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 9 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 ,  ...

  • 14 genes correlated to 'DISTANT.METASTASIS'.

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

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

    • TICAM2|353376 ,  AATF|26574 ,  MED24|9862 ,  PSMD3|5709 ,  PGAP3|93210 ,  ...

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

    • ZNHIT2|741 ,  OVCH1|341350 ,  PAIP2B|400961

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

    • NIPAL4|348938 ,  RTN4RL1|146760

  • 4 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • TDRD12|91646 ,  RB1|5925 ,  HAP1|9001 ,  PDE12|201626

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

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
DISTANT METASTASIS ANOVA test N=14        
LYMPH NODE METASTASIS ANOVA test N=11        
COMPLETENESS OF RESECTION ANOVA test N=3        
NUMBER OF LYMPH NODES Spearman correlation test N=2 higher number.of.lymph.nodes N=2 lower number.of.lymph.nodes N=0
NEOPLASM DISEASESTAGE ANOVA test N=4        
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=6.7)
  censored N = 151
  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: 'DISTANT.METASTASIS'

14 genes related to 'DISTANT.METASTASIS'.

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

DISTANT.METASTASIS Labels N
  M0 130
  M1 19
  M1A 5
  M1B 1
  MX 33
     
  Significant markers N = 14
List of top 10 genes differentially expressed by 'DISTANT.METASTASIS'

Table S8.  Get Full Table List of top 10 genes differentially expressed by 'DISTANT.METASTASIS'

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 S3.  Get High-res Image As an example, this figure shows the association of RB1|5925 to 'DISTANT.METASTASIS'. P value = 2.06e-11 with ANOVA analysis.

Clinical variable #6: 'LYMPH.NODE.METASTASIS'

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

Table S9.  Basic characteristics of clinical feature: 'LYMPH.NODE.METASTASIS'

LYMPH.NODE.METASTASIS Labels N
  N0 117
  N1 27
  N1A 12
  N1B 7
  N1C 1
  N2 16
  N2A 2
  N2B 9
  NX 1
     
  Significant markers N = 11
List of top 10 genes differentially expressed by 'LYMPH.NODE.METASTASIS'

Table S10.  Get Full Table List of top 10 genes differentially expressed by 'LYMPH.NODE.METASTASIS'

ANOVA_P Q
TICAM2|353376 6.89e-14 1.24e-09
AATF|26574 9.606e-11 1.73e-06
MED24|9862 2.549e-09 4.6e-05
PSMD3|5709 4.174e-09 7.53e-05
PGAP3|93210 1.424e-08 0.000257
POP4|10775 5.76e-08 0.00104
STARD3|10948 1.205e-07 0.00217
C17ORF37|84299 2.65e-07 0.00478
PNMA5|114824 6.407e-07 0.0116
ERBB2|2064 1.36e-06 0.0245

Figure S4.  Get High-res Image As an example, this figure shows the association of TICAM2|353376 to 'LYMPH.NODE.METASTASIS'. P value = 6.89e-14 with ANOVA analysis.

Clinical variable #7: 'COMPLETENESS.OF.RESECTION'

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

Table S11.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 116
  R1 1
  R2 2
  RX 22
     
  Significant markers N = 3
List of 3 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S12.  Get Full Table List of 3 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
ZNHIT2|741 2.712e-11 4.89e-07
OVCH1|341350 3.741e-08 0.000675
PAIP2B|400961 2.41e-07 0.00435

Figure S5.  Get High-res Image As an example, this figure shows the association of ZNHIT2|741 to 'COMPLETENESS.OF.RESECTION'. P value = 2.71e-11 with ANOVA analysis.

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

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

Table S13.  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 S14.  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 S6.  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.

Clinical variable #9: 'NEOPLASM.DISEASESTAGE'

4 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S15.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 32
  STAGE II 17
  STAGE IIA 55
  STAGE IIB 3
  STAGE IIC 1
  STAGE III 9
  STAGE IIIA 5
  STAGE IIIB 24
  STAGE IIIC 12
  STAGE IV 12
  STAGE IVA 13
  STAGE IVB 1
     
  Significant markers N = 4
List of 4 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S16.  Get Full Table List of 4 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
TDRD12|91646 1.083e-08 0.000195
RB1|5925 1.949e-08 0.000352
HAP1|9001 8.102e-08 0.00146
PDE12|201626 1.338e-06 0.0241

Figure S7.  Get High-res Image As an example, this figure shows the association of TDRD12|91646 to 'NEOPLASM.DISEASESTAGE'. P value = 1.08e-08 with ANOVA analysis.

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 = 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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