Keywords
colorectal carcinoma - Her2/neu - Ki-67 - tumor budding - tumor-infiltrating lymphocytes
Introduction
Colorectal carcinoma is the third most common cancer diagnosed worldwide. It is also
the second most common cause of cancer-related mortality.[1] The scenario remains rather bleak even though several novel screening and diagnostic
modalities have come into practice. In India, colorectal carcinoma accounts for 3%
of all cancers, but the incidence rates are rising alarmingly.[2] Novel prognostic markers are being explored every day to combat this deadly disease.
Tumor budding index has emerged as one of the most important prognostic parameters
not only in colorectal carcinoma, but also in carcinomas of the head and neck, breast,
esophagus, and lung.[3] Determination of tumor budding in colon cancer has played an important role in defining
therapeutic strategies, especially regarding adjuvant and neoadjuvant therapies.[4]
Her2 (human epidermal growth factor receptor 2) is a proto-oncogene found on chromosome
17q21 and it encodes the protein, Her2, which is responsible for the proliferatioxn
and differentiation of cells. Her2 is a therapeutic target in breast and gastric carcinoma
patients, who show Her2 amplification or overexpression.[5] The role of Her2 in colorectal carcinoma is still being explored worldwide.[6]
Ki67 labeling index is an important prognostic parameter in different malignancies,
including breast carcinoma and neuroendocrine tumors. However, the prognostic role
of Ki67 labeling index in colorectal carcinoma has been noted with limited success
in a few studies.[7]
The main aims of the present study were to find out the tumor budding index, Her2
status, and Ki67 labeling index in colorectal carcinoma patients and also to evaluate
the correlation between these parameters and various clinicopathological aspects of
the disease.
Materials and Methods
The study was performed after obtaining approval from the Institutional Ethical Committee
(Ref no. – MC/KOL/IEC/NON-SPON/873/01/2021, dated July 1, 2021).
A total of 41 histologically proven colorectal cancer patients attending the surgery
and radiotherapy departments of the institution, over 18 months, for follow-up after
surgery, were included in this study. They were subjected to a questionnaire for the
collection of relevant clinical information.
The hematoxylin and eosin-stained histopathological slides were reviewed and relevant
details including tumor budding index and tumor-infiltrating lymphocytes (TIL) score
were recorded. A tumor bud is defined as single cells or small clusters of cells (up
to 4 cells in a cluster) within the tumor or at the invasive front of the tumor.[8] In the present study, intratumoral tumor buds were not considered. All the sections
taken from the tumor were first screened under low power objective (×10) to determine
the areas with the highest number of tumor buds at the invasive front. The tumor budding
index was recorded as an average of 10 high-power fields (HPF) (×40) and it was designated
“low” if the score was less than 10 tumor buds/10 HPF and “high” when it was ≥ 10/10
HPF.[8] The cases in which there was heavy lymphocytic infiltration at the invasive front,
obscuring the tumor buds, immunostain (PanCK) was used, to highlight them and facilitate
scoring. In the present study, PanCK was used in 9 cases (21.9%).
TIL in the stroma of the tumor was also assessed at the invasive front of the tumor.
The assessment was done according to the modified Klintrup–Makinen score and the stromal
mononuclear cell infiltration at the invasive front was categorized into dour groups
(excluding areas of crush artifact, necrosis, and hyalinization): 0–no increase in
inflammatory cells; 1–patchy increase in inflammatory cells but no destruction of
invading cancer cell islands; 2–band-like inflammatory cell infiltration with some
destruction of cancer cell islands; and 3–very prominent inflammatory cell infiltration
forming cup-like zone at the invasive margin with destruction of cancer cell islands.[9]
The formalin-fixed paraffin-embedded blocks were collected and relevant sections were
stained with Her2/neu and Ki67 immunostains. Standardized immunoperoxidase techniques
were used for staining using Her2-EP3 (Pathinsitu) and Ki-MIB1 (Pathinsitu) clones
for Her2/neu and Ki67, respectively. Her2/neu scoring was done based on the system
used to evaluate HercepTest: [6] 0–no immunoreactivity or immunoreactivity in < 10%
of tumor cells (negative); 1+ – faint weak immunoreactivity in > 10% of tumor cells
but only a portion of the membrane is positive (incomplete) (negative); 2+ – weak
to moderate complete membrane immunoreactivity in > 10% of tumor cells (weak positive);
and 3+ – moderate to strong complete membrane immunoreactivity in > 10% of tumor cells
(positive).
Ki67 index was evaluated by calculating the percentage of tumor cells showing positive
nuclear staining. When < 25% positively stained tumor cells were found, it was considered
as low Ki67-Li (labeling index) and a percentage of ≥ 25% as high Ki67-Li.[7]
All the relevant data were tabulated and analyzed to assess the correlation between
tumor budding index, Her2/neu, Ki67 scores, and different clinicopathological features
of colorectal carcinoma. The collected data was tabulated in a spreadsheet using Microsoft
Excel 2019 and then statistical analysis was carried out using IBM SPSS statistics
for Windows, version 26.0. Graphs were prepared using the GraphPad Prism for Windows,
Version 9.0. Survival analysis was conducted to find the survival distributions of
the patients according to the tumor budding index, HER2/neu, and KI-67 scores. Survival
analysis was done with SPSS software by Kaplan–Meier analysis. A p-value of less than 0.05 was considered significant (p < 0.05).
Results
Among the 41 colorectal carcinoma patients included in the study, 34 (82.9%) were
males and 7 (17.1%) were females. The age range was from 28 to 92 years, with a mean
of 55.83 ± 16.37 years. Colon was involved in 30 (73%) patients and pure rectal carcinoma
occurred in 11 (27%) patients. Well-differentiated carcinoma was reported in 7 patients
(17.1%), moderately differentiated in 23 (56.1%), and poorly differentiated in 11
patients (26.8%). The tumors were staged as pT2 in 9 patients, pT3 in 25, and pT4
in 7 patients. Analysis of the tumor budding index showed high budding in 25 (61%)
and low budding in 16 patients (39%). There was a significant correlation between
tumor budding and the pT stage of the tumor (p-value: 0.001). There were 3 (7.3%) Her2/neu positive cases. There was no significant
correlation found between Her2/neu expression and different clinicopathological parameters
of colorectal carcinoma. High Ki67 expression was found in 23 patients (56.1%) and
18 (43.9%) patients showed low Ki67 score. A significant correlation was found between
Ki-67 expression status and TIL score (p: 0.008) ([Table 1]).
Table 1
Correlation between tumor budding score, Her2/neu status, Ki-67 score, and different
clinicopathological parameters
|
Clinicopathological features.
|
Tumor budding score
|
HER2/neu status
|
Ki67 score
|
|
High tumor budding
|
Low tumor budding
|
HER2/neu negative
|
HER2/neu positive
|
High Ki67 score
|
Low Ki67 score
|
|
Gender
|
|
|
|
|
|
|
|
Male
|
19 (55.9%)
|
15 (44.1%)
|
31 (91.2%)
|
3 (8.8%)
|
20 (58.8%)
|
14 (41.2%)
|
|
Female
|
6 (85.7%)
|
1 (14.3%)
|
7 (100%)
|
0 (0%)
|
3 (42.9%)
|
4 (57.1%)
|
|
Tumor site
|
|
|
|
|
|
|
|
Ascending colon
|
1 (50%)
|
1 (50%)
|
2 (100%)
|
0 (0%)
|
1 (50%)
|
1 (50%)
|
|
Ascending colon and part of transverse colon
|
2 (100%)
|
0 (0%)
|
2 (100%)
|
0 (0%)
|
0 (0%)
|
2 (100%)
|
|
Caecum
|
6 (75%)
|
2 (25%)
|
6 (75%)
|
2 (25%)
|
5 (62.5%)
|
3 (37.5%)
|
|
Caecum and part of ascending colon
|
0 (0%)
|
1 (100%)
|
1 (100%)
|
0 (0%)
|
0 (0%)
|
1 (100%)
|
|
Rectum
|
6 (54.5%)
|
5 (45.5%)
|
10 (90.9%)
|
1 (9.1%)
|
6 (54.5%)
|
5 (45.5%)
|
|
Sigmoid colon
|
8 (61.5%)
|
5 (38.5%)
|
13 (100%)
|
0 (0%)
|
7 (53.8%)
|
6 (46.2%)
|
|
Sigmoid colon and part of upper rectum
|
1 (50%)
|
1 (50%)
|
2 (100%)
|
0 (0%)
|
2 (100%)
|
0 (0%)
|
|
Transverse colon
|
1 (50%)
|
1 (50%)
|
2 (100%)
|
0 (0%)
|
2 (100%)
|
0 (0%)
|
|
Tumor grade
|
|
|
|
|
|
|
|
G1
|
5 (71.4%)
|
2 (28.6%)
|
7 (100%)
|
0 (0%)
|
3 (42.9%)
|
4 (57.1%)
|
|
G2
|
14 (60.9%)
|
9 (39.1%)
|
22 (95.7%)
|
1 (4.3%)
|
13 (56.5%)
|
10 (43.5%)
|
|
G3
|
6 (54.5%)
|
5 (45.5%)
|
9 (81.8%)
|
2 (18.2%)
|
7 (63.6%)
|
4 (36.4%)
|
|
Tumor stage
|
|
|
|
|
|
|
|
pT
|
|
|
|
|
|
|
|
pT1
|
0 (0%)[a]
|
0 (0%)[a]
|
0 (0%)
|
0 (0%)
|
0 (0%)
|
0 (0%)
|
|
pT2
|
1 (11.11%)[a]
|
8 (88.89%)[a]
|
8 (88.89%)
|
1 (11.11%)
|
4 (44.44%)
|
5 (55.56%)
|
|
pT3
|
19 (76%)[a]
|
6 (24%)[a]
|
24 (96.00%)
|
1 (4.0%)
|
17 (68%)
|
8 (32%)
|
|
pT4
|
5 (71.43%)[a]
|
2 (28.57%)[a]
|
6 (85.71%)
|
1 (14.29%)
|
2 (28.57%)
|
5 (71.43%)
|
|
pN
|
|
|
|
|
|
|
|
pN0
|
19 (67.86%)
|
9 (32.14%)
|
25 (89.29%)
|
3 (10.71%)
|
17 (60.71%)
|
11 (39.29%)
|
|
pN1
|
3 (42.86%)
|
4 (57.14%)
|
7 (100%)
|
0 (0%)
|
3 (42.86%)
|
4 (57.14%)
|
|
pN2
|
3 (50%)
|
3 (50%)
|
6 (100.0%)
|
0 (0%)
|
3 (50%)
|
3 (50%)
|
|
Dukes' staging
|
|
|
|
|
|
|
|
A
|
1 (16.7%)
|
5 (83.3%)
|
5 (83.3%)
|
1 (16.7%)
|
4 (66.7%)
|
2 (33.3%)
|
|
B
|
17 (77.3%)
|
5 (22.7%)
|
20 (90.9%)
|
2 (9.1%)
|
13 (59.1%)
|
9 (40.9%)
|
|
C
|
6 (60%)
|
4 (40%)
|
10 (100%)
|
0 (0%)
|
5 (50%)
|
5 (50%)
|
|
D
|
1 (33.3%)
|
2 (66.7%)
|
3 (100%)
|
0 (0%)
|
1 (33.3%)
|
2 (66.7%)
|
|
AJCC staging
|
|
|
|
|
|
|
|
I
|
1 (16.7%)
|
5 (83.3%)
|
5 (83.3%)
|
1 (16.7%)
|
4 (66.7%)
|
2 (33.3%)
|
|
II
|
17 (77.3%)
|
5 (22.7%)
|
20 (90.9%)
|
2 (9.1%)
|
13 (59.1%)
|
9 (40.9%)
|
|
III
|
6 (60%)
|
4 (40%)
|
10 (100%)
|
0 (0%)
|
5 (50%)
|
5 (50%)
|
|
IV
|
1 (33.3%)
|
2 (66.7%)
|
3 (100%)
|
0 (0%)
|
1 (33.3%)
|
2 (66.7%)
|
|
LVI
|
|
|
|
|
|
|
|
Absent
|
17 (56.7%)
|
13 (43.3%)
|
27 (90%)
|
3 (10%)
|
18 (60%)
|
12 (40%)
|
|
Present
|
8 (72.7%)
|
3 (27.3%)
|
11 (100%)
|
0 (0%)
|
5 (45.5%)
|
6 (54.5%)
|
|
PNI
|
|
|
|
|
|
|
|
Absent
|
20 (55.6%)
|
16 (44.4%)
|
33 (91.7%)
|
3 (8.3%)
|
21 (58.3%)
|
15 (41.7%)
|
|
Present
|
5 (100%)
|
0 (0%)
|
5 (100%)
|
0 (0%)
|
2 (40%)
|
3 (60%)
|
|
Tumor-infiltrating lymphocytes (TIL score)
|
High tumor budding
|
Low tumor budding
|
HER2 negative
|
HER2 positive
|
High Ki
|
Low Ki
|
|
Absent
|
5 (38.5%)
|
8 (61.5%)
|
13 (100%)
|
0 (0%)
|
6 (46.2%)[a]
|
7 (53.8%)[a]
|
|
Marked
|
7 (87.5%)
|
1 (12.5%)
|
7 (87.5%)
|
1 (12.5%)
|
7 (87.5%)[a]
|
1 (12.5%)[a]
|
|
Mild
|
8 (66.7%)
|
4 (33.3%)
|
11 (91.7%)
|
1 (8.3%)
|
3 (25.0%)[a]
|
9 (75%)[a]
|
|
Moderate
|
5 (62.5%)
|
3 (37.5%)
|
7 (87.5%)
|
1 (12.5%)
|
7 (87.5%)[a]
|
1 (12.5%)[a]
|
|
Vitality status
|
|
|
|
|
|
|
|
Alive
|
17 (60.71%)
|
11 (39.29%)
|
2 (7.14%)
|
26 (92.32%)
|
18 (64.29%)
|
10 (35.71%)
|
|
Deceased
|
8 (61.54%)
|
5 (38.46%)
|
1 (7.69%)
|
12 (92.31%)
|
5 (38.46%)
|
8 (61.54%)
|
Abbreviations: AJCC, American Joint Committee on Cancer; LVI, lymphovascular invasion;
PNI, perineural invasion.
a Denotes statistically significant parameters.
Discussion
The clinicopathological parameters of colon carcinoma found in the present study were
comparable with those noted by Gianni et al in their work.[5]
Graham et al found a strong correlation between tumor budding and late stage of disease.[10] In the present study, a significant correlation between tumor budding and the pT
stage of the tumor was noted. It has been suggested that tumor budding may be used
as an indicator to decide therapeutic measures in cases of adenocarcinoma originating
in a polyp. Further, the decision of adjuvant treatment in stage II carcinoma and
that of neoadjuvant therapy when colorectal carcinoma is diagnosed in preoperative
biopsies, rests, at least partially on tumor bud count.[8]
Melling et al concluded in their study that a high Ki67 index is a biomarker that
may be used to determine the prognosis of patients with advanced stage of colorectal
carcinoma.[7] Heidari et al and Farzand et al reported that Her2 expression may be used as prognostic
marker of colorectal carcinoma.[11]
[12] No significant correlation was found between Her2/neu expression and different clinicopathological
aspects of the patients in the present study, similar to that reported by Kilicarslan
et al.[13]
A significant correlation was noted between Ki-67 expression status and TIL score
in the present study. Jakubowska et al were of the opinion that TIL should be reported
in all cases of colorectal carcinoma since they are an indicator of disease progression
and prognosis.[14] Idos et al also noted that TIL and their subsets may be used as prognostic markers
in colorectal carcinoma.[15]
Conclusion
Colorectal carcinoma is one of the most frequent cancers and still a major cause of
cancer mortality, although the treatment regime has continuously improved. HER2/neu,
Ki67, and tumor budding are very effective in treatment and prognostic purposes. There
was a significant correlation of tumor budding with pathological T stage, Dukes' stage,
and American Joint Committee on Cancer stage. A significant correlation was also obtained
between Ki 67 score and TIL status.
It may be concluded that the prognostic importance of tumor budding is fairly well
established. Increased tumor buds are significantly correlated with the advanced stage
of colorectal carcinoma. Considering the small sample size of the present study, there
is a need to explore the prognostic values of HER2/neu and Ki-67 further, in larger
cohorts, in the future.[3]
[4]
[8]
