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 496 genes and 9 clinical features across 868 samples, statistically thresholded by Q value < 0.05, 6 clinical features related to at least one genes.

  • 1 gene correlated to 'Time to Death'.

    • HSA-MIR-874

  • 26 genes correlated to 'AGE'.

    • HSA-MIR-424 ,  HSA-MIR-31 ,  HSA-MIR-598 ,  HSA-MIR-542 ,  HSA-MIR-99A ,  ...

  • 301 genes correlated to 'HISTOLOGICAL.TYPE'.

    • HSA-MIR-210 ,  HSA-MIR-301A ,  HSA-MIR-616 ,  HSA-MIR-501 ,  HSA-MIR-3653 ,  ...

  • 3 genes correlated to 'DISTANT.METASTASIS'.

    • HSA-MIR-2276 ,  HSA-MIR-374C ,  HSA-MIR-3174

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

    • HSA-MIR-374C ,  HSA-MIR-874 ,  HSA-MIR-197 ,  HSA-MIR-92A-1 ,  HSA-MIR-574 ,  ...

  • 9 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • HSA-MIR-143 ,  HSA-MIR-210 ,  HSA-LET-7F-2 ,  HSA-MIR-200A ,  HSA-MIR-374C ,  ...

  • No genes correlated to 'GENDER', 'RADIATIONS.RADIATION.REGIMENINDICATION', and 'NUMBER.OF.LYMPH.NODES'.

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=1 shorter survival N=1 longer survival N=0
AGE Spearman correlation test N=26 older N=2 younger N=24
GENDER t test   N=0        
HISTOLOGICAL TYPE ANOVA test N=301        
RADIATIONS RADIATION REGIMENINDICATION t test   N=0        
DISTANT METASTASIS ANOVA test N=3        
LYMPH NODE METASTASIS ANOVA test N=6        
NUMBER OF LYMPH NODES Spearman correlation test   N=0        
NEOPLASM DISEASESTAGE ANOVA test N=9        
Clinical variable #1: 'Time to Death'

One gene related to 'Time to Death'.

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

Time to Death Duration (Months) 0-223.4 (median=18.2)
  censored N = 714
  death N = 95
     
  Significant markers N = 1
  associated with shorter survival 1
  associated with longer survival 0
List of one gene significantly associated with 'Time to Death' by Cox regression test

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

HazardRatio Wald_P Q C_index
HSA-MIR-874 1.56 1.448e-05 0.0072 0.601

Figure S1.  Get High-res Image As an example, this figure shows the association of HSA-MIR-874 to 'Time to Death'. four curves present the cumulative survival rates of 4 quartile subsets of patients. P value = 1.45e-05 with univariate Cox regression analysis using continuous log-2 expression values.

Clinical variable #2: 'AGE'

26 genes related to 'AGE'.

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

AGE Mean (SD) 58.47 (13)
  Significant markers N = 26
  pos. correlated 2
  neg. correlated 24
List of top 10 genes significantly correlated to 'AGE' by Spearman correlation test

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

SpearmanCorr corrP Q
HSA-MIR-424 -0.2292 8.548e-12 4.24e-09
HSA-MIR-31 -0.2155 3.211e-10 1.59e-07
HSA-MIR-598 -0.1939 8.58e-09 4.24e-06
HSA-MIR-542 -0.1847 4.309e-08 2.12e-05
HSA-MIR-99A -0.1839 4.908e-08 2.41e-05
HSA-MIR-381 -0.1813 7.735e-08 3.8e-05
HSA-MIR-652 -0.1663 8.55e-07 0.000419
HSA-LET-7C -0.1637 1.269e-06 0.000621
HSA-MIR-202 -0.1998 2.237e-06 0.00109
HSA-MIR-450B -0.1518 7.253e-06 0.00353

Figure S2.  Get High-res Image As an example, this figure shows the association of HSA-MIR-424 to 'AGE'. P value = 8.55e-12 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'GENDER'

No gene related to 'GENDER'.

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

GENDER Labels N
  FEMALE 859
  MALE 9
     
  Significant markers N = 0
Clinical variable #4: 'HISTOLOGICAL.TYPE'

301 genes related to 'HISTOLOGICAL.TYPE'.

Table S6.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  INFILTRATING DUCTAL CARCINOMA 677
  INFILTRATING LOBULAR CARCINOMA 111
  MEDULLARY CARCINOMA 4
  MIXED HISTOLOGY (PLEASE SPECIFY) 26
  MUCINOUS CARCINOMA 10
  OTHER SPECIFY 40
     
  Significant markers N = 301
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

Table S7.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
HSA-MIR-210 3.587e-38 1.78e-35
HSA-MIR-301A 5.299e-28 2.62e-25
HSA-MIR-616 1.558e-24 7.7e-22
HSA-MIR-501 1.458e-23 7.19e-21
HSA-MIR-3653 3.212e-23 1.58e-20
HSA-MIR-197 5.929e-23 2.91e-20
HSA-MIR-301B 1.929e-22 9.45e-20
HSA-MIR-1307 4.027e-22 1.97e-19
HSA-MIR-324 6.042e-22 2.95e-19
HSA-MIR-1306 9.848e-22 4.8e-19

Figure S3.  Get High-res Image As an example, this figure shows the association of HSA-MIR-210 to 'HISTOLOGICAL.TYPE'. P value = 3.59e-38 with ANOVA analysis.

Clinical variable #5: 'RADIATIONS.RADIATION.REGIMENINDICATION'

No gene related to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

Table S8.  Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 210
  YES 658
     
  Significant markers N = 0
Clinical variable #6: 'DISTANT.METASTASIS'

3 genes related to 'DISTANT.METASTASIS'.

Table S9.  Basic characteristics of clinical feature: 'DISTANT.METASTASIS'

DISTANT.METASTASIS Labels N
  CM0 (I+) 2
  M0 771
  M1 14
  MX 81
     
  Significant markers N = 3
List of 3 genes differentially expressed by 'DISTANT.METASTASIS'

Table S10.  Get Full Table List of 3 genes differentially expressed by 'DISTANT.METASTASIS'

ANOVA_P Q
HSA-MIR-2276 1.961e-07 9.72e-05
HSA-MIR-374C 4.281e-06 0.00212
HSA-MIR-3174 3.358e-05 0.0166

Figure S4.  Get High-res Image As an example, this figure shows the association of HSA-MIR-2276 to 'DISTANT.METASTASIS'. P value = 1.96e-07 with ANOVA analysis.

Clinical variable #7: 'LYMPH.NODE.METASTASIS'

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

Table S11.  Basic characteristics of clinical feature: 'LYMPH.NODE.METASTASIS'

LYMPH.NODE.METASTASIS Labels N
  N0 255
  N0 (I+) 22
  N0 (I-) 131
  N0 (MOL+) 1
  N1 103
  N1A 129
  N1B 32
  N1C 2
  N1MI 24
  N2 50
  N2A 53
  N3 18
  N3A 30
  N3B 2
  N3C 1
  NX 15
     
  Significant markers N = 6
List of 6 genes differentially expressed by 'LYMPH.NODE.METASTASIS'

Table S12.  Get Full Table List of 6 genes differentially expressed by 'LYMPH.NODE.METASTASIS'

ANOVA_P Q
HSA-MIR-374C 3.314e-08 1.64e-05
HSA-MIR-874 4.3e-08 2.13e-05
HSA-MIR-197 2.88e-05 0.0142
HSA-MIR-92A-1 5.184e-05 0.0256
HSA-MIR-574 6.08e-05 0.0299
HSA-MIR-92A-2 6.799e-05 0.0334

Figure S5.  Get High-res Image As an example, this figure shows the association of HSA-MIR-374C to 'LYMPH.NODE.METASTASIS'. P value = 3.31e-08 with ANOVA analysis.

Clinical variable #8: 'NUMBER.OF.LYMPH.NODES'

No gene related to 'NUMBER.OF.LYMPH.NODES'.

Table S13.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 2.21 (4.4)
  Significant markers N = 0
Clinical variable #9: 'NEOPLASM.DISEASESTAGE'

9 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S14.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 72
  STAGE IA 67
  STAGE IB 7
  STAGE II 8
  STAGE IIA 294
  STAGE IIB 195
  STAGE III 2
  STAGE IIIA 124
  STAGE IIIB 25
  STAGE IIIC 41
  STAGE IV 14
  STAGE TIS 1
  STAGE X 17
     
  Significant markers N = 9
List of 9 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S15.  Get Full Table List of 9 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
HSA-MIR-143 5.031e-09 2.5e-06
HSA-MIR-210 1.406e-08 6.96e-06
HSA-LET-7F-2 1.133e-07 5.6e-05
HSA-MIR-200A 3.455e-06 0.0017
HSA-MIR-374C 1.038e-05 0.0051
HSA-MIR-125B-2 4.096e-05 0.0201
HSA-MIR-338 6.775e-05 0.0332
HSA-LET-7A-2 8.604e-05 0.0421
HSA-LET-7A-1 9.022e-05 0.044

Figure S6.  Get High-res Image As an example, this figure shows the association of HSA-MIR-143 to 'NEOPLASM.DISEASESTAGE'. P value = 5.03e-09 with ANOVA analysis.

Methods & Data
Input

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

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

  • Number of patients = 868

  • Number of genes = 496

  • Number of clinical features = 9

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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