Introduction:
Breast cancer is a heterogeneous disease both histologically & molecularly [1]. It is the most common malignant tumor and is also the second most common cause of carcinoma death in women worldwide [2].Different molecular subtypes have been described [1].
The standard histopathological characteristics remain the most useful prognostic factors although more molecular & genomic markers are emerging [3]. Histological characteristics include histological types, tumor size, nodal status, grade, hormone receptor expression & HER -2 over expression status [4].
Tumor budding is defined as tumor cells which detach from the cohesive tumor mass. They can be either a single tumor cell or small clusters. This can be seen within the tumor called intra tumoral budding or at the periphery called as peripheral tumor budding [5].
At molecular level, budded cells exhibit epithelial mesenchymal transition(EMT) like changes and they are postulated as the histological representation of EMT. EMT is a multistep dynamic cellular phenomenon in which epithelial cells lose their cell-cell adhesion & gain migratory & invasive trails which are typical of mesenchymal cells [4,6].
EMT is trans-differentiation of epithelial cells into mesenchymal cells. During this process the cells lose junctions and apical-basal polarity, reorganise their cytoskeleton and reprogramme gene expression. This gives the cells an invasive phenotype [7].
Tumor budding is a general invasive indicator, an early step in cancer metastasis & considered as a poor prognostic factor in carcinoma. It is used as the main prognostic factor in colon cancer but it is now gaining popularity in other tumor types such as breast, pancreas, oesophagus & larynx. There is limited literature on the role of tumor budding in breast cancer [8]. This study aims to examine the association of tumor budding & clinicopathological characteristics in breast cancer.
Materials and Methods
This study was an observational analytical study which was conducted in the department of pathology at Adichunchanagiri Institute of Medical Sciences over a period of 18 months from October 2018 to March 2020. All the surgically resected breast specimens received in histopathology, department of Pathology, diagnosed of primary invasive ductal carcinoma breast were included in the study. Patients who had undergone neoadjuvant chemotherapy and radiotherapy, or with concurrent malignancy at other sites or with previous history of malignancy were excluded from the study.
Brief clinical history including age, site of lesion, laterality, unifocal or multifocal, local examination findings, systemic examination and clinical diagnosis was recorded. Ultrasound findings was recorded wherever available. Formalin fixed, grossed, paraffin embedded blocks for the tumor, peri-tumoral area and lymph nodes was prepared and stained with H & E for mounting and histopathology analysis and grading was done by Nottingham modification of the Scarf Bloom Richardson method as grade 1, grade 2 and grade 3.
Tumor budding was counted in the maximum invasive area. Tumor budding was defined as the presence of single tumor cells or small clusters of cells in the tumor stroma [9].The buds was counted at 40x magnification in 10 hotspots (densest area). The tumor budding was categorized into low grade if the average number of tumor buds was <4 in 10 HPF and high grade if the average number of tumor buds was >4 in 10 HPF(fig 1). Immunostaining for marker cytokeratin was done and tumor budding was counted and categorized as low and high tumor budding (fig 2).
Results
Among the 50 cases, majority of cases were in the age group of 35-40 years(26%), presented right sided tumor (60%), had tumor size <5cm (100%), were in grade 2 and 3 (78%), had lymphovascular invasion (54%), necrosis(60%) and had lymph node involvement (56%). The distribution of cases according to various baseline clinicopathological characteristics are listed in Table 1.
| Table 1 : Distribution of cases based on various clinicopathological characteristics |
| Features |
Observation |
Frequency(%) |
Age |
4th grade |
26% |
Laterality |
Right |
60% |
Left |
40% |
Tumor size |
<2 |
4% |
>5 |
24% |
2-5 |
72% |
Grade |
1 |
22% |
2 |
44% |
3 |
34% |
Lymphovascular invasion |
Yes |
54% |
No |
46% |
lymph node involvement |
Present |
44 |
Absent |
56 |
Necrosis |
Present |
60 |
Absent |
40 |
Staging |
pT1 |
4 |
pT2 |
56 |
pT3 |
32 |
pT4 |
8 |
Correlation between high tumor budding and clinicopathological characteristics are depicted in Table 2.
Cases presenting with lymphovascular invasion (graph1), presence of necrosis (graph 2) and lymph node involvement showed high tumor budding. High tumor budding was also associated with higher stage of the tumor. Few other clinicopathological characteristics like age of the patient, tumor laterality, focality and grade of the tumor did not correlate with tumor budding.
| Table 2: Correlation of clinicopathological variables with high tumor budding and low tumor budding |
| Features |
High tumor budding |
Low tumor budding |
P value |
48% |
52% |
Grade |
1 |
8 |
14 |
0.484 |
2 |
20 |
24 |
3 |
20 |
14 |
Focality |
Uni |
2 |
0 |
0.48 |
Multi |
46 |
52 |
Margins |
positive |
12 |
14 |
0.87 |
negative |
36 |
38 |
LV invasion |
present |
40 |
30 |
0.048 |
absent |
8 |
22 |
staging |
pT1 |
2 |
2 |
0.023 |
pT2 |
18 |
38 |
pT3 |
24 |
8 |
pT4 |
4 |
4 |
LN involvement |
yes |
46 |
40 |
0.045 |
no |
2 |
12 |
Necrosis |
Yes |
36 |
24 |
0.038 |
no |
12 |
28 |
Tumor size |
mean |
5.79 cm |
4.04 cm |
0.016 |
 |
 |
Graph 1: Correlation of high tumor budding and low tumor budding with lymphovascular invasion |
Graph 2: Correlation of high tumor budding and low tumor budding with necrosis |
 |
Fig 1: Tumor budding in invasive breast carcinoma. (a) Tissue section of invasive carcinoma of no special type displaying less than 4 tumor bud (Black arrow) at the invasive front of the tumor. (H and E, ×400).(b) Tissue section of invasive carcinoma of no special type showing more than 4 tumor buds (Black arrows) at the invasive front of the tumor. (H and E, ×400). (c) Section of invasive ductal carcinoma showing inflammatory infiltrate mainly macrophages and few fibroblasts mimicking tumor buds (H & E ×200). |
 |
Fig 2: IHC staining of tumor budding in invasive breast carcinoma. (a) Tissue section of invasive carcinoma of no special type displaying less than 4 tumor bud (Black arrow) at the invasive front of the tumor. (Pan CK,× 400). (b)Tissue section of invasive carcinoma of no special type showing more than 4 tumor buds (Black arrows) at the invasive front of the tumor. (Pan CK,× 400). |
Discussion
Tumor budding has been assumed to represent cancer cells caught in the process of invasion. The process of metastasis begins with detachment of cells from the tumor bulk which is followed by infiltration through contiguous tissues into small blood vessels. These cells then travel through the circulation to remote locations where they extravasate and may finally establish colonies of metastatic disease [10].
To date, consensus on the definition and quantification of tumor budding has not been reached. Besides the counting method proposed by Ueno et al., other criteria, such as the rapid bud count method and scoring method have been used. However, despite the lack of standardization, nearly all the reported evaluating systems presented with a good reproducibility [11].
Recent studies have stated that focal myoepithelial cell degeneration- induced auto immunoreactions trigger the disruption of the capsule which selectively start monoclonal proliferation of tumor stem cells [12-19]. After the disruption of the capsule there might be a localised loss of tumor suppressors which promote tumor growth and escape the cascade of programmed cell death [20,21]. Following this there might be an increase in permeability of nutrients, growth factors and altered oxygen level for the proliferation of the stem cells [22,23].
There is a localised increase in leukocyte infiltration which directly exports growth factors to tumor cells through direct physical contact [24,25].When there is direct tumor stromal contact there is activation of stromal MMP and reduced expression of E-cadherin and other cell surface adhesion molecules which in turn promotes epithelial mesenchymal transition [26,27].
EMT is a physiologic processes that play a key role in embryonic development, wound healing, and tissue repair. Aberrant activation of EMT is considered to be a hallmark of cancer metastasis. The invasive front of the lesions display a striking disorganization at the tumor architecture level (i.e., loss of glandular aspect for differentiated carcinomas and loss of trabecular aspect for undifferentiated carcinomas, respectively) along with a dedifferentiation at the cellular level. These changes ultimately result in isolated tumor cells showing a uniform phenotype, irrespective of the differentiation of the main tumor mass. The phenotype attain features compatible with cell motility with loss of cell junctions and basement membrane and developing cytoplasmic microfilaments and pseudopodia which invade the host tissues through locomotion. This is known as epithelial mesenchymal transition. Tumor budding has been associated with poor cancer outcomes [28].
Over the last few years, numerous studies have established tumor budding to be an independent predictor of lymph node positivity, lymphatic invasion, higher grade of tumor, local and distant metastasis, and poor prognosis among patients with all pathological stages of Colorectal Cancer [29]. Whether tumor budding can be considered a relevant factor in the prognosis of breast carcinomas is a new area of research. In this study, we aimed to determine if tumor budding is correlated with clinicopathological characteristics of breast cancer and if it can be used as a prognostic marker.
Most of the studies on tumor budding compared in the table 3 including the present study had Invasive ductal carcinoma as the primary lesion. In most of the studies clustering of cases was seen in fifth decade and above. However our study and studies by Agarwal R et al [30].and Liang F et al [4] showed clustering of cases in 4th decade. This may be due to the late presentation of cases in other studies.
In the present study, tumor buds were counted in 10 different fields in line with previous studies [1,10,31]. In contrast Sriwidyani NP et al., [6] Gujam FJA et al. [3] and Liang F et al. [4] counted tumor buds in only 5 fields. Karamitopoulou E et al. [32] have done a study on colorectal carcinoma for a proposal for using 10-high-power-fields scoring method for assessment of tumor budding. It would be appropriate that tumor buds are counted in ten fields for accurate stratification of cases into low and high tumor budding groups.
The present study used the cut off as <4/10 HPF for low tumor budding and >4/10 HPF for high tumor budding in line with studies by Salhia B et al., [1] Gabal SM et al. [31]. However the cut offs for tumor budding in other studies are not uniform. This type of discrepancy might be due to the different methods used for tumor budding in different studies [1,7,9,31].
Similar to the study by Liang F et al., [4] we have used both H&E and IHC for counting the tumor buds for better visualization, so the grading of buds into high and low tumor budding would be appropriate. Previous studies on breast carcinoma have shown that IHC staining is a conclusive marker for tumor budding as it excludes inflammatory cells, fibroblasts and other artifacts [1,4,7].
In the present study there was significant correlation of tumor budding with tumor size. High tumor budding showed mean tumor size of 5.79cm and low tumor budding showed mean size of 4.04cm( p= 0.016). Gabal SM et al [31], Salhia et al [1] and Gujam FJA et al.[3] found poor correlation with tumor size and tumor budding. The reason might be due to the different counting methods used to count tumor buds [1].
Similar to the findings of previous studies [1,4,6,7], the present study showed significant correlation with the primary tumor staging and tumor budding (p=0.023). In the present study most(n=12/24) of the high tumor budding cases were in pT3 stage where as most (n=19/26) of the low tumor budding cases were in pT2 stage.
Lymphovascular invasion showed a significant correlation with the tumor budding (p=0.048) which is in line with most previous studies. Lymph node staging also showed significant correlation with the tumor budding (p=0.045). The higher staging of lymph node showed more cases with high tumor budding. Similarly Kumarguru BN et al., [9] Masilamani S et al. [10] and Salhia B et al. [1] also showed significant correlation with the lymph node staging and tumor budding. This observation certainly suggests that tumor budding can be considered as a poor prognostic factor.
Similar to study by Kumarguru BN et al. [7] present study also showed significant correlation between tumor budding and necrosis (p=0.038). Among the 24 cases of high tumor budding, 18 of them had necrosis. 6 cases among the high tumor budding group did not show necrosis.
When overall grade was taken into consideration, it did not show significant correlation with the tumor budding(p=0.484). Only one study i.e by Sriwidyani NP et al. [6] showed significant correlation with tumor budding.
Surgically resected margins did not show significant correlation with the tumor budding (p=0.877). The only other study that has studied the correlation of the surgical margins with tumor budding did not find any significant correlation [31]. This may be explained by improved surgical resections with adequate tumor free margins.
| Table 3: Comparison of various clinicopathological characteristics with other studies |
| Parameters |
Present Study |
Kumarguru BN et al.[30] |
Agarwal R et al.[31] |
Masilamani S et al.[25] |
Gabal SM et al.[24] |
Sriwidyani NP et al.[8] |
Gujam FJA et al.[5] |
Salhia B et al.[3] |
Liang F et al.[6] |
Total no of cases |
50 |
50 |
40 |
107 |
61 |
70 |
474 |
148 |
160 |
Age |
Forth decade |
Sixth decade |
Forth decade |
53.7(median) |
53.1(median) |
48.6 |
>50 |
61(median) |
>35 |
Lesion |
IDC (NOS) |
IDC (NOS) |
Primary BC |
IDC |
IDC-NOS |
IDC (NOS) |
IDC- NOS |
IDC-NOS |
IDC-NOS |
Power of objective |
40x |
40x |
20x |
10x |
20x |
40x |
20x |
40x |
20x |
No of fields |
10 |
10 |
|
10 |
10 |
5 |
5 |
10 |
5 |
TB cut off value |
>4 |
>20 |
>10 |
>/-10 |
>/-4 |
>10 |
>20 |
>4 |
>7 |
Staining |
H&E and IHC (pan cytokeratin) |
H&E |
H&E and IHC (pan cytokeratin) |
H&E |
H&E |
IHC (cytokeratin) |
H&E |
IHC (pan-cytokeratin) |
H&E, IHC (pan cytokeratin) |
Tumor budding did not show significant correlation with tumor focality (p=0.48). Only Study by Gabal SM et al. [31] included correlation of tumor focality with tumor budding.
Though the studies on tumor budding have been heterogenous with regard to study design, cut offs used to define tumor budding and have had discrepant results, most studies, similar to the present study, have found tumor budding to correlate with the features of tumor aggressiveness. Tumor budding, is indeed, an emerging prognostic factor in invasive breast cancer.
The limitations of the present study are low sample size, and as there was a limited study period follow up of the patients was not possible in order to know the outcome of the worse prognostic factors.
Conclusion
Significant association of high tumor budding with aggresive clinicopathological parameters implicates its usefulness as an important prognostic marker in breast cancer. Similarly Cytokeratin stain can be used as a surrogate marker for determining budding status. Further studies involving large sample size and with sufficient standardization in future is recommended to ensure incorporated in the reporting of breast carcinoma.
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