Δ133p53 and Δ133p53/FLp53 ratio significantly increased in ESCC tissues
As there is no specific antibody for the Δ133p53 isoform , we first examined the mRNA abundance of Δ133p53 in 42 pairs of tumor and adjacent normal tissues to determine whether Δ133p53 was associated with ESCC progression. Δ133p53 isoform significantly increased in cancerous tissues (0.10 ± 0.09 vs 0.05 ± 0.03, P = 0.0019) (Fig. 1A). We simultaneously detected mRNA expression of FLp53 in these paired samples and confirmed that FLp53 is significantly decreased in cancerous tissues of ESCC patients (0.06 ± 0.04 vs 0.17 ± 0.14, P < 0.0001) (Fig. 1B). In addition, we also noticed that the expression ratio of Δ133p53 to FLp53 significantly increased in cancerous tissues (2.40 ± 3.16 vs 0.50 ± 0.64, P = 0.0006) (Fig. 1C).
Elevated tissue Δ133p53/FLp53 ratio predicts poor outcome of ESCC patients
To confirm the association of Δ133p53 and FLp53 with clinical outcomes of ESCC patients, we then measured Δ133p53 and FLp53 expression in 180 FFPE ESCC tissue samples. The optimal cut-off values of Δ133p53, FLp53 and Δ133p53/FLp53 ratio for predicting the prognosis of ESCC were estimated by X-tile 3.6.1 software (Fig. S1). ESCC patients were divided into two groups (Δ133p53 < 0.0425 and ≥ 0.0425; FLp53 < 0.0195 and ≥ 0.0195; Δ133p53/FLp53 ratio < 2.6470 and ≥ 2.6740). The clinicopathological characteristics of the ESCC patients divided by the cut-off values of Δ133p53, FLp53 and Δ133p53/FLp53 ratio are shown in Table 1. Our results indicated that the elevated Δ133p53 was signiﬁcantly associated with increased TNM stage (P = 0.0011), differentiation grade (P = 0.0030) and recurrence (P = 0.0371), although it had no significant correlations with other clinicopathological features such as age, gender, and BMI index (Table 1). Decreased FLp53 was significantly associated with male gender (P = 0.0166), higher BMI (P = 0.0078), lower tumor location (P = 0.0295), increased TNM stage (P < 0.0001), differentiation grade (P < 0.0001) and recurrence P < 0.0001) (Table 1). Increased Δ133p53/FLp53 ratio was significantly associated with Lower tumor location (P = 0.0091), increased TNM stage (P < 0.0001), differentiation grade (P < 0.0001) and recurrence P < 0.0001) (Table 1).
The median survival time of all patients was 25.47 months (range 0.3–60 months), 106 patients (58.89%) were censored and 74 patients (41.11%) died during our follow-up period. Kaplan-Meier analysis revealed that increased tissue abundance of Δ133p53 (χ2 = 4.83, P = 0.0279) (Fig. 1D) and decreased p53 expression (χ2 = 27.76, P < 0.0001) (Fig. 1E), as well as increased Δ133p53/FLp53 ratio (χ2 = 42.34, P < 0.0001) (Fig. 1F) were significantly related to worse OS. By the time of analysis, 149 of 180 ESCC patients had regular follow-up checks for recurrence after surgery. Recurrence occurred in 61 of 149 patients, with a median follow-up time of 13.4 months (range 0.8–42 months), and 88 patients had clear evidence of no recurrence. Tissue Δ133p53 and Δ133p53/FLp53 ratio were significantly increased in recurrent patients compared with non-recurrent ones (P = 0.0032 and P < 0.0001, respectively), and FLp53 was decreased in recurrent patients (P = 0.0023) (Fig. S2). Kaplan-Meier analysis revealed that increased Δ133p53 (χ2 = 4.69, P = 0.0304) (Fig. 1G) showed a weak correlation with poor PFS, while decreased p53 expression (χ2 = 15.53, P < 0.0001) (Fig. 1H) and increased Δ133p53/FLp53 ratio (χ2 = 39.18, P < 0.0001) (Fig. 1I) were significantly related to worse PFS. Compared with tissue Δ133p53 and FLp53 expression, tissue Δ133p53/FLp53 ratio showed the strongest correlation with OS and PFS of ESCC patients.
Serum Δ133p53/FLp53 ratio shows prognostic significance in ESCC patients
p53 could exist as circulating DNA or mRNA in cancers [28, 31], we next further evaluated Δ133p53 and FLp53 mRNA expression in serum of 180 ESCC patients. Pearson’s correlation coefficient analysis revealed that serum Δ133p53, FLp53 and Δ133p53/FLp53 ratio were positively correlated with their tissue expression, and Pearson’s correlation coefficients were 0.7019 (P < 0.0001) (Fig. 2A), 0.4030 (P < 0.0001) (Fig. 2B) and 0.4137 (P < 0.0001) (Fig. 2C), respectively. ESCC patients were divided into two groups (Δ133p53 < 0.0870 and ≥ 0.0870; FLp53 < 0.0300 and ≥ 0.0300; Δ133p53/FLp53 ratio < 2.9160 and ≥ 2.9160) by X-tile 3.6.1 software (Fig. S3). Correlation analysis revealed that the elevated serum Δ133p53 was signiﬁcantly associated with male gender (P = 0.0105), increased M stage (P = 0.0086), N stage (P = 0.0184) and TNM stage (P = 0.0008), as well as recurrence (P = 0.0005) (Table 2). Increased Δ133p53/FLp53 ratio was significantly associated with high BMI index (P = 0.0287), increased M stage (P < 0.0001), N stage (P = 0.0133), TNM stage (P < 0.0001), differentiation grade (P = 0.0131) and recurrence (P < 0.0001) (Table 2). Whereas decreased serum FLp53 was only associated with increased TNM stage (P = 0.0276) and recurrence (P = 0.0007) (Table 2).
Serum Δ133p53 and Δ133p53/FLp53 ratio were significantly increased in recurrent patients compared with non-recurrent ones (P = 0.0002 and P < 0.0001, respectively) (Figs. S4A and S4C), FLp53 was decreased in recurrent patients (P = 0.0001) (Fig. S4B). However, survival analysis indicated that increased serum Δ133p53 showed no association with OS (χ2 = 2.89, P = 0.0890) (Fig. 2D) and PFS (χ2 = 2.93, P = 0.0871) (Fig. 2E). Decreased p53 expression correlated with decreased OS (χ2 = 5.80, P = 0.0160) (Fig. 2F) and PFS (χ2 = 10.27, P = 0.0014) (Fig. 2G); and increased Δ133p53/FLp53 ratio correlated with poor OS (χ2 = 17.26, P < 0.0001) (Fig. 2H) and PFS (χ2 = 56.16, P < 0.0001) (Fig. 2I). Compared with serum Δ133p53 and FLp53 expression, serum Δ133p53/FLp53 ratio showed the strongest correlation with OS and PFS of ESCC patients.
Tissue and serum Δ133p53/FLp53 ratio are poor independent prognostic factors for ESCC
The univariate survival analysis indicated that tissue and serum Δ133p53/FLp53 ratio were correlated with OS (Table 3) and PFS (Table 4) of ESCC patients. Furthermore, the multivariate analyses identified that the tissue Δ133p53/FLp53 ratio was an independent prognostic factor for OS (HR = 3.864; 95% CI = 2.293–6.511, P < 0.0001) (Table 3) and PFS (HR = 2.283; 95% CI = 1.387–3.760, P < 0.0001) (Table 4) of ESCC patients. The serum Δ133p53/FLp53 ratio was also an independent prognostic factor for OS (HR = 2.503; 95% CI = 1.465–4.276, P = 0.0011) (Table 3) and PFS (HR = 3.230; 95% CI = 1.947–5.359, P < 0.0001) (Table 3) of ESCC patients. Besides, M stage and recurrence were also predictive indicators for OS and PFS. However, tissue and serum Δ133p53 or FLp53 were not independent prognostic factors for OS and PFS of ESCC patients. The one exception is tissue FLp53 (data not shown).
Tissue and serum Δ133p53/FLp53 ratios show no difference in prognostic performance for OS and PFS
We also conducted a ROC analysis of tissue and serum Δ133p53/FLp53 ratio. The AUC values of tissue and serum Δ133p53/FLp53 ratio in the Cox regression model for OS were 0.695 (P = 0.0351) and 0.641 (P = 0.0360), respectively (Fig. 3A), and 0.692 (P = 0.0337 and 0.649 (P = 0.0340) for PFS, respectively (Fig. 3B). Prognostic performance of tissue and serum Δ133p53/FLp53 ratio showed no statistical difference for OS and PFS (P = 0.2066 and P = 0.3207, respectively).
The dynamic change of serum Δ133p53/FLp53 ratio after surgery and its association with recurrence
To evaluate whether serum Δ133p53/FLp53 ratio dynamically correlates with treatment response, we further measured its postoperative level in 180 ESCC patients within 72 h after resection. Postoperative serum Δ133p53/FLp53 ratio significantly dropped after surgery (4.31 ± 7.69 vs 2.81 ± 4.12, P = 0.0098) (Fig. 4A). Based on changes between preoperative and postoperative serum Δ133p53/FLp53 ratio, patients were divided into four groups: I, persistent high levels at the two time points; II, preoperative high followed by postoperative low; III, preoperative low followed by postoperative high, and IV, preoperative low followed by postoperative low, using the same cutoff value of preoperative serum Δ133p53/FLp53 ratio (2.9160). Persistent high levels of serum Δ133p53/FLp53 ratio predicted poor OS (Fig. 4B), higher recurrence rates (Fig. 4C) and worse PFS (Fig. 4D) in ESCC patients. Patients in group I showed significantly shorter time to relapse (TTR) and higher recurrence rates than group IV (median TTR of 5.57 months vs not reached; recurrence of 27/28 vs 14/88, P < 0.0001), as well as group III (median TTR of 5.57 months vs 29.4 months; recurrence of 27/28 vs 4/13, P < 0.0001), while there was a propensity of increased recurrence compared with group II (median TTR of 5.57 months vs 8.07 months; recurrence of 27/28 vs 16/20; P = 0.0643) (Fig. 4C). Compared with group IV, patients in groups I and II had significantly shorter TTR and higher recurrence rates (all P < 0.0001, Fig. 4C).
Twelve patients with no history of radio- or chemo-therapy after tumor resection were selected for measurement of real-time changes of serum Δ133p53/FLp53 ratio in each time of follow-up (tumor recurrence was monitored by CT scans every 4 months in the first two years). Six patients were confirmed to have lung, brain or lymph node metastasis (Fig. 4E), and the other 6 showed no evidence of recurrence (Fig. 4F). The serum Δ133p53/FLp53 ratios were higher than 2.9160 at the time of detected recurrence in 6 patients and even increased prior to radiologic progression in 2/6 patients, while they were lower than 2.9160 in 4/6 patients with no recurrence (Fig. 4G). In addition, healthy donors exhibited significantly lower serum Δ133p53/FLp53 ratios than did ESCC patients preoperatively, mostly lower than 2.9160 (Fig. S4D). The best cutoff value of the serum Δ133p53/FLp53 ratio to distinguish ESCC patients from healthy donors was 0.6798 (Sensitivity: 76.11%; Specificity: 77.55%, AUC = 0.8376), which was also less than 2.9160 (Fig. S4E). These results indicate the potential usefulness of serum Δ133p53/FLp53 ratio as a prognostic predictor for ESCC, and may also reflect recurrence risk in a real-time manner.