3.1. Endostar enhanced NACT short-term treatment effects
In this study, the recently efficacy of endostar+NACT was evaluated, it was showed that endostar+NACT resulted in a complete response (CR) rate of 79.1(38/48),a partial response (PR) rate of 10.4% (5/48) and a stable disease (SD) rates 6.3%(3/48). NACT alone results in a CR rate of 52.1% (25/48), a PR rate of 20.1% (10/48) and a stable disease (SD) rates 4.2%(2/48).So chemotherapeutic response rates(RR) in endostar group and NACT group were 89.6% vs.72.9% respectively, there was obviously difference between the 2 groups(Fisher's exact test;α<0.05,P=0.0124).The disease control rates (DCR) in endostar group and NACT group were 95.8% vs.77.1%, respectively. There was obviously difference between the 2 groups (Fisher's exact test,α<0.05,P=0.0447).This results indicated endostar increased chemotherapeutic agents effects in recent efficacy compared with chemotherapy alone,see Table 3.
3.2. Endostar enhanced NACT long-term efficacy
To following up long-term efficacy, the patients were summed up for 38 to 57 months,average 49 months. The average following up time was 49.6 months (region; 38.3–67.3 months).
The 5-year overall survival(OS) rates in endostar group and NACT group were 83.3% and 70.8%, respectively,this proved obviously different between 2 groups (fisher's exact test,α<0.05,P=0.026).These included 8 patients died in endostar group and 14 patients died in NACT group.
The 2-year progression free survival(PFS) rates in endostar group and NACT group were 96.4% and 81.6%.There was statistically significant between two group(Fisher's exact test,P =0.0014,α<0.05).
The 2-year local relapse free rates in the two groups were 96.4% and 87.7% separately (Fisher's exact test,α<0.05,P=0.0398).These included 2 patients local relapse in endostar group and 6 patients local relapse in NACT group.
The 2-year distance metastasis free survival rates in the two groups were 100% and 93.8% separately (Fisher's exact test,α<0.05,P=0.0289),There was statistically significant between two groups. These included no patients distance metastasis in endostar group and 3 patients in NACT group.
The survival rates of patients were summed up as following curves. The results indicated that chemotherapy combined with endostar obviously enhanced long-term effects than chemotherapy alone. see Fig.2, Table 3.
3.3. Endostar reduced NACT toxicity
To assess chemotherapy adverse responses, Common Terminology Criteria Adverse Events Version 4.0 from National Cancer Institute (NCI) was adopted.The general adverse effects included myelosuppression,hair loss, gastrointestinal reactions, weightlessness, and oral ulcer were decreased.Dysfunction of vital organs was not found in all patients,see Table 4.
In endostar group,the incidenc of myelosuppression(such as leukocytopenia) in grades 0,1 and 2 were 55.0%,18.0%,and 27.0%,differently.In NACT alone group, the incidence of myelosuppressionin in grades 0, 1, 2, and 3 were 8%, 20.5%, 50.3%,and 21.2%,differently.There were statistical difference between 2 groups (Chi-square=63.16,P<0.0001,α<0.05).
In endostar group,the incidence of gastrointestinal reactions in grades 0,1,and 2 were 38.0%,49.0%,and 13.0%,separately.The incidence gastrointestinal reactions in grades 2,3 and 4 were 75.4%,17.5% and 7.1% in NACT group,differently. There was distinctly difference between 2 groups(Chi-square=32.08,P < 0.0001,α<0.05).
In the endostar group, the incidence of weight loss in grades 1 and 2 were 75.2%, and 24.8% separately.In the NACT alone group, the incidence weight loss in grades 0 and 1 were 89.8.%,10.2%,seperately.There was obviously difference between 2 groups (Chi-square=164.7, P < 0.0001,α<0.05).
In endostar group, the incidence of oral ulcer in grades 0, 1 were 78.0%, 22.0%,separately. In NACT group, the incidence of oral ulcer in grades 0, 1, 2 were 46.3%, 35.5% and 18.2%, seperately. The two groups had obviously difference (Chi-square=29.22,P<0.0001,α<0.05).
In endostar group,the incidence of hair loss in grades 0,1,and 2 were 45.0%,49.0%,and 6.0%,separately. In the chemotherapy alone group, the incidence of hair loss in grades 0, 1, 2 were 23.5%, 56.4% and 21.1%, seperately.The two groups had obviously difference (Chi-square=15.92,P=0.0003,α<0.05).
These results confirmed endostar relieved NACT adverse reactions compared with NACT alone.
3.4. Endostar enhanced NACT tumor volume reduction effects
The change of tumor volume was monitored by MRI, it showed the tumor volume was 5.48±0.36 cm3 and 3.10±0.32cm3 (n1=48) before and after treatment in endostar group respectively.In contrast, the tumor volume was 5.39±0.45 cm3 and 4.51±0.25cm3 (n2=48) before and after treatment in NACT group.There was distinctly different between two groups before and after treatment(two-way ANOVA,F(1,188)=201.2,P<0.0001).The same result could be found from the maximum diameter basing on MRI mesurement.So it exhibited that tumor volumes and maximum diameters further declined in endostar group than NACT group, see Fig.4. As measured by tumor volume and maximum diameter changes,endostar enhance the effects of NACT for cervical cancer patients.
3.5. Treatment efficacy of endostar determined by DCE‑MRI
In this study,DCE-MRI was used to obtain cervical cancer perfusion and permeability status.
The receiver operating characteristic (ROC) determines the optimal cutoff value for DCE-MRI detection.The critical point has the shortest distance from the top left corner to the ROC curve.On this point the tangent line of the ROC curve intersects with the ROC curve, the sum of sensitivity and specificity value is the highest at the same time with the least sum of false positives and false negatives value.
ROC analysis for DCE-MRI parameters were carried out between endostar+NACT and NACT group before and after treatment.Before treatment, ROC analysis showed area under the curve values of Ktrans, Ve, and kep (Ktrans/ve) in endostar group were 0.35±0.02,0.56±0.03,0.59±0.08 separately.These results were regarded as baseline parameters.
One week after endostar+NACT treatment,ROC analysis was performed for indicators of DEC-MRI Ktrans,Ve, kep separately.ROC analysis via unpaired t test showed area under the curve values were 0.54±0.03(t=36.51,P<0.0001,n=48),0.82±0.04(t=36.03,P<0.0001),0.62±0.05(t=1.469,P=0.1452) for Ktrans,Ve,kep, separately.There were significant different before and after treatment for Ktrans and Ve value.
In NACT group,the values of Ktrans,Ve and kep were 0.37±0.08, 0.63±0.15, 0.59±0.11vs.0.39±0.10(t=1.082,P=0.282),0.59±0.12(t=1.443,P=0.1524),0.66±0.23(t=1.902,P=0.602) before and after treatment. There were no significant differences before and after treatment, see Fig. 4A, 5A.
When the cutoff of Ktrans value was 0.425(ml/min), the sensitivity, specificity were 81.25(95% CI: 54.35-95.95),93.75 (95% CI: 69.77- 99.84) separately, see Fig. 4B.
When the cutoff of Ve value was 0.525(ml/ml), the sensitivity, specificity were 87.5(95% CI: 61.65-98.45), 93.75 (95% CI: 69.65- 97.94) separately, see Fig. 5B.
When the cutoff of kep value was 0.325(ml/min),the sensitivity, specificity were 53.85(95% CI: 25.13-80.78), 84.62 (95%CI:54.55-98.08) respectively.
The above results proved DEC-MRI was non-invasive method that could be used to predict for cervical cancer treatment effects and monitor NACT effects in real time. Endostar improved tumor perfusion and permeability and increased chemotherapy efficacy.
3.6. Vascular maturation and cell proliferation
Before the treatment, CD31 staining rates were 85.54±6.32vs.83.36±7.63 in endostar group and NACT group respectively,there was no significantly different between two groups(t test, P=0.1362, t=1.503).One week after treatment,CD31 staining rates in endostar group and NACT group were 45.43±4.35vs. 78.24±6.54,respectively,This confirmed microvessel density in endostar group were relatively reduced than that of NACT group.There was significant different between two group(Two-wayANOVA,F(1,188)=486.3,P<0.0001).
α-SMA staining were 13.38±4.46vs.15.45±5.37(n1=n2=48) before treatment. Seven day after treatment,α-SMA label were 37.35±5.34 vs.13.67±4.74 in endostar group and NACT group, showed higher than NACT group.There was dramatically different between two groups (two-way ANOVA, F(1,188)=149.9, P<0.0001). This prove before treatment,tumor pericyte density was at the same level,after treatment, pericyte rates exhibited an obviously increase in endostar group.
The change of α-SMA and CD31 ratio resulted in a higher vascular maturity index (VMI) in endostar group(before treatment:0.15±0.04vs.0.18±0.05;after treatment:0.28±0.05vs.0.19±0.06, n1=n2=48).There was observably different between two groups(two-way ANOVA,F(1,188)=64.42,P<0.0001).While α-SMA label and VMI were higher in endostar group.This proved endostar made tumor vascular structure more maturely than that of NACT group.
Meanwhile,before the treatment, Ki67 positive staining exhibited the same proliferative level in endostar group and NACT group(42.5±3.3vs. 43.6±5.4,n1=n2=48),there was no significantly different between two groups(t test,P=0.2315,t=1.204). Seven days after treatment, Ki67 positive staining rates in endostar group and NACT group were 23.32±4.25% vs. 35.36 ± 4.67 %, respectively, there were significant different between two group(Two-way ANOVA,F(1,188)=486.3,P<0.0001).This proved the tumor proliferative rates in endostar group were obviously reduced than NACT group,see Fig.6.
3.7 VEGF-Notch signal pathway detection
The genetic expression of VEGF-Notch signal pathway was analyzed by real-time PCR. The elements of these including VEGFR,Notch1,Notch1,4,Dll4,Jagged and hes-1 were obviously down regulated in endostar group except VEGF, comparing to NACT group.This was also confirmed from the protein expression of VEGFR,Jagged-1,Dll4,Notch1,4 and hes-1 by western blot analysis,this proved endostar blocked VEGF-Notch connection via interdicting VEGFR (P<0.01),see Fig.7.