The expression pattern of FAM19A5 in thyroid cancer patients
Through using R software, clinical data of 513 patients were collected from the TCGA database, including the patient’s age, gender, histological type, clinical stage, and TNM classification, as well as radiation therapy, residual tumor, vital status, sample type overall survival and disease-specific survival (Table 1). Subsequently, in order to analyze the expression pattern of FAM19A5, we analyzed the expression of FAM19A5 in 52 healthy individuals and 513 patients. As shown in Fig. 1, FAM19A5 expression was significantly higher in tumor tissues than that in normal tissues (P < 2.2e-16). In addition, differences in FAM19A5 expression was found according to N classification (P = 0.019), sample type (P = 0.0022).
Table 1
baseline data of thyroid cancer patients
characteristics | Numbers of cases |
age | |
< 50 | 286(55.75) |
>=50 | 227(44.25) |
gender | |
FEMALE | 373(72.71) |
MALE | 140(27.29) |
histological_type |
NA | 9(1.75) |
Classical/usual | 366(71.35) |
Follicular | 102(19.88) |
Tall | 36(7.02) |
stage | |
NA | 2(0.39) |
I | 289(56.34) |
II | 52(10.14) |
III | 113(22.03) |
IV | 57(11.11) |
T_classification |
T1 | 143(27.88) |
T2 | 169(32.94) |
T3 | 176(34.31) |
T4 | 23(4.48) |
TX | 2(0.39) |
N_classification |
N0 | 230(44.83) |
N1 | 233(45.42) |
NX | 50(9.75) |
M_classification |
NA | 1(0.19) |
M0 | 286(55.75) |
M1 | 9(1.75) |
MX | 217(42.3) |
radiation_therapy |
NA | 20(3.9) |
NO | 178(34.7) |
YES | 315(61.4) |
residual_tumor |
NA | 32(6.24) |
R0 | 393(76.61) |
R1 | 54(10.53) |
R2 | 4(0.78) |
RX | 30(5.85) |
vital_status | |
DECEASED | 16(3.12) |
LIVING | 497(96.88) |
sample_type |
Metastatic | 8(1.56) |
Primary Tumor | 505(98.44) |
FAM19A5 | |
high | 78(15.2) |
low | 435(84.8) |
Diagnostic significance of FAM19A5 expression in thyroid cancer
To evaluate the diagnostic significance of FAM19A5 expression, we established ROC curve using the expression value of thyroid cancer patients and healthy individuals from TCGA. We found that FAM19A5 expression had diagnostic value overall (AUC = 0.914; Fig. 2A). Subsequently, we analyzed the diagnostic value of FAM19A5 expression in different stages of thyroid cancer, including stage I (AUC = 0.923; Fig. 2B), stage II (AUC = 0.918; Fig. 2C), stage III (AUC = 0.895; Fig. 2D) and stage IV (AUC = 0.904; Fig. 2E).
Correlation between FAM19A5 expression and clinical characteristics of thyroid cancer.
To further analyze the correlation between FAM19A5 expression and clinical characteristics, we divided patients into two groups (high expression and low expression of FAM19A5 expression) according to the ROC curve threshold. As shown in Table 2, the chi-square test indicated low FAM19A5 expression was significantly associated with histological type (P = 0.011), stage, (P = 0.020) T classification (P = 0.019), M classification (P = 0.014), and vital status (P = 0.030).
Table 2
the association between FAM19A5 and clinical parameters
| | | FAM19A5 mRNA expression | | |
parameter | variable | N | high | % | low | % | χ2 | p-value |
age | < 50 | 286 | 42 | (53.85) | 244 | (56.09) | 0.060 | 0.807 |
| >=50 | 227 | 36 | (46.15) | 191 | (43.91) | | |
gender | FEMALE | 373 | 52 | (66.67) | 321 | (73.79) | 1.353 | 0.245 |
| MALE | 140 | 26 | (33.33) | 114 | (26.21) | | |
histological_type | Classical/usual | 366 | 49 | (63.64) | 317 | (74.24) | 9.068 | 0.011 |
| Follicular | 102 | 25 | (32.47) | 77 | (18.03) | | |
| Tall | 36 | 3 | (3.9) | 33 | (7.73) | | |
stage | I | 289 | 37 | (47.44) | 252 | (58.2) | 11.090 | 0.020 |
| II | 52 | 16 | (20.51) | 36 | (8.31) | | |
| III | 113 | 17 | (21.79) | 96 | (22.17) | | |
| IV | 57 | 8 | (10.26) | 49 | (11.32) | | |
T_classification | T1 | 143 | 14 | (17.95) | 129 | (29.66) | 11.594 | 0.019 |
| T2 | 169 | 36 | (46.15) | 133 | (30.57) | | |
| T3 | 176 | 22 | (28.21) | 154 | (35.4) | | |
| T4 | 23 | 6 | (7.69) | 17 | (3.91) | | |
| TX | 2 | 0 | (0) | 2 | (0.46) | | |
N_classification | N0 | 230 | 38 | (48.72) | 192 | (44.14) | 1.265 | 0.490 |
| N1 | 233 | 31 | (39.74) | 202 | (46.44) | | |
| NX | 50 | 9 | (11.54) | 41 | (9.43) | | |
M_classification | M0 | 286 | 33 | (42.31) | 253 | (58.29) | 8.161 | 0.014 |
| M1 | 9 | 3 | (3.85) | 6 | (1.38) | | |
| MX | 217 | 42 | (53.85) | 175 | (40.32) | | |
radiation_therapy | NO | 178 | 22 | (29.33) | 156 | (37.32) | 1.429 | 0.232 |
| YES | 315 | 53 | (70.67) | 262 | (62.68) | | |
residual_tumor | R0 | 393 | 54 | (75) | 339 | (82.89) | 2.617 | 0.319 |
| R1 | 54 | 11 | (15.28) | 43 | (10.51) | | |
| R2 | 4 | 1 | (1.39) | 3 | (0.73) | | |
| RX | 30 | 6 | (8.33) | 24 | (5.87) | | |
vital_status | DECEASED | 16 | 6 | (7.69) | 10 | (2.3) | 4.708 | 0.030 |
| LIVING | 497 | 72 | (92.31) | 425 | (97.7) | | |
sample_type | Metastatic | 8 | 0 | (0) | 8 | (1.84) | 0.505 | 0.477 |
| Primary Tumor | 505 | 78 | (100) | 427 | (98.16) | | |
Effect of FAM19A5 expression on overall survival in thyroid cancer
We performed survival analysis to evaluate the effect of FAM19A5 expression on the overall survival of thyroid cancer patients. As shown in Fig. 3, Kaplan-Meier curves showed that the patients of high FAM19A5 expression significantly had the poor overall survival (P = 0.013). Subgroups analysis found that high FAM19A5 expression significantly affects patient OS in stage III (P = 0.0043), stage III/IV (P = 0.0022), T3 (P < 0.0001), M0 (P = 0.038) and older (P = 0.018).
High FAM19A5 expression is an independent risk factor for patient’s prognosis.
To assess the prognostic significance of FAM19A5 expression in thyroid cancer, univariate Cox analysis and multivariate Cox analysis were performed. We selected potential variables that make sense in univariate analysis to conduct multivariable Cox analysis (Table 3). We found that high FAM19A5 expression is an independent risk factor for poor OS (hazard ratio [HR] = 2.93, 95% confidence interval [CI]:1.06–8.09, P = 0.039).
Table 3
univariate and multivariate analysis of overall survival
| Overall Survival |
| Univariate analysis | Multivariate analysis |
Parameters | Hazard Ratio | CI95 | Pvalue | Hazard Ratio | CI95 | Pvalue |
age | 1.2E + 09 | 0-Inf | 0.997 | | | |
gender | 1.98 | 0.72–5.47 | 0.189 | | | |
histological_type | 0.4 | 0.12–1.38 | 0.147 | | | |
stage | 2.53 | 1.59–4.05 | 0.000 | 2.12 | 1.22–3.67 | 0.007 |
T_classification | 2.61 | 1.4–4.86 | 0.003 | 1.31 | 0.68–2.51 | 0.423 |
N_classification | 0.91 | 0.44–1.86 | 0.793 | | | |
M_classification | 2.12 | 0.86–5.23 | 0.104 | | | |
radiation_therapy | 0.62 | 0.26–1.5 | 0.293 | | | |
residual_tumor | 1.67 | 1.1–2.54 | 0.016 | 1.25 | 0.77–2.03 | 0.374 |
FAM19A5 | 3.36 | 1.22–9.25 | 0.019 | 2.93 | 1.06–8.11 | 0.039 |
Effect of FAM19A5 expression on disease specific survival in thyroid cancer
We performed survival analysis to evaluate the effect of FAM19A5 expression on the disease specific survival of thyroid cancer patients. As shown in Fig. 4, Kaplan-Meier curves showed that the patients of high FAM19A5 expression significantly had the poor disease specific survival (P = 0.0021). Subgroups analysis found that high FAM19A5 expression significantly affects patient DSS in stage III (P = 0.00056), stage III/IV (P = 0.00085), T3 (P < 0.0001), male (P = 0.004) and older (P = 0.00081).
High FAM19A5 expression is an independent risk factor for patient’s prognosis.
To assess the prognostic significance of FAM19A5 expression in thyroid cancer, univariate Cox analysis and multivariate Cox analysis were performed. We selected potential variables that make sense in univariate analysis to conduct multivariable Cox analysis (Tables 3 and 4). We found that high FAM19A5 expression is an independent risk factor for poor OS (hazard ratio [HR] = 6.5, 95% confidence interval [CI]:1.44–29.48, P = 0.015).
Table 4
univariate and multivariate analysis of disease specific survival
| Disease Specific Survival |
| Univariate analysis | Multivariate analysis |
| Hazard Ratio | CI95 | Pvalue | Hazard Ratio | CI95 | Pvalue |
age | 1.1E + 09 | 0-Inf | 0.998 | | | |
gender | 4.07 | 0.9-18.33 | 0.067 | | | |
histological_type | 0.27 | 0.04–1.82 | 0.177 | | | |
stage | 4.19 | 1.67–10.48 | 0.002 | 2.7 | 0.96–7.58 | 0.06 |
T_classification | 7.36 | 2.25–24.07 | 0.001 | 2.66 | 0.79–8.91 | 0.114 |
N_classification | 0.64 | 0.22–1.83 | 0.408 | | | |
M_classification | 2.54 | 0.65–9.98 | 0.182 | | | |
radiation_therapy | 1.4 | 0.29–6.69 | 0.676 | | | |
residual_tumor | 1.78 | 0.99–3.22 | 0.056 | | | |
FAM19A5 | 7.4 | 1.65–33.12 | 0.009 | 6.5 | 1.44–29.48 | 0.015 |
As shown in Table 5, GSEA revealed significant differences in the enrichment of the MSigDB Collection (NOM P < .05, false discovery rate [FDR] < 0.25). We chose the most essential signaling pathways based on NES (Table 5, Fig. 5). Figure 5 shows that nitrogen metabolism, alzheimers disease, and vascular muscle contraction were enriched in low FAM19A5 expression phenotype, respectively.
Table 5
Gene set in high FAM19A5 patients
NAME | SIZE | ES | NES | NOM p-val |
KEGG_NITROGEN_METABOLISM | 23 | -0.60217 | -1.53977 | 0.012766 |
KEGG_ALZHEIMERS_DISEASE | 157 | -0.38693 | -1.53467 | 0.043222 |
KEGG_VASCULAR_SMOOTH_MUSCLE_CONTRACTION | 114 | -0.48842 | -1.49213 | 0.041575 |
IHC and qRT‑PCR result.
Comparing the IHC experimental pictures obtained in the HPA database, it can
be seen that the expression of FAM19A5 in thyroid cancer tissues was signifcantly higher than that in normal thyroid tissues (Fig. 6A,B). We utilized qRT-PCR to validate the FAM19A5 expression in thyroid cancer tissues and found the FAM19A5 expression was up-regulated in the thyroid cancer (N = 3) compared with normal thyroid tissues (N = 3; P < 0.001) (Fig. 6C).