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 18061 genes and 12 clinical features across 243 samples, statistically thresholded by Q value < 0.05, 10 clinical features related to at least one genes.

  • 82 genes correlated to 'PRIMARY.SITE.OF.DISEASE'.

    • MTERF|7978 ,  CLDN18|51208 ,  ZNF12|7559 ,  C19ORF20|91978 ,  ACVR2B|93 ,  ...

  • 33 genes correlated to 'GENDER'.

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

  • 210 genes correlated to 'HISTOLOGICAL.TYPE'.

    • USPL1|10208 ,  AQP3|360 ,  NR6A1|2649 ,  MRPS31|10240 ,  SLC25A36|55186 ,  ...

  • 6 genes correlated to 'PATHOLOGY.T'.

    • FSTL3|10272 ,  RAB6B|51560 ,  IGLON5|402665 ,  GADD45B|4616 ,  TRPC1|7220 ,  ...

  • 41 genes correlated to 'PATHOLOGY.N'.

    • HAP1|9001 ,  MAP6|4135 ,  ATP5B|506 ,  SUSD5|26032 ,  ZBTB20|26137 ,  ...

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

    • TDRD12|91646 ,  RB1|5925 ,  FAM171A1|221061 ,  ZNF558|148156 ,  ZNF675|171392 ,  ...

  • 18 genes correlated to 'TUMOR.STAGE'.

    • HAP1|9001 ,  CAMK2B|816 ,  TCHH|7062 ,  BEND3|57673 ,  UNG|7374 ,  ...

  • 15 genes correlated to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

    • ATP5L2|267020 ,  C1ORF74|148304 ,  GAPDHS|26330 ,  CDH22|64405 ,  KLHDC8A|55220 ,  ...

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

    • ZNHIT2|741 ,  OVCH1|341350

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

    • NIPAL4|348938 ,  CRLF1|9244 ,  AMIGO2|347902 ,  LOC100126784|100126784 ,  C5ORF23|79614 ,  ...

  • 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        
PRIMARY SITE OF DISEASE t test N=82 rectum N=53 colon N=29
GENDER t test N=33 male N=13 female N=20
HISTOLOGICAL TYPE ANOVA test N=210        
PATHOLOGY T Spearman correlation test N=6 higher pT N=6 lower pT N=0
PATHOLOGY N Spearman correlation test N=41 higher pN N=34 lower pN N=7
PATHOLOGICSPREAD(M) ANOVA test N=12        
TUMOR STAGE Spearman correlation test N=18 higher stage N=15 lower stage N=3
RADIATIONS RADIATION REGIMENINDICATION t test N=15 yes N=4 no N=11
COMPLETENESS OF RESECTION ANOVA test N=2        
NUMBER OF LYMPH NODES Spearman correlation test N=14 higher number.of.lymph.nodes N=12 lower number.of.lymph.nodes N=2
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=6.1)
  censored N = 197
  death N = 27
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

No gene related to 'AGE'.

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

AGE Mean (SD) 64.97 (13)
  Significant markers N = 0
Clinical variable #3: 'PRIMARY.SITE.OF.DISEASE'

82 genes related to 'PRIMARY.SITE.OF.DISEASE'.

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

PRIMARY.SITE.OF.DISEASE Labels N
  COLON 192
  RECTUM 49
     
  Significant markers N = 82
  Higher in RECTUM 53
  Higher in COLON 29
List of top 10 genes differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

Table S4.  Get Full Table List of top 10 genes differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

T(pos if higher in 'RECTUM') ttestP Q AUC
MTERF|7978 7.34 4.368e-12 7.89e-08 0.7176
CLDN18|51208 -6.65 2.774e-10 5.01e-06 0.676
ZNF12|7559 7.04 3.543e-10 6.4e-06 0.772
C19ORF20|91978 -7.03 6.574e-10 1.19e-05 0.7865
ACVR2B|93 6.62 1.39e-09 2.51e-05 0.7537
ZNF737|100129842 6.21 3.614e-09 6.53e-05 0.6783
ZNF470|388566 6.15 4.095e-09 7.39e-05 0.6642
IRF2BP2|359948 6.42 4.296e-09 7.76e-05 0.7379
ZNF628|89887 -6.32 9.634e-09 0.000174 0.7516
SHROOM4|57477 6.08 1.093e-08 0.000197 0.7068

Figure S1.  Get High-res Image As an example, this figure shows the association of MTERF|7978 to 'PRIMARY.SITE.OF.DISEASE'. P value = 4.37e-12 with T-test analysis.

Clinical variable #4: 'GENDER'

33 genes related to 'GENDER'.

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

GENDER Labels N
  FEMALE 115
  MALE 128
     
  Significant markers N = 33
  Higher in MALE 13
  Higher in FEMALE 20
List of top 10 genes differentially expressed by 'GENDER'

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

T(pos if higher in 'MALE') ttestP Q AUC
ZFY|7544 49.76 2.759e-108 4.98e-104 0.9995
XIST|7503 -43.26 3.441e-108 6.21e-104 0.9996
KDM5D|8284 61.45 1.296e-95 2.34e-91 1
RPS4Y1|6192 41.8 6.403e-91 1.16e-86 1
PRKY|5616 31.54 7.705e-84 1.39e-79 0.9958
EIF1AY|9086 46.79 4.691e-74 8.47e-70 1
DDX3Y|8653 52.66 6.485e-71 1.17e-66 1
USP9Y|8287 45.64 3.853e-65 6.95e-61 1
NLGN4Y|22829 35.24 9.159e-60 1.65e-55 0.9994
TSIX|9383 -22.39 9.878e-53 1.78e-48 0.9989

Figure S2.  Get High-res Image As an example, this figure shows the association of ZFY|7544 to 'GENDER'. P value = 2.76e-108 with T-test analysis.

Clinical variable #5: 'HISTOLOGICAL.TYPE'

210 genes related to 'HISTOLOGICAL.TYPE'.

Table S7.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  COLON ADENOCARCINOMA 168
  COLON MUCINOUS ADENOCARCINOMA 24
  RECTAL ADENOCARCINOMA 47
  RECTAL MUCINOUS ADENOCARCINOMA 2
     
  Significant markers N = 210
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

Table S8.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
USPL1|10208 1.867e-10 3.37e-06
AQP3|360 2.608e-10 4.71e-06
NR6A1|2649 3.571e-10 6.45e-06
MRPS31|10240 3.899e-10 7.04e-06
SLC25A36|55186 7.641e-10 1.38e-05
CSE1L|1434 1.653e-09 2.99e-05
AMACR|23600 2.01e-09 3.63e-05
ZNF12|7559 2.13e-09 3.85e-05
RPIA|22934 2.177e-09 3.93e-05
NUFIP1|26747 2.249e-09 4.06e-05

Figure S3.  Get High-res Image As an example, this figure shows the association of USPL1|10208 to 'HISTOLOGICAL.TYPE'. P value = 1.87e-10 with ANOVA analysis.

Clinical variable #6: 'PATHOLOGY.T'

6 genes related to 'PATHOLOGY.T'.

Table S9.  Basic characteristics of clinical feature: 'PATHOLOGY.T'

PATHOLOGY.T Mean (SD) 2.88 (0.69)
  N
  T0 1
  T1 8
  T2 43
  T3 157
  T4 34
     
  Significant markers N = 6
  pos. correlated 6
  neg. correlated 0
List of 6 genes significantly correlated to 'PATHOLOGY.T' by Spearman correlation test

Table S10.  Get Full Table List of 6 genes significantly correlated to 'PATHOLOGY.T' by Spearman correlation test

SpearmanCorr corrP Q
FSTL3|10272 0.3098 8.412e-07 0.0152
RAB6B|51560 0.3055 1.211e-06 0.0219
IGLON5|402665 0.3083 1.306e-06 0.0236
GADD45B|4616 0.3038 1.389e-06 0.0251
TRPC1|7220 0.3024 1.57e-06 0.0283
NGF|4803 0.3021 2.168e-06 0.0391

Figure S4.  Get High-res Image As an example, this figure shows the association of FSTL3|10272 to 'PATHOLOGY.T'. P value = 8.41e-07 with Spearman correlation analysis.

Clinical variable #7: 'PATHOLOGY.N'

41 genes related to 'PATHOLOGY.N'.

Table S11.  Basic characteristics of clinical feature: 'PATHOLOGY.N'

PATHOLOGY.N Mean (SD) 0.57 (0.76)
  N
  N0 143
  N1 57
  N2 40
     
  Significant markers N = 41
  pos. correlated 34
  neg. correlated 7
List of top 10 genes significantly correlated to 'PATHOLOGY.N' by Spearman correlation test

Table S12.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.N' by Spearman correlation test

SpearmanCorr corrP Q
HAP1|9001 0.3462 6.191e-08 0.00112
MAP6|4135 0.3322 1.362e-07 0.00246
ATP5B|506 -0.3244 2.766e-07 0.005
SUSD5|26032 0.3243 2.948e-07 0.00532
ZBTB20|26137 0.3232 3.075e-07 0.00555
CRLF1|9244 0.3315 3.186e-07 0.00575
CAMK2B|816 0.335 3.618e-07 0.00653
NACAD|23148 0.3208 3.809e-07 0.00688
LOC100126784|100126784 0.3205 4.615e-07 0.00833
GALNTL1|57452 0.3158 6.578e-07 0.0119

Figure S5.  Get High-res Image As an example, this figure shows the association of HAP1|9001 to 'PATHOLOGY.N'. P value = 6.19e-08 with Spearman correlation analysis.

Clinical variable #8: 'PATHOLOGICSPREAD(M)'

12 genes related to 'PATHOLOGICSPREAD(M)'.

Table S13.  Basic characteristics of clinical feature: 'PATHOLOGICSPREAD(M)'

PATHOLOGICSPREAD(M) Labels N
  M0 167
  M1 23
  M1A 6
  M1B 1
  MX 42
     
  Significant markers N = 12
List of top 10 genes differentially expressed by 'PATHOLOGICSPREAD(M)'

Table S14.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGICSPREAD(M)'

ANOVA_P Q
TDRD12|91646 5.18e-11 9.36e-07
RB1|5925 3.032e-10 5.47e-06
FAM171A1|221061 1.454e-09 2.63e-05
ZNF558|148156 3.261e-09 5.89e-05
ZNF675|171392 5.534e-09 9.99e-05
TMEM185A|84548 1.037e-08 0.000187
ZNF845|91664 8.688e-08 0.00157
CCDC122|160857 1.751e-07 0.00316
ZNF28|7576 3.354e-07 0.00605
C13ORF31|144811 4.289e-07 0.00774

Figure S6.  Get High-res Image As an example, this figure shows the association of TDRD12|91646 to 'PATHOLOGICSPREAD(M)'. P value = 5.18e-11 with ANOVA analysis.

Clinical variable #9: 'TUMOR.STAGE'

18 genes related to 'TUMOR.STAGE'.

Table S15.  Basic characteristics of clinical feature: 'TUMOR.STAGE'

TUMOR.STAGE Mean (SD) 2.38 (0.94)
  N
  Stage 1 42
  Stage 2 91
  Stage 3 66
  Stage 4 32
     
  Significant markers N = 18
  pos. correlated 15
  neg. correlated 3
List of top 10 genes significantly correlated to 'TUMOR.STAGE' by Spearman correlation test

Table S16.  Get Full Table List of top 10 genes significantly correlated to 'TUMOR.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
HAP1|9001 0.3707 1.13e-08 0.000204
CAMK2B|816 0.3676 3.753e-08 0.000678
TCHH|7062 0.3419 1.356e-07 0.00245
BEND3|57673 -0.3358 1.704e-07 0.00308
UNG|7374 -0.3334 2.112e-07 0.00381
TMEM59L|25789 0.3334 6.765e-07 0.0122
IGLON5|402665 0.3219 7.618e-07 0.0138
SHC1|6464 0.3165 9.084e-07 0.0164
PIM1|5292 0.3148 1.043e-06 0.0188
GADD45B|4616 0.3133 1.177e-06 0.0212

Figure S7.  Get High-res Image As an example, this figure shows the association of HAP1|9001 to 'TUMOR.STAGE'. P value = 1.13e-08 with Spearman correlation analysis.

Clinical variable #10: 'RADIATIONS.RADIATION.REGIMENINDICATION'

15 genes related to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

Table S17.  Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 5
  YES 238
     
  Significant markers N = 15
  Higher in YES 4
  Higher in NO 11
List of top 10 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S18.  Get Full Table List of top 10 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

T(pos if higher in 'YES') ttestP Q AUC
ATP5L2|267020 -8.68 7.927e-14 1.39e-09 0.7267
C1ORF74|148304 -9.76 1.951e-10 3.43e-06 0.8067
GAPDHS|26330 -11.7 3.979e-09 6.98e-05 0.8832
CDH22|64405 8.09 3.995e-08 0.000701 0.9074
KLHDC8A|55220 -10.48 4.052e-08 0.000711 0.8471
SLC25A4|291 10.24 9.826e-08 0.00172 0.837
NCRNA00105|80161 -7.45 1.863e-07 0.00327 0.7067
SLC22A11|55867 -8.46 2.836e-07 0.00498 0.7325
NEUROD1|4760 7.8 4.017e-07 0.00705 0.8141
CLDN8|9073 -10.55 5.517e-07 0.00968 0.8579

Figure S8.  Get High-res Image As an example, this figure shows the association of ATP5L2|267020 to 'RADIATIONS.RADIATION.REGIMENINDICATION'. P value = 7.93e-14 with T-test analysis.

Clinical variable #11: 'COMPLETENESS.OF.RESECTION'

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

Table S19.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 151
  R1 1
  R2 2
  RX 25
     
  Significant markers N = 2
List of 2 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S20.  Get Full Table List of 2 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
ZNHIT2|741 1.714e-13 3.1e-09
OVCH1|341350 3.444e-09 6.22e-05

Figure S9.  Get High-res Image As an example, this figure shows the association of ZNHIT2|741 to 'COMPLETENESS.OF.RESECTION'. P value = 1.71e-13 with ANOVA analysis.

Clinical variable #12: 'NUMBER.OF.LYMPH.NODES'

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

Table S21.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 2.25 (5.2)
  Significant markers N = 14
  pos. correlated 12
  neg. correlated 2
List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

Table S22.  Get Full Table List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
NIPAL4|348938 0.3677 3.47e-08 0.000627
CRLF1|9244 0.3647 1.098e-07 0.00198
AMIGO2|347902 0.3403 3.81e-07 0.00688
LOC100126784|100126784 0.3363 6.002e-07 0.0108
C5ORF23|79614 0.334 6.819e-07 0.0123
PRSS35|167681 0.3333 7.238e-07 0.0131
SNCG|6623 0.33 8.914e-07 0.0161
TMEM59L|25789 0.3362 1.363e-06 0.0246
TCHH|7062 0.326 1.735e-06 0.0313
CAP2|10486 0.3214 1.765e-06 0.0319

Figure S10.  Get High-res Image As an example, this figure shows the association of NIPAL4|348938 to 'NUMBER.OF.LYMPH.NODES'. P value = 3.47e-08 with Spearman correlation analysis. The straight line presents the best linear regression.

Methods & Data
Input

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

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

  • Number of patients = 243

  • Number of genes = 18061

  • Number of clinical features = 12

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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