Correlation between miRseq expression and clinical features
Overview
Introduction

This pipeline uses various statistical tests to identify miRs whose expression levels correlated to selected clinical features.

Summary

Testing the association between 455 genes and 7 clinical features across 481 samples, statistically thresholded by Q value < 0.05, 6 clinical features related to at least one genes.

  • 56 genes correlated to 'Time to Death'.

    • HSA-MIR-223 ,  HSA-MIR-130B ,  HSA-MIR-34C ,  HSA-MIR-21 ,  HSA-MIR-10B ,  ...

  • 1 gene correlated to 'AGE'.

    • HSA-MIR-590

  • 12 genes correlated to 'GENDER'.

    • HSA-MIR-100 ,  HSA-MIR-455 ,  HSA-MIR-708 ,  HSA-MIR-599 ,  HSA-MIR-30A ,  ...

  • 21 genes correlated to 'DISTANT.METASTASIS'.

    • HSA-MIR-106B ,  HSA-MIR-193A ,  HSA-MIR-155 ,  HSA-MIR-625 ,  HSA-MIR-28 ,  ...

  • 2 genes correlated to 'LYMPH.NODE.METASTASIS'.

    • HSA-MIR-10B ,  HSA-MIR-223

  • 39 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • HSA-MIR-139 ,  HSA-MIR-625 ,  HSA-LET-7I ,  HSA-MIR-28 ,  HSA-MIR-155 ,  ...

  • No genes correlated to 'KARNOFSKY.PERFORMANCE.SCORE'

Results
Overview of the results

Complete statistical result table is provided in Supplement Table 1

Table 1.  Get Full Table This table shows the clinical features, statistical methods used, and the number of genes that are significantly associated with each clinical feature at Q value < 0.05.

Clinical feature Statistical test Significant genes Associated with                 Associated with
Time to Death Cox regression test N=56 shorter survival N=49 longer survival N=7
AGE Spearman correlation test N=1 older N=1 younger N=0
GENDER t test N=12 male N=3 female N=9
KARNOFSKY PERFORMANCE SCORE Spearman correlation test   N=0        
DISTANT METASTASIS t test N=21 m1 N=16 m0 N=5
LYMPH NODE METASTASIS ANOVA test N=2        
NEOPLASM DISEASESTAGE ANOVA test N=39        
Clinical variable #1: 'Time to Death'

56 genes related to 'Time to Death'.

Table S1.  Basic characteristics of clinical feature: 'Time to Death'

Time to Death Duration (Months) 0.1-111 (median=35.2)
  censored N = 323
  death N = 155
     
  Significant markers N = 56
  associated with shorter survival 49
  associated with longer survival 7
List of top 10 genes significantly associated with 'Time to Death' by Cox regression test

Table S2.  Get Full Table List of top 10 genes significantly associated with 'Time to Death' by Cox regression test

HazardRatio Wald_P Q C_index
HSA-MIR-223 1.64 2.721e-13 1.2e-10 0.652
HSA-MIR-130B 2 9.949e-12 4.5e-09 0.653
HSA-MIR-34C 1.28 5.06e-10 2.3e-07 0.644
HSA-MIR-21 2.1 1.033e-09 4.7e-07 0.659
HSA-MIR-10B 0.56 6.213e-09 2.8e-06 0.366
HSA-MIR-101-1 0.56 1.314e-08 5.9e-06 0.393
HSA-MIR-1248 1.41 4.348e-08 2e-05 0.619
HSA-MIR-18A 1.53 1.027e-07 4.6e-05 0.618
HSA-MIR-3614 1.61 1.914e-07 8.6e-05 0.616
HSA-MIR-138-2 1.42 2.924e-07 0.00013 0.651

Figure S1.  Get High-res Image As an example, this figure shows the association of HSA-MIR-223 to 'Time to Death'. four curves present the cumulative survival rates of 4 quartile subsets of patients. P value = 2.72e-13 with univariate Cox regression analysis using continuous log-2 expression values.

Clinical variable #2: 'AGE'

One gene related to 'AGE'.

Table S3.  Basic characteristics of clinical feature: 'AGE'

AGE Mean (SD) 60.58 (12)
  Significant markers N = 1
  pos. correlated 1
  neg. correlated 0
List of one gene significantly correlated to 'AGE' by Spearman correlation test

Table S4.  Get Full Table List of one gene significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-590 0.1846 4.661e-05 0.0212

Figure S2.  Get High-res Image As an example, this figure shows the association of HSA-MIR-590 to 'AGE'. P value = 4.66e-05 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'GENDER'

12 genes related to 'GENDER'.

Table S5.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 163
  MALE 318
     
  Significant markers N = 12
  Higher in MALE 3
  Higher in FEMALE 9
List of top 10 genes differentially expressed by 'GENDER'

Table S6.  Get Full Table List of top 10 genes differentially expressed by 'GENDER'

T(pos if higher in 'MALE') ttestP Q AUC
HSA-MIR-100 8.16 7.964e-15 3.62e-12 0.7301
HSA-MIR-455 -5.38 1.374e-07 6.24e-05 0.6593
HSA-MIR-708 5.39 1.444e-07 6.54e-05 0.6566
HSA-MIR-599 -5.05 8.276e-07 0.000374 0.6747
HSA-MIR-30A -4.36 1.707e-05 0.0077 0.6143
HSA-MIR-30C-2 -4.36 1.805e-05 0.00812 0.6148
HSA-MIR-31 4.34 1.967e-05 0.00883 0.6288
HSA-MIR-500B -4.18 3.773e-05 0.0169 0.6111
HSA-MIR-204 -4.14 4.445e-05 0.0199 0.641
HSA-MIR-328 -4.01 7.342e-05 0.0327 0.608

Figure S3.  Get High-res Image As an example, this figure shows the association of HSA-MIR-100 to 'GENDER'. P value = 7.96e-15 with T-test analysis.

Clinical variable #4: 'KARNOFSKY.PERFORMANCE.SCORE'

No gene related to 'KARNOFSKY.PERFORMANCE.SCORE'.

Table S7.  Basic characteristics of clinical feature: 'KARNOFSKY.PERFORMANCE.SCORE'

KARNOFSKY.PERFORMANCE.SCORE Mean (SD) 88.33 (23)
  Score N
  0 2
  70 1
  80 3
  90 13
  100 17
     
  Significant markers N = 0
Clinical variable #5: 'DISTANT.METASTASIS'

21 genes related to 'DISTANT.METASTASIS'.

Table S8.  Basic characteristics of clinical feature: 'DISTANT.METASTASIS'

DISTANT.METASTASIS Labels N
  M0 405
  M1 76
     
  Significant markers N = 21
  Higher in M1 16
  Higher in M0 5
List of top 10 genes differentially expressed by 'DISTANT.METASTASIS'

Table S9.  Get Full Table List of top 10 genes differentially expressed by 'DISTANT.METASTASIS'

T(pos if higher in 'M1') ttestP Q AUC
HSA-MIR-106B 5.7 6.381e-08 2.9e-05 0.6738
HSA-MIR-193A 5.57 9.469e-08 4.3e-05 0.6407
HSA-MIR-155 5.4 3.791e-07 0.000172 0.6873
HSA-MIR-625 5.4 4.328e-07 0.000196 0.6887
HSA-MIR-28 5.22 7.987e-07 0.00036 0.659
HSA-LET-7I 5.15 1.024e-06 0.000461 0.6667
HSA-MIR-144 -5.19 1.063e-06 0.000477 0.6849
HSA-MIR-130B 5.06 1.575e-06 0.000706 0.6683
HSA-MIR-27A 4.74 5.384e-06 0.00241 0.6273
HSA-MIR-21 4.69 7.295e-06 0.00325 0.6458

Figure S4.  Get High-res Image As an example, this figure shows the association of HSA-MIR-106B to 'DISTANT.METASTASIS'. P value = 6.38e-08 with T-test analysis.

Clinical variable #6: 'LYMPH.NODE.METASTASIS'

2 genes related to 'LYMPH.NODE.METASTASIS'.

Table S10.  Basic characteristics of clinical feature: 'LYMPH.NODE.METASTASIS'

LYMPH.NODE.METASTASIS Labels N
  N0 222
  N1 18
  NX 241
     
  Significant markers N = 2
List of 2 genes differentially expressed by 'LYMPH.NODE.METASTASIS'

Table S11.  Get Full Table List of 2 genes differentially expressed by 'LYMPH.NODE.METASTASIS'

ANOVA_P Q
HSA-MIR-10B 8.35e-06 0.0038
HSA-MIR-223 3.598e-05 0.0163

Figure S5.  Get High-res Image As an example, this figure shows the association of HSA-MIR-10B to 'LYMPH.NODE.METASTASIS'. P value = 8.35e-06 with ANOVA analysis.

Clinical variable #7: 'NEOPLASM.DISEASESTAGE'

39 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S12.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 228
  STAGE II 51
  STAGE III 125
  STAGE IV 77
     
  Significant markers N = 39
List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S13.  Get Full Table List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
HSA-MIR-139 6.337e-13 2.88e-10
HSA-MIR-625 4.138e-11 1.88e-08
HSA-LET-7I 2.711e-09 1.23e-06
HSA-MIR-28 3.663e-09 1.66e-06
HSA-MIR-155 4.302e-09 1.94e-06
HSA-MIR-21 6.453e-09 2.9e-06
HSA-MIR-144 7.579e-09 3.4e-06
HSA-MIR-130B 1.04e-08 4.66e-06
HSA-MIR-486 1.299e-08 5.81e-06
HSA-MIR-130A 4.558e-08 2.03e-05

Figure S6.  Get High-res Image As an example, this figure shows the association of HSA-MIR-139 to 'NEOPLASM.DISEASESTAGE'. P value = 6.34e-13 with ANOVA analysis.

Methods & Data
Input

  • Expresson data file = KIRC-TP.miRseq_RPKM_log2.txt

  • Clinical data file = KIRC-TP.clin.merged.picked.txt

  • Number of patients = 481

  • Number of genes = 455

  • Number of clinical features = 7

Survival analysis

For survival clinical features, Wald's test in univariate Cox regression analysis with proportional hazards model (Andersen and Gill 1982) was used to estimate the P values using the 'coxph' function in R. Kaplan-Meier survival curves were plot using the four quartile subgroups of patients based on expression levels

Correlation analysis

For continuous numerical clinical features, Spearman's rank correlation coefficients (Spearman 1904) and two-tailed P values were estimated using 'cor.test' function in R

Student's t-test analysis

For two-class clinical features, two-tailed Student's t test with unequal variance (Lehmann and Romano 2005) was applied to compare the log2-expression levels between the two clinical classes using 't.test' function in R

ANOVA analysis

For multi-class clinical features (ordinal or nominal), one-way analysis of variance (Howell 2002) was applied to compare the log2-expression levels between different clinical classes using 'anova' function in R

Q value calculation

For multiple hypothesis correction, Q value is the False Discovery Rate (FDR) analogue of the P value (Benjamini and Hochberg 1995), defined as the minimum FDR at which the test may be called significant. We used the 'Benjamini and Hochberg' method of 'p.adjust' function in R to convert P values into Q values.

References
[1] Andersen and Gill, Cox's regression model for counting processes, a large sample study, Annals of Statistics 10(4):1100-1120 (1982)
[2] Spearman, C, The proof and measurement of association between two things, Amer. J. Psychol 15:72-101 (1904)
[3] Lehmann and Romano, Testing Statistical Hypotheses (3E ed.), New York: Springer. ISBN 0387988645 (2005)
[4] Howell, D, Statistical Methods for Psychology. (5th ed.), Duxbury Press:324-5 (2002)
[5] Benjamini and Hochberg, Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society Series B 59:289-300 (1995)
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