Mirna21 Expression in the Breast Cancer Tumor Tissue is Independent of Neoadjuvant Chemotherapy

Figures & data

Table 1 Clinical and Pathological Characteristics of Patients with Breast Cancer

Variables	All Patients (n=55)	Chemo Naïve (n=27)	NAC (n=28)	p-value
Mean ± SE
Age (y.o.)	54.2±1.9	54.3±2.8	48.1±2.1	0.001
Pathological tumor size (cm)	3.44±0.31	4.00±0.44	2.89±0.41	0.087
Clinical tumor size (cm)	4.67±0.31	3.56±0.37	5.75±0.43	0.001
Frequency n (%)
Histologic types				0.589
Tubular carcinoma	1 (1.9)	1 (3.7)	-
NST	52 (94.5)	25 (92.6)	27 (96.4)
ILC	2 (3.8)	1 (3.7)	1 (3.6)
Molecular subtypes				0.216
Luminal A	7 (12.7)	6 (22.2)	1 (3.6)
Luminal B	28 (50.9)	12 (44.5)	16 (57.1)
HER-2	12 (21.8)	5 (18.5)	7 (25.0)
Triple-negative	8 (14.6)	4 (14.8)	4 (14.3)
TNM staging				0.002
I	9 (16.4)	8 (29.6)	1 (3.6)
II	29 (52.7)	16 (59.3)	13 (46.4)
III	17 (30.9)	3 (11.1)	14 (50.0)
Grade				0.338
1	4 (8.4)	3 (11.1)	1 (4.3)
2	22 (45.8)	14 (51.9)	9 (39.1)
3	22 (45.8)	10 (37.0)	13 (56.5)
Lymph node involvement				0.995
None	35 (63.6)	17 (63.0)	18 (64.3)
1 to 3	14 (25.5)	7 (25.9)	7 (25.0)
≥ 4	6 (10.9)	3 (11.1)	3 (10.7)

Notes: Clinical and pathological characteristics of 55 patients with breast cancer analyzed in this study (27 chemo naïve and 28 treated with NAC). Statistical analysis comparing chemo naïve versus NAC was performed for age, clinical and pathologic tumor size, histological types and grades, molecular subtypes, TNM staging system and axillary status using the Mann–Whitney U-test and Pearson’s chi-squared test. Patients treated with NAC were younger and presented with larger tumors and more advanced disease (TNM stages II and III). Most of the patients in both groups presented NST and luminal A and B molecular breast cancer subtypes. No difference was observed between the groups in terms of axillary status.

Abbreviations: NST, invasive carcinoma of nonspecial type; ILC, invasive lobular carcinoma; HER-2, HER-2 overexpression; TNM, tumor-node-metastasis breast cancer staging; NAC, neoadjuvant chemotherapy.

Figure 1 Patterns of miRNA-21 Expression in Breast Cancer Tissue. (A) miRNA-21 expression in tumor tissue and in breast tissue from controls. miRNA-21/RNU6B was calculated using the 2^−ΔΔCt method. The expression values are presented as the ΔΔCq value on a log2 scale. (B) Absolute quantification of miRNA-21 expression, presented as the means of the Cq values. Statistical analysis was performed using a generalized linear model. Data are presented as the mean ± SE. Black dots represent outliers.

Table 2 miRNA-21 Expression According to Breast Cancer Prognostic Factors

Breast Cancer Prognostic Factors	miRNA-21/RNU6B	miRNA-21/Cq
Molecular subtypes
Control	1.76±0.50	25.0±1.0
Luminal A	6.03±1.89 ^a	19.6±0.9 ^a
Luminal B	3.60±0.61 ^a	19.6±0.5 ^a
HER-2	6.03±1.44 ^a	20.1±0.7 ^a
Triple-negative	4.07±1.19	18.1±0.8 ^a
p-value	0.021	<0.001
TNM staging
Control	1.76±0.50	25.0±1.0
Stage I	4.52±1.28 ^a	19.2±0.8 ^a
Stage II	4.33±0.73 ^a	19.0±0.4 ^a
Stage III	4.99±1.06 ^a	20.6±0.6 ^a
p-value	0.02	<0.001
Grade
Control	1.76±0.50	25.0±1.0
Grade 1	4.35±2.11	19.4±1.1 ^a
Grade 2	3.91±0.71 ^a	19.8±0.5 ^a
Grade 3	5.04±0.93 ^a	18.5±0.4 ^a
p-value	0.019	<0.001
Lymph node involvement
Control	1.76±0.50	25.0±1.0
None	5.15±0.78 ^a	19.9±0.4 ^a
1 to 3	6.06±1.51 ^a	18.8±0.6 ^a
≥ 4	5.34±1.96	18.6±0.9 ^a
p-value	0.007	<0.001

Notes:^ap<0.05 versus control. Upregulated levels of miRNA-21 were observed for all patients with breast cancer in comparison to the control group, regardless of molecular subtypes, TNM staging, grade and lymph node involvement. The expression of miRNA-21 was calculated using the 2^−ΔΔCt method (miRNA-21/RNU6B) and means of the absolute miRNA-21 Cq values (miRNA-21/Cq). Statistical analysis was performed using a generalized linear model, and the values are presented as the mean ± SE. ^ap<0.05 versus control.

Figure 2 Correlation of miRNA-21 Expression with Tumor Size and Response to Neoadjuvant Chemotherapy. (A) Correlation between miRNA-21 and tumor size (cm) (Spearman’s rank correlation coefficient). (B) miRNA-21 expression considering exposure to NAC (or not) and achievement of PCR (miRNA-21/RNU6B was calculated using the 2^−ΔΔCt method). The expression values are presented as the ΔΔCq value on a log2 scale (generalized linear model) (C) miRNA-21 expression based on the means of the Cq values and considering exposure to NAC and achievement of PCR (generalized linear model). Data are presented as the mean ± SE.

Abbreviations: PCR, pathological complete response; NAC, neoadjuvant chemotherapy; Naïve, chemotherapy naïve.

Table 3 Prognostic Factors and miRNA-21 Expression in Patients Treated with Neoadjuvant Chemotherapy

Breast Cancer Prognostic Factors	Controls	Naïve	NAC	p-value	Controls	Naïve	NAC	p-value
	miRNA-21/RNU6B				miRNA-21/Cq
Molecular subtypes				0.044				<0.001
Control	1.76±0.50				25.0±1.0	-	-
Luminal A	–	6.37±2.17 ^a	–		–	19.3±0.9 ^a	-
Luminal B	–	4.08±1.13 ^a	3.30±0.71 ^a		–	18.3±0.6 ^a	20.6±0.6 ^a, b
HER-2	–	6.16±2.29 ^a	5.95±1.87 ^a		–	18.1±0.9 ^a	21.5±0.9 ^a, b
Triple-negative	–	3.56±1.71	4.37±1.62		–	18.2±1.2 ^a	18.2±0.9 ^a
TNM staging				0.008				<0.001
Control	1.76±0.50	–	–		25.0±1.0	–	–
Stage I	-	4.69±1.36 ^a	–		-	19.2±0.8 ^a	–
Stage II	-	5.71±1.30 ^a	2.97±0.67 ^b		-	18.2±0.5 ^a	20.0±0.6^a,b
Stage III	-	2.24±1.06	5.64±1.28 ^a, b		-	18.2±1.2^a	21.1±0.6 ^a, b
Grade								<0.001
Control	1.76±0.50	–	–		25.0±1.0	–	–
Grade 1	–	1.57±0.65	–		–	17.3±0.9 ^a	–
Grade 2	–	1.42±0.27	0.62±0.15		–	19.0±0.5 ^a	26.0±2.4 ^a, b
Grade 3	–	1.35±0.31	1.11±0.22		–	18.1±0.5 ^a	21.2±0.6 ^a
Lymph node involvement				0.031				0.002
Control	1.76±0.50	–	–		25.0±1.0	–	–
None	-	6.29±1.34 ^a	4.08±0.84 ^a		–	18.4±0.5 ^a	21.4±0.5 ^a
1 to 3	-	7.21±2.58 ^a	5.08±1.74		–	18.8±0.8 ^a	18.8±0.8 ^a
≥ 4	–	3.09±1.53	7.61±3.84		–	18.0±1.1 ^a	19.2±1.2 ^a

Notes:^ap<0.05 versus control and ^bversus naïve. Comparison of miRNA-21 expression in the tumor tissue of patients with breast cancer treated or not with NAC and control samples was performed for prognostic factors: molecular subtypes, TNM staging, grade and lymph node involvement. The expression of miRNA-21 was calculated utilizing the 2^−ΔΔCt method and the means of the absolute miRNA-21 Cq values. Statistical analysis was performed using a generalized linear model. Values are presented as the mean ± SE. ^ap<0.05 versus control and ^bversus naïve.