INTRODUCTION:

The breast is the major site for cancer incidence and the most common raison for cancer death among women with an estimated 240,000 new cases among US women in 2013¹. According to National Center of Cancer (NCC) in Syria, the incidence of breast cancer was 75.2 women for 100,000 in 2007. In Syria, higher incidence was remarked in coastal regions, especially in Lattakia. Recently, great interest in detection of early prognostic factors had been remarked with increased incidence of breast cancer and with its associated enormous treatment costs, morbidity and mortality among women².

In recent years, control of breast cancer and strategies of preventing are the most effective procedures for reducing number of death and amelioration of prognosis of patients³. Breast cancer prevention researches need molecularly targeted approach for success treatment and less toxic drugs.

Tumor size, histological grade, stage, lymph nodes metastasis and other clinical or pathological factors are important but still insufficient as early factors for detection or for prediction of future response to hormone-therapy or chemotherapy in many cases. Estrogen receptor ER-expression, Progesterone receptor PR-expression and human epidermal growth factor receptor 2 Her's-2-expressions are so variety according to age, stage, involved lymph nodes and other characteristic of tumor or patients. In fact, women with Triple Negative (TN) expression (ER, PR and Her-2 receptors expression are negative) or negative ER-expression present a different response to hormone-therapy⁴. In addition, it's better to select women at high risk who have high pN staging, advanced stage, large tumor size, positive Her-2-expression, and negative ER-expression and (TN) patients. Therefore, cases must be studied carefully and targeted chemotherapy must be used for women who might benefit most from this therapy with maximum safe preventive and minimum various toxic -effects².

Recent studies had been investigated molecular Breast Cancer Subtypes (BCSs) with different expression status of ER, PR receptors and Her-2 receptor. In fact, these subtypes vary in patients remarkably, overall survival (OS) and response to radio/chemotherapy were also different^4,5. Additionally, Her-2 overexpression with ER-negative is present in approximately 20-30% of all human breast cancer, while Triple- Negative breast cancer TN is present in approximately 15-20% of all diagnosed cases⁶. Research studies in vitro and in vivo have shown that patients with Her-2 overexpression were positive for ER-receptor, which propose a bishormone-treatemnt⁷. Therefore, development of effective protocol of treatment for these heterogenic forms of disease or particular subtypes proposes a great challenge⁸.

Histological inflammation is remarked in breast cancer with the infiltration of macrophages in invasive tumors, resulting increasing poor prognosis, and reducing overall survival in many patients⁹-¹¹. Therefore, many cases of resistance to hormone therapy or chemotherapy might benefit of reducing serum CRP levels or other inflammation mediators. Inflammation theory is widely studied, and these mediators could be markers in patients at high risk among postmenopausal women which suggest the role of age¹².

Recently, many studies approved that the incidence of ER-negative breast cancer has a poor prognosis and often occurs in postmenopausal women¹³. In addition, more than 80% of positive Her-2 receptor expression patients were 46 years and older, which support those HER's-2 positive breast cancers are more aggressive and poor survival rate in postmenopausal women^14.

The incidence of breast cancer has declined in recent years in the United States and worldwide. However, the decreased incidence was remarked especially in women aged more than 50 years, and this amelioration was more evident in women positive for estrogen receptor (ER) than in ER-negative cancers¹⁵. Therefore, there is a great interest to study ER-negative breast cancer patients as a risk group for hormone therapy resistance in addition to advanced age.

The aim of this study is firstly to classify 60-breast cancer patients into Breast Cancer Subtypes (BCSs) according to expression status of ER, PR receptors and Her-2 receptor as molecularly research approach and its relation to other prognostic classical factors, and secondary, to identify patients at high risk who might resist to future hormone-therapy.

MATERIAL AND METHODS:

1. Population of study (patients and excluded individuals):

Sixty female-patients were selected for this study from September 2015 until December 2016 among women admitted in National hospital of Jableh (Jableh- Syria) and diagnosed as breast cancer. Basic blood investigations, chest x-ray, ECG and CT scan were done for all the patients. Core needle biopsy was done; the majority of patients underwent a total or a subtotal mastectomy with auxiliary dissection.

Patients with acute inflammation state, cardiovascular diseases, and Type-1, or Type-2 diabetes were excluded.

2. Classification of patients according to pathological TNM staging:

The patients were then assessed according to the pathological TNM staging according to the American Joint Committee on Cancer (AJCC) breast cancer staging system:

1. Tumor size: (T1 = ≤2 cm, T2 = 2.1-5 cm, T3 =>5 cm, T4 = chest wall or skin infiltration

2. Nodal staging: (pN1 = 1-3 nodes, pN2 = 4-9 nodes, pN3= > 9 nodes).

3. Metastasis: presence (M0) or absence (M1) of metastasis.

Data were then obtained prospectively from pathology reports which included tumor size, stage, histologic grade, and number of lymph node excised.

3. ER, PR and Her-2/ neu receptors expression assessment by IHC and FISH analysis:

Estrogen receptor ER and progesterone receptor PR expression as positive or negative were confirmed by Immunohistochemical analysis (IHC) and considered positive if more than 10% of tumor cells were positively (weak or strong).

HER-2/neu receptor expression was confirmed by Fluorescent In-Situ Hybridation (FISH) analysis, and only score II or score III (intense membrane staining in more than 10% of tumor cells) were considered positive, while tumor cells with score 0, or I were negative.

4. Breast Cancer Subtypes (BCSs) classification according to ER, PR and Her-2 expression:

Patients were classified as Breast Cancer Subtypes (BCSs) according to ER, PR and Her-2/ neu receptors expression as following into five subtypes:

1. Triple Positive TP (ER+, PR +and Her-2 +).

2. Triple Negative TN (ER-, PR- and Her-2).

3. Luminal A (Her-2 -, ER+ and /or PR+).

4. Luminal B (Her-2 +, ER+ and /or PR+).

5. Her-2+ (ER-, PR-).

5. Statistical analysis:

Descriptive statistics using frequencies (%) for categorical data were calculated. The chi-square test was used to compare percentages between groups.

All analyses were completed using the Statistical Package for the Social Sciences (SPSS) version 18 (IBM Corporation). As the analysis involves multiple subgroups, significance was set at the 5% level (P. value < 0.05).

All analysis was done using the same method in the same laboratory and same pathologist. A written consent was signed by every patient and approved by the Institutional Ethical Board.

RESULTS:

1. Staging and Histological diagnosis of patients:

All patients selected for this study were diagnosed as Invasive Ductal Carcinoma IDC (n=54, 90%) which was the common histologic type, Invasive Lobular Carcinoma ILC (n=4, 6.67%), and Mucinous carcinoma MC (n=2, 3.33%) as shown in table 1.

Patients were divided according to Histologic Grade as following: Grade I: (n=1, 1.67%), Grade II (n=50, 83.33%), and Grade III: (n=9, 15%).

Stage distribution was done as following: stage I: 3 patients (5%), stage II: 28 patients (46.67%), stage III: 29 patients (48.33%), while there was no patient with stage IV as advanced stage among patients selected. Histological diagnosis is shown in table 1.

Table 1: Histological diagnosis and Staging of patients:

Diagnosis		N=60
		Number of patients	%
Diagnosis	IDC	54	90
	ILC	4	6.67
	Mucinous C	2	3.33
Histologic Grade	I	1	1.67
	II	50	83.33
	III	9	15
Stage	I	3	5
	II	28	46.67
	III	29	48.33

2. Characteristics of patients and tumor status:

Mean age of patients selected for this study at diagnosis was 58 years (range 32 to 77 years). Patients were then distributed by age into two groups: group (1): younger than 46 years (n=28, 46, 7%), and group (2): patients aged 46 years and older (n=32, 53.3%). The age of 46 years was suggested as the age of beginning of disorders or menopausal statue in the majority of patients as a common case in oriental society.

The mean size of tumors was 2.38cm with range from 1.2 to 11cm. Patients were distributed into three groups according to tumor size: T1 (n=14, 23.33%), T2 (n=37, 61.67%), and T3 (n=9, 15%). The major of patients were with tumor size ≥2.1 cm.

Concerning pN staging, 49 patients were with positive lymph node metastasis (81.67%), while 11 patients were with no metastasis to lymph nodes (18.33%).

The major pN staging was pN2 with 4-9 positive lymph nodes (n=33, 55%).

All characteristics of patients and tumor status are summarized in table 2 and in figure 1.

Table 2: Characteristics of patients and tumor status:

Variable		Number of patients	%
Age, years	<46	28	46.7
Age, years	≥46	32	53,3
Tumor Size ( cm )	≤2	14	23.33
	2.1 to 5	37	61.67
	>5	9	15
Nodal stage, pN	pN0	11	18.33
	pN1:1 to 3	12	20
	pN2: 4 to 9	33	55
	pN 3: >9	4	6.67

(A)

(B)

(C)

Figure 1: Characteristics of patients and tumor status: distribution of patients according to age in years (A), positive lymph nodes (B) and tumor size in cm (C).

3. ER-receptor, PR-receptors and Her-2/neu receptor expression:

Concerning hormone receptor (ER, PR) and Her-2 expression, our results found that 41 patients were ER-receptor positive (68.33%), 43 patients were PR-receptor positive (71.67%), and 44 patients were Her-2 receptor positive (73.33%) as shown in table 3.

Table 3: ER-receptor, PR-receptors and Her-2/neu receptor expression

Hormone receptor status		Number of patients	%
ER status	Negative	19	31.67
ER status	Positive	41	68.33
PR status	Negative	17	28.33
PR status	Positive	43	71.67
Her-2 status	Negative	16	26.67
Her-2 status	Positive	44	73.33

37 out of 41 patients ER-receptor positive were also positive for PR- receptors (90.24%), while 6 out of 19 ER-receptor negative were positive for PR- receptor expression (31.58%), and the difference was strongly significant (P.value <0.0001).

33 out of 41 patients ER-receptor positive were positive for Her-2/neu receptors expression (80.49%), while 11 out of 19 patients ER-receptor negative were positive for Her-2/neu receptors expression (57.89%) and the difference was not statistically significant (P,value =0.066).

Table 4: Relation of Her-2/neu expression with hormone receptor status

P . value	No. of patients with negative Her-2 N, (%)	No. of patients with positive Her-2 N, (%)	Hormon receptor
0.066			Estrogen receptor status
	8 (19.51)	33 (80.49)	Positive (41)
	8 (42.11)	11(57.89)	Negative (19)
0.321			Progesterone receptor status
	13 (30.23)	30 (82.35)	Positive (43)
	3 (17.65)	14 (69.77)	Negative (17)

30 out of 44 patients Her-2/neu-receptor positive were positive for PR-receptor expression (68.18%),while 13 out of 16 patients Her-2/neu-receptor negative were positive for PR-receptor expression (81.25%) and the difference was not statistically significant (P,value = 0.321). Relation shown in table 4.

4. Breast Cancer Subtypes (BCSs) status according to ER, PR and Her-2/neu receptor expression:

Patients were classified as Breast Cancer Subtypes (BCSs) according to ER, PR and Her-2/ neu receptors expression into five subtypes as shown in figure 2.

The major number of patients were with TP (n=29, 48.33%), there were 3 patients with TN (5%), 13 patients with luminal A and 5 patients with luminal B (8.33%). 10 patients were positive for Her-2 expression and negative for ER/PR receptor expression as fifth type (16.67%).

Our results show also that 19 patients were negative ER-receptor expression (31.67%), 17 patients were negative for PR-receptor expression (28.33%), and there were 16 patients negative for Her-2 expression (26.67%).

These negative subtypes were important to predictive response to chemotherapy.

Figure 2: Breast Cancer Subtypes ( BCSs) classification according to ER, PR and Her-2 receptor expression

5. Relation of Breast Cancer Subtypes (BCSs) and tumor characteristics:

Our results show that the relation between Breast Cancer Subtypes BCSs and stage was statistically important as shown in table 4: 13 patients TP were SII, 15 patients TP were SIII. We found also that 10 patients were Luminal A and SIII and the difference was important (P. value 0.046).

Although the major number of patients were TP (n=18) and were ≥ 46 years, there was no relation between BCSs and age (P.value=0.369). Our results show that no relation was found between BCSs and tumor size (P. value= 0.074) or diagnosis (P.value= 0.1).

Additionally, our results show that the major number of patients were with grade II and TP (n=22) or luminal A (n=13) in comparison with patients GI and TP/ luminal B (n=0), and the difference was not statistically important (P.value= 0.079).

Relation between BCSs and tumor characteristics shown in table 5.

Concerning pN staging, we had been redistributed patients into two groups as following:

1. Patients with positive lymph nodes 1-4 lymph nodes.

2. Patients with positive lymph nodes >4 lymph nodes.

Our results show that the major number of patients were TP and with pN >4 lymph nodes (n=22), and this difference was statistically important (P. value= 0.013)

Concerning relation between hormone status and pNstaging, 31 out of 43 PR-receptor positive patients (83.78%) compared with 6 out of 17 PR- receptor negative patients (16.22%), had lymph node >4, the relation between lymph nodes and PR-receptor statue was statistically significant (p,value =0.008).

Our results show that 28 out of 41 ER-receptor positive patients (75.68%) had lymph node >4 compared with 9 out of 19 ER- receptor negative patients (24.32%). The relation between lymph node involvement and ER- receptor expression was not statistically significant (P = 0.121). In addition, 28 out of 44 Her-2/neu-receptor positive patients (75.68%) compared with 9 out of 16 Her-2/ neu- receptor negative patients (24.32%) had lymph node >4, and this relation was not statistically significant (P=0.603). Figure 3 shows the relation between Hormone receptor expression and lymph node status.

Table 5: Relation between BCSs and tumor characteristics

		TP	Luminal B	Her-2 + (ER- PR-)	TN	Luminal A	P. value
		Number of patients
Age, year	<46	11	2	4	2	9	0.369
	≥46	18	3	6	1	4
Tumor Size,	T1: ≤2cm	9	1	1	0	3	0.074
	T2: 2.1 to 5cm	18	2	9	1	7
	T3: >5cm	2	2	0	2	3
Diagnosis	IDC	27	3	9	3	12	0.1
	ILC	0	2	1	0	1
	Mucinous	2	0	0	0	0
Grade	I	0	1	0	0	0	0.079
	II	22	4	9	2	13
	III	7	0	1	1	0
Stage	I	1	0	1	0	1	0.046
	II	13	3	7	3	2
	III	15	2	2	0	10

In addition, our results show that patients positive for PR-receptor, ER-receptor expression and for Her-2 expression were with pN>4 lymph nodes. These results suggest advanced cases, but good prognosis for future anti-hormone/ chemotherapy. Results show also that there were patients negative for hormone receptor expression and with less than 4 lymph nodes as complicated cases for chemotherapy. Relation between hormone –receptor status and pN staging shown in figure 3.

Figure 3: Relation between hormone –receptor status and pN staging. Patients positive for PR-receptor, ER-receptor expression and for Her-2 expression were with pN>4 lymph nodes.

DISCUSSION:

In Syria as well as worldwide, the incidence of breast cancer increases relatively with enormous costs and increased mortality rate among women. Additionally, resistances to endocrine-therapy or chemotherapy are remarked in many cases. Therefore, there is a great need to identify patients who might benefit most from these treatments with less side-effect.

Recently, there is evidence that Cancer Stem Cells (CSCs) are relatively resistant to chemo-, radio-, or to endocrine therapies, and breast cancer was the first solid tumor in which CSCs were identified. Up to 25% of breast cancer overexpression Her-2 was with higher rate of recurrence and mortality. In fact, Her-2 receptor overexpression promotes tumor formation and metastasis by increasing the proportion of CSCs and their self-renewal and invasive properties. In addition, Her-2 overexpression increases IL-6 expression via STAT3/Akt/NFkB signaling pathway and enhancing BCSCs activity^{16, 17}.

Recent researches had been investigated a novel approach: elimination of BCSCs by using Repertaxin which is a combination of CXCR1/CXCR2 inhibitors with Her-2 target therapy. These findings propose the inflammation hypothesis, and the role of inflammation mediators such as CRP, CRPhs, IL-8, and IL-6 as serum markers, in addition to classical tumor markers^10,
18.

Expression of ER-receptor, PR-receptor, and Her-2 receptor vary in patients and response to hormone-therapy varies also according to these receptors-expressions. In recent studies, Her-2 overexpression with ER-negative is present in 20-30% of all breast cancer, while Triple- Negative breast cancer (TN) is present in approximately 15-20% of all diagnosed cases⁶. Therefore, modulation of protocol of treatment must be studied for each patient according to breast cancer subtypes.

This study aimed firstly to characterize 60 female- patients of breast cancer who admitted in National hospital of Jableh- Syria as a homogenate population of study. Breast Cancer Subtypes (BCSs) Characterization was assessed according to ER, PR receptor and to Her-2 receptor expression status. Our secondary aim was to study relation between these BCSs and other risk factors such as lymph nodes, tumor size, stage, grade and histological diagnosis. In addition, we were interesting to identify the patients at high risk who might resist to endocrine-therapy.

Data show that 90% of patients were diagnosed as invasive ductal carcinoma (IDC), 83.33% of patients were with grade II. Concerning stage, results show that the major of patients were with stage II (46.67%), or stage III (48.33%), while only 5% of cases were with stage I. These findings present a high histological homogeneity in study population concerning histological diagnosis, stage and grade with no advanced cases such as stage IV.

Results have been investigated that triple positive breast cancer (TP: ER+, PR +, and Her-2 +) was presented in approximately 48.33% of patients. Triple negative breast cancer (TN: ER-, PR- and Her-2-) was presented in 5 % of all diagnosed cases, while other studies remarked triple negative breast cancer (TN) in approximately 15-20% of tumor diagnosed⁶. Luminal A: (Her-2 -, ER+ and /or PR+) was presented in 21.66% of cases, and Luminal B: (Her-2 +, ER+ and /or PR+) was presented only in 8.33% of all cases. The fifth subtype Her-2+ (ER-, PR-) was presented in 16.67% of patients, which proposes a subtype of high risk; overexpression of Her-2 with negative-ER and negative-PR receptors expression¹⁹. These results were investigated by Suh et al, in 2002; they proposed prevention and treatment of experimental breast cancer with the combination of a new selective estrogen receptor modulator (arzoxifene, and a new rexinoid) as a new target hormone-therapy and according to hormone receptor expression¹⁹. Many other researches support this hypothesizes for studying cases carefully concerning hormone-receptors expression status.

Concerning negative hormone-receptor expression, our results show also that 32.67%, 28.33% of patients were negative for ER, PR- receptor expression respectively, and 26.67% of patient present negative Her-2 expression as shown in table 3. Additional results as shown in table 4 present that patients positive for Her-2 expression were also positive for ER, PR-receptors as the major group, while negative Her-2 patients were negative for ER (42.11%) and for PR receptors (17.65%). Positive ER- receptor tumors were associated significantly with positive PR-receptor expression, and this present a high possibility to response to classic hormone therapy⁸.These results propose additional modulation of hormone therapy because of expected resistance and suggest different protocol for these patients at high risk.

All these results concerning histological diagnosis and hormone receptor expression propose high variation in breast cancer subtypes (BCSs) regarding homogeneity in histological diagnosis, stage, and grade. Therefore, it's so interesting to study carefully patients according to ER, PR receptor expression, or Her-2 expression for each patient.

This study has shown that the mean age of patients was 58 years, and 46.7% of patients aged younger than 46 years, while 53.3% aged older than 46 years; the distribution of patients between these two groups of age was approximately the same. Regarding the relation of age with BCSs, we found that the difference of age; younger or older than 46 years was not clear enough, while patients presented with luminal A were younger than 46 years, but the difference was not statistically important, and this present these patients as a group at high risk as shown in table 5. These results correlate with many studies which approved that breast cancer occurs in younger as well as in aged women²⁰. Different biologic behavior of breast cancer in young women may be due to progesterone receptor positive status; elevated breast cancer mortality in women younger than age 40 years compared with older women is attributed to poorer survival in early-stage disease^21,22. In fact, these differences were not clear enough and additional studies should focus on association between age and tumor characterizes.

Although relation of Breast Cancer Subtypes (BCSs) with tumor size, diagnosis or with grade were not important statistically (P. value>0.05), it was clear that the most number of patients with tumor size T2, IDC and grade II were TP as shown in table 5.

Results concerning relation of BCSs and tumor characteristics show that patients staged as stage II were TP (n=13) and Her-2 + (n=15), while patients presented with stage III were TP (n=15) and luminal A (n=10) respectively, and this suggest that stage III presents with negative Her-2 expression; as group of high risk which might resist to future hormone-therapy. Our results did not find any patient presented as stage I or III and TN. These differences between BCSs and stage were statistically important (P. value= 0.046) as shown in table 5.

For studying the relation of BCSs and lymph nodes metastasis, our results show that breast cancer presented with pN>4 lymph nodes were positive for PR-receptor, ER-receptor expression and for Her-2 expression. These results suggest advanced cases and high risk of metastasis and this presents more aggressive status, but good prognosis for future anti-hormone therapy²³. In addition, we found also that breast cancer presented with lymph nodes less than 4 were negative for hormone receptor expression as especially complicated cases for chemotherapy^{24, 25}. In fact, many studies indicate that it's difficult to know number of lymph nodes should be removed in breast cancer patients, and lymph nodes staging is not sufficient. Therefore the number of lymph nodes examined varies depending upon the surgeon’s decision, technique used, the patient’s anatomy, and the completeness of the pathological examination²⁶. It is also not clear the minimal number of nodes that should be examined to establish lymph node metastasis²⁷.

Results concerning relation of lymph nodes and PR-receptor expression indicate that the difference was statistically important with PR- receptor expression, and this could present an indicator for response to future anti PR-receptor therapy.

Recently, studies have revealed that other parameters related to the status of lymph nodes, such as number of removed lymph nodes (RLNs), number of negative of lymph nodes (NLNs), and ratio of involved to removed nodes (lymph node ratio, LNR), which impact the prognosis of breast cancer patients^28. Many results show that pN stage and NLN count have a better prognostic value compared to LNR in patients with breast cancer²⁹. This proposes that additional studies should focus on association between number of negative lymph nodes and tumor characterizes. Therefore, it would be interesting to evaluate other serum biomarkers such as CRPhs, IL-6 as inflammatory markers beside all clinical and pathological factors.

In summary, patients have almost same histological diagnosis; 90% of patients were diagnosed as invasive ductal carcinoma (IDC), and although this great histological homogeneity, majority of patients presents high variety of ER-receptor, PR-receptor and Her-2 receptor expression as heterogenic breast cancer subtypes BCSs (TP, TN, Luminal A, Luminal B, and Her-2+). Therefore, it would be interesting to identify breast cancer subtypes at high risk who might resist to future therapy, especially patients with overexpression Her-2 and negative ER, PR-receptor expression.

These finding propose different strategies of prevention and different protocols of radio /chemo- therapy according to different predictive response. Therefore, it would be very important to follow up patients within treatment to evaluate the prognostic value of baseline hormone expression at the time of diagnosis with recurrence of breast cancer, overall survival (OS), disease-free survival (DFS) and death from breast cancer.

CONCLUSION:

Patients present a high variety of ER, PR, and Her-2 receptors expression as breast cancer sybtypes (BCSs); overexpression of Her-2 receptor and negative ER-receptor expression were identified as breast cancer patients at high risk who might resist to future hormone therapy, and the difference was statistically important with stage. Triple positive breast cancer (TP) were presented with lymph nodes more than 4, and triple negative breast cancer (TN) presents another challenge in decision of protocol of treatment.

ACKNOWLEDGMENTS:

We thank Laboratory of Pathology-Lattakia- Syria and for National Hospital of Jableh (Jableh-Lattakia-Syria).

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Received on 09.11.2017 Modified on 29.12.2017

Research J. Pharm. and Tech 2018; 11(8): 3703-3710.

DOI: 10.5958/0974-360X.2018.00680.7