The Prognostic Impact of Tumor Border Configuration, Tumor Budding and Tumor Stroma Ratio in Colorectal Carcinoma
Lamia Sabry ABOELNASR, Hala Said EL-REBEY, Asmaa Shams El Dein MOHAMED, Asmaa Gaber ABDOU
Department of Pathology, Faculty of Medicine, Menoufia University, MENOUFIA, EGYPT
Keywords: Colorectal cancer, Tumor budding, Tumor stroma ratio, Tumor border, Prognosis
Tumor border configuration, tumor budding and tumor stroma ratio are reliable histopathological parameters that play a central
role in the invasion-metastasis cascade. This study aimed to investigate the prognostic impact of these parameters and a new combined score in
Material and Method: A cohort of 103 colorectal cancer surgical specimens was retrospectively evaluated for tumor border configuration, tumor
budding and tumor stroma ratio using H&E sections. A combined risk score was then constructed to divide cases into low risk-tumors and high
Results: Infiltrating tumor border, high tumor budding, low tumor stroma ratio and high combined risk score were associated with positive
lymph node involvement, presence of metastasis, high tumor grade, lymphovascular invasion, poor overall survival and short recurrence-free
survival. Infiltrating tumor border, high tumor budding and high combined risk score were associated with advanced T stage. High tumor
budding, and low tumor stroma ratio were associated with perineural invasion. Infiltrating tumor border was associated with increased tumor
size and conventional adenocarcinoma, high tumor budding and low tumor stroma ratio. Low tumor stroma ratio was associated with high tumor
budding. On multivariate survival analysis, tumor stroma ratio was found to be an independent predictor for overall survival and recurrence-free
Conclusion: Tumor border configuration, tumor budding, tumor stroma ratio and the newly constructed combined risk score are potential
predictors of outcome in colorectal cancer patients, suggesting that their incorporation in the routine histopathological evaluation could be
useful in determining the prognosis of colorectal cancer cases.
Colorectal cancer (CRC) is one of the most common
cancers worldwide. According to GLOBOCAN 2020
data, CRC is the third most frequently diagnosed cancer
in the world representing 10% of all cancer diagnoses 1
Surgical resection is the primary treatment modality for
early stage CRC. The most effective postsurgical tool for
assessing prognosis is the histopathologic analysis of the
resected specimen including TNM-classification according
to the American Joint Committee on Cancer (AJCC) 2
However, studies revealed that patients’ outcome may vary
considerably even within the same tumor stage 3
recognition, standardization, and reporting of further
histomorphological prognostic features are important
clues for more accurate stratification and individualized
Tumor border configuration (TBC) has been reported to
have prognostic significance that is independent of stage
4. According to Jass, TBC is classified in a two-tier
system as either infiltrating or pushing. Tumors with an
infiltrating growth pattern often show signs of epithelial–
mesenchymal transition (EMT), which can be identified
histologically by the presence of “tumor buds” 5. Tumor
budding (TB) can be defined as the presence of isolated
single cells or small cell clusters of less than five cells at the
invasive front of tumor <6>. TB is another representative
of EMT where the cells display migratory and invasive
properties through losing intracellular and cell-matrix
contacts mediated by E-cadherin 7. The recent dataset for
histopathological reporting of CRC by the Royal College of
Pathologists recommended TB reporting 8. Regarding its
prognostic impact in CRC, some studies showed its poor
prognostic role while others denied 9,10.
Importantly, stromal cells also actively participate in EMT
process. They play a central role in cancer initiation and
invasion-metastasis-cascade 11. Tumor stroma ratio
(TSR) is an estimate of the proportion of epithelial and
stromal cells. Studies have shown a strong association
between high stromal content and poor prognosis in
different cancer types 12.
TBC, TB and TSR are highly producible, reliable and
convenient histopathological parameters. However, their
universal acceptance as reportable factors has been held
back due to lack of studies, variation in methods and
controversial results. This study aimed to investigate the
prognostic impact of TBC, TB, TSR and a new combined
score in CRC.
|Patients and Samples
This retrospective study included 103 primary CRC cases.
Inclusion criteria were as follows: curative surgical colectomy
with lymphadenectomy specimens that were diagnosed
by histopahology as adenocarcinoma. Exclusion criteria
were as follows: cases with incomplete clinicopathologic
records, lost follow-up or cases that received neoadjuvant
chemotherapy or radiotherapy. All cases that met these criteria,
through the period between 2015 and 2019, were included.
All formalin-fixed, paraffin embedded tissue blocks
were retrieved from the archival material of the Pathology
Department, Menoufia University.
This study was approved by the Ethics Committee of the
Faculty of Medicine, Menoufia University, Egypt. Patient
demographics, and data including tumor location and size
were obtained from the original pathology reports.
Four μm-thick sections were cut from all representative
tissue blocks and all sections were stained by H&E. The
mean number of evaluated tissue slides containing the
tumoral areas was 4 (range 4-5) slides for each case and
the selected slides contained at least 75% tumor tissue. All
slides were re-evaluated independently by 2 experienced
pathologists (L.S.A and A.S.E) for assessment of pathologic
stage, histopathologic type, tumor grade, presence of
lymphovascular invasion (LVI) and perineural invasion
(PI) according to the 2019 WHO classification of tumours
of the digestive system 2. Studied CRC cases included
both conventional and mucinous adenocarcinoma cases.
Regarding TBC, TB score and TSR, all slides were scanned
to select the single most appropriate slide that highly met
the recommended criteria for each parameter assessment
as mentioned later.
Tumor Border Configuration (TBC)
The H&E slide selected for TBC assessment was the one
showing the part of tumor with greater depth of invasion
(i.e. slides used routinely to assess T stage). According
to Jass, an infiltrating TBC was defined as dissection of
malignant growth in the form of irregular clusters or cords
through the bowel wall with diminished desmoplastic
stromal response 5. In contrast, margins were considered
pushing when they were reasonably well circumscribed
with a clear delineation of the tumor invasive front and
absence of widely dissecting tumor glands in the muscularis
propria or mesenteric adipose tissue 13.
Tumor Budding (TB)
First, the H&E stained sections were examined with a
×10 objective lens, and the slide showing an area of the
invasive margin with the highest density of tumor buds
was subjectively selected (hot-spot sampling). Then, the
number of tumor buds was counted in 10 HPFs, in a field
that measured 0.785 mm2. In sections with less than 10
HPFs available, buds were counted in as many adjacent
HPFs as possible, and the mean number of buds was
calculated according to this number of examined fields.
As recommended by the International Tumor Budding
Consensus Conference (ITBCC), the TB score was
reported by using a 3-tiered system (low, 0–4 tumor buds;
intermediate, 5–9 tumor buds; high, 10 or more tumor
Tumor Stroma Ratio (TSR)
The H&E slide representing the deepest invasive front
was selected from each case. In case of more slides to be
representative for the deepest invasive front, slides were
scanned using the ×10 objective lens to select the area with
the highest stromal percentage, which was considered
decisive 14. Then, an area where both tumor and stromal
tissue are present within the field was selected using a ×20
objective lens. Tumor cells had to be present at all borders
of the selected field (north-east-south-west) as described by
Huijbers et al. 15. TSR was estimated per microscopic field
and scored into two groups as high TSR (low stroma as ≤
50%) and low TSR (high stroma > 50%). Areas of necrosis,
mucin, major vascular structures and muscle tissue were
visually excluded from the scoring.
Construction of A New Combined Risk Score (CRS)
Infiltrating TBC, TB score > 5 (median) and low TSR were
categorized as risk items. Final categories were as follows:
low risk-tumors with ≤ 1 risk item and high risk-tumors
with > 1 risk items.
Overall survival (OS) was calculated from the date of
surgery to either the date of death or the last followup.
Recurrence-free survival (RFS) was calculated from
the date of surgery until the date of recurrence based on
typical imaging appearance and evidenced by positive
colonoscopic biopsy findings.
Statistical analysis was performed using SPSS software
version 22.0 (IBM SPSS Inc. IL, USA). Analyses of
associations between the assessed histomorphologic
variables and other clinicopathological variables were
carried out by using χ2-tests. The Kaplan–Meier method and
log rank test were used for survival analysis. Cox regression
analysis was used to perform multivariable analysis of
TBC, TB and TSR. A p value less than 0.05 was considered
significant. For inter-observer variability analysis, Kappa
(K) values were generated, and agreement was reported as moderate, substantial, and almost perfect for Κ values of
0.41–0.60, 0.61–0.80, and 0.81–1, respectively 16.
|Clinicopathologic Data of the Studied CRC (Table I)
The age of the studied cases ranged between 25 and 85 years
with a median of 57 years and a mean± SD of 55± 13 years.
The greatest dimension ranged between 2 and 21 cm with
a mean± SD of 5.98± 3.36 cm and 6 cm as a median value.
TBC, TB Score, TSR and CRS in Studied CRC Cases
Sixty-nine cases (67%) showed infiltrating TBC while 34
cases (33%) showed pushing TBC (Figure 1A,B). Forty-five
cases (43.7%) showed low TB score, 35 cases (34%) showed
intermediate TB score and 23 cases (22.3%) showed high
TB score (Figure 1C,D). Sixty-seven cases (65%) showed
high TSR, while 36 cases (35%) showed low TSR (Figure
2A,B). Fifty-six cases (54.4%) had high CRS while 47 cases
(45.6%) had low CRS.
Click Here to Zoom
|Figure 1: A) A case of colonic adenocarcinoma showing infiltrating tumor border configuration in the form of malignant growth
dissecting muscularis propria (highlighted by a red circle) (H&E x100), B) A case of colonic adenocarcinoma with a pushing tumor
border configuration (H&E x40), C) Foci of tumor budding at the invasive tumor margin (highlighted by red circles) (H&E x100),
D) Foci of tumor budding at the invasive tumor margin (highlighted by red circles) (H&E x200).
Inter-Observer Reproducibility of TBC, TB and TSR
Relationship Between Investigated Parameters
(TBC, TB, TSR and CRS) and Other Studied
Clinicopathological Parameters in CRC Cases
Infiltrating TBC was significantly associated with increased
tumor size (p=0.009), advanced T stage (p<0.001), positive
lymph node (LN) involvement (p<0.001), presence of
metastasis (p=0.03), conventional adenocarcinoma (p=
0.001), high tumor grade (p<0.001) and LVI (p=0.017)
Click Here to Zoom
|Table I: Clinicopathological characteristics of studied colorectal cancer (CRC) cases, relationship with tumor border configuration
(TBC) and tumor budding (TB) score.
High TB score was significantly associated with advanced
T stage (p<0.001), positive LN involvement (p=0.007),
presence of metastasis (p<0.001), high tumor grade
(p<0.001), LVI (p=0.007) and PI (p=0.02) (Table I).
Low TSR was significantly associated with positive LN
involvement (p=0.001), presence of metastasis (p<0.001),
high tumor grade (p=0.006), LVI (p=0.005) and PI
(p=0.009) (Table II).
Click Here to Zoom
|Table II: Relationship of tumor stroma ratio (TSR) and combined risk score (CRS) with clinicopathological characteristics.
High CRS was significantly associated with advanced T
stage, positive LN involvement, positive LVI (p<0.001 for
each), presence of metastasis (p=0.026) and high tumor
grade (p=0.005) (Table II).
Relationship Between Investigated Parameters
(TBC, TB, TSR)
Infiltrating TBC was significantly associated with low TSR
(p= 0.03), and high TB score (p<0.001). Moreover, high TB
score was associated with low TSR (p<0.001) (Figure 3A-C).
Click Here to Zoom
|Figure 2: A) A case of colonic adenocarcinoma with malignant glands at 4 borders showing high tumor stroma ratio (TSR) (low stromal
content < 50%) (H&E x200), B) Low TSR (high stromal content > 50%) (H&E x200).
Click Here to Zoom
|Figure 3: A) Infiltrating tumor border configuration (TBC) was significantly associated with low tumor stroma ratio (TSR) (p=0.03),
B) Infiltrating TBC was significantly associated with high tumor budding (TB) score (p<0.001), C) High TB score was associated with
low TSR (p<0.001).
The Impact of Investigated Parameters (TBC, TB, TSR
and CRS) on Survival
Univariate analysis of OS showed the bad prognostic impact
of infiltrating TBC (p=0.015), high TB score (p> 0.001)
and low TSR (p<0.001) (Figure 4A-C). Infiltrating TBC
(p=0.001), high TB score (p>0.001) and low TSR (p<0.001)
were significantly associated with short RFS (Figure 5A-C).
High CRS was significantly associated with poor OS
(p>0.001) and short RFS (p<0.001) (Figure 6A,B).
Click Here to Zoom
|Figure 4: Kaplan-Meier survival curve demonstrating the impact of tumor border configuration (TBC) (p=0.015) (A), tumor budding
(TB) score (p<0.001) (B) and tumor stroma ratio (TSR) (p<0.001) on overall survival (C).
Click Here to Zoom
|Figure 5: Kaplan-Meier survival curve demonstrating the impact of tumor border configuration (TBC) (p=0.001) (A), tumor budding
(TB) score (p<0.001) (B) and tumor stroma ratio (TSR) (C) on recurrence-free survival.
Click Here to Zoom
|Figure 6: Kaplan-Meier survival curve demonstrating the impact of combined risk score (CRS) on overall survival (p<0.001) (A) and
on recurrence-free survival (B).
On multivariate survival analysis, TSR was shown to be an
independent predictor for OS and RFS (p=0.001) (p<0.001),
respectively (Table III).
Click Here to Zoom
|Table III: Multivariate COX regression analysis of overall survival and recurrence-free survival for the investigated parameters in
studied CRC cases.
The present study showed a poor prognostic impact of
infiltrating TBC on OS and RFS, compared to pushing
TBC. Infiltrating TBC had a significant association with
adverse prognostic pathologic parameters such as large
tumor size, advanced T stage, positive LN involvement,
presence of metastasis, high tumor grade and LVI. These
findings were in agreement with Morikawa et al. who
observed that the infiltrating growth pattern was associated
with worse prognosis among stage I-III CRC patients,
independent of other clinical, pathologic, and molecular
. Interestingly, the configuration of
the invasive margin correlates with molecular alterations
in CRC. Specifically, a well-demarcated, pushing tumor
border is a feature frequently seen in MMR-deficient
. In contrast, an infiltrating tumor border
is significantly more frequent in tumors with activating
. While MMR-deficient CRC generally has a favorable outcome, BRAF is an independent
predictor of an aggressive clinical course 19
indicates that constitutive activation of BRAF may increase the migratory and invasive capacity of human colon cancer
. This could contribute to the poor prognostic
impact observed in CRC-cases with infiltrating TBC.
Moreover, the prognostic impact of TBC may refer to hostrelated
factors that influence the appearance of the tumor
border in CRC. Halvorsen and Seim described a marked
absence of peritumoral inflammation in patients with
an infiltrating TBC 21. In contrast, CRC-cases with a
pushing border have a well-characterized association with
dense peritumoral inflammatory infiltrate. Importantly, it
is well-known that the density of peritumoral inflammatory
response reflects the efficiency of anti-tumor host response,
which may be a possible confounding factor of the good
prognostic impact of pushing TBC 18,22.
Interestingly, unlike other studies that showed no
correlation between TBC and histopathologic type of CRC,
the present study showed a significant association between
pushing TBC and mucinous colonic adenocarcinoma
(MCA). This may be referred to the molecular profile
stating that most of MCA occurs in patients with hereditary
nonpolyposis CRC (HNPCC or Lynch syndrome) and
thus represents high-level MSI (MSI-H) tumors which
are known for their pushing margin configuration 23.
Messerini et al. also reported a positive correlation between
MSI-H MCA and expanding growth pattern 24. Reported prominent host immune response in MSI-H MCA may
justify their decreased invasive potential represented in
pushing TBC 25. In agreement with our observation,
Hacking et al. reported that most of MCA cases had low
TB score which was significantly associated with pushing
TBC 26. Further research studies are recommended to
investigate in depth the molecular characteristics of MCA
in correlation to their histopathological features.
The present study demonstrated that high TB score was
correlated with poor OS and short RFS. A high TB score
showed a significant association with adverse prognostic
pathologic parameters as advanced T stage, positive LN
involvement, presence of metastasis, high tumor grade,
LVI and PI. These findings were in agreement with Wyk et
al. and Eriksen et al. who referred that to dedifferentiation
of cells that tend to lose adhesion, dissociate and be more
aggressive 9,10. There is a close relationship between
TB and the process of EMT. In this transitional process,
budding cells lose intracellular and cell-matrix contacts
mediated by E-cadherin, migrate through the extracellular
matrix, invade lymphovascular structures and form
metastatic tumor colonies in lymph nodes and at distant
The present study demonstrated the independent
prognostic impact of TSR regarding both OS and RFS.
This is in accordance with previous studies that reported
the adverse prognostic impact of increased stromal
component in both early disease and advanced colon
cancer 9,14,29. Furthermore, low TSR was correlated
with increased invasive and aggressive potential of CRC
through its significant association with positive LN
involvement, metastasis, high tumor grade, LVI and PI.
Similar correlations were reported by Eriksen et al. and
These findings may owe to the capability of stromal
mesenchymal cells to orchestrate the invasion-metastasiscascade
11. Several secreted molecular regulators of
stromal cells have a pro-tumorigenic role. For example,
upregulation of heat shock factor 1 (HSF1), Yes-associated
protein 1 (YAP1), Stromelysin 1 and stromal-derived
exosomes have emerged as mediators of cancer progression
through enhancing cancer cell motility, invasion, metabolic
reprogramming and inducing cancer stem cell features 31
As patients with stage II colon cancer have highly variable
outcomes, TSR is a useful tool to select patients who are
at risk of developing recurrence of disease or metastases.
Huijbers et al. investigated the TSR next to the ASCO
criteria; they found that the TSR improved the ASCO
criteria and reclassified 14% of the patients as high‐risk.
This suggests that adjuvant therapy might be considered in
stage II patients with low TSR 15.
A significant association between TBC, TB and TSR was
found with an infiltrating TBC related to increasing TB
score and a higher fraction of stroma (low TSR). Eriksen et
al. also observed a significant correlation between the mean
number of buds and TSR with an increasing number of TB
related to a lower TSR 9. Park et al. found an association
between low TSR and the presence of an infiltrating
invasive margin 32. Wang et al. reported that cases with
high TB score had predominantly infiltrating TBC 33.
This is in accordance with the consideration that the three
parameters reflect the histopathologic translation of EMT
where cancer cells assume a mesenchymal phenotype
characterized by increased migratory capacity, invasiveness,
increased resistance to apoptosis and increased production
of extracellular matrix (ECM) components 28,34.
The integration of TBC, TB and TSR into an objective
CRS model enhanced the prognostic impact of these
parameters regarding OS and RFS. Furthermore, high CRS
was significantly associated with advanced T stage, positive
LN involvement, positive LVI, presence of metastasis
and high tumor grade. These findings were in agreement
with Dourado et al. who investigated a combined model
of TB and TSR in oral squamous cell carcinoma 35.
Interestingly, the CRS model has included both cancer cell
features (TB, TBC) and stromal features (TSR). The present
study is considered the first one to construct a combined
model of TBC, TB and TSR and investigate its prognostic
impact in CRC. Further studies are recommended to be
conducted on larger cohorts for more validation.
Although assessment of TBC, TB and TSR was an easy
method to apply, there were practical challenging difficulties.
Peritumoral inflammatory response might be difficult to
differentiate from TB, and may sometimes obscure the
underlying budding. In such cases, immunohistochemistry
staining for cytokeratin may help to highlight TB.
Furthermore, in case of a stromal percentage at or around
the cut-off point of 50%, consulting a second observer
could be of help when in doubt. Overall, the inter-observer
agreement was in a clinically useful and applicable range
for the 3 parameters, ranging from substantial agreement
in the setting of TSR to almost perfect in assessment of
TBC and TB, in accordance with earlier studies using
the same method (36,37). This high inter-observer
agreement enhances the importance of adherence to a
standardized scoring system and standardized protocol in
the management of challenging settings during assessment
In conclusion, TBC, TB score and TSR are highly reproducible,
reliable and convenient parameters that could be
easily assessed in H&E stained slides and included in routine
histopathologic reports. The incorporation of these
features into a CRS covering both epithelial and stromal
features of tumor might be used to improve the stratification
of CRC patients into low risk and high risk regarding
Conflict of Interest
All authors confirm that that there are no conflicts of interest.
Concept: LSA, AGA, Design: LSA, ASM, HSE, AGA, Data collection
or processing: LSA, ASM, HSE, Analysis or Interpretation: LSA,
ASM, HSE, AGA, Literature search: LSA, Writing: LSA, AGA,
Approval: LSA, ASM, HSE, AGA.
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