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 18041 genes and 10 clinical features across 192 samples, statistically thresholded by Q value < 0.05, 7 clinical features related to at least one genes.

  • 30 genes correlated to 'GENDER'.

    • XIST|7503 ,  ZFY|7544 ,  KDM5D|8284 ,  RPS4Y1|6192 ,  PRKY|5616 ,  ...

  • 33 genes correlated to 'HISTOLOGICAL.TYPE'.

    • SETD7|80854 ,  OSR2|116039 ,  RNF125|54941 ,  PAFAH1B1|5048 ,  MAP3K6|9064 ,  ...

  • 1 gene correlated to 'PATHOLOGY.N'.

    • PDE12|201626

  • 14 genes correlated to 'PATHOLOGICSPREAD(M)'.

    • RB1|5925 ,  TDRD12|91646 ,  S100Z|170591 ,  FAM171A1|221061 ,  ZNF845|91664 ,  ...

  • 4 genes correlated to 'TUMOR.STAGE'.

    • UNG|7374 ,  HAP1|9001 ,  TCHH|7062 ,  BEND3|57673

  • 4 genes correlated to 'COMPLETENESS.OF.RESECTION'.

    • ZNHIT2|741 ,  OVCH1|341350 ,  PNPLA6|10908 ,  MORN4|118812

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

    • NIPAL4|348938 ,  RTN4RL1|146760

  • No genes correlated to 'Time to Death', 'AGE', and 'PATHOLOGY.T'.

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=30 male N=13 female N=17
HISTOLOGICAL TYPE t test N=33 colon mucinous adenocarcinoma N=27 colon adenocarcinoma N=6
PATHOLOGY T Spearman correlation test   N=0        
PATHOLOGY N Spearman correlation test N=1 higher pN N=0 lower pN N=1
PATHOLOGICSPREAD(M) ANOVA test N=14        
TUMOR STAGE Spearman correlation test N=4 higher stage N=2 lower stage N=2
COMPLETENESS OF RESECTION ANOVA test N=4        
NUMBER OF LYMPH NODES Spearman correlation test N=2 higher number.of.lymph.nodes N=2 lower number.of.lymph.nodes 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) 0.1-129.1 (median=6.6)
  censored N = 150
  death N = 25
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

No gene related to 'AGE'.

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

AGE Mean (SD) 65.4 (14)
  Significant markers N = 0
Clinical variable #3: 'GENDER'

30 genes related to 'GENDER'.

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

GENDER Labels N
  FEMALE 92
  MALE 100
     
  Significant markers N = 30
  Higher in MALE 13
  Higher in FEMALE 17
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
XIST|7503 -40.35 1.064e-87 1.92e-83 1
ZFY|7544 42.8 8.828e-84 1.59e-79 0.9991
KDM5D|8284 54.8 1.049e-76 1.89e-72 1
RPS4Y1|6192 37.74 1.333e-74 2.4e-70 1
PRKY|5616 27.6 3.198e-65 5.77e-61 0.9949
NLGN4Y|22829 30.73 3.779e-50 6.81e-46 0.9991
TMSB4Y|9087 27.11 1.541e-48 2.78e-44 0.9899
DDX3Y|8653 43.18 5.888e-48 1.06e-43 1
EIF1AY|9086 39.28 6.612e-48 1.19e-43 1
USP9Y|8287 38.97 1.278e-44 2.3e-40 1

Figure S1.  Get High-res Image As an example, this figure shows the association of XIST|7503 to 'GENDER'. P value = 1.06e-87 with T-test analysis.

Clinical variable #4: 'HISTOLOGICAL.TYPE'

33 genes related to 'HISTOLOGICAL.TYPE'.

Table S5.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  COLON ADENOCARCINOMA 168
  COLON MUCINOUS ADENOCARCINOMA 24
     
  Significant markers N = 33
  Higher in COLON MUCINOUS ADENOCARCINOMA 27
  Higher in COLON ADENOCARCINOMA 6
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

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

T(pos if higher in 'COLON MUCINOUS ADENOCARCINOMA') ttestP Q AUC
SETD7|80854 6.38 7.503e-08 0.00135 0.7748
OSR2|116039 6.1 1.155e-07 0.00208 0.7393
RNF125|54941 6.33 1.523e-07 0.00275 0.7847
PAFAH1B1|5048 6.32 1.597e-07 0.00288 0.7914
MAP3K6|9064 6.21 1.755e-07 0.00316 0.782
KCTD15|79047 5.98 2.745e-07 0.00495 0.7579
TOX|9760 6.28 2.935e-07 0.00529 0.8046
CAMTA2|23125 5.92 3.084e-07 0.00556 0.7542
MEIS2|4212 6.03 3.693e-07 0.00666 0.7862
NAA20|51126 -5.89 4.026e-07 0.00726 0.7527

Figure S2.  Get High-res Image As an example, this figure shows the association of SETD7|80854 to 'HISTOLOGICAL.TYPE'. P value = 7.5e-08 with T-test analysis.

Clinical variable #5: 'PATHOLOGY.T'

No gene related to 'PATHOLOGY.T'.

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

PATHOLOGY.T Mean (SD) 2.9 (0.68)
  N
  T0 1
  T1 5
  T2 33
  T3 126
  T4 27
     
  Significant markers N = 0
Clinical variable #6: 'PATHOLOGY.N'

One gene related to 'PATHOLOGY.N'.

Table S8.  Basic characteristics of clinical feature: 'PATHOLOGY.N'

PATHOLOGY.N Mean (SD) 0.53 (0.73)
  N
  N0 117
  N1 47
  N2 27
     
  Significant markers N = 1
  pos. correlated 0
  neg. correlated 1
List of one gene significantly correlated to 'PATHOLOGY.N' by Spearman correlation test

Table S9.  Get Full Table List of one gene significantly correlated to 'PATHOLOGY.N' by Spearman correlation test

SpearmanCorr corrP Q
PDE12|201626 -0.3448 1.033e-06 0.0186

Figure S3.  Get High-res Image As an example, this figure shows the association of PDE12|201626 to 'PATHOLOGY.N'. P value = 1.03e-06 with Spearman correlation analysis.

Clinical variable #7: 'PATHOLOGICSPREAD(M)'

14 genes related to 'PATHOLOGICSPREAD(M)'.

Table S10.  Basic characteristics of clinical feature: 'PATHOLOGICSPREAD(M)'

PATHOLOGICSPREAD(M) Labels N
  M0 130
  M1 19
  M1A 5
  M1B 1
  MX 33
     
  Significant markers N = 14
List of top 10 genes differentially expressed by 'PATHOLOGICSPREAD(M)'

Table S11.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGICSPREAD(M)'

ANOVA_P Q
RB1|5925 2.057e-11 3.71e-07
TDRD12|91646 1.979e-09 3.57e-05
S100Z|170591 3.505e-09 6.32e-05
FAM171A1|221061 8.725e-09 0.000157
ZNF845|91664 9.899e-09 0.000179
ZNF675|171392 7.073e-08 0.00128
ZNF558|148156 8.032e-08 0.00145
CCDC122|160857 1.616e-07 0.00291
TMEM185A|84548 2.397e-07 0.00432
C13ORF31|144811 7.189e-07 0.013

Figure S4.  Get High-res Image As an example, this figure shows the association of RB1|5925 to 'PATHOLOGICSPREAD(M)'. P value = 2.06e-11 with ANOVA analysis.

Clinical variable #8: 'TUMOR.STAGE'

4 genes related to 'TUMOR.STAGE'.

Table S12.  Basic characteristics of clinical feature: 'TUMOR.STAGE'

TUMOR.STAGE Mean (SD) 2.38 (0.92)
  N
  Stage 1 31
  Stage 2 77
  Stage 3 50
  Stage 4 25
     
  Significant markers N = 4
  pos. correlated 2
  neg. correlated 2
List of 4 genes significantly correlated to 'TUMOR.STAGE' by Spearman correlation test

Table S13.  Get Full Table List of 4 genes significantly correlated to 'TUMOR.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
UNG|7374 -0.3691 2.732e-07 0.00493
HAP1|9001 0.3758 2.986e-07 0.00539
TCHH|7062 0.3665 4.195e-07 0.00757
BEND3|57673 -0.3567 7.203e-07 0.013

Figure S5.  Get High-res Image As an example, this figure shows the association of UNG|7374 to 'TUMOR.STAGE'. P value = 2.73e-07 with Spearman correlation analysis.

Clinical variable #9: 'COMPLETENESS.OF.RESECTION'

4 genes related to 'COMPLETENESS.OF.RESECTION'.

Table S14.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 116
  R2 2
  RX 22
     
  Significant markers N = 4
List of 4 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S15.  Get Full Table List of 4 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
ZNHIT2|741 5.337e-12 9.63e-08
OVCH1|341350 3.741e-08 0.000675
PNPLA6|10908 1.631e-06 0.0294
MORN4|118812 2.553e-06 0.0461

Figure S6.  Get High-res Image As an example, this figure shows the association of ZNHIT2|741 to 'COMPLETENESS.OF.RESECTION'. P value = 5.34e-12 with ANOVA analysis.

Clinical variable #10: 'NUMBER.OF.LYMPH.NODES'

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

Table S16.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 1.98 (4.9)
  Significant markers N = 2
  pos. correlated 2
  neg. correlated 0
List of 2 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

Table S17.  Get Full Table List of 2 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
NIPAL4|348938 0.3922 1.231e-07 0.00222
RTN4RL1|146760 0.3636 1.831e-06 0.033

Figure S7.  Get High-res Image As an example, this figure shows the association of NIPAL4|348938 to 'NUMBER.OF.LYMPH.NODES'. P value = 1.23e-07 with Spearman correlation analysis. The straight line presents the best linear regression.

Methods & Data
Input

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

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

  • Number of patients = 192

  • Number of genes = 18041

  • Number of clinical features = 10

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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