Triple Negative Breast Cancer (TNBC) Prognostic Value of Micro RNA-454 Expression for Overall Survival

Breast cancer is the most common type of cancer and the primary cause of death among women who have been diagnosed with cancer. Luminal A, Luminal B, HER2/neu+, and Triple Negative Breast Cancer are the four subtypes that are used to classify breast cancer. Currently, there is no specific targeted therapy for triple-negative breast cancer (TNBC), resulting in a brief median recurrence time and unfavorable prognosis. Recent studies have demonstrated that miRNA-454 plays an oncogenic role in several different malignancies, and this role is associated with the growth, invasion, and metastasis of tumors. We analyzed miRNA-454 expression in 50 paraffin-embedded tissue samples from TNBC patients at Dr. Sardjito Hospital Yogyakarta utilizing qRT-PCR. The purpose of this research was to determine whether or not the expression of miRNA-454 is useful to forecast survival in patients with stage I-III transformational breast cancer. Methods: This investigation was a retrospective cohort study utilizing data from clinical TNBC patients at Dr. Sardjito Hospital in Yogyakarta from 2014 to 2017. Fifty patients were incorporated into the analysis. Data on clinicopathological features were documented. The results of the univariate analysis showed that patients with high miRNA-454 expression had a higher probability of dying within two years compared to those with low miRNA-454 expression (79.2% versus 53.8%; HR = 4.334; P = 0.037). Multivariate analysis indicated that elevated miRNA-454 expression heightened the 2-year mortality risk, with a hazard ratio of 3.086 (95% CI: 1.082-8.806; p=0.035). Conclusions: The expression of MiRNA 454 serves as an independent prognostic indicator for survival in patients with triple-negative breast cancer (TNBC). Patients with triple-negative breast cancer with elevated miRNA-454 expression demonstrate a reduced survival rate. The expression of miRNA-454 is also a factor that can be used to predict the therapeutic response in TNBC.

Introduction

Breast cancer ranks highest among most cancers in women, with an incidence of 19.18/100,000 population and a mortality of 12.75/100,000 population [1]. Triple-negative (TNBC), HER2/neu+, Luminal A, and Luminal B are the four molecular subtypes of breast cancer. Triple-negative breast cancer (TNBC) is defined by the lack of estrogen receptors (ER), progesterone receptors (PR), and HER2 receptors in the tumors. Triple-negative breast cancer is a diverse condition. This has become a clinical challenge because there has not yet been a targeted therapy for TNBC [2].

MicroRNA regulates various biological processes, including cell survival, apoptosis, and invasion, to induce TNBC cells. Various miRs exhibit differential expressions in TNBC, suggesting that miR may be a potential prognostic biomarker for breast cancer. Identifying the appropriate prognostic biomarker for TNBC patients enables the selection of therapeutic regimens that might offer significant advantages to patients [3].

The miR is an endogenous RNA composed of 23 nucleotides that serve as a crucial gene regulator by binding to mRNA protein-coding genes and directly inhibiting post-transcriptional proteins. Multiple studies indicate that miR-454 expression is associated with tumor proliferation, invasion, and metastasis in many malignancies. In hepatocellular carcinoma, the suppression of miR-454 suppresses cell proliferation, invasion, and epithelial-mesenchymal transition [4]. Four Elevated miR-454 expression induces substantial cell proliferation, colony formation, invasion, and cell cycle progression in uveal melanoma cells [5]. Five Gliomas exhibiting strong miR-454 expression had a much poorer prognosis than those with low miR-454 expression. These findings indicate that miR-454 may be a novel predictive biomarker [6]. Nevertheless, limited research has assessed the correlation between miR-454 and clinical outcomes in breast cancer. A study indicated that miR-454-3p regulation was elevated in breast cancer [7].

Materials and Method

This retrospective cohort study utilized clinical data from TNBC patients diagnosed between 2014 and 2017. The initial data collection of TNBC patients was conducted at the Tulip Cancer Centre, Dr. Sardjito Hospital Yogyakarta, against TNBC patients diagnosed and undergoing treatment between January 1, 2014, and December 2017. The follow-up period was extended until December 2019. The paraffin block data was collected at the Pathology Anatomy Dr. Sardjito Hospital, FKKMK UGM, Waskita, and CITO Laboratory. The qRT-PCR analysis was performed to evaluate miR-454 expression at the Pathology Anatomy Laboratory FKKMK UGM.

The study subjects were patients diagnosed with non-metastatic TNBC after surgery who underwent chemotherapy treatment at the Tulip Cancer Centre Dr. Sardjito Hospital Yogyakarta between 1 January 2014 and December 2017 and met the research criteria.

Data analysis was conducted with univariate, bivariate, multivariate, and survival analysis using SPSS 22 software. Univariate analysis is presented with frequency distribution, mean ± standard deviation (SD), or median. Bivariate analysis between clinicopathological variables with miR-454 expression using the Chi-Square or Fisher Exact test.

Results

Patient Clinical Characteristics and minRNA Expression of 454

The data for 1683 breast cancer patients diagnosed at Dr. Sardjito Hospital Yogyakarta between 2014 and 2017 were retrieved from the Cancer Registry. Out of 106 patients, 50 were deemed eligible to participate in the trial. Fifty patients with TNBC had their miR-454 expression studied on paraffin tissue blocks, and the results showed a 2-year survival rate.

The survival of the study subjects was monitored until December 2019. The median follow-up duration was 29.46 months (95% CI: 27.11–31.81). The average survival duration for 50 subjects was 32.18 months (about 2 and a half years), with a 2-year survival rate of 66%.

The median age of the 50 subjects was 50. Twenty-five (50%) TNBC patients were younger than 50. Out of the 50 subjects, 22 (44%) had an overweight body mass index (Table I).

Table I. Baseline Characteristics of Research Subjects

Variables	Frequency (%)
Age (years)
≤50 years old	25 (50)
>50 years old	25 (50)
Body mass index
Underweight (BMI ≤ 18.5)	6 (12)
Normoweight (BMI 18.5–24.9)	22 (44)
Overweight (BMI ≥ 25)	22 (44)
Histology grade
1 or 2	11 (22)
3	32 (64)
Not known	7 (14)
Tumor size (T)
≤2 cm	1 (2)
>2–≤5 cm	13 (26)
>5 cm	36 (72)
Lymph node involvement (N)
Negative	17 (34)
Positive	33 (66)
Clinical stage
II	16 (32)
III	34 (68)
Chemotherapy
Anthracycline based	35 (70)
Non-anthracycline based	12 (24)
Not known	3 (6)

Most patients had high-grade tumors, 32 (64%) subjects. Most patients were a tumor size >5 cm, 36 (72%) subjects. Most patients had positive lymph nodes, 36 (64%). Most patients were with stage III, 33 (66%) subjects. Most patients received anthracycline-based chemotherapy, 35 (70%) subjects (Table I).

The clinicopathological features and the expression of miR-454 did not differ significantly (Table II).

Table II. Relationship Between miR-454 Expression and Baseline Characteristics

Variable	Total	miR-454 expression		p-value
		Low N (%)	High N (%)
Age (years)
≤50 years old	25	12 (48)	13 (52)	1.00
>50 years old	25	12 (48)	13 (52)
Body mass index
Underweight (BMI ≤ 18.5)	6	4 (67)	2 (33)	0.621
Normoweight (BMI 18.5–24.9)	22	10 (45)	12 (55)
Overweight (BMI ≥ 25)	22	10 (45)	12 (55)
Histology grade
1 or 2	11	6 (55)	5 (45)	0.870
3	32	15 (47)	17 (53)
Not known	7	3 (43)	4 (57)
Tumor size (T)
≤2 cm	1	1 (100)	0 (0)	0.486
>2 cm but ≤5	13	7 (54)	6 (46)
>5 cm	36	16 (44)	20 (56)
Lymph node involvement (N)
Negative	17	8 (47)	9 (53)	0.924
Positive	33	16 (48)	17 (52)
Clinical stage
II	16	9 (56)	7 (44)	0.423
III	34	15 (44)	19 (56)
Type of chemotherapy
Anthracycline based	35	14 (40)	21 (60)	0.097
Non-anthracycline based	12	7 (58.3)	5 (41.7)
Not known	3	3 (100)	0 (0)

The Relationship Between miR-454 Expression and Clinical Outcomes in Patients with Triple-Negative Breast Cancer (TNBC)

In this study of 50 TNBC patients, 17 patients (34%) succumbed, while 33 patients (66%) remained alive. The median overall survival rates at two years were not determined due to the number of deaths not reaching 50% of cases (Fig. 1).

Fig. 1. Kaplan-Meier survival curves over 2 years.

The survival analysis utilizing the log-rank test indicated that elevated expression of miR-454 in tumor tissue correlates with a statistically significant increase in 2-year mortality risk (HR = 4.334; p = 0.037). This study reports a 2-year survival rate of 79.2% for TNBC patients exhibiting low miR-454 expression, compared to 53.8% for those with high miR-454 expression (p = 0.037) (Fig. 2).

Fig. 2. Kaplan-Meier curve miRNA-454 expression and 2-year survival: miR-454 expression with 2 years overall survival: Low miR-454 expression: 79.29%, High miR-454 expression: 53.8%, and p = 0.037.

Overall survival in TNBC patients was also analyzed with other prognostic variables including age, body mass index, histology grade, tumor size, lymph node involvement, clinical stage, and type of chemotherapy (Fig. 3).

Fig. 3. Kaplan-Meier curve of overall survival for two years and lymph node involvement.

The research revealed that there were variations in the 2-year overall survival based on the clinical stage and lymph node involvement variables (p = 0.027 and p = 0.042). Involvement of lymph nodes and survival outcomes Two-year survival Negative lymph nodes: 88.2%; Positive lymph nodes: 54.5%; p = 0.027.

Univariate and Multivariate Analysis of Prognostic Factors in TNBC Patients

Lymph nodes positive compared to lymph nodes negative had HR 4.556 (95% CI: 1.040–19.948, p = 0.044), and clinical stage III compared to clinical stage II had HR 4.101 (95% CI: 0.937–17.956; p = 0.061) (Table III).

Table III. Univariate Analysis of 2-Year Overall Survival Prognostic Factors

Variable	Overall survival (%)	p-value
Age (years)		0.330
≤50 years old	60%
>50 years old	72%
Body mass index		0.663
Underweight (BMI ≤ 18.5)	83.3%
Normal weight (BMI 18.5–24.9)	59.1%
Overweight (BMI ≥ 25)	68.2%
Histology grade		0.752
1 or 2	72.7%
3	62.5%
Not known	71.4%
Tumor size		0.379
≤2 cm	0%
>2 cm but ≤5	76.9%
>5 cm	63.9%
Lymph node involvement (N)		0.027
Negative	88.2%
Positive	54.5%
Clinical stage		0.042
I–II	87.5%
III	55.9%
Types of chemotherapy		0.101
Anthracycline based	57.1%
Non anthracycline based	91.7%
Not known	66.7%
miR-454 expression		0.037
Low	79.2%
High	53.8%

The multivariate analysis using Cox regression is displayed in Table IV. The multivariate analysis results indicated that lymph node involvement was inconsistent and significantly impacted the survival of patients with triple-negative breast cancer, yielding a significance value of 0.044 and a hazard ratio of 4.556 (95% CI = 1.040–19.948). Furthermore, the expression of Micro RNA 454 exhibited variability, significantly impacting the survival of patients with triple-negative breast cancer, with a p-value of 0.047 and a hazard ratio of 2.888 (95% CI = 1.014–8.221).

Table IV. The Hazard Ratio of 2-Year Overall Survival Prognostic Factors

Variable	Overall survival
	HR (95% CI)	p-value
Age	0.621 (0.236–1.634)	0.335
Body mass index	1.149 (0.561–2.355)	0.704
Histology grade	1.085 (0.499–2.358)	0.837
Tumor size	1.109 (0.422–2.915)	0.833
Lymph node involvement	4.556 (1.040–19.948)	0.044
Clinical stage	4.101 (0.937–17.956)	0.061
Type of chemotherapy	0.400 (0.127–1.254)	0.116
MiR-454 expression	2.888 (1.014–8.221)	0.047

Discussion

The average survival duration for 50 participants with TNBC in this research was 32.895 months (about 2 and a half years), with a 2-year survival rate of 66%. The American Cancer Society reports that the five-year survival rate for individuals with triple-negative breast cancer (TNBC) is 65%. In Malaysia, research indicates that TNBC subtypes are associated with a diminished prognosis, exhibiting a 5-year survival rate of 76% and a mean survival length of 4.5 years (4,015–4,977). In Malaysia, research indicates that TNBC subtypes are associated with an unfavorable prognosis, with a 5-year survival rate of 76% and a mean survival length of 4.5 years (4,015–4,977). [8]. In Australia, the 5-year survival rate for TNBC patients was 72%, with a median time to death of 3.55 years; 92% of fatalities occurred within 5 years. Recurrence may manifest early (median 1.6 years), with 74% of patients developing distant metastases during their initial recurrence. The local recurrence rate is 4.5%, signifying a low occurrence [9].

In our analysis, most patients exhibited high-grade tumor histology (32 patients, 64%), big tumor size (36 patients, 72%), lymph node involvement (36 patients, 64%), and advanced clinical stage (33 patients, 66%). Research has shown that, in comparison to other subtypes, patients with TNBC displayed the following characteristics: a higher percentage of individuals under 35 years (13.98% vs 5.18%, p = 0.012), an increased incidence of familial breast cancer history (11.49% vs 4.54%, p = 0.031), a greater occurrence of tumors measuring 5 cm in diameter (12.73% vs 5.49%, p = 0.44), a larger proportion of patients with positive lymph nodes (64.6% vs 48%, p = 0.011), a higher prevalence of tumors classified as clinical stage III (27.95% vs 14.25%, p = 0.007), and an increased rate of tumors with histological grade 3 (21.43% vs 11.70%, p = 0.028) [10].

This research revealed clinically significant survival rates associated with lymph node involvement (2-year survival rate for N negative: N positive = 88.2%: 54.5%, p = 0.027) and clinical stage (2-year survival rate for stage II: stage III = 87.5%: 55.9%, p = 0.042). The hazard ratios were HR 4.556 (95% CI: 1.040–19.948, p = 0.044) and HR 4.101 (95% CI: 0.937–17.956; p = 0.061) for the occurrence of death, respectively. Reported lymph node involvement related to recurrence-free period (5-year recurrence-free period for N0–1: N2–3 = 83.9%: 67.5%, p = 0.004), survival rate (5-year survival rate for N0–1: N2–3 = 91.8%: 78.6%, p = 0.038), and distant metastasis-free period (5-year distant metastasis-free period for N0–1: N2–3 = 87.3%: 73.1%, p = 0.002). Histology grade has no significant relationship with recurrence-free period, survival rate, or distant metastasis-free period [11]. Individuals exhibiting nodal involvement demonstrate an elevated mortality risk associated with breast cancer (HR = 5.64, p = 0.008). The tumor size and clinical stage variables did not demonstrate statistically significant effects on survival rate (HR = 3.13, 95%CI: 0.30–32.24, p = 0.337; HR = 2.41, 95%CI: 0.33–17.25, p = 0.381) [12]. A study involving 1601 breast cancer patients, including 180 with the TNBC subtype, and a follow-up period of 8.1 years demonstrated no correlation between tumor size and nodal status among TNBC patients. Small tumors in triple-negative breast cancer (TNBC) exhibit significant nodal positivity; 55% of women with tumors measuring ≤1 cm have at least one positive lymph node [13].

Analyzed the correlation between miR-454 expression and clinical prognosis to assess the clinical significance of microRNA in breast cancer. Comparable findings regarding the association between miR-454 and survival rates in the TNBC subtype were observed. [9]. Our study found that the 2-year survival rate for TNBC patients with low miR-454 expression was 79.2%, while for those with high miR-454 expression, it was 53.8%. Research by Cao Z.G. et al. and our study demonstrated that elevated miR-454 expression correlates with poorer clinical outcomes in TNBC. MiR-454 serves as an independent and effective prognostic factor for the clinical outcomes in triple-negative breast cancer (TNBC). Demonstrated that miR-454 is significantly expressed in the tumor tissue of patients with triple-negative breast cancer (TNBC). MiR-454 may serve as a potential prognostic indicator in triple-negative breast cancer (TNBC) [14].

Cell proliferation has been reported to be enhanced by MiR-454, which functions as an oncogenic microRNA by targeting PTEN. PTEN, a primary negative regulator of PI3K activity, is frequently mutated in cancer and is classified as a tumor suppressor gene. PTEN mutations in triple-negative breast cancer (TNBC) are significantly correlated with unfavorable prognosis. The hypothesis posits that miR-454 may inhibit the expression of Smad4 or PTEN by directly binding to the 3′UTR, thereby functioning as an oncogenic microRNA [15].

The PI3K/AKT pathway is critical in various biological processes, regulating downstream pathways such as cell proliferation, apoptosis, and the cell cycle. The activation of the PI3K/AKT pathway in TNBC has been attributed to mutations in the PTEN gene. PTEN is recognized as the second most frequently mutated tumor suppressor gene in cancer and is crucial for regulating proliferation, apoptosis, and cell cycles in various tumor cell types. Recent studies indicate that the PI3K/AKT pathway is a critical mechanism through which PTEN exerts its antioncogenic function. [16]. The findings suggest that miR-454 facilitates the proliferation of TNBC cells through the modulation of the PI3K/AKT signaling pathway. MiR-454 functions as an oncogene, influencing tumorigenesis and the malignant progression of triple-negative breast cancer (TNBC) [17]. MiR-454 is implicated in forecasting the response to various chemotherapy agents. Previous studies indicate that microRNA plays a role in the mechanism of chemoresistance by affecting target genes or signaling pathways [18].

The prevalence of BRCA1 and BRCA2 germline mutations in patients with TNBC is only approximately 10%–15%. Data indicates a significantly higher proportion of TNBC patients with somatic mutations in this gene, which is associated with the loss of homologous recombination. This condition exhibits several characteristics typical of sporadic TNBC, resembling tumors with BRCA1/2 mutations, a phenomenon called “BRCAness”. In tumors exhibiting BRCA1/2 deficiency, whether due to germline or somatic mutations, homologous recombination is impaired due to the loss of proteins critical for DNA repair [19].

Numerous studies demonstrate that the response to DNA damage is significantly influenced by PTEN and BRCA1. The activation of AKT exhibits an inverse relationship with BRCA1 expression in breast cancer. In contrast, BRCA1 deficiency may also activate the oncogenic signaling pathway AKT [20]. Examining the interplay between BRCA1 and PTEN will enhance the formulation of agents and strategies for improved cancer treatment.

Elevated levels of microRNAs, including miR-146a, miR-146b-5p, miR-182, and miR-181a/b, have been shown to reduce the control of BRCA1 expression. This decrease affects BRCA1-mediated proliferation and homologous recombination, which are associated with susceptibility to cisplatin and PARP inhibitors by compromising the repair processes for double-strand DNA damage, thereby acting as a hallmark of “BRCAness.” [21].

Conclusion

The expression of MiR-454 is an independent prognostic factor for the 2-year survival rate of non-metastatic TNBC patients. Patients with triple-negative breast cancer exhibiting elevated miR-454 expression demonstrate a reduced 2-year survival rate relative to those with lower miR-454 expression levels. The expression MiR-454 may serve as a predictive factor for therapeutic response in triple-negative breast cancer (TNBC).