Article Figures & Data
Figures
- Figure 1.
Comparison of the sensitivity and specificity of prognosis by the integrated mRNA–lncRNA signature, the mRNA-only signature, and the traditional clinicopathologic factors in the training (n = 165) and validation (n = 101) sets. Time-dependent ROC curves were plotted to assess the efficacy of the signatures, with AUCs reported. We also calculated the performances of the combined traditional prognostic factors, including tumor size, grade, and number of involved lymph nodes. All factors were coded as categorical variables. Limited by the follow-up time, we could only perform analysis up to two years.
- Figure 2.
Estimates of RFS according to the scores calculated by the integrated mRNA–lncRNA signature and the mRNA-only signature in the training (n = 165) and the validation (n = 101) sets. A, Kaplan–Meier analysis of RFS according to the scores calculated by the integrated mRNA–lncRNA signature and the mRNA-only signature. B, patients were stratified according to the receipt of the taxane-based chemotherapy to validate the interaction between each risk group and the taxane-based chemotherapy using multivariate Cox proportional hazards regression analysis adjusted for clinicopathologic factors in Table 2. The low-risk group was used as reference. The bars represent the HRs in different set and the lines represent the 95% CI. If the limit of 95% CI outranges the scale on the x-axis, the data were shown as arrows.
- Figure 3.
Biologic function of the lncRNAs HIST2H2BC and SNRPEP4 incorporated in the integrated signature. A, cell proliferation was determined by CCK-8 assay after transfection with siRNAs for 48 hours. The results are shown as the percentage of optical density (OD) with negative control (NC) as reference. B, representative light microscopic images of migrated cells through the Transwell chamber (magnification, 100×). The number of migrated cells was calculated and compared between each siRNA with NC. C, effect of lncRNAs on the resistance to paclitaxel. The results were assessed by CCK-8 assay and the relative cell viability was calculated. All results are represented as the mean ± SD from three independent experiments. Notes: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Tables
- Table 1.
Clinicopathologic characteristics of TNBC patients according to the integrated RNA signatures in two sets
Training set Validation set mRNA signature mRNA–lncRNA signature mRNA signature mRNA–lncRNA signature Characteristics N High risk (%) Low risk (%) High risk (%) Low risk (%) N High risk (%) Low risk (%) High risk (%) Low risk (%) Age, y Median 55 50 56 50 56 53.5 53.5 53.5 54 53 IQR 46–61 43–58 49.8–62 43–59 49–61.3 44.8–59 45.5–58.3 44.8–60.3 46.5–59 44–59 ≤50 68 38 (55.9) 30 (44.1) 34 (50.0) 34 (50.0) 39 19 (48.7) 20 (51.3) 20 (51.3) 19 (48.7) >50 97 31 (32.3) 66 (68.8) 31 (32.3) 66 (68.8) 62 26 (41.9) 36 (58.1) 32 (51.6) 30 (48.4) Menopause Yes 101 37 (36.6) 64 (63.4) 35 (34.7) 66 (65.3) 64 26 (40.6) 38 (59.4) 31 (48.4) 33 (51.6) No 64 32 (50.0) 32 (50.0) 30 (46.9) 34 (53.1) 32 14 (43.8) 18 (56.3) 16 (50.0) 16 (50.0) Unknown 0 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 5 5 (100.0) 0 (0.0) 5 (100.0) 0 (0.0) Tumor size, cm ≤2 58 23 (39.7) 35 (60.3) 19 (32.8) 39 (67.2) 36 17 (47.2) 19 (52.8) 19 (52.8) 17 (47.2) >2 104 44 (42.3) 60 (57.7) 45 (43.3) 59 (56.7) 65 28 (43.1) 37 (56.9) 33 (50.8) 32 (49.2) Unknown 3 2 (66.7) 1 (33.3) 1 (33.3) 2 (66.7) 0 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Tumor grade I–II 32 9 (28.1) 23 (71.9) 10 (31.3) 22 (68.8) 31 15 (48.4) 16 (51.6) 18 (58.1) 13 (41.9) III 104 48 (46.2) 56 (53.8) 45 (43.3) 59 (56.7) 70 30 (42.9) 40 (57.1) 34 (48.6) 36 (51.4) Unknown 29 12 (41.4) 17 (58.6) 10 (34.5) 19 (65.5) 0 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Positive LNs ≤3 115 47 (40.9) 68 (59.1) 44 (38.3) 71 (61.7) 82 38 (46.3) 44 (53.7) 44 (53.7) 38 (46.3) >3 50 22 (44.0) 28 (56.0) 21 (42.0) 29 (58.0) 19 7 (36.8) 12 (63.2) 8 (42.1) 11 (57.9) Chemotherapy Taxane 124 52 (41.9) 72 (58.1) 48 (38.7) 76 (61.3) 66 29 (43.9) 37 (56.1) 34 (51.5) 32 (48.5) Non-taxane 27 12 (44.4) 15 (55.6) 12 (44.4) 15 (55.6) 35 16 (45.7) 19 (54.3) 18 (51.4) 17 (48.6) Unknown 14 5 (35.7) 9 (64.3) 5 (35.7) 9 (64.3) 0 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Radiotherapy Yes 50 23 (46.0) 27 (54.0) 21 (42.0) 29 (58.0) 39 19 (48.7) 20 (51.3) 21 (53.8) 18 (46.2) No 103 41 (39.8) 62 (60.2) 41 (39.8) 62 (60.2) 42 18 (42.9) 24 (57.1) 21 (50.0) 21 (50.0) Unknown 12 5 (41.7) 7 (58.3) 3 (25.0) 9 (75.0) 20 8 (40.0) 12 (60.0) 10 (50.0) 10 (50.0) Follow-up time, mo Median 13.9 12.6 14.2 11.7 14.3 18.5 18.5 18.3 18 19.2 IQR 8.6–21.1 8.4–19.3 9.3–22.2 8.2–18.4 9.5–22.6 15–26.2 13.6–26.1 15.1–26.5 13.7–25.7 15.2–27.5 RFS event 22 17 5 17 5 9 7 2 8 1 Abbreviations: IQR, interquartile range; LN, lymph node; mo, months; RFS, recurrence-free survival; y, year.
- Table 2.
Multivariate Cox proportional hazards regression analysis of the derived RNA signatures and traditional characteristics with RFS
mRNA-only signature Integrated mRNA–lncRNA signature Training set Validation set Training set Validation set Variablea HR (95% CI) P HR (95% CI) P HR (95% CI) P HR (95% CI) P Age (≤50 y as reference) 1.06 (0.16–6.95) 0.949 1.29 (0.10–16.09) 0.845 1.01 (0.09–11.11) 0.995 1.18 (0.11–12.90) 0.890 Menopause (no as reference) 0.67 (0.12–3.73) 0.646 0.43 (0.03–5.77) 0.521 0.63 (0.07–5.38) 0.672 0.56 (0.05–6.18) 0.635 Tumor grade (≤II as reference) 1.61 (0.40–6.45) 0.501 0.71 (0.10–5.00) 0.729 1.79 (0.44–7.31) 0.416 0.91 (0.14–6.09) 0.920 Tumor size (≤2 cm as reference) 1.13 (0.34–3.70) 0.846 4.45 (0.48–41.47) 0.189 0.78 (0.20–2.99) 0.712 4.72 (0.51–43.68) 0.171 Positive LNs (≤3 as reference) 1.95 (0.53–7.24) 0.319 2.48 (0.53–11.53) 0.247 2.37 (0.61–9.16) 0.211 3.38 (0.70–16.31) 0.130 Radiotherapy (no as reference) 4.03 (0.93–17.39) 0.062 3.87 (0.64–23.49) 0.141 3.79 (0.83–17.31) 0.086 3.58 (0.63–20.33) 0.151 Chemotherapy (non-taxane as reference) 0.49 (0.15–1.57) 0.228 1.22 (0.25–5.95) 0.804 0.69 (0.17–2.74) 0.593 1.41 (0.27–7.26) 0.683 Signature (low-risk as reference) 4.46 (1.34–14.91) 0.015 6.31 (1.20–33.26) 0.030 10.00 (2.53–39.47) 0.001 14.04 (1.56–126.71) 0.019 Abbreviation: LN, lymph node.
↵aAdjusted by Cox proportional hazards models including age, menopausal status, tumor grade, tumor size, positive lymph nodes, radiotherapy, chemotherapy, and integrated RNA signature.
- Table 3.
Multivariate Cox proportional hazards regression analysis of RFS, including interaction of signatures with adjuvant chemotherapy
mRNA signature mRNA–lncRNA signature Training set Validation set Training set Validation set Variablea HR (95% CI) P HR (95% CI) P HR (95% CI) P HR (95% CI) P Chemotherapy (non-taxane as reference) 0.49 (0.15–1.57) 0.228 1.22 (0.25–5.95) 0.804 0.69 (0.17–2.74) 0.593 1.41 (0.27–7.26) 0.683 Signature (low-risk as reference) 4.46 (1.34–14.91) 0.015 6.31 (1.20–33.26) 0.030 10.00 (2.53–39.47) 0.001 14.04 (1.56–126.71) 0.019 Interaction 1.82 (0.62–5.37) 0.277 4.14 (0.93–18.48) 0.063 5.74 (1.54–21.33) 0.009 4.46 (1.00–19.88) 0.05 ↵aAdjusted by Cox proportional hazards models including clinical variables as categorized in Table 2. Here, we present only three items: chemotherapy, signature, and the interaction between them. Other parameters (age, menopausal status, tumor grade, tumor size, positive lymph nodes and radiotherapy) are not shown.
Supplementary Data
- Supplementary table 1 - Supplementary Table 1. Selected mRNAs and lncRNAs by comparing the expression profiles in 33 paired tumor and adjacent normal tissues of triple-negative breast cancer.
- Supplementary figures - Supplementary Figure 1. Kaplan-Meier estimates of recurrence-free survival (RFS) according to the expression of every ten mRNA/lncRNA incorporated in the integrated signature in the training set (n=165). RNA expression level was coded as high or low according to the median expression level. Supplementary Figure 2. Validation of the expression of each mRNA/lncRNA incorporated in the integrated signature in the 33 paired tumor and normal breast tissues from the training set using quantitative real-time polymerase chain reaction (qRT-PCR). Supplementary Figure 3. Flowchart of the study design, patient selection and analytical strategy. Supplementary Figure 4. Details of the filtration procedures. For lncRNAs, either lncRNAs with fold change >1.5 and P<0.05 in survival analysis or fold change >2 and P<0.1 were included. Supplementary Figure 5. Down regulations of the HIST2H2BC and SNRPEP4 enhanced apoptosis induced by paclitaxel. Supplementary Figure 6. Flow cytometric analyses of cell cycle distribution in control and siRNA knockdown breast cancer cells.
Supplementary Data
- Supplementary table 1 - Supplementary Table 1. Selected mRNAs and lncRNAs by comparing the expression profiles in 33 paired tumor and adjacent normal tissues of triple-negative breast cancer.
- Supplementary figures - Supplementary Figure 1. Kaplan-Meier estimates of recurrence-free survival (RFS) according to the expression of every ten mRNA/lncRNA incorporated in the integrated signature in the training set (n=165). RNA expression level was coded as high or low according to the median expression level. Supplementary Figure 2. Validation of the expression of each mRNA/lncRNA incorporated in the integrated signature in the 33 paired tumor and normal breast tissues from the training set using quantitative real-time polymerase chain reaction (qRT-PCR). Supplementary Figure 3. Flowchart of the study design, patient selection and analytical strategy. Supplementary Figure 4. Details of the filtration procedures. For lncRNAs, either lncRNAs with fold change >1.5 and P<0.05 in survival analysis or fold change >2 and P<0.1 were included. Supplementary Figure 5. Down regulations of the HIST2H2BC and SNRPEP4 enhanced apoptosis induced by paclitaxel. Supplementary Figure 6. Flow cytometric analyses of cell cycle distribution in control and siRNA knockdown breast cancer cells.
Volume 22, Issue 7
