TABIQUERÍA Y FALSOS TECHOS

ovarian cancer

The prognostic values of KLK mRNA expression and clinical parameters for the patient outcome (PFS/OS) were estimated by univariate and multivariate Cox regression analyses. In univariate Cox regression analysis (Table 15, Table 16), residual tumor mass and ascites fluid volume are univariate predictors both for PFS and OS.Patients with residual tumor mass (>0 mm) have a significantly higher risk of disease relapse and cancer-related death, compared to the tumor-free cases. A larger ascites fluid volume (>500 ml) predicts worse PFS and OS.

In HGSOC, KLK4 mRNA expression (Table 15) represents an unfavorable predictive factor for OS (HR: 2.28, 95%CI: 1.38-3.76, p=0.001), indicating an approximately two- fold increased probability of cancer-related death in the KLK4 high-expressing group. However, no significant association was observed between KLK4 mRNA levels and PFS. Regarding KLK5 (Table 16), elevated mRNA levels are significantly associated

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with shortened PFS (HR: 1.60, 95% CI: 1.01-2.55, p=0.047), but not with OS. Elevated KLK7 mRNA levels (Table 16) present an unfavorable prognostic value for PFS (HR: 1.75, 95% CI: 1.07-2.84, p=0.025) and show a trend towards significance in case of OS (HR: 1.66, 95% CI: 0.99-2.79, p=0.055).

Table 15. Univariate Cox regression analysis of KLK4 mRNA expression and clinical parameters with patient survival in advanced high-grade serous ovarian cancer

Clinical parameters PFS OS

Noa _{HR (95% CI)}b _{p No}a _{HR (95% CI)}b _p

Age 0.627 0.348

≤ 60 years 43 1 50 1

> 60 years 65 1.12 (0.70-1.79) 76 1.27 (0.77-2.08)

Residual tumor mass <0.001 <0.001

0 mm 59 1 64 1

> 0 mm 49 2.53 (1.60-4.02) 60 3.76 (2.18-6.48)

Ascites fluid volume 0.018 0.011

≤ 500 ml 63 1 72 1 > 500 ml 39 1.78 (1.10-2.87) 47 1.93 (1.16-3.18) KLK4 mRNAc _{0.121 0.001} low 55 1 62 1 high 52 1.44 (0.91-2.78) 63 2.28 (1.38-3.76) a _{Number of patients;}

b _{HR: hazard ratio (CI: confidence interval) of univariate Cox regression analysis;} c _{Dichotomized into low and high levels by the 50}th _percentile;

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Table 16. Univariate Cox regression analysis of KLK5 and KLK7 mRNA expression and clinical parameters with patient survival in advanced high-grade serous ovarian cancer Clinical parameters PFS OS Noa _{HR (95% CI)}b _{p No}a _{HR (95% CI)}b _p Age 0.762 0.414 ≤ 60 years 43 1 49 1 > 60 years 62 1.08 (0.67-1.72) 70 1.24 (0.74-2.06)

Residual tumor mass <0.001 <0.001

0 mm 58 1 60 1

> 0 mm 47 2.41 (1.53-3.90) 57 3.80 (2.17-6.65)

Ascites fluid volume 0.019 0.005

≤ 500 ml 61 1 66 1 > 500 ml 38 1.79 (1.10-2.90) 46 2.10 (1.25-3.54) KLK5 mRNAc _{0.047 0.269} low 62 1 73 1 high 42 1.60 (1.01-2.55) 45 1.33 (0.80-2.20) KLK7 mRNAd _{0.025 0.055} low 73 1 82 1 high 32 1.75 (1.07-2.84) 37 1.66 (0.99-2.79) a _{Number of patients;}

b _{HR: hazard ratio (CI: confidence interval) of univariate Cox regression analysis;} c _{Dichotomized into low and high levels by the 67}th _percentile;

d _{Dichotomized into low and high levels by the 67}th _percentile;

Bold values indicate statistical significance (p≤0.05); values in italics indicate a trend towards significance.

Furthermore, the impact of KLKs on clinical outcome was validated by the respective Kaplan-Meier survival analysis. The patients in the KLK4 mRNA high group (Figure 9) have a worse OS, compared to the low subgroup. KLK5 overexpression (Figure 10) as well as KLK7 overexpression (Figure 11) were significantly correlated with shortened PFS.

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Figure 9. Kaplan–Meier survival analysis concerning KLK4 mRNA expression in patients with advanced high-grade serous ovarian cancer

Patients with elevated KLK4 mRNA levels display a significantly worse OS (p=0.001, B) but not PFS (A), compared with those with low KLK4 mRNA levels.

Figure 10. Kaplan–Meier survival analysis concerning KLK5 mRNA expression in patients with advanced high-grade serous ovarian cancer

Patients with elevated KLK5 mRNA levels display a significantly shortened PFS (p=0.041, A) but not OS (B), compared with those with low KLK5 mRNA levels.

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Figure 11. Kaplan–Meier survival analysis concerning KLK7 mRNA expression in patients with advanced high-grade serous ovarian cancer

Patients with elevated KLK7 mRNA levels display a significantly worse PFS (p = 0.021, A) and show a trend towards significance in case of OS (B), compared with those with low KLK7 mRNA levels.

Multivariate Cox regression analysis was performed to assess the independent prognostic value of the clinical parameters and KLK mRNA expression in HGSOC. First, a base model was established, consisting of the established clinical parameters age, residual tumor mass, and ascites fluid volume. Here, residual tumor mass is the only independent predictive marker for the outcome, while ascites fluid volume loses its prognostic value. Next, the KLKs were individually added to the base model, summarized in Table 17-19. When subjected to multivariate Cox regression analysis, KLK4 mRNA expression retains the unfavorable predictive power for OS (HR: 2.31, 95% CI: 1.27-4.20, p=0.006; Table 17). KLK5 mRNA expression may have a trend towards significance for PFS (HR: 1.53, 95% CI: 0.93-2.51, p=0.095; Table 18). In case of KLK7 (Table 19), mRNA expression represents an unfavorable predictor for PFS (HR: 2.19, 95% CI: 1.23-3.89, p=0.007) and OS (HR: 1.94, 95% CI: 1.06-3.55, p=0.032). To sum up, KLK4 and KLK7 mRNA expression were demonstrated to be independent biomarkers for patient outcome in HGSOC.

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Table 17. Multivariate Cox regression analysis of KLK4 mRNA expression and clinical parameters with patient survival in advanced high-grade serous ovarian cancer

The biological marker KLK4 mRNA was added to the base model of clinical parameters, which included age, residual tumor mass, and ascites fluid volume. Significant p-values (p≤0.05) are indicated in bold.

a _{Number of patients;}

b _{HR: hazard ratio (CI: confidence interval) of multivariable Cox regression analysis;} c _{Dichotomized into low and high levels by 50}th _percentile.

Clinical parameters PFS OS

Noa _{HR (95% CI)}b _{p No}a _{HR (95% CI)}b _p

Age 0.733 0.470

≤ 60 years 41 1 47 1

> 60 years 60 0.92 (0.56-1.51) 69 1.22 (0.72-2.07)

Residual tumor mass 0.002 <0.001

0 mm 58 1 63 1

> 0 mm 43 2.36 (1.38-4.05) 53 3.58 (1.90-6.74)

Ascites fluid volume 0.474 0.911

≤ 500 ml 63 1 71 1

> 500 ml 38 1.22 (0.71-2.10) 45 1.03 (0.58-1.86)

KLK4 mRNAc _{0.284 0.006}

low 52 1 58 1

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Table 18. Multivariate Cox regression analysis of KLK5 mRNA expression and clinical parameters with patient survival in advanced high-grade serous ovarian cancer

The biological marker KLK4 mRNA was added to the base model of clinical parameters, which included age, residual tumor mass, and ascites fluid volume. Significant p-values (p≤0.05) are indicated in bold.

a _{Number of patients;}

b _{HR: hazard ratio (CI: confidence interval) of multivariable Cox regression analysis;} c _{Dichotomized into low (tertiles 1+2) and high (tertile 3) levels by 67}th _percentile.

Clinical parameters PFS OS

Noa _{HR (95% CI)}b _{p No}a _{HR (95% CI)}b _p

Age 0.592 0.660

≤ 60 years 41 1 46 1

> 60 years 57 0.87 (0.53-1.44) 63 1.13 (0.65-1.96)

Residual tumor mass 0.005 <0.001

0 mm 57 1 59 1

> 0 mm 41 2.20 (1.17-3.81) 50 3.29 (1.69-6.41)

Ascites fluid volume 0.363 0.605

≤ 500 ml 60 1 64 1

> 500 ml 38 1.33 (0.76-2.31) 45 1.18 (0.64-1.91)

KLK5 mRNAc _{0.095 0.718}

low 59 1 69 1

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Table 19. Multivariate Cox regression analysis of KLK7 mRNA expression and clinical parameters with patient survival in advanced high-grade serous ovarian cancer

The biological marker KLK4 mRNA was added to the base model of clinical parameters, which included age, residual tumor mass, and ascites fluid volume. Significant p-values (p≤0.05) are indicated in bold.

a _{Number of patients;}

b _{HR: hazard ratio (CI: confidence interval) of multivariable Cox regression analysis;} c _{Dichotomized into low (tertiles 1+2) and high (tertile 3) levels by 67}th _percentile.

4.1.5 Validation of the association of KLK mRNA expression with patients survival