Differential Expression Levels of Plasma microRNAs in Neuroblastoma Patients Identified by Next-Generation Sequencing

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Table 1. Sample Characteristics (n = 68)

Age

Sex

Stage

Outcome

MYCN

Sample

Plasma

Tissue samples

(corresponding samples)1

Total

31

37 (27)

<=12 m

10

12 (9)

13 -17 m

5 (3)

≥ 18m

17

20 (15)

Male

22

25 (19)

Female

12 (8)

L1

11

12 (10)

L2

8 (5)

11

15 (11)

MS

2 (1)

Dead

10

16 (10)

Alive

21

21 (17)

Non-amplified

25

26 (21)

Amplified

11 (6)

( ) = available corresponding tissue samples to plasma samples

Figure 1. Workflow of the current study. Stage group : INRGSS stage M and non M. Outcome group : deceased and alive.

MYCN group : amplified and non-amplified.

A.

Normalized expression

2500

p = 0.017

2000

1500

1000

500

Alive outcome Deceased

B.

1800

1600

Tissue

1400

1200

1000

800

Alive

600

Deceased

400

200

500

1000

1500

2000

2500

Plasma

Figure 2. Results of analysis in outcome group.

A. Plasma level of the miRNA significantly correlated with outcome,

miR-92a-3p.

B. Correlation plot of miR-92a-3p levels in plasma and tissue samples.

Spearman rho = 0.308.

A.

2000

P = 0.002

Normalized expression

1800

1600

1400

1200

1000

800

600

400

200

L1, L2, MS

stage

B.

900

800

Tissue

700

600

500

400

L1, L2 & MS

300

200

100

500

1000

1500

2000

Plasma

Figure 3. Results of analysis in INRGSS stage group. A. Plasma level

of the miRNA correlated with INRGSS stage, miR-375.

B. Correlation plot of miR-375 levels in plasma and tissue samples.

Spearman rho = 0.545.

10

15

20

25

500

1000

1500

2000

2500

MYCN

Ampilified

Non-Amplified

Amplified

Non-AmplifiedMYCN

p = 0.006

p = 0.007

D.

B.

18

16

14

12

10

1800

1600

1400

1200

1000

800

600

400

200

500

Plasma

1500

Plasma

1000

Non-amplified

2000

20

Non-amplified

Amplified

2500

Amplified

Figure 4. Plasma level of the miRNA significantly correlated with MYCN status, miR-92a-3p. B. Correlation plot of miR-92a-3p levels in plasma

and tissue samples. Spearman rho = 0.255. C. Plasma level of the miRNA significantly correlated with MYCN status, miR-99a-5p.

D. Correlation plot of miR-99a5p levels in plasma and tissue samples. Spearman rho = 0.212.

C.

Normalized expression

Normalized expression

Tissue

Tissue

A.

Figure 5. Receiver operating characteristic curve of the combination of miR-92a-3p, miR-375, and miR-99a-5p levels in

plasma samples (AUC = 0.726, p = 0.001, 95 % CI = 0.612-0.841, sensitivity = 77 %, specificity = 56.7 %)

Figure 6. Kaplan-Meier plot of outcome based on miR-92a-3p levels. The 5-year overall survival rate was 89% for

patients with low miR-92a-3p expression levels and 45 % for patients with high miR-92a-3p expression levels.

Low/high expression levels were defined as expression levels below or above the average of plasma miR-92a-3p levels

in all cases (mean = 732.15 counts).

...