Head and Neck Squamous Cell Carcinoma: Correlation between gene methylation status and clinical features
Maintained by Juok Cho (Broad Institute)
Overview
Introduction

This pipeline uses various statistical tests to identify genes whose promoter methylation levels correlated to selected clinical features.

Summary

Testing the association between 17421 genes and 11 clinical features across 299 samples, statistically thresholded by Q value < 0.05, 7 clinical features related to at least one genes.

- 2 genes correlated to 'Time to Death'.

- ZNF266 , WIPI1

- 4 genes correlated to 'AGE'.

- SLC35D3 , HAND1 , XKR6 , PRSS27

- 5 genes correlated to 'GENDER'.

- KIF4B , FRG1B , SLC22A3 , NLRP2 , RPA1

- 7 genes correlated to 'PATHOLOGY.N'.

- SLC47A2 , AVPI1 , FAM185A , ARHGAP27 , ESRRA , ...

- 7 genes correlated to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

- FLII , ZCCHC17 , RTN4IP1 , CKS2 , RPL7L1 , ...

- 3 genes correlated to 'NUMBERPACKYEARSSMOKED'.

- PHOSPHO2 , RAB21 , MMADHC

- 5 genes correlated to 'TOBACCOSMOKINGHISTORYINDICATOR'.

- C17ORF82 , THAP11 , SLC12A7 , ACCN4 , KCNC3

- No genes correlated to 'PATHOLOGY.T', 'TUMOR.STAGE', 'STOPPEDSMOKINGYEAR', and 'YEAROFTOBACCOSMOKINGONSET'.

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=2 | shorter survival | N=1 | longer survival | N=1
AGE | Spearman correlation test | N=4 | older | N=4 | younger | N=0
GENDER | t test | N=5 | male | N=2 | female | N=3
PATHOLOGY T | Spearman correlation test | N=0 |  |  |  |
PATHOLOGY N | Spearman correlation test | N=7 | higher pN | N=7 | lower pN | N=0
TUMOR STAGE | Spearman correlation test | N=0 |  |  |  |
RADIATIONS RADIATION REGIMENINDICATION | t test | N=7 | yes | N=1 | no | N=6
NUMBERPACKYEARSSMOKED | Spearman correlation test | N=3 | higher numberpackyearssmoked | N=0 | lower numberpackyearssmoked | N=3
STOPPEDSMOKINGYEAR | Spearman correlation test | N=0 |  |  |  |
TOBACCOSMOKINGHISTORYINDICATOR | ANOVA test | N=5 |  |  |  |
YEAROFTOBACCOSMOKINGONSET | Spearman correlation test | N=0 |  |  |  |

Clinical variable #1: 'Time to Death'

2 genes related to 'Time to Death'.

Table S1. Basic characteristics of clinical feature: 'Time to Death'

Time to Death | Duration (Months) | 0.1-210.9 (median=14.8)
| censored | N = 179
| death | N = 117
| Significant markers | N = 2
| associated with shorter survival | 1
| associated with longer survival | 1

List of 2 genes significantly associated with 'Time to Death' by Cox regression test

Table S2. Get Full Table List of 2 genes significantly associated with 'Time to Death' by Cox regression test

| HazardRatio | Wald_P | Q | C_index
--- | --- | --- | --- | ---
ZNF266 | 8001 | 2.455e-07 | 0.0043 | 0.608
WIPI1 | 0 | 7.547e-07 | 0.013 | 0.454

Figure S1. Get High-res Image As an example, this figure shows the association of ZNF266 to 'Time to Death'. four curves present the cumulative survival rates of 4 quartile subsets of patients. P value = 2.46e-07 with univariate Cox regression analysis using continuous log-2 expression values.

Clinical variable #2: 'AGE'

4 genes related to 'AGE'.

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

AGE | Mean (SD) | 61.14 (12)
| Significant markers | N = 4
| pos. correlated | 4
| neg. correlated | 0

List of 4 genes significantly correlated to 'AGE' by Spearman correlation test

Table S4. Get Full Table List of 4 genes significantly correlated to 'AGE' by Spearman correlation test

| SpearmanCorr | corrP | Q
--- | --- | --- | ---
SLC35D3 | 0.3072 | 5.932e-08 | 0.00103
HAND1 | 0.2824 | 6.878e-07 | 0.012
XKR6 | 0.2808 | 8.002e-07 | 0.0139
PRSS27 | 0.2672 | 2.774e-06 | 0.0483

Figure S2. Get High-res Image As an example, this figure shows the association of SLC35D3 to 'AGE'. P value = 5.93e-08 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'GENDER'

5 genes related to 'GENDER'.

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

GENDER | Labels | N
| FEMALE | 81
| MALE | 218
| Significant markers | N = 5
| Higher in MALE | 2
| Higher in FEMALE | 3

List of 5 genes differentially expressed by 'GENDER'

Table S6. Get Full Table List of 5 genes differentially expressed by 'GENDER'

| T(pos if higher in 'MALE') | ttestP | Q | AUC
--- | --- | --- | --- | ---
KIF4B | -11.42 | 1.288e-21 | 2.24e-17 | 0.8553
FRG1B | -5.97 | 2.708e-08 | 0.000472 | 0.7198
SLC22A3 | 5.3 | 2.257e-07 | 0.00393 | 0.5962
NLRP2 | 5.07 | 1.269e-06 | 0.0221 | 0.6906
RPA1 | -4.91 | 1.524e-06 | 0.0265 | 0.5609

Figure S3. Get High-res Image As an example, this figure shows the association of KIF4B to 'GENDER'. P value = 1.29e-21 with T-test analysis.

Clinical variable #4: 'PATHOLOGY.T'

No gene related to 'PATHOLOGY.T'.

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

PATHOLOGY.T | Mean (SD) | 2.92 (1)
|  | N
| T1 | 23
| T2 | 77
| T3 | 59
| T4 | 101
| Significant markers | N = 0

Clinical variable #5: 'PATHOLOGY.N'

7 genes related to 'PATHOLOGY.N'.

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

PATHOLOGY.N | Mean (SD) | 1.03 (0.96)
|  | N
| N0 | 99
| N1 | 32
| N2 | 96
| N3 | 5
| Significant markers | N = 7
| pos. correlated | 7
| neg. correlated | 0

List of 7 genes significantly correlated to 'PATHOLOGY.N' by Spearman correlation test

Table S9. Get Full Table List of 7 genes significantly correlated to 'PATHOLOGY.N' by Spearman correlation test

| SpearmanCorr | corrP | Q
--- | --- | --- | ---
SLC47A2 | 0.356 | 2.457e-08 | 0.000428
AVPI1 | 0.3295 | 2.805e-07 | 0.00489
FAM185A | 0.3168 | 8.333e-07 | 0.0145
ARHGAP27 | 0.3158 | 9.112e-07 | 0.0159
ESRRA | 0.3039 | 2.415e-06 | 0.0421
VAMP8 | 0.3031 | 2.559e-06 | 0.0446
BTNL8 | 0.3018 | 2.853e-06 | 0.0497

Figure S4. Get High-res Image As an example, this figure shows the association of SLC47A2 to 'PATHOLOGY.N'. P value = 2.46e-08 with Spearman correlation analysis.

Clinical variable #6: 'TUMOR.STAGE'

No gene related to 'TUMOR.STAGE'.

Table S10. Basic characteristics of clinical feature: 'TUMOR.STAGE'

TUMOR.STAGE | Mean (SD) | 3.29 (0.98)
|  | N
| Stage 1 | 16
| Stage 2 | 47
| Stage 3 | 40
| Stage 4 | 152
| Significant markers | N = 0

Clinical variable #7: 'RADIATIONS.RADIATION.REGIMENINDICATION'

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

Table S11. Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION | Labels | N
| NO | 75
| YES | 224
| Significant markers | N = 7
| Higher in YES | 1
| Higher in NO | 6

List of 7 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S12. Get Full Table List of 7 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

| T(pos if higher in 'YES') | ttestP | Q | AUC
--- | --- | --- | --- | ---
FLII | 5.7 | 4.461e-08 | 0.000777 | 0.693
ZCCHC17 | -5.65 | 1.054e-07 | 0.00184 | 0.7057
RTN4IP1 | -5.29 | 4.899e-07 | 0.00853 | 0.6849
CKS2 | -5.17 | 8.851e-07 | 0.0154 | 0.6911
RPL7L1 | -5.08 | 1.176e-06 | 0.0205 | 0.6839
EHMT2 | -5.02 | 1.234e-06 | 0.0215 | 0.6693
TMEM57 | -5.01 | 1.786e-06 | 0.0311 | 0.6988

Figure S5. Get High-res Image As an example, this figure shows the association of FLII to 'RADIATIONS.RADIATION.REGIMENINDICATION'. P value = 4.46e-08 with T-test analysis.

Clinical variable #8: 'NUMBERPACKYEARSSMOKED'

3 genes related to 'NUMBERPACKYEARSSMOKED'.

Table S13. Basic characteristics of clinical feature: 'NUMBERPACKYEARSSMOKED'

NUMBERPACKYEARSSMOKED | Mean (SD) | 50.21 (38)
| Significant markers | N = 3
| pos. correlated | 0
| neg. correlated | 3

List of 3 genes significantly correlated to 'NUMBERPACKYEARSSMOKED' by Spearman correlation test

Table S14. Get Full Table List of 3 genes significantly correlated to 'NUMBERPACKYEARSSMOKED' by Spearman correlation test

| SpearmanCorr | corrP | Q
--- | --- | --- | ---
PHOSPHO2 | -0.3744 | 9.191e-07 | 0.016
RAB21 | -0.3667 | 1.596e-06 | 0.0278
MMADHC | -0.3597 | 2.59e-06 | 0.0451

Figure S6. Get High-res Image As an example, this figure shows the association of PHOSPHO2 to 'NUMBERPACKYEARSSMOKED'. P value = 9.19e-07 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #9: 'STOPPEDSMOKINGYEAR'

No gene related to 'STOPPEDSMOKINGYEAR'.

Table S15. Basic characteristics of clinical feature: 'STOPPEDSMOKINGYEAR'

STOPPEDSMOKINGYEAR | Mean (SD) | 1994.58 (14)
| Significant markers | N = 0

Clinical variable #10: 'TOBACCOSMOKINGHISTORYINDICATOR'

5 genes related to 'TOBACCOSMOKINGHISTORYINDICATOR'.

Table S16. Basic characteristics of clinical feature: 'TOBACCOSMOKINGHISTORYINDICATOR'

TOBACCOSMOKINGHISTORYINDICATOR | Labels | N
| CURRENT REFORMED SMOKER FOR < OR = 15 YEARS | 82
| CURRENT REFORMED SMOKER FOR > 15 YEARS | 53
| CURRENT SMOKER | 95
| LIFELONG NON-SMOKER | 58
| Significant markers | N = 5

List of 5 genes differentially expressed by 'TOBACCOSMOKINGHISTORYINDICATOR'

Table S17. Get Full Table List of 5 genes differentially expressed by 'TOBACCOSMOKINGHISTORYINDICATOR'

| ANOVA_P | Q
--- | --- | ---
C17ORF82 | 1.005e-06 | 0.0175
THAP11 | 1.125e-06 | 0.0196
SLC12A7 | 2.403e-06 | 0.0419
ACCN4 | 2.674e-06 | 0.0466
KCNC3 | 2.776e-06 | 0.0483

Figure S7. Get High-res Image As an example, this figure shows the association of C17ORF82 to 'TOBACCOSMOKINGHISTORYINDICATOR'. P value = 1e-06 with ANOVA analysis.

Clinical variable #11: 'YEAROFTOBACCOSMOKINGONSET'

No gene related to 'YEAROFTOBACCOSMOKINGONSET'.

Table S18. Basic characteristics of clinical feature: 'YEAROFTOBACCOSMOKINGONSET'

YEAROFTOBACCOSMOKINGONSET | Mean (SD) | 1964.45 (12)
| Significant markers | N = 0

Methods & Data
Input

- Expresson data file = HNSC-TP.meth.for_correlation.filtered_data.txt

- Clinical data file = HNSC-TP.clin.merged.picked.txt

- Number of patients = 299

- Number of genes = 17421

- Number of clinical features = 11

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.

Download Results

This is an experimental feature. The full results of the analysis summarized in this report can be downloaded from the TCGA Data Coordination Center.

- Analysis Results (MD5 checksum)

- Auxiliary Data (MD5 checksum)

- MAGE-TAB File (MD5 checksum)

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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