sensitivity and specificity for discriminating
between survivors and nonsurvivors.
ROC curves were constructed to define the budding cutoff
with the best predictive ability for anti-EGFR therapy
nonresponse in a population of metastatic CRC.

Role of Immunohistochemistry in the Evaluation of
Budding

Some authors have attempted to eliminate the subjectivity inherent in bud counting by using immunohistochemistry with anti-cytokeratin antibodies to
highlight buds and distinguish them from surrounding
stromal cells.12,13,22,23,31,32,38,40,43,44,48,51,54,55 The use of
immunohistochemistry in the assessment of budding
is somewhat controversial: some authors argue that the
evaluation of budding should be limited to H&Estained slides because of the cost and impracticality of
performing immunohistochemistry in routine cases,
while others argue that immunohistochemistry should
be used routinely to improve the accuracy and
reproducibility of bud counts. In addition to cost
considerations, the use of immunohistochemistry may
also require the establishment of a separate cutoff for
defining positive budding. One study comparing the
bud counts obtained on H&E to those obtained with
immunohistochemistry, not surprisingly, found significantly higher counts with the latter.31 Indeed, the few
studies that have attempted to define an optimal
budding threshold on anti-cytokeratin-stained slides
have reported higher cutoffs than those found to be
prognostically significant on H&E (summarized in
Table 2).
Currently, the role for immunohistochemistry in the
evaluation of tumor budding is unclear, and further
studies are needed to assess the relationship between
bud counts obtained on H&E vs those obtained with
anti-cytokeratin antibodies, and to determine the most
prognostically useful cutoff for bud counting with both
H&E and anti-cytokeratin antibodies.
Interobserver Variability in the Assessment of Tumor
Budding

In light of these myriad sources of subjectivity,
one would expect the evaluation of budding to
Modern Pathology (2012) 25, 1315–1325

49 buds in 20 field
(0.95 mm2)

25 buds in 25 field
(0.785 mm2)
16 buds in 40 field
(area not specified)
414 buds in 40 field
(area not specified)

suffer from suboptimal interobserver variability.
A few studies on tumor budding have incorporated
an assessment of interobserver variability, with
reported kappa values ranging from 0.41 to
0.938.15,27,32,34,38,41,48 Kappa values, a commonly used
measure of interobserver agreement, can be interpreted
as follows: o0.20, poor; 0.21–0.40, fair; 0.41–0.60,
moderate; 0.61–0.80, good; and 40.80, very good.56
Intuitively, one would expect the use of
immunohistochemistry to improve interobserver
variability, but studies assessing interobserver variability on immunohistochemically stained slides
have reported kappa values ranging from 0.53 to
0.874,32,38,48 similar to those reported by authors
assessing tumour budding on H&E.15,27,32,34,41 Only
one study has directly compared interobserver
variability between assessments performed on H&E
and immunohistochemically stained slides among
the same group of pathologists: when Suzuki et al32
evaluated budding with both H&E and immunohistochemistry, they showed only a modest
improvement in interobserver variability when
anti-cytokeratin antibodies were used (k ¼ 0.41 with
H&E, and k ¼ 0.53 with immunohistochemistry).

Practical considerations in the assessment and reporting of tumor budding
Opinions are divided as to whether tumor budding
should be recorded in the pathological assessment
of colorectal carcinoma. Although the College of
American Pathologists has not included budding in
its checklist of reportable features, both the Association of Directors of Anatomic and Surgical Pathology
and the Union for International Cancer Control
recommend its inclusion in routine reporting.57,58
Our institutional policy is to report the presence
or absence of tumor budding in all malignant
polyps and colorectal cancer resection specimens.

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B Mitrovic et al

Although we recognize the limitations posed by a
lack of definitional uniformity, we feel that its
assessment is important both for the purposes of
data gathering, and, in the setting of malignant
polyps, for guiding treatment decisions.
Until universally accepted criteria are established,
we apply those of Ueno et al which are not only of
proven prognostic significance, but are also easily
applied and are the most widely used criteria in the
budding literature. We report tumor budding as
present if Z10 groups of o5 cells are counted in
a 20 objective field (ie, Ueno’s so-called ‘highgrade budding’).27 In borderline cases, where only
5–10 definite buds/20 field are identified but there
are additional possible tumor cells that cannot be
confidently differentiated from stroma or histiocytes, a cytokeratin stain is performed. If this
confirms the impression of additional tumor cells,
bringing the count to Z10 buds, we report as
positive for tumor budding. However, we caution
against the routine use of cytokeratin stains in cases
where bud counts on H&E do not approach 10/
20 objective. In our experience, counts by cytokeratin immunohistochemistry are substantially
higher than those on H&E and the limited data
suggest that much higher cutoffs are needed to reach
prognostic significance.23,38,48 As tumor budding
is most prominent at the invasive tumor front, we
concentrate on, but do not restrict our assessment to,
this area.
At scanning power, clues to the presence of tumor
budding include:
(a) infiltrating growth pattern;
(b) marked irregularity at invasive front; and
(c) blurring of the interface between tumor and
underlying stroma.
The presence of these features prompts close
evaluation at medium and high magnification.
In our experience, there are several scenarios in
which tumor-budding assessment is challenging:
(a) abundance of histiocytes and activated lymphocytes at invasive front (Figure 2a);
(b) prominent stromal reaction (Figure 2c);
(c) glandular fragmentation associated with a
marked acute inflammatory infiltrate;
(d) tumour fragments floating in mucin pools; and
(e) fragmented glands with surrounding retraction
artifact (Figure 2e)
In scenarios (a) and (b), a cytokeratin stain is used
to distinguish between tumor cells and stromal/
inflammatory cells (Figures 2b and d). In scenarios
(c) to (e), the fragmented cells are excluded from bud
counts.
We believe it is particularly important to report
tumour budding in the setting of malignant polyps,
as this finding increases the risk of nodal metastasis
to around 15–20%,26 and should prompt consideration of a segmental resection with lymphadenectomy. As such, we make a point of commenting not

only on its presence, but on its associated risk of
nodal metastasis (as we do for lymphovascular
invasion and a poorly differentiated component).
Our approach will undoubtedly need to be refined
once optimal evidence-based guidelines are established. However, based on the available evidence,
and in an area lacking standardization, this strategy
provides institutional guidelines and consistency
for reporting of tumour budding.

Conclusions and future directions
There is strong evidence to suggest that tumor budding is an adverse prognostic factor that can help to
stratify patients into more meaningful risk groups
than TNM staging alone, and, even more importantly, has the potential to guide decision making.
Its presence in T1 colorectal carcinoma, when
evaluated in conjunction with other prognostically
significant clinical and histopathological features,
can identify a subset of high-risk patients requiring
segmental resection including nodes. In the setting
of Stage II colorectal carcinoma, the presence of
budding has been associated with significantly
worse outcomes, and if this subset of patients is
shown to derive a survival benefit from chemotherapy, the evaluation of budding could potentially
assume an important role in treatment algorithms in
Stage II colorectal carcinoma.
In order for the considerable prognostic power
of tumor budding to be fully realized, consensus
criteria for its evaluation must be established, both
to guide further research in this area and to provide
the practicing pathologist with guidelines for its
reporting. With respect to setting these criteria,
studies focusing on budding should be designed to
determine, if possible, the optimal quantitative
cutoff for defining clinically significant tumor
budding. Tumor budding is a continuous variable;
thus, any cutoff is bound to be at least somewhat
arbitrary, but an attempt must be made at defining
the budding threshold that will provide the most
clinically relevant prognostic and predictive information. Furthermore, authors reporting on tumor
budding should provide more detailed information
regarding the qualitative and quantitative criteria
used to evaluate budding in order to allow for
meaningful comparisons among different studies.
Finally, the role of immunohistochemistry in the
evaluation of budding needs to be clarified.
Although it is probably unnecessary and impractical
to perform immunohistochemistry on all colorectal
carcinoma cases, there may be certain situations,
particularly in the context of a prominent peritumoral inflammatory reaction, where a cytokeratin
stain may reveal buds that are obscured on H&E.
Criteria for immunohistochemical evaluation therefore also need to be established.
The greatest obstacle to the adoption of budding
as a routinely reportable feature is the lack of
Modern Pathology (2012) 25, 1315–1325

Tumor budding

1324

B Mitrovic et al

well-defined, evidence-based criteria for its assessment. Given the overwhelming evidence that tumor
budding is one of the most powerful prognostic
factors at our disposal, it is incumbent on the GI
pathology community to move swiftly to address
and remove the barriers to its universal reporting.

Acknowledgement
We thank Dr Masanori Tanaka (Department of
Pathology, City Hospital, Hirosaki, Japan) for assistance in obtaining and translating Japanese articles.

Disclosure/conflict of interest
The authors declare no conflict of interest.

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