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 593 genes and 6 clinical features across 23 samples, statistically thresholded by Q value < 0.05, 2 clinical features related to at least one genes.

  • 1 gene correlated to 'PRIMARY.SITE.OF.DISEASE'.

    • HSA-MIR-184

  • 1 gene correlated to 'LYMPH.NODE.METASTASIS'.

    • HSA-MIR-16-2

  • No genes correlated to 'Time to Death', 'AGE', 'GENDER', and 'NEOPLASM.DISEASESTAGE'.

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=0        
AGE Spearman correlation test   N=0        
PRIMARY SITE OF DISEASE ANOVA test N=1        
GENDER t test   N=0        
LYMPH NODE METASTASIS ANOVA test N=1        
NEOPLASM DISEASESTAGE ANOVA test   N=0        
Clinical variable #1: 'Time to Death'

No gene related to 'Time to Death'.

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

Time to Death Duration (Months) 4.9-78.4 (median=13.8)
  censored N = 5
  death N = 9
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

No gene related to 'AGE'.

Table S2.  Basic characteristics of clinical feature: 'AGE'

AGE Mean (SD) 59.43 (13)
  Significant markers N = 0
Clinical variable #3: 'PRIMARY.SITE.OF.DISEASE'

One gene related to 'PRIMARY.SITE.OF.DISEASE'.

Table S3.  Basic characteristics of clinical feature: 'PRIMARY.SITE.OF.DISEASE'

PRIMARY.SITE.OF.DISEASE Labels N
  DISTANT METASTASIS 3
  REGIONAL CUTANEOUS OR SUBCUTANEOUS TISSUE (INCLUDES SATELLITE AND IN-TRANSIT METASTASIS) 6
  REGIONAL LYMPH NODE 14
     
  Significant markers N = 1
List of one gene differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

Table S4.  Get Full Table List of one gene differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

ANOVA_P Q
HSA-MIR-184 7.481e-05 0.0444

Figure S1.  Get High-res Image As an example, this figure shows the association of HSA-MIR-184 to 'PRIMARY.SITE.OF.DISEASE'. P value = 7.48e-05 with ANOVA analysis.

Clinical variable #4: 'GENDER'

No gene related to 'GENDER'.

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

GENDER Labels N
  FEMALE 12
  MALE 11
     
  Significant markers N = 0
Clinical variable #5: 'LYMPH.NODE.METASTASIS'

One gene related to 'LYMPH.NODE.METASTASIS'.

Table S6.  Basic characteristics of clinical feature: 'LYMPH.NODE.METASTASIS'

LYMPH.NODE.METASTASIS Labels N
  N0 11
  N1 1
  N1A 2
  N1B 3
  N2B 2
  N3 2
     
  Significant markers N = 1
List of one gene differentially expressed by 'LYMPH.NODE.METASTASIS'

Table S7.  Get Full Table List of one gene differentially expressed by 'LYMPH.NODE.METASTASIS'

ANOVA_P Q
HSA-MIR-16-2 2.311e-05 0.0137

Figure S2.  Get High-res Image As an example, this figure shows the association of HSA-MIR-16-2 to 'LYMPH.NODE.METASTASIS'. P value = 2.31e-05 with ANOVA analysis.

Clinical variable #6: 'NEOPLASM.DISEASESTAGE'

No gene related to 'NEOPLASM.DISEASESTAGE'.

Table S8.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 4
  STAGE IA 1
  STAGE II 1
  STAGE IIB 2
  STAGE IIC 3
  STAGE III 1
  STAGE IIIA 2
  STAGE IIIB 2
  STAGE IIIC 4
     
  Significant markers N = 0
Methods & Data
Input

  • Expresson data file = SKCM-WT.miRseq_RPKM_log2.txt

  • Clinical data file = SKCM-WT.clin.merged.picked.txt

  • Number of patients = 23

  • Number of genes = 593

  • Number of clinical features = 6

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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