Correlation between mRNAseq expression and clinical features
Overview
Introduction

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

Summary

Testing the association between 18307 genes and 9 clinical features across 134 samples, statistically thresholded by Q value < 0.05, 4 clinical features related to at least one genes.

  • 11 genes correlated to 'GENDER'.

    • USP9Y|8287 ,  PRKY|5616 ,  RPS4Y1|6192 ,  XIST|7503 ,  CYORF15A|246126 ,  ...

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

    • TRIM25|7706

  • 7 genes correlated to 'NUMBER.OF.LYMPH.NODES'.

    • PRR15L|79170 ,  C6ORF141|135398 ,  BAT2|7916 ,  TTYH1|57348 ,  FBXO5|26271 ,  ...

  • 7 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • SLC39A5|283375 ,  LIMD1|8994 ,  BAAT|570 ,  PXT1|222659 ,  FAM35B2|439965 ,  ...

  • No genes correlated to 'Time to Death', 'AGE', 'KARNOFSKY.PERFORMANCE.SCORE', 'NUMBERPACKYEARSSMOKED', and 'DISTANT.METASTASIS'.

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        
GENDER t test N=11 male N=9 female N=2
KARNOFSKY PERFORMANCE SCORE Spearman correlation test   N=0        
NUMBERPACKYEARSSMOKED Spearman correlation test   N=0        
DISTANT METASTASIS ANOVA test   N=0        
LYMPH NODE METASTASIS ANOVA test N=1        
NUMBER OF LYMPH NODES Spearman correlation test N=7 higher number.of.lymph.nodes N=4 lower number.of.lymph.nodes N=3
NEOPLASM DISEASESTAGE ANOVA test N=7        
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) 0.1-131.2 (median=7)
  censored N = 93
  death N = 34
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

No gene related to 'AGE'.

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

AGE Mean (SD) 67.43 (11)
  Significant markers N = 0
Clinical variable #3: 'GENDER'

11 genes related to 'GENDER'.

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

GENDER Labels N
  FEMALE 35
  MALE 99
     
  Significant markers N = 11
  Higher in MALE 9
  Higher in FEMALE 2
List of top 10 genes differentially expressed by 'GENDER'

Table S4.  Get Full Table List of top 10 genes differentially expressed by 'GENDER'

T(pos if higher in 'MALE') ttestP Q AUC
USP9Y|8287 35.67 3.32e-45 6.07e-41 1
PRKY|5616 24.81 1.109e-40 2.03e-36 0.9982
RPS4Y1|6192 42.28 1.026e-38 1.88e-34 1
XIST|7503 -21.86 1.745e-37 3.19e-33 0.9924
CYORF15A|246126 29.8 5.354e-32 9.79e-28 1
DDX3Y|8653 35.53 5.772e-29 1.06e-24 1
KDM5D|8284 40.02 8.781e-29 1.61e-24 1
ZFY|7544 27.38 3.684e-27 6.74e-23 1
TSIX|9383 -15.59 6.296e-23 1.15e-18 0.9873
NLGN4Y|22829 18.25 1.908e-22 3.49e-18 0.9885

Figure S1.  Get High-res Image As an example, this figure shows the association of USP9Y|8287 to 'GENDER'. P value = 3.32e-45 with T-test analysis.

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

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

Table S5.  Basic characteristics of clinical feature: 'KARNOFSKY.PERFORMANCE.SCORE'

KARNOFSKY.PERFORMANCE.SCORE Mean (SD) 78.21 (16)
  Significant markers N = 0
Clinical variable #5: 'NUMBERPACKYEARSSMOKED'

No gene related to 'NUMBERPACKYEARSSMOKED'.

Table S6.  Basic characteristics of clinical feature: 'NUMBERPACKYEARSSMOKED'

NUMBERPACKYEARSSMOKED Mean (SD) 36.29 (23)
  Significant markers N = 0
Clinical variable #6: 'DISTANT.METASTASIS'

No gene related to 'DISTANT.METASTASIS'.

Table S7.  Basic characteristics of clinical feature: 'DISTANT.METASTASIS'

DISTANT.METASTASIS Labels N
  M0 77
  M1 5
  MX 51
     
  Significant markers N = 0
Clinical variable #7: 'LYMPH.NODE.METASTASIS'

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

Table S8.  Basic characteristics of clinical feature: 'LYMPH.NODE.METASTASIS'

LYMPH.NODE.METASTASIS Labels N
  N0 75
  N1 14
  N2 27
  N3 5
  NX 10
     
  Significant markers N = 1
List of one gene differentially expressed by 'LYMPH.NODE.METASTASIS'

Table S9.  Get Full Table List of one gene differentially expressed by 'LYMPH.NODE.METASTASIS'

ANOVA_P Q
TRIM25|7706 6.343e-07 0.0116

Figure S2.  Get High-res Image As an example, this figure shows the association of TRIM25|7706 to 'LYMPH.NODE.METASTASIS'. P value = 6.34e-07 with ANOVA analysis.

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

7 genes related to 'NUMBER.OF.LYMPH.NODES'.

Table S10.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 1.88 (3.7)
  Significant markers N = 7
  pos. correlated 4
  neg. correlated 3
List of 7 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

Table S11.  Get Full Table List of 7 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
PRR15L|79170 0.4991 1.457e-07 0.00267
C6ORF141|135398 -0.5036 2.674e-07 0.00489
BAT2|7916 -0.4878 3.03e-07 0.00555
TTYH1|57348 0.4776 5.768e-07 0.0106
FBXO5|26271 -0.4774 5.845e-07 0.0107
ANXA9|8416 0.4581 1.859e-06 0.034
ERP27|121506 0.4538 2.385e-06 0.0437

Figure S3.  Get High-res Image As an example, this figure shows the association of PRR15L|79170 to 'NUMBER.OF.LYMPH.NODES'. P value = 1.46e-07 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #9: 'NEOPLASM.DISEASESTAGE'

7 genes related to 'NEOPLASM.DISEASESTAGE'.

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

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 1
  STAGE II 37
  STAGE III 43
  STAGE IV 48
     
  Significant markers N = 7
List of 7 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S13.  Get Full Table List of 7 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
SLC39A5|283375 5.365e-09 9.82e-05
LIMD1|8994 6.675e-09 0.000122
BAAT|570 7.634e-09 0.00014
PXT1|222659 4.345e-08 0.000795
FAM35B2|439965 1.864e-07 0.00341
TTC36|143941 2.265e-07 0.00415
HAO2|51179 1.897e-06 0.0347

Figure S4.  Get High-res Image As an example, this figure shows the association of SLC39A5|283375 to 'NEOPLASM.DISEASESTAGE'. P value = 5.36e-09 with ANOVA analysis.

Methods & Data
Input

  • Expresson data file = BLCA-TP.uncv2.mRNAseq_RSEM_normalized_log2.txt

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

  • Number of patients = 134

  • Number of genes = 18307

  • 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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