Correlation between miRseq expression and clinical features
Overview
Introduction

This pipeline uses various statistical tests to identify miRs whose log2 expression levels correlated to selected clinical features.

Summary

Testing the association between 498 miRs and 12 clinical features across 993 samples, statistically thresholded by P value < 0.05 and Q value < 0.3, 10 clinical features related to at least one miRs.

  • 6 miRs correlated to 'Time to Death'.

    • HSA-MIR-874 ,  HSA-MIR-30A ,  HSA-MIR-328 ,  HSA-MIR-1307 ,  HSA-MIR-99B ,  ...

  • 54 miRs correlated to 'AGE'.

    • HSA-MIR-424 ,  HSA-MIR-31 ,  HSA-MIR-99A ,  HSA-MIR-381 ,  HSA-MIR-598 ,  ...

  • 46 miRs correlated to 'NEOPLASM.DISEASESTAGE'.

    • HSA-MIR-210 ,  HSA-MIR-374C ,  HSA-MIR-301A ,  HSA-MIR-143 ,  HSA-MIR-455 ,  ...

  • 11 miRs correlated to 'PATHOLOGY.T.STAGE'.

    • HSA-MIR-758 ,  HSA-MIR-127 ,  HSA-MIR-9-2 ,  HSA-MIR-150 ,  HSA-MIR-9-1 ,  ...

  • 9 miRs correlated to 'PATHOLOGY.N.STAGE'.

    • HSA-MIR-10A ,  HSA-MIR-3613 ,  HSA-MIR-455 ,  HSA-MIR-92A-2 ,  HSA-MIR-577 ,  ...

  • 109 miRs correlated to 'PATHOLOGY.M.STAGE'.

    • HSA-MIR-16-1 ,  HSA-MIR-628 ,  HSA-MIR-424 ,  HSA-MIR-3170 ,  HSA-MIR-550A-2 ,  ...

  • 393 miRs correlated to 'HISTOLOGICAL.TYPE'.

    • HSA-MIR-210 ,  HSA-MIR-1306 ,  HSA-MIR-130B ,  HSA-MIR-301A ,  HSA-MIR-197 ,  ...

  • 37 miRs correlated to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

    • HSA-MIR-3605 ,  HSA-MIR-3607 ,  HSA-MIR-1271 ,  HSA-MIR-940 ,  HSA-MIR-3647 ,  ...

  • 12 miRs correlated to 'NUMBER.OF.LYMPH.NODES'.

    • HSA-MIR-577 ,  HSA-MIR-10A ,  HSA-MIR-3613 ,  HSA-MIR-221 ,  HSA-MIR-92A-2 ,  ...

  • 105 miRs correlated to 'RACE'.

    • HSA-MIR-660 ,  HSA-MIR-1304 ,  HSA-MIR-20A ,  HSA-MIR-93 ,  HSA-MIR-103-1 ,  ...

  • No miRs correlated to 'GENDER', and 'ETHNICITY'.

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 miRs that are significantly associated with each clinical feature at P value < 0.05 and Q value < 0.3.

Clinical feature Statistical test Significant miRs Associated with                 Associated with
Time to Death Cox regression test N=6 shorter survival N=5 longer survival N=1
AGE Spearman correlation test N=54 older N=9 younger N=45
NEOPLASM DISEASESTAGE Kruskal-Wallis test N=46        
PATHOLOGY T STAGE Spearman correlation test N=11 higher stage N=3 lower stage N=8
PATHOLOGY N STAGE Spearman correlation test N=9 higher stage N=1 lower stage N=8
PATHOLOGY M STAGE Kruskal-Wallis test N=109        
GENDER Wilcoxon test   N=0        
HISTOLOGICAL TYPE Kruskal-Wallis test N=393        
RADIATIONS RADIATION REGIMENINDICATION Wilcoxon test N=37 yes N=37 no N=0
NUMBER OF LYMPH NODES Spearman correlation test N=12 higher number.of.lymph.nodes N=1 lower number.of.lymph.nodes N=11
RACE Kruskal-Wallis test N=105        
ETHNICITY Wilcoxon test   N=0        
Clinical variable #1: 'Time to Death'

6 miRs related to 'Time to Death'.

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

Time to Death Duration (Months) 0-234.3 (median=21.4)
  censored N = 858
  death N = 114
     
  Significant markers N = 6
  associated with shorter survival 5
  associated with longer survival 1
List of 6 miRs differentially expressed by 'Time to Death'

Table S2.  Get Full Table List of 6 miRs significantly associated with 'Time to Death' by Cox regression test

HazardRatio Wald_P Q C_index
HSA-MIR-874 1.5 1.304e-05 0.0065 0.586
HSA-MIR-30A 0.77 6.289e-05 0.031 0.379
HSA-MIR-328 1.37 9.714e-05 0.048 0.586
HSA-MIR-1307 1.35 0.0001386 0.069 0.621
HSA-MIR-99B 1.46 0.0003144 0.16 0.586
HSA-MIR-148B 1.5 0.0004165 0.21 0.581
Clinical variable #2: 'AGE'

54 miRs related to 'AGE'.

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

AGE Mean (SD) 58.66 (13)
  Significant markers N = 54
  pos. correlated 9
  neg. correlated 45
List of top 10 miRs differentially expressed by 'AGE'

Table S4.  Get Full Table List of top 10 miRs significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-424 -0.2092 3.708e-11 1.85e-08
HSA-MIR-31 -0.2111 6.072e-11 3.02e-08
HSA-MIR-99A -0.2024 1.629e-10 8.08e-08
HSA-MIR-381 -0.1941 9.348e-10 4.63e-07
HSA-MIR-598 -0.1903 1.91e-09 9.43e-07
HSA-MIR-652 -0.1842 6.351e-09 3.13e-06
HSA-LET-7C -0.1771 2.37e-08 1.17e-05
HSA-MIR-542 -0.1692 9.875e-08 4.85e-05
HSA-MIR-125B-1 -0.1607 4.273e-07 0.000209
HSA-MIR-202 -0.1954 6.796e-07 0.000332
Clinical variable #3: 'NEOPLASM.DISEASESTAGE'

46 miRs related to 'NEOPLASM.DISEASESTAGE'.

Table S5.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 80
  STAGE IA 79
  STAGE IB 10
  STAGE II 9
  STAGE IIA 335
  STAGE IIB 223
  STAGE III 2
  STAGE IIIA 136
  STAGE IIIB 26
  STAGE IIIC 58
  STAGE IV 16
  STAGE TIS 1
  STAGE X 17
     
  Significant markers N = 46
List of top 10 miRs differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S6.  Get Full Table List of top 10 miRs differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
HSA-MIR-210 9.842e-09 4.9e-06
HSA-MIR-374C 3.241e-06 0.00161
HSA-MIR-301A 5.311e-06 0.00263
HSA-MIR-143 6.606e-06 0.00327
HSA-MIR-455 6.644e-06 0.00328
HSA-MIR-130B 7.034e-06 0.00347
HSA-LET-7F-2 8.67e-06 0.00427
HSA-MIR-1247 1.569e-05 0.0077
HSA-MIR-484 1.706e-05 0.00836
HSA-MIR-222 1.73e-05 0.00846
Clinical variable #4: 'PATHOLOGY.T.STAGE'

11 miRs related to 'PATHOLOGY.T.STAGE'.

Table S7.  Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'

PATHOLOGY.T.STAGE Mean (SD) 1.92 (0.72)
  N
  1 263
  2 576
  3 115
  4 36
     
  Significant markers N = 11
  pos. correlated 3
  neg. correlated 8
List of top 10 miRs differentially expressed by 'PATHOLOGY.T.STAGE'

Table S8.  Get Full Table List of top 10 miRs significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-758 -0.1506 1.999e-06 0.000995
HSA-MIR-127 -0.1255 7.465e-05 0.0371
HSA-MIR-9-2 0.1183 0.0001911 0.0948
HSA-MIR-150 -0.117 0.0002253 0.112
HSA-MIR-9-1 0.1159 0.0002574 0.127
HSA-MIR-382 -0.1158 0.0002627 0.13
HSA-MIR-409 -0.1153 0.0002805 0.138
HSA-MIR-411 -0.1155 0.0003019 0.148
HSA-MIR-3662 0.1456 0.0003397 0.166
HSA-MIR-181C -0.1117 0.0004284 0.21
Clinical variable #5: 'PATHOLOGY.N.STAGE'

9 miRs related to 'PATHOLOGY.N.STAGE'.

Table S9.  Basic characteristics of clinical feature: 'PATHOLOGY.N.STAGE'

PATHOLOGY.N.STAGE Mean (SD) 0.77 (0.91)
  N
  0 470
  1 329
  2 109
  3 68
     
  Significant markers N = 9
  pos. correlated 1
  neg. correlated 8
List of 9 miRs differentially expressed by 'PATHOLOGY.N.STAGE'

Table S10.  Get Full Table List of 9 miRs significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-10A 0.1558 1.001e-06 0.000499
HSA-MIR-3613 -0.1319 3.586e-05 0.0178
HSA-MIR-455 -0.1283 5.859e-05 0.0291
HSA-MIR-92A-2 -0.1283 5.863e-05 0.0291
HSA-MIR-577 -0.1389 0.0001132 0.0559
HSA-MIR-17 -0.1143 0.000346 0.171
HSA-MIR-19B-1 -0.1115 0.000504 0.248
HSA-MIR-548B -0.1272 0.0005354 0.263
HSA-MIR-19B-2 -0.1106 0.00054 0.265
Clinical variable #6: 'PATHOLOGY.M.STAGE'

109 miRs related to 'PATHOLOGY.M.STAGE'.

Table S11.  Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'

PATHOLOGY.M.STAGE Labels N
  CM0 (I+) 2
  M0 843
  M1 16
  MX 132
     
  Significant markers N = 109
List of top 10 miRs differentially expressed by 'PATHOLOGY.M.STAGE'

Table S12.  Get Full Table List of top 10 miRs differentially expressed by 'PATHOLOGY.M.STAGE'

ANOVA_P Q
HSA-MIR-16-1 3.068e-12 1.53e-09
HSA-MIR-628 2.232e-09 1.11e-06
HSA-MIR-424 1.847e-08 9.16e-06
HSA-MIR-3170 4.332e-08 2.14e-05
HSA-MIR-550A-2 6.214e-08 3.07e-05
HSA-MIR-3607 9.118e-08 4.5e-05
HSA-MIR-345 2.061e-07 0.000101
HSA-MIR-23B 3.009e-07 0.000148
HSA-MIR-361 3.905e-07 0.000191
HSA-MIR-3174 5.152e-07 0.000252
Clinical variable #7: 'GENDER'

No miR related to 'GENDER'.

Table S13.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 982
  MALE 11
     
  Significant markers N = 0
Clinical variable #8: 'HISTOLOGICAL.TYPE'

393 miRs related to 'HISTOLOGICAL.TYPE'.

Table S14.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  INFILTRATING CARCINOMA NOS 1
  INFILTRATING DUCTAL CARCINOMA 735
  INFILTRATING LOBULAR CARCINOMA 165
  MEDULLARY CARCINOMA 5
  MIXED HISTOLOGY (PLEASE SPECIFY) 28
  MUCINOUS CARCINOMA 14
  OTHER SPECIFY 44
     
  Significant markers N = 393
List of top 10 miRs differentially expressed by 'HISTOLOGICAL.TYPE'

Table S15.  Get Full Table List of top 10 miRs differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
HSA-MIR-210 1.176e-40 5.86e-38
HSA-MIR-1306 1.238e-33 6.15e-31
HSA-MIR-130B 6.44e-33 3.19e-30
HSA-MIR-301A 2.101e-32 1.04e-29
HSA-MIR-197 7.001e-32 3.46e-29
HSA-MIR-328 1.129e-31 5.57e-29
HSA-MIR-505 2.187e-31 1.08e-28
HSA-MIR-616 5.565e-31 2.73e-28
HSA-MIR-301B 7.534e-31 3.69e-28
HSA-MIR-186 9.976e-31 4.88e-28
Clinical variable #9: 'RADIATIONS.RADIATION.REGIMENINDICATION'

37 miRs related to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

Table S16.  Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 278
  YES 715
     
  Significant markers N = 37
  Higher in YES 37
  Higher in NO 0
List of top 10 miRs differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S17.  Get Full Table List of top 10 miRs differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

W(pos if higher in 'YES') wilcoxontestP Q AUC
HSA-MIR-3605 70216 5.313e-10 2.65e-07 0.6285
HSA-MIR-3607 74547 9.293e-10 4.62e-07 0.625
HSA-MIR-1271 74602 5.432e-07 0.000269 0.6038
HSA-MIR-940 74171 1.885e-06 0.000933 0.5991
HSA-MIR-3647 80330 2.654e-06 0.00131 0.5959
HSA-MIR-551B 58147 4.088e-06 0.00202 0.6012
HSA-MIR-3620 9498 4.163e-06 0.00205 0.6541
HSA-MIR-3677 80556 6.926e-06 0.0034 0.5919
HSA-MIR-101-2 117574 7.378e-06 0.00362 0.5915
HSA-MIR-3928 70619 8.127e-06 0.00397 0.5943
Clinical variable #10: 'NUMBER.OF.LYMPH.NODES'

12 miRs related to 'NUMBER.OF.LYMPH.NODES'.

Table S18.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 2.33 (4.6)
  Significant markers N = 12
  pos. correlated 1
  neg. correlated 11
List of top 10 miRs differentially expressed by 'NUMBER.OF.LYMPH.NODES'

Table S19.  Get Full Table List of top 10 miRs significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-577 -0.1634 2.264e-05 0.0113
HSA-MIR-10A 0.1423 3.519e-05 0.0175
HSA-MIR-3613 -0.1362 7.608e-05 0.0377
HSA-MIR-221 -0.1291 0.0001775 0.0879
HSA-MIR-92A-2 -0.1266 0.0002365 0.117
HSA-MIR-222 -0.1265 0.0002381 0.117
HSA-MIR-29A -0.1233 0.0003437 0.169
HSA-MIR-197 -0.1232 0.0003468 0.17
HSA-MIR-2114 -0.1546 0.0003617 0.177
HSA-LET-7G -0.1214 0.0004255 0.208
Clinical variable #11: 'RACE'

105 miRs related to 'RACE'.

Table S20.  Basic characteristics of clinical feature: 'RACE'

RACE Labels N
  AMERICAN INDIAN OR ALASKA NATIVE 1
  ASIAN 57
  BLACK OR AFRICAN AMERICAN 138
  WHITE 709
     
  Significant markers N = 105
List of top 10 miRs differentially expressed by 'RACE'

Table S21.  Get Full Table List of top 10 miRs differentially expressed by 'RACE'

ANOVA_P Q
HSA-MIR-660 2.079e-16 1.04e-13
HSA-MIR-1304 3.098e-16 1.54e-13
HSA-MIR-20A 4.23e-13 2.1e-10
HSA-MIR-93 1.119e-12 5.54e-10
HSA-MIR-103-1 4.587e-11 2.27e-08
HSA-MIR-17 5.524e-11 2.72e-08
HSA-MIR-361 7.203e-11 3.54e-08
HSA-MIR-185 9.8e-11 4.81e-08
HSA-MIR-500B 1.089e-10 5.34e-08
HSA-MIR-128-2 3.682e-10 1.8e-07
Clinical variable #12: 'ETHNICITY'

No miR related to 'ETHNICITY'.

Table S22.  Basic characteristics of clinical feature: 'ETHNICITY'

ETHNICITY Labels N
  HISPANIC OR LATINO 36
  NOT HISPANIC OR LATINO 795
     
  Significant markers N = 0
Methods & Data
Input

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

  • Clinical data file = BRCA-TP.merged_data.txt

  • Number of patients = 993

  • Number of miRs = 498

  • Number of clinical features = 12

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

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

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

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] Howell, D, Statistical Methods for Psychology. (5th ed.), Duxbury Press:324-5 (2002)
[4] Lehmann and Romano, Testing Statistical Hypotheses (3E ed.), New York: Springer. ISBN 0387988645 (2005)
[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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