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 18025 genes and 12 clinical features across 571 samples, statistically thresholded by Q value < 0.05, 11 clinical features related to at least one genes.

  • 7 genes correlated to 'AGE'.

    • MTERF|7978 ,  MGA|23269 ,  AMH|268 ,  CNTD2|79935 ,  ZNF75A|7627 ,  ...

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

    • DRD5|1816 ,  ZNF141|7700 ,  ZNF530|348327 ,  C19ORF46|163183 ,  MAP7D2|256714 ,  ...

  • 54 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • C5ORF23|79614 ,  C13ORF15|28984 ,  C2CD4A|145741 ,  ZNF845|91664 ,  RGL2|5863 ,  ...

  • 94 genes correlated to 'PATHOLOGY.T.STAGE'.

    • SCG2|7857 ,  C5ORF23|79614 ,  SPP1|6696 ,  ZFHX4|79776 ,  LOC100126784|100126784 ,  ...

  • 121 genes correlated to 'PATHOLOGY.N.STAGE'.

    • TEAD3|7005 ,  RGL2|5863 ,  GSR|2936 ,  C5ORF23|79614 ,  CASP1|834 ,  ...

  • 307 genes correlated to 'PATHOLOGY.M.STAGE'.

    • OR6W1P|89883 ,  NBPF15|284565 ,  POTEE|445582 ,  SPIN2A|54466 ,  LOC100132832|100132832 ,  ...

  • 42 genes correlated to 'GENDER'.

    • XIST|7503 ,  ZFY|7544 ,  RPS4Y1|6192 ,  DDX3Y|8653 ,  KDM5D|8284 ,  ...

  • 943 genes correlated to 'HISTOLOGICAL.TYPE'.

    • AQP3|360 ,  PLAGL2|5326 ,  SLC19A3|80704 ,  POFUT1|23509 ,  ASCL2|430 ,  ...

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

    • DCDC2B|149069 ,  HIST1H2AE|3012 ,  ZNF813|126017 ,  MSLNL|401827 ,  BEST3|144453 ,  ...

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

    • LOC150776|150776 ,  RRN3P2|653390 ,  AGAP11|119385 ,  PMS2L2|5380 ,  PMS2CL|441194 ,  ...

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

    • C5ORF23|79614 ,  CXCL2|2920 ,  TEAD3|7005 ,  SHC1|6464 ,  CASP1|834 ,  ...

  • No genes correlated to 'Time to Death'

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=7 older N=3 younger N=4
PRIMARY SITE OF DISEASE t test N=126 rectum N=93 colon N=33
NEOPLASM DISEASESTAGE ANOVA test N=54        
PATHOLOGY T STAGE Spearman correlation test N=94 higher stage N=89 lower stage N=5
PATHOLOGY N STAGE Spearman correlation test N=121 higher stage N=79 lower stage N=42
PATHOLOGY M STAGE ANOVA test N=307        
GENDER t test N=42 male N=16 female N=26
HISTOLOGICAL TYPE ANOVA test N=943        
RADIATIONS RADIATION REGIMENINDICATION t test N=8 yes N=4 no N=4
COMPLETENESS OF RESECTION ANOVA test N=396        
NUMBER OF LYMPH NODES Spearman correlation test N=79 higher number.of.lymph.nodes N=43 lower number.of.lymph.nodes N=36
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) 0.1-140.4 (median=12.1)
  censored N = 381
  death N = 72
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

7 genes related to 'AGE'.

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

AGE Mean (SD) 66.48 (13)
  Significant markers N = 7
  pos. correlated 3
  neg. correlated 4
List of 7 genes significantly correlated to 'AGE' by Spearman correlation test

Table S3.  Get Full Table List of 7 genes significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
MTERF|7978 -0.2448 3.486e-09 6.28e-05
MGA|23269 -0.2417 5.098e-09 9.19e-05
AMH|268 0.2354 2.233e-08 0.000402
CNTD2|79935 0.2033 1.025e-06 0.0185
ZNF75A|7627 -0.2029 1.085e-06 0.0196
MRE11A|4361 -0.1987 1.789e-06 0.0322
LOC154761|154761 0.1978 1.974e-06 0.0356

Figure S1.  Get High-res Image As an example, this figure shows the association of MTERF|7978 to 'AGE'. P value = 3.49e-09 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'PRIMARY.SITE.OF.DISEASE'

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

Table S4.  Basic characteristics of clinical feature: 'PRIMARY.SITE.OF.DISEASE'

PRIMARY.SITE.OF.DISEASE Labels N
  COLON 414
  RECTUM 153
     
  Significant markers N = 126
  Higher in RECTUM 93
  Higher in COLON 33
List of top 10 genes differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

Table S5.  Get Full Table List of top 10 genes differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

T(pos if higher in 'RECTUM') ttestP Q AUC
DRD5|1816 -8.59 3.233e-15 5.83e-11 0.7293
ZNF141|7700 7.5 3.205e-13 5.78e-09 0.6642
ZNF530|348327 7.54 3.538e-13 6.38e-09 0.6713
C19ORF46|163183 6.85 2.393e-11 4.31e-07 0.629
MAP7D2|256714 6.88 2.497e-11 4.5e-07 0.6465
ELAVL2|1993 6.72 7.466e-11 1.35e-06 0.663
SHROOM4|57477 6.43 3.404e-10 6.13e-06 0.6314
ZNF662|389114 6.39 5.436e-10 9.79e-06 0.659
HOXC6|3223 -6.45 6.863e-10 1.24e-05 0.6777
MBP|4155 -6.33 8.251e-10 1.49e-05 0.6552

Figure S2.  Get High-res Image As an example, this figure shows the association of DRD5|1816 to 'PRIMARY.SITE.OF.DISEASE'. P value = 3.23e-15 with T-test analysis.

Clinical variable #4: 'NEOPLASM.DISEASESTAGE'

54 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S6.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 96
  STAGE IA 1
  STAGE II 39
  STAGE IIA 157
  STAGE IIB 11
  STAGE IIC 3
  STAGE III 28
  STAGE IIIA 20
  STAGE IIIB 72
  STAGE IIIC 49
  STAGE IV 57
  STAGE IVA 22
  STAGE IVB 1
     
  Significant markers N = 54
List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S7.  Get Full Table List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
C5ORF23|79614 1.095e-09 1.97e-05
C13ORF15|28984 5.555e-09 1e-04
C2CD4A|145741 5.857e-09 0.000106
ZNF845|91664 1.739e-08 0.000313
RGL2|5863 2.679e-08 0.000483
BEND3|57673 3.343e-08 0.000602
TYMS|7298 3.346e-08 0.000603
NPR3|4883 3.661e-08 0.00066
TEAD3|7005 4.32e-08 0.000778
TMEM185A|84548 4.503e-08 0.000811

Figure S3.  Get High-res Image As an example, this figure shows the association of C5ORF23|79614 to 'NEOPLASM.DISEASESTAGE'. P value = 1.1e-09 with ANOVA analysis.

Clinical variable #5: 'PATHOLOGY.T.STAGE'

94 genes related to 'PATHOLOGY.T.STAGE'.

Table S8.  Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'

PATHOLOGY.T.STAGE Mean (SD) 2.87 (0.64)
  N
  0 1
  1 18
  2 99
  3 388
  4 63
     
  Significant markers N = 94
  pos. correlated 89
  neg. correlated 5
List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

Table S9.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
SCG2|7857 0.2497 1.839e-09 3.31e-05
C5ORF23|79614 0.2493 3.78e-09 6.81e-05
SPP1|6696 0.2422 4.884e-09 8.8e-05
ZFHX4|79776 0.243 5.917e-09 0.000107
LOC100126784|100126784 0.2552 6.912e-09 0.000125
CSRP2|1466 0.2389 7.971e-09 0.000144
SOX11|6664 0.2388 1.341e-08 0.000242
NALCN|259232 0.2477 1.925e-08 0.000347
HTR2B|3357 0.237 2.557e-08 0.000461
RBP7|116362 0.2299 3.346e-08 0.000603

Figure S4.  Get High-res Image As an example, this figure shows the association of SCG2|7857 to 'PATHOLOGY.T.STAGE'. P value = 1.84e-09 with Spearman correlation analysis.

Clinical variable #6: 'PATHOLOGY.N.STAGE'

121 genes related to 'PATHOLOGY.N.STAGE'.

Table S10.  Basic characteristics of clinical feature: 'PATHOLOGY.N.STAGE'

PATHOLOGY.N.STAGE Mean (SD) 0.61 (0.78)
  N
  0 324
  1 139
  2 104
     
  Significant markers N = 121
  pos. correlated 79
  neg. correlated 42
List of top 10 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

Table S11.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
TEAD3|7005 0.2777 1.675e-11 3.02e-07
RGL2|5863 0.2617 2.468e-10 4.45e-06
GSR|2936 -0.2592 3.683e-10 6.64e-06
C5ORF23|79614 0.2643 4.087e-10 7.37e-06
CASP1|834 -0.2458 3.025e-09 5.45e-05
COQ2|27235 -0.2435 4.248e-09 7.65e-05
NARS|4677 -0.2429 4.655e-09 8.39e-05
C13ORF15|28984 0.2427 4.823e-09 8.69e-05
MINPP1|9562 -0.2408 6.372e-09 0.000115
PDE12|201626 -0.2391 8.222e-09 0.000148

Figure S5.  Get High-res Image As an example, this figure shows the association of TEAD3|7005 to 'PATHOLOGY.N.STAGE'. P value = 1.68e-11 with Spearman correlation analysis.

Clinical variable #7: 'PATHOLOGY.M.STAGE'

307 genes related to 'PATHOLOGY.M.STAGE'.

Table S12.  Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'

PATHOLOGY.M.STAGE Labels N
  M0 431
  M1 68
  M1A 8
  M1B 1
  MX 54
     
  Significant markers N = 307
List of top 10 genes differentially expressed by 'PATHOLOGY.M.STAGE'

Table S13.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGY.M.STAGE'

ANOVA_P Q
OR6W1P|89883 5.902e-17 1.06e-12
NBPF15|284565 2.399e-12 4.32e-08
POTEE|445582 4.869e-12 8.78e-08
SPIN2A|54466 1.376e-11 2.48e-07
LOC100132832|100132832 2.025e-11 3.65e-07
TUBA4B|80086 3.085e-11 5.56e-07
FUNDC2P2|388965 3.492e-11 6.29e-07
UBE2NL|389898 3.687e-11 6.64e-07
ANXA2P3|305 5.05e-11 9.1e-07
LOC344967|344967 6.563e-11 1.18e-06

Figure S6.  Get High-res Image As an example, this figure shows the association of OR6W1P|89883 to 'PATHOLOGY.M.STAGE'. P value = 5.9e-17 with ANOVA analysis.

Clinical variable #8: 'GENDER'

42 genes related to 'GENDER'.

Table S14.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 269
  MALE 302
     
  Significant markers N = 42
  Higher in MALE 16
  Higher in FEMALE 26
List of top 10 genes differentially expressed by 'GENDER'

Table S15.  Get Full Table List of top 10 genes differentially expressed by 'GENDER'

T(pos if higher in 'MALE') ttestP Q AUC
XIST|7503 -51.23 7.302e-190 1.32e-185 0.9971
ZFY|7544 49.68 1.52e-147 2.74e-143 0.9959
RPS4Y1|6192 43.43 2.756e-144 4.97e-140 0.9969
DDX3Y|8653 47.8 2.087e-107 3.76e-103 0.9983
KDM5D|8284 43.85 5.396e-93 9.72e-89 0.998
TSIX|9383 -25.43 1.725e-88 3.11e-84 0.9622
PRKY|5616 23.79 1.113e-77 2.01e-73 0.9343
NLGN4Y|22829 30.17 4.559e-77 8.21e-73 0.9844
CYORF15A|246126 30.63 1.132e-59 2.04e-55 0.9878
UTY|7404 33.15 7.167e-59 1.29e-54 0.9918

Figure S7.  Get High-res Image As an example, this figure shows the association of XIST|7503 to 'GENDER'. P value = 7.3e-190 with T-test analysis.

Clinical variable #9: 'HISTOLOGICAL.TYPE'

943 genes related to 'HISTOLOGICAL.TYPE'.

Table S16.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  COLON ADENOCARCINOMA 359
  COLON MUCINOUS ADENOCARCINOMA 54
  RECTAL ADENOCARCINOMA 138
  RECTAL MUCINOUS ADENOCARCINOMA 12
     
  Significant markers N = 943
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

Table S17.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
AQP3|360 3.536e-22 6.37e-18
PLAGL2|5326 8.037e-22 1.45e-17
SLC19A3|80704 2.148e-20 3.87e-16
POFUT1|23509 1.999e-19 3.6e-15
ASCL2|430 9.923e-19 1.79e-14
LY6G6D|58530 7.85e-18 1.41e-13
SNHG11|128439 8.963e-18 1.62e-13
CKAP4|10970 4.65e-17 8.38e-13
MAPRE2|10982 8.997e-17 1.62e-12
DDX27|55661 1.094e-16 1.97e-12

Figure S8.  Get High-res Image As an example, this figure shows the association of AQP3|360 to 'HISTOLOGICAL.TYPE'. P value = 3.54e-22 with ANOVA analysis.

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

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

Table S18.  Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 9
  YES 562
     
  Significant markers N = 8
  Higher in YES 4
  Higher in NO 4
List of 8 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S19.  Get Full Table List of 8 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

T(pos if higher in 'YES') ttestP Q AUC
DCDC2B|149069 12.24 1.448e-11 2.59e-07 0.7616
HIST1H2AE|3012 -12.97 5.796e-11 1.04e-06 0.8225
ZNF813|126017 -9.02 1.476e-07 0.00264 0.7259
MSLNL|401827 8.73 3.533e-07 0.00632 0.7912
BEST3|144453 11.51 1.338e-06 0.024 0.8916
ZNF701|55762 -8.19 1.464e-06 0.0262 0.7519
CTBP1|1487 8.73 1.526e-06 0.0273 0.8486
ZNF586|54807 -7.77 2.04e-06 0.0365 0.7479

Figure S9.  Get High-res Image As an example, this figure shows the association of DCDC2B|149069 to 'RADIATIONS.RADIATION.REGIMENINDICATION'. P value = 1.45e-11 with T-test analysis.

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

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

Table S20.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 409
  R1 5
  R2 34
  RX 28
     
  Significant markers N = 396
List of top 10 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S21.  Get Full Table List of top 10 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
LOC150776|150776 4.745e-13 8.55e-09
RRN3P2|653390 6.817e-13 1.23e-08
AGAP11|119385 1.512e-12 2.73e-08
PMS2L2|5380 1.744e-12 3.14e-08
PMS2CL|441194 1.768e-12 3.19e-08
GUSBL2|375513 2.295e-12 4.14e-08
GPR89C|728932 3.935e-12 7.09e-08
LOC728875|728875 4.361e-12 7.86e-08
POLR2J4|84820 4.433e-12 7.99e-08
NSUN5P1|155400 5.94e-12 1.07e-07

Figure S10.  Get High-res Image As an example, this figure shows the association of LOC150776|150776 to 'COMPLETENESS.OF.RESECTION'. P value = 4.75e-13 with ANOVA analysis.

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

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

Table S22.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 2.24 (4.8)
  Significant markers N = 79
  pos. correlated 43
  neg. correlated 36
List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

Table S23.  Get Full Table List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
C5ORF23|79614 0.2675 8.031e-10 1.45e-05
CXCL2|2920 -0.261 8.543e-10 1.54e-05
TEAD3|7005 0.2607 8.917e-10 1.61e-05
SHC1|6464 0.2574 1.472e-09 2.65e-05
CASP1|834 -0.2566 1.651e-09 2.97e-05
GSR|2936 -0.2549 2.132e-09 3.84e-05
C2CD4A|145741 -0.2517 3.423e-09 6.17e-05
RGL2|5863 0.2489 5.218e-09 9.4e-05
MINPP1|9562 -0.246 7.922e-09 0.000143
NPR3|4883 0.2665 8.323e-09 0.00015

Figure S11.  Get High-res Image As an example, this figure shows the association of C5ORF23|79614 to 'NUMBER.OF.LYMPH.NODES'. P value = 8.03e-10 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.merged_data.txt

  • Number of patients = 571

  • Number of genes = 18025

  • 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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