Introduction: Breast carcinoma is a malignancy that forms in the cells of the breast. Breast carcinoma is one of the non-communicable diseases (NCDs) that has become a major public health issue. Globally, cancer is a feeding cause of death in which deaths due to breast cancer accounts for 15% of all cancer deaths among women Negative node breast carcinoma simply means breast cancer without lymph node involvement. Objectives: To study the association of tumour size, evaluated after histopathological studies with different clinicobiological parameters like age, histological grade, tumour markers such as p53, ki67 and bc1-2. Methods: This study was conducted from Jan 2021-June 2022 at department of pathology, Medical College, Kolkata, West Bengal, India. Total 30 patients were included in this study. Statistical data were analysed by using Microsoft Excel and SPSS V.20 software. Results: In the study group analyzed, pathological tumor size ranged from 1.1 to 9cm. Tumors in 13cases exceeded 5cm while the rest was below 5cm.We showed that the pathological size was significantly associated with age greater than or equal to 50years than the one who were less than 50years of age(t=5,P<0.001),and histological grade III VS I(P<0.001). Also we found a significant difference for p53(P<0.001) positivity and ki-67(P<0.01). Conclusions: It was found that tumor size was significantly associated with age over 50years,histological grade 3 and increased immunohistochemical expression of ki-67 and p53,all of which support its prognostic value. |
In Breast cancer pathogenesis, studies have shown that mutations and abnormal changes in the genes are responsible for cellular growth regulation, homeostasis and maintenance of health. Novel biomarkers such as ki67, p53, bcl-2 are thus emerging tools for classifying breast carcinomas, guiding therapy and predicting treatment response and prognosis.
BcI-2 being an ant apoptotic gene plays an ant apoptotic role, resulting malignant cell accumulation leading to a more aggressive course, poor clinical outcome or resistance to therapy in most tumour expressing bcl-2, However, in breast carcinoma it has a favourable outcome.
P53 on the other hand play a role in tumorigenesis by its effect on cell proliferation. Breast cancer comprises the second most common neoplasm in humans after lung cancer.1
The human breast consists of a branching ductal network consisting of an inner layer of polarized luminal epithelial cells, and an outer layer of myoepithelial cells. The ductal network terminates in lobular units commonly called the terminal duct lobular units (TDLUs).2
TDLUs produce milk and are the primary source of most breast cancer precursors and cancers. In 2012 the name for the most common type of breast cancer was changed from invasive ductal carcinoma, noswise specified (NOS) (2003) to invasive carcinoma of no special type (NST).3 Special types of breast cancer account for up to 25% of all breast cancers. The latest edition of the World Health Organization recognizes at least 17 different histological special types.4 Tumor size, lymph node involvement, histologic type, histologic grade, and a receptor (estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2)) expression status by immunohistochemistry (IHC) have been well established as prognostic and predictive factors for breast cancers.5
Triple-negative breast cancer (TNBC) constitutes around 15% of all breast cancer cases and is characterized by tumors that do not express estrogen receptor (ER), progesterone receptor (PR), and do not overexpress human epidermal growth factor receptor 2 (HER2). As a result, they do not benefit from hormonal or trastuzumab-based therapy. Patients with TNBC have worse overall survival than patients with non-TNBC 6.
Domestic dogs (Canis lupus familiaris) are excellent models of human complicated diseases for many reasons, including their approachability and popular role in different cultures. Many similar characteristics in human and dog cancers including, spontaneous development, clinical presentation, tumor heterogeneity, disease progression, and response to standard therapies have promoted the approval of this comparative model as an alternative to mice. Breast cancer represents the second most frequent neoplasm in sexually intact female dogs after skin cancer. Canine mammary tumors are a naturally occurring heterogenous group of cancers that have several features in common with human breast cancer. These similarities include etiology, signaling pathway activation and histological classification. Female dogs typically have five pairs of mammary glands, which are called thoracic (2 pairs), abdominal (2 pairs), and inguinal glands (1 pair). The appearance of canine mammary tumors (CMT) in female dogs under the age of two is rare but increases remarkably for over six years old. Old age, mixed breed, and large size lead to its development and reflect malignancy risk factors.7
Maiti et al.8 studied 70 cases of CMTs. Forty-eight cases had solitary growth and 36 tumors were pedunculated and 34 were sessile. Thirty-eight mammary growths were ulcerated and inflamed and the remaining 32 were intact and subcutaneous. The most affected mammary glands were caudal abdominal and inguinal (4th and 5th). The majority of mammary gland tumors in female dogs are of epithelial origin, and approximately 50% are malignant.9 The incidence of CMT in female dogs spayed before their first heat is 0.05% but increases to 8% or 26% if spayed after the first or second heat, respectively.10
This aim of this study was to study the expression of ki67, p53 and bcl-2 in association with tumour size in a node negative breast carcinoma.
This present institutional based observational prospective study was carried out in Department of Pathology, Medical College, Kolkata, West Bengal, India between Jan 2021-June 2022.
Study population and sampling technique : All clinically suspected cases of breast carcinoma attending surgery OPD of Medical College, Kolkata and further histopathologically proven as node negative breast carcinoma in the Department of Pathology, Medical College, and Kolkata was included in our study. Based on past records we expect 30 cases given within the period of data collection fulfilling the Inclusion and exclusion criteria.
Inclusion criteria : Age between 25-80 years and Histopathologically proven cases of node negative breast carcinoma was included in our study.
Exclusion criteria : Age below 25years, and histopathologically proven node positive breast ca cases will not be included in the study.
Study variables: Patient particulars: (age, sex, occupation, socio-economic status, h/o, intake of any oral contraceptives, particularly in our case tumor size expression with age of the patient has been analyzed. Gross examination findings were tumor size, location, lymph node status. Histopathological findings were diagnosis with histological subtype, tumor characteristics (tumor grade, lympho-vascular invasion, pTNM stage). Immunohistochemistry findings were p53, ki67 and bcl-2 expression in histopathological samples of node negative breast carcinoma
Detailed history noted and clinical examination of the patients are done according to the questionnaire formed. Histopathological examination and irnmunohistochemistry analysis (p53, ki67 and bci--2) was done. Then results were analyzed at the end of data collection.
Specimen of breast followed by modified radical mastectomy are received in Dept of Pathology in 10% Neutral buffered formalin. Specimens after thorough breadloafing are fixed in 10% Neutral Buffered formalin for 24 hours and subjected to gross examination. Histopathological examination is performed on the Haematoxylin and Eosin-stained sections of formalin-fixed, paraffin-embedded tissues from the breast specimens.
examination and investigation
Data Analysis plan- The data was tabulated in Microsoft Excel software and analysed with SPSS V.20 software. An alpha level of 5% has been taken that is if any p value is <0.05, it was considered as significant.
Table 1: Comparison of Tumor Sizes according to the Age Groups (n=30)
Age Groups |
n |
Mean±S.D. |
Median |
Minimum |
Maximum |
P value |
<50 years |
14 |
2.54±1.29 |
2.3 |
1.1 |
6 |
<0.001 |
≥ 50 years |
16 |
5.48±1.86 |
5.55 |
1.5 |
9 |
|
Overall |
30 |
51±7.35 |
50 |
40 |
65 |
|
The overall mean age of the patients included in the present study was 51±7.35 years Comparisons by the Independent sample t-test indicated that there was a statistically significant difference between the two age groups and thereby implying very strong evidence that the mean tumor size was greater in patients ≥ 50 years than the ones who were<50 years of age(P<0.001). (Table 1)
Table 2:Comparison of Tumor Sizes according to the Grades of Tumor by the one-way ANOVA test
Grade of Tumor |
n |
Mean±S.D. |
Median |
Minimum |
Maximum |
F value |
P value |
Grade I |
9 |
1.97±0.7 |
2.2 |
1.1 |
3 |
F (2, 27) = 19 |
<0.001** |
Grade II |
11 |
4.06±1.56 |
4 |
1.8 |
6.5 |
||
Grade III |
10 |
6.08±1.82 |
6.1 |
2.2 |
9 |
n:Samples size per group
Comparisons were performed using One-way ANOVA Test to compare the tumor sizes according to the Grades of Tumor and it showed a statistically significant difference [F (2, 27) = 19, P=0.01]. (Table 2)
Table 3: Comparison of Tumor Sizes according to the Lympho-vascular invasion status
Lymphovascular invasion |
n |
Mean±S.D. |
Median |
Minimum |
Maximum |
t value |
P value |
Positive |
6 |
5.93±0.55 |
6.1 |
5 |
6.5 |
2.5 |
0.02* |
Negative |
24 |
3.65±2.21 |
3 |
1.1 |
9 |
n:Samples size per group
Comparisons by the Independent sample t-test indicated that the mean tumor size was greater in patients who had a Lympho-vascular invasion than the ones who didn’t and the difference was statistically significant (t= 2.5,P=0.02). (Table 3)
Table 4:Comparison of Tumor Sizes according to the Grade of KI-67 expression by the one-way ANOVA test
KI Grade |
n |
Mean±S.D. |
Median |
Minimum |
Maximum |
F value |
P value |
Grade I |
12 |
2.94±2.09 |
2.5 |
1.1 |
9 |
F (2, 27) = 5 |
0.01** |
Grade II |
7 |
3.94±1.76 |
4.2 |
1.5 |
6.1 |
||
Grade III |
11 |
5.48±1.85 |
6 |
2 |
8 |
n:Samples size per group
Comparisons were performed using One-way ANOVA Test to compare the tumor sizes according to the Grades of Ki-67 expression and it showed a statistically significant difference [F (2, 27) = 5, P=0.01]. (Table 4)
p53 |
n |
Mean±S.D. |
Median |
Minimum |
Maximum |
t value |
P value |
Positive |
11 |
6.01±0.89 |
6 |
5 |
8 |
4.8 |
<0.001** |
Negative |
19 |
3.01±1.94 |
2.5 |
1.1 |
9 |
||
Bcl-2 |
|
|
|
|
|
|
|
Positive |
14 |
3.64±2.4 |
3.05 |
1.1 |
9 |
1.1 |
0.28 |
Negative |
16 |
4.52±1.96 |
5 |
2 |
8 |
||
ER |
|
|
|
|
|
|
|
Positive |
17 |
3.72±2.29 |
2.5 |
1.1 |
9 |
1.1 |
0.27 |
Negative |
13 |
4.62±2.01 |
5 |
1.5 |
8 |
||
PR |
|
|
|
|
|
|
|
Positive |
17 |
3.72±2.29 |
2.5 |
1.1 |
9 |
1.1 |
0.27 ns |
Negative |
13 |
4.62±2.01 |
5 |
1.5 |
8 |
n:Samples size per group
Comparisons by the Independent sample t-test indicated that the mean tumor size was greater in patients who had a positive p53 expression than the ones with a negative p53 expression and the difference was statistically significant(t=4.8,P<0.001). Comparisons by the Independent sample t-test indicated that the mean tumor size was greater in patients who had a negative Bcl-2 expression than the ones with a positive Bcl-2 expression but the difference was not statistically significant(t=1.1,P=0.28). Comparisons by the Independent sample t-test indicated that the mean tumor size was greater in patients who had a negative ER expression than the ones with a positive ER expression but the difference was not statistically significant(t=1.1,P=0.27). Comparisons by the Independent sample t-test indicated that the mean tumor size was greater in patients who had a negative PR expression than the ones with a positive PR expression but the difference was not statistically significant(t=1.1,P=0.27). (Table 5)
Table 6: Distribution according to different parameters.
Side |
Frequency |
Percent |
TNM |
Frequency |
Percent |
Left |
14 |
46.7% |
T1cN0Mx |
1 |
3.3% |
Right |
16 |
53.3% |
T1N0Mx |
9 |
30.0% |
Histopathological types |
|
|
T2N0Mx |
11 |
36.7% |
Encapsulated papillary ca with invasion |
1 |
3.3% |
T3N0Mx |
9 |
30.0% |
IDC, NOS |
6 |
20.0% |
Lymph vascular invasion |
|
|
IDC, NST |
20 |
66.7% |
Negative |
24 |
80.0% |
Met aplastic carcinoma |
3 |
10.0% |
Positive |
6 |
20.0% |
Grade KI |
|
|
P53 |
|
|
1 |
12 |
40.0% |
Negative |
19 |
63.3% |
2 |
7 |
23.3% |
Positive |
11 |
36.7% |
3 |
11 |
36.7% |
|
|
|
In our study, 14 (46.7%) patients had Left sided tumor and 16 (53.3%) patients had Right sided tumor. In our study, 6 (20.0%) patients had IDC, NOS and 20 (66.7%) patients had IDC, NST, 3 (10.0%) patients had met aplastic carcinoma. In our study, 9 (30.0%) patients had T1N0Mx, 11 (36.7%) patients had T2N0Mx, and 9 (30.0%) patients had T3N0Mx. In our study, 9 (30.0%) patients had Lymph vascular invasion. In our study, 11 (36.7%) patients had P53. In our study, 12 (63.3%) patients had 1, 7 (23.3%) patients had 2, 11 (36.7%) patients had 3 in grade KI. (Table 6)
Table 7 : Distribution of mean of different parameters.
|
Number |
Mean |
SD |
Minimum |
Maximum |
Median |
Age |
30 |
50.7000 |
7.3539 |
40.0000 |
65.0000 |
50.0000 |
Ki-67 |
30 |
25.0000 |
19.0734 |
10.0000 |
80.0000 |
20.0000 |
Grade ki |
30 |
1.9667 |
.8899 |
1.0000 |
3.0000 |
2.0000 |
Mean Age (years) (mean±s.d.) of patients was 50.7000± 7.3539. Mean Ki-67 (mean±s.d.) of patients was 25.0000± 19.0734. Mean Grade ki (mean±s.d.) of patients was 1.9667± .8899. (Table 7)
This study was conducted from Jan 2021-June 2022 at department of pathology, Medical College, Kolkata. 30 patients were included in this study.
In our study, we analyzed possible associations between tumor size and clinico biological factors commonly used in daily clinical practice in patients with breast IDCs without axillary lymph node involvement, that is focusing exclusively on the size. Following analysis of 30cases, a statistically significant association was found between tumor size and age over 50years, advanced histological grade, high cell proliferation and immunohistochemical expression of p53.
Tumor size is a classical parameter of tumor biology and is directly related to a greater chance for regional axillary involvement, a greater number of invaded nodes and greater possibility of recurrence and death. Its prognostic value can be seen in cases with and without axillary lymph node involvement, being very important in the absence of regional spread because it may help identify patients with high or low risk of recurrence.11
In our study, there is a very strong evidence that the mean tumor size was greater in patients ≥ 50 years than the ones who were<50 years of age (t= 5, P<0.001).
In a study it was found that women older than 60years had an increased tumor size when were from a hospital breast unit, but not if they were from screening campaigns, which highlights the importance of the patient’s origin and supports the possible cause of greater tumor size being a delayed diagnosis.12
Histological grade is a classic parameter of breast tumor biology and is associated with survival and TNM classification, being of particular relevance in cases without axillary lymph node involvement, which allows patients to be stratified for certain therapies. Larger tumors with histological grade3 is a fact consistent with that described by other authors in their study.13
In our study, there was a highly statistically significant difference between Grade I and Grade III and thereby implying very strong evidence that the mean tumor size was greater in Grade III than Grade I(P=0.01).
An association between increased tumor size and immunohistochemical expression of ki-67 and p53. Cell proliferation is a prognostic factor in cases without axillary lymph node involvement and correlates with various biological factors, including p53.14
Silvestrini et al. demonstrated that tumor cell Proliferation is an important predictor of axillary relapse in elderly patients with ER positive breast cancer and could help to identify patients who should undergo axillary surgery.15
In our study, the mean tumor size was greater in patients who had a positive p53 expression than the ones with a negative p53 expression and the difference was statistically significant (t=4.8, P<0.001).
Relationship between tumor size and hormonal receptors were analyzed but there was no significant differences when the expression of ER, PR were considered.
Sharma M et al 16 (2016) observed that to correlate clinicopathological and immunohistochemical profile of BRCA1 positive and non BRCA1 breast cancer patients with ER, PR, BCL2, P53 and Ki-67 to gain more insight into biological characteristics of breast cancer to emphasize the need of genetic testing for BRCA1 in blood relatives of patients with BRCA1 mutation. All cases were subjected to immunohistochemistry for BRCA1, ER, PR, P53, BCL2 and Ki-67 expression. Grade II Tumors constituted the maximum (67%). The most common age group was 41-60 years (62%). BRCA1 positivity was seen in 27/70 cases (38%). BRCA1 positive cases tend to present at higher stage than BRCA1 negative cases showing significantly greater tumor size (p< 0.001) and lymph node involvement (p =0.001).
Mansouri H et al17 (2019) this study associated Ki-67, p53, and BCL-2 markers with clinical histopathological (CH) features using currently available limited data on these markers in Tanzania. Retrospective chart review study was conducted among females with confirmed breast cancer (BC) at Muhimbili National Hospital in Tanzania between 2016 and 2017. Inclusion criteria were met by 76 patients with a mean age of 51.32 ± 14.28 years. Of these, 86.4% were stage III and IV, whereas 83.5% cases had grade 2 and grade 3. Upon immunostaining, 85.5% and 57.9% were Ki-67 and BCL-2 positive respectively.
Ameh-Mensah C et al18 (2021) found that little is known about the role of apoptosis in the tumorigenesis and prognosis of breast cancer in Ghana. Chemotherapeutic drug efficacy partially relates to apoptosis induction, rendering it a vital target in cancer therapy with unique biomarker opportunities that have not been exploited. Aberrations in this pathway are central to tumorigenesis, tumor progression, overall tumor growth, and regression during treatment therapies. Antiapoptotic bcl-2 (gene) and p53 are known to play roles in apoptosis while Ki-67 is a proliferative marker. The aim of their study is to determine the association of bcl-2 (protein) with p53 and Ki-67 in 203 consecutive breast cancer cases over a 10-year period.
Atwa AM et al19 (2022) showed that to evaluate whether p53, cyclin A and ki67 immunohistochemical (IHC) assay can be used as predictors for Wilms’ tumor (WT) unfavorable outcomes. It is a non-concurrent cohort study including patients who underwent nephrectomy for WT from January 2000 to December 2015 in a tertiary referral center. Over a 5- year follow-up, unfavorable events, including relapse and cancer-specific mortality (CSM), were recorded. P53, cyclin A, and ki67 IHC assay were carried out for formalin-fixed paraffin-embedded WT samples.
In breast IDCs without axillary lymph node involvement, tumor size was significantly associated exclusively with age over 50years, histological grade 3 and increased immunohistochemical expression of ki-67 and p53, all of which support its prognostic value.
Acknowledgements : Authors would like to acknowledge the patients who participated in this research study.
Funding: No funding sources
Conflict of interest: None declared
Ethical approval: The study was approved by the institutional ethics committee