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 18308 genes and 13 clinical features across 450 samples, statistically thresholded by Q value < 0.05, 10 clinical features related to at least one genes.

  • 30 genes correlated to 'AGE'.

    • AHRR|57491 ,  C11ORF84|144097 ,  PLA1A|51365 ,  GPR15|2838 ,  C9ORF140|89958 ,  ...

  • 5 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • ZNF540|163255 ,  FAM117A|81558 ,  PTGDS|5730 ,  RBP5|83758 ,  CR2|1380

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

    • C14ORF139|79686 ,  ST8SIA1|6489 ,  RASGRP2|10235 ,  FAM125B|89853 ,  PTGDS|5730 ,  ...

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

    • RCOR3|55758 ,  ARID4A|5926 ,  DAPK2|23604 ,  ZNF483|158399 ,  EPM2AIP1|9852 ,  ...

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

    • CUL4A|8451 ,  DCUN1D2|55208 ,  AMFR|267 ,  SLC35F2|54733 ,  TMCO3|55002 ,  ...

  • 92 genes correlated to 'GENDER'.

    • PRKY|5616 ,  ZFY|7544 ,  RPS4Y1|6192 ,  XIST|7503 ,  DDX3Y|8653 ,  ...

  • 35 genes correlated to 'HISTOLOGICAL.TYPE'.

    • A4GNT|51146 ,  CREB3L3|84699 ,  REG4|83998 ,  BTNL8|79908 ,  MUC6|4588 ,  ...

  • 1 gene correlated to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

    • PGAM2|5224

  • 3 genes correlated to 'YEAROFTOBACCOSMOKINGONSET'.

    • ANKRD33|341405 ,  GPR15|2838 ,  HIST1H2BG|8339

  • 1 gene correlated to 'COMPLETENESS.OF.RESECTION'.

    • PAX2|5076

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

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=30 older N=14 younger N=16
NEOPLASM DISEASESTAGE ANOVA test N=5        
PATHOLOGY T STAGE Spearman correlation test N=88 higher stage N=2 lower stage N=86
PATHOLOGY N STAGE Spearman correlation test N=9 higher stage N=1 lower stage N=8
PATHOLOGY M STAGE ANOVA test N=49        
GENDER t test N=92 male N=34 female N=58
KARNOFSKY PERFORMANCE SCORE Spearman correlation test   N=0        
HISTOLOGICAL TYPE ANOVA test N=35        
RADIATIONS RADIATION REGIMENINDICATION t test N=1 yes N=1 no N=0
NUMBERPACKYEARSSMOKED Spearman correlation test   N=0        
YEAROFTOBACCOSMOKINGONSET Spearman correlation test N=3 higher yearoftobaccosmokingonset N=3 lower yearoftobaccosmokingonset N=0
COMPLETENESS OF RESECTION ANOVA test N=1        
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) 0-224 (median=12.7)
  censored N = 298
  death N = 116
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

30 genes related to 'AGE'.

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

AGE Mean (SD) 65.48 (9.7)
  Significant markers N = 30
  pos. correlated 14
  neg. correlated 16
List of top 10 genes significantly correlated to 'AGE' by Spearman correlation test

Table S3.  Get Full Table List of top 10 genes significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
AHRR|57491 -0.2769 8.566e-09 0.000157
C11ORF84|144097 -0.2658 3.294e-08 0.000603
PLA1A|51365 0.2581 8.426e-08 0.00154
GPR15|2838 -0.2622 1.383e-07 0.00253
C9ORF140|89958 -0.2528 1.56e-07 0.00286
CYP1A1|1543 -0.3075 1.861e-07 0.00341
METTL7B|196410 0.2469 3.069e-07 0.00562
EDA2R|60401 0.2457 3.551e-07 0.0065
THRB|7068 0.2444 4.109e-07 0.00752
PRSS12|8492 0.2424 5.298e-07 0.00969

Figure S1.  Get High-res Image As an example, this figure shows the association of AHRR|57491 to 'AGE'. P value = 8.57e-09 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'NEOPLASM.DISEASESTAGE'

5 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S4.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 3
  STAGE IA 111
  STAGE IB 131
  STAGE IIA 41
  STAGE IIB 64
  STAGE IIIA 66
  STAGE IIIB 11
  STAGE IV 22
     
  Significant markers N = 5
List of 5 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S5.  Get Full Table List of 5 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
ZNF540|163255 3.803e-07 0.00696
FAM117A|81558 5.27e-07 0.00965
PTGDS|5730 6.371e-07 0.0117
RBP5|83758 7.553e-07 0.0138
CR2|1380 1.826e-06 0.0334

Figure S2.  Get High-res Image As an example, this figure shows the association of ZNF540|163255 to 'NEOPLASM.DISEASESTAGE'. P value = 3.8e-07 with ANOVA analysis.

Clinical variable #4: 'PATHOLOGY.T.STAGE'

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

Table S6.  Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'

PATHOLOGY.T.STAGE Mean (SD) 1.86 (0.74)
  N
  1 139
  2 251
  3 40
  4 18
     
  Significant markers N = 88
  pos. correlated 2
  neg. correlated 86
List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

Table S7.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
C14ORF139|79686 -0.2805 1.529e-09 2.8e-05
ST8SIA1|6489 -0.2687 7.519e-09 0.000138
RASGRP2|10235 -0.268 8.274e-09 0.000151
FAM125B|89853 -0.2677 8.611e-09 0.000158
PTGDS|5730 -0.2621 1.805e-08 0.00033
GP1BA|2811 -0.2643 1.953e-08 0.000358
CCL19|6363 -0.2583 3.036e-08 0.000556
MAL|4118 -0.2552 4.339e-08 0.000794
MS4A1|931 -0.2554 4.347e-08 0.000796
PPP1R16B|26051 -0.2546 4.663e-08 0.000853

Figure S3.  Get High-res Image As an example, this figure shows the association of C14ORF139|79686 to 'PATHOLOGY.T.STAGE'. P value = 1.53e-09 with Spearman correlation analysis.

Clinical variable #5: 'PATHOLOGY.N.STAGE'

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

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

PATHOLOGY.N.STAGE Mean (SD) 0.52 (0.77)
  N
  0 285
  1 85
  2 68
  3 2
     
  Significant markers N = 9
  pos. correlated 1
  neg. correlated 8
List of 9 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

Table S9.  Get Full Table List of 9 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
RCOR3|55758 -0.2425 2.602e-07 0.00476
ARID4A|5926 -0.2422 2.712e-07 0.00497
DAPK2|23604 -0.2418 2.84e-07 0.0052
ZNF483|158399 -0.2398 3.557e-07 0.00651
EPM2AIP1|9852 -0.2343 6.692e-07 0.0122
MASP2|10747 -0.2303 1.074e-06 0.0197
LRRC37A2|474170 -0.2272 1.463e-06 0.0268
DBH|1621 -0.2265 2.035e-06 0.0372
MRPS12|6183 0.2235 2.19e-06 0.0401

Figure S4.  Get High-res Image As an example, this figure shows the association of RCOR3|55758 to 'PATHOLOGY.N.STAGE'. P value = 2.6e-07 with Spearman correlation analysis.

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

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

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

PATHOLOGY.M.STAGE Labels N
  M0 310
  M1 18
  M1A 1
  M1B 3
  MX 114
     
  Significant markers N = 49
List of top 10 genes differentially expressed by 'PATHOLOGY.M.STAGE'

Table S11.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGY.M.STAGE'

ANOVA_P Q
CUL4A|8451 2.095e-12 3.84e-08
DCUN1D2|55208 1.923e-09 3.52e-05
AMFR|267 1.995e-09 3.65e-05
SLC35F2|54733 2.589e-09 4.74e-05
TMCO3|55002 9.808e-09 0.00018
SEZ6|124925 1.466e-08 0.000268
PROZ|8858 1.633e-08 0.000299
LAMP1|3916 1.642e-08 3e-04
TFDP1|7027 2.609e-08 0.000477
INSM1|3642 3.868e-08 0.000708

Figure S5.  Get High-res Image As an example, this figure shows the association of CUL4A|8451 to 'PATHOLOGY.M.STAGE'. P value = 2.1e-12 with ANOVA analysis.

Clinical variable #7: 'GENDER'

92 genes related to 'GENDER'.

Table S12.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 244
  MALE 206
     
  Significant markers N = 92
  Higher in MALE 34
  Higher in FEMALE 58
List of top 10 genes differentially expressed by 'GENDER'

Table S13.  Get Full Table List of top 10 genes differentially expressed by 'GENDER'

T(pos if higher in 'MALE') ttestP Q AUC
PRKY|5616 51.7 1.777e-179 3.25e-175 0.9997
ZFY|7544 62.54 4.691e-159 8.59e-155 0.9982
RPS4Y1|6192 57.02 1.875e-133 3.43e-129 0.9974
XIST|7503 -41.44 1.198e-114 2.19e-110 0.9798
DDX3Y|8653 57.39 1.319e-99 2.41e-95 0.9981
KDM5D|8284 51.93 3.621e-86 6.63e-82 0.9991
USP9Y|8287 51.33 1.214e-84 2.22e-80 0.9997
NLGN4Y|22829 30.74 1.387e-62 2.54e-58 0.9921
UTY|7404 37.85 3.765e-46 6.89e-42 0.9971
TSIX|9383 -19.26 1.63e-42 2.98e-38 0.9586

Figure S6.  Get High-res Image As an example, this figure shows the association of PRKY|5616 to 'GENDER'. P value = 1.78e-179 with T-test analysis.

Clinical variable #8: 'KARNOFSKY.PERFORMANCE.SCORE'

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

Table S14.  Basic characteristics of clinical feature: 'KARNOFSKY.PERFORMANCE.SCORE'

KARNOFSKY.PERFORMANCE.SCORE Mean (SD) 83.9 (23)
  Significant markers N = 0
Clinical variable #9: 'HISTOLOGICAL.TYPE'

35 genes related to 'HISTOLOGICAL.TYPE'.

Table S15.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  LUNG ACINAR ADENOCARCINOMA 13
  LUNG ADENOCARCINOMA MIXED SUBTYPE 92
  LUNG ADENOCARCINOMA- NOT OTHERWISE SPECIFIED (NOS) 287
  LUNG BRONCHIOLOALVEOLAR CARCINOMA MUCINOUS 4
  LUNG BRONCHIOLOALVEOLAR CARCINOMA NONMUCINOUS 17
  LUNG CLEAR CELL ADENOCARCINOMA 2
  LUNG MICROPAPILLARY ADENOCARCINOMA 3
  LUNG MUCINOUS ADENOCARCINOMA 2
  LUNG PAPILLARY ADENOCARCINOMA 18
  LUNG SIGNET RING ADENOCARCINOMA 1
  LUNG SOLID PATTERN PREDOMINANT ADENOCARCINOMA 4
  MUCINOUS (COLLOID) CARCINOMA 7
     
  Significant markers N = 35
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

Table S16.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
A4GNT|51146 1.304e-12 2.39e-08
CREB3L3|84699 1.905e-12 3.49e-08
REG4|83998 3.594e-11 6.58e-07
BTNL8|79908 1.102e-09 2.02e-05
MUC6|4588 1.105e-08 0.000202
MIA|8190 2.042e-08 0.000374
DPCR1|135656 4.451e-08 0.000815
SOX10|6663 6.948e-08 0.00127
SPOCD1|90853 7.322e-08 0.00134
INSC|387755 9.415e-08 0.00172

Figure S7.  Get High-res Image As an example, this figure shows the association of A4GNT|51146 to 'HISTOLOGICAL.TYPE'. P value = 1.3e-12 with ANOVA analysis.

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

One gene related to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

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

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 21
  YES 429
     
  Significant markers N = 1
  Higher in YES 1
  Higher in NO 0
List of one gene differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S18.  Get Full Table List of one gene differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

T(pos if higher in 'YES') ttestP Q AUC
PGAM2|5224 6.41 6.066e-07 0.0111 0.7533

Figure S8.  Get High-res Image As an example, this figure shows the association of PGAM2|5224 to 'RADIATIONS.RADIATION.REGIMENINDICATION'. P value = 6.07e-07 with T-test analysis.

Clinical variable #11: 'NUMBERPACKYEARSSMOKED'

No gene related to 'NUMBERPACKYEARSSMOKED'.

Table S19.  Basic characteristics of clinical feature: 'NUMBERPACKYEARSSMOKED'

NUMBERPACKYEARSSMOKED Mean (SD) 42.09 (27)
  Significant markers N = 0
Clinical variable #12: 'YEAROFTOBACCOSMOKINGONSET'

3 genes related to 'YEAROFTOBACCOSMOKINGONSET'.

Table S20.  Basic characteristics of clinical feature: 'YEAROFTOBACCOSMOKINGONSET'

YEAROFTOBACCOSMOKINGONSET Mean (SD) 1964.96 (12)
  Significant markers N = 3
  pos. correlated 3
  neg. correlated 0
List of 3 genes significantly correlated to 'YEAROFTOBACCOSMOKINGONSET' by Spearman correlation test

Table S21.  Get Full Table List of 3 genes significantly correlated to 'YEAROFTOBACCOSMOKINGONSET' by Spearman correlation test

SpearmanCorr corrP Q
ANKRD33|341405 0.3859 3.938e-07 0.00721
GPR15|2838 0.3145 1.132e-06 0.0207
HIST1H2BG|8339 0.3083 1.527e-06 0.0279

Figure S9.  Get High-res Image As an example, this figure shows the association of ANKRD33|341405 to 'YEAROFTOBACCOSMOKINGONSET'. P value = 3.94e-07 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #13: 'COMPLETENESS.OF.RESECTION'

One gene related to 'COMPLETENESS.OF.RESECTION'.

Table S22.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 309
  R1 10
  R2 4
  RX 16
     
  Significant markers N = 1
List of one gene differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S23.  Get Full Table List of one gene differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
PAX2|5076 1.008e-06 0.0185

Figure S10.  Get High-res Image As an example, this figure shows the association of PAX2|5076 to 'COMPLETENESS.OF.RESECTION'. P value = 1.01e-06 with ANOVA analysis.

Methods & Data
Input

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

  • Clinical data file = LUAD-TP.merged_data.txt

  • Number of patients = 450

  • Number of genes = 18308

  • Number of clinical features = 13

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

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

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

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] Howell, D, Statistical Methods for Psychology. (5th ed.), Duxbury Press:324-5 (2002)
[4] Lehmann and Romano, Testing Statistical Hypotheses (3E ed.), New York: Springer. ISBN 0387988645 (2005)
[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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