Correlation between miRseq expression and clinical features
Thyroid Adenocarcinoma (Primary solid tumor)
23 September 2013  |  analyses__2013_09_23
Maintainer Information
Citation Information
Maintained by Juok Cho (Broad Institute)
Cite as Broad Institute TCGA Genome Data Analysis Center (2013): Correlation between miRseq expression and clinical features. Broad Institute of MIT and Harvard. doi:10.7908/C14Q7SCN
Overview
Introduction

This pipeline uses various statistical tests to identify miRs whose expression levels correlated to selected clinical features.

Summary

Testing the association between 514 genes and 15 clinical features across 476 samples, statistically thresholded by Q value < 0.05, 13 clinical features related to at least one genes.

  • 2 genes correlated to 'Time to Death'.

    • HSA-MIR-376A-1 ,  HSA-MIR-181A-2

  • 6 genes correlated to 'AGE'.

    • HSA-MIR-1229 ,  HSA-MIR-376A-1 ,  HSA-MIR-181A-2 ,  HSA-MIR-362 ,  HSA-MIR-625 ,  ...

  • 38 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • HSA-MIR-139 ,  HSA-MIR-210 ,  HSA-MIR-425 ,  HSA-MIR-146B ,  HSA-MIR-369 ,  ...

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

    • HSA-MIR-139 ,  HSA-MIR-126 ,  HSA-MIR-363 ,  HSA-MIR-338 ,  HSA-MIR-30C-2 ,  ...

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

    • HSA-MIR-146B ,  HSA-MIR-21 ,  HSA-MIR-204 ,  HSA-MIR-7-2 ,  HSA-MIR-222 ,  ...

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

    • HSA-MIR-3677 ,  HSA-MIR-136 ,  HSA-MIR-589 ,  HSA-MIR-151 ,  HSA-MIR-1274B

  • 2 genes correlated to 'GENDER'.

    • HSA-MIR-651 ,  HSA-MIR-361

  • 172 genes correlated to 'HISTOLOGICAL.TYPE'.

    • HSA-MIR-21 ,  HSA-MIR-146B ,  HSA-MIR-7-2 ,  HSA-MIR-204 ,  HSA-MIR-345 ,  ...

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

    • HSA-MIR-888 ,  HSA-MIR-374A ,  HSA-MIR-660 ,  HSA-MIR-324 ,  HSA-MIR-20B ,  ...

  • 71 genes correlated to 'EXTRATHYROIDAL.EXTENSION'.

    • HSA-MIR-30A ,  HSA-MIR-363 ,  HSA-MIR-148B ,  HSA-MIR-21 ,  HSA-MIR-126 ,  ...

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

    • HSA-MIR-7-2 ,  HSA-MIR-21 ,  HSA-MIR-1179 ,  HSA-MIR-511-2 ,  HSA-MIR-382 ,  ...

  • 1 gene correlated to 'MULTIFOCALITY'.

    • HSA-MIR-132

  • 3 genes correlated to 'TUMOR.SIZE'.

    • HSA-MIR-9-2 ,  HSA-MIR-183 ,  HSA-MIR-9-1

  • No genes correlated to 'RADIATIONEXPOSURE', and 'COMPLETENESS.OF.RESECTION'.

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=6 older N=3 younger N=3
NEOPLASM DISEASESTAGE ANOVA test N=38        
PATHOLOGY T STAGE Spearman correlation test N=15 higher stage N=4 lower stage N=11
PATHOLOGY N STAGE t test N=59 class1 N=29 class0 N=30
PATHOLOGY M STAGE ANOVA test N=5        
GENDER t test N=2 male N=0 female N=2
HISTOLOGICAL TYPE ANOVA test N=172        
RADIATIONS RADIATION REGIMENINDICATION t test N=10 yes N=7 no N=3
RADIATIONEXPOSURE t test   N=0        
EXTRATHYROIDAL EXTENSION ANOVA test N=71        
COMPLETENESS OF RESECTION ANOVA test   N=0        
NUMBER OF LYMPH NODES Spearman correlation test N=56 higher number.of.lymph.nodes N=35 lower number.of.lymph.nodes N=21
MULTIFOCALITY t test N=1 unifocal N=0 multifocal N=1
TUMOR SIZE Spearman correlation test N=3 higher tumor.size N=1 lower tumor.size N=2
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) 0-158.8 (median=14.2)
  censored N = 458
  death N = 13
     
  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
HSA-MIR-376A-1 3.9 4.821e-05 0.025 0.885
HSA-MIR-181A-2 0.33 5.05e-05 0.026 0.171

Figure S1.  Get High-res Image As an example, this figure shows the association of HSA-MIR-376A-1 to 'Time to Death'. four curves present the cumulative survival rates of 4 quartile subsets of patients. P value = 4.82e-05 with univariate Cox regression analysis using continuous log-2 expression values.

Clinical variable #2: 'AGE'

6 genes related to 'AGE'.

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

AGE Mean (SD) 47.11 (16)
  Significant markers N = 6
  pos. correlated 3
  neg. correlated 3
List of 6 genes significantly correlated to 'AGE' by Spearman correlation test

Table S4.  Get Full Table List of 6 genes significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-1229 0.2165 1.654e-05 0.0085
HSA-MIR-376A-1 0.256 2.055e-05 0.0105
HSA-MIR-181A-2 -0.1898 3.068e-05 0.0157
HSA-MIR-362 0.1885 3.491e-05 0.0178
HSA-MIR-625 -0.1855 4.656e-05 0.0237
HSA-MIR-3622A -0.1952 8.492e-05 0.0432

Figure S2.  Get High-res Image As an example, this figure shows the association of HSA-MIR-1229 to 'AGE'. P value = 1.65e-05 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'NEOPLASM.DISEASESTAGE'

38 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S5.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 270
  STAGE II 50
  STAGE III 106
  STAGE IV 2
  STAGE IVA 41
  STAGE IVC 5
     
  Significant markers N = 38
List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

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

ANOVA_P Q
HSA-MIR-139 8.104e-12 4.17e-09
HSA-MIR-210 3.644e-09 1.87e-06
HSA-MIR-425 2.405e-07 0.000123
HSA-MIR-146B 2.529e-07 0.000129
HSA-MIR-369 3.19e-07 0.000163
HSA-MIR-154 6.341e-07 0.000323
HSA-MIR-31 9.001e-07 0.000457
HSA-MIR-376C 1.324e-06 0.000671
HSA-MIR-126 1.91e-06 0.000967
HSA-MIR-487B 3.328e-06 0.00168

Figure S3.  Get High-res Image As an example, this figure shows the association of HSA-MIR-139 to 'NEOPLASM.DISEASESTAGE'. P value = 8.1e-12 with ANOVA analysis.

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

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

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

PATHOLOGY.T.STAGE Mean (SD) 2.12 (0.88)
  N
  1 138
  2 160
  3 157
  4 19
     
  Significant markers N = 15
  pos. correlated 4
  neg. correlated 11
List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

Table S8.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-139 -0.269 2.651e-09 1.36e-06
HSA-MIR-126 -0.257 1.386e-08 7.11e-06
HSA-MIR-363 -0.2318 3.457e-07 0.000177
HSA-MIR-338 -0.1965 1.649e-05 0.00843
HSA-MIR-30C-2 -0.1942 2.068e-05 0.0105
HSA-MIR-145 -0.1937 2.163e-05 0.011
HSA-MIR-92B 0.1931 2.305e-05 0.0117
HSA-MIR-3074 -0.193 2.321e-05 0.0118
HSA-MIR-7-2 -0.1952 2.389e-05 0.0121
HSA-MIR-20B -0.191 2.89e-05 0.0146

Figure S4.  Get High-res Image As an example, this figure shows the association of HSA-MIR-139 to 'PATHOLOGY.T.STAGE'. P value = 2.65e-09 with Spearman correlation analysis.

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

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

Table S9.  Basic characteristics of clinical feature: 'PATHOLOGY.N.STAGE'

PATHOLOGY.N.STAGE Labels N
  class0 216
  class1 213
     
  Significant markers N = 59
  Higher in class1 29
  Higher in class0 30
List of top 10 genes differentially expressed by 'PATHOLOGY.N.STAGE'

Table S10.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGY.N.STAGE'

T(pos if higher in 'class1') ttestP Q AUC
HSA-MIR-146B 7.47 6.66e-13 3.42e-10 0.6692
HSA-MIR-21 7.18 3.462e-12 1.78e-09 0.6751
HSA-MIR-204 -6.7 7.104e-11 3.64e-08 0.6694
HSA-MIR-7-2 -6.67 8.57e-11 4.38e-08 0.6767
HSA-MIR-222 6.65 1.215e-10 6.19e-08 0.656
HSA-MIR-1179 -6.54 1.888e-10 9.61e-08 0.6756
HSA-MIR-511-2 6.31 6.938e-10 3.52e-07 0.6683
HSA-MIR-221 6.11 2.72e-09 1.38e-06 0.6328
HSA-MIR-375 5.96 5.828e-09 2.95e-06 0.6347
HSA-MIR-363 -5.85 9.813e-09 4.96e-06 0.6508

Figure S5.  Get High-res Image As an example, this figure shows the association of HSA-MIR-146B to 'PATHOLOGY.N.STAGE'. P value = 6.66e-13 with T-test analysis.

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

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

Table S11.  Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'

PATHOLOGY.M.STAGE Labels N
  M0 258
  M1 8
  MX 209
     
  Significant markers N = 5
List of 5 genes differentially expressed by 'PATHOLOGY.M.STAGE'

Table S12.  Get Full Table List of 5 genes differentially expressed by 'PATHOLOGY.M.STAGE'

ANOVA_P Q
HSA-MIR-3677 4.137e-06 0.00213
HSA-MIR-136 1.883e-05 0.00966
HSA-MIR-589 4.511e-05 0.0231
HSA-MIR-151 5.035e-05 0.0257
HSA-MIR-1274B 8.659e-05 0.0442

Figure S6.  Get High-res Image As an example, this figure shows the association of HSA-MIR-3677 to 'PATHOLOGY.M.STAGE'. P value = 4.14e-06 with ANOVA analysis.

Clinical variable #7: 'GENDER'

2 genes related to 'GENDER'.

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

GENDER Labels N
  FEMALE 350
  MALE 126
     
  Significant markers N = 2
  Higher in MALE 0
  Higher in FEMALE 2
List of 2 genes differentially expressed by 'GENDER'

Table S14.  Get Full Table List of 2 genes differentially expressed by 'GENDER'

T(pos if higher in 'MALE') ttestP Q AUC
HSA-MIR-651 -4.98 1.368e-06 0.000703 0.6502
HSA-MIR-361 -4.39 1.649e-05 0.00846 0.6171

Figure S7.  Get High-res Image As an example, this figure shows the association of HSA-MIR-651 to 'GENDER'. P value = 1.37e-06 with T-test analysis.

Clinical variable #8: 'HISTOLOGICAL.TYPE'

172 genes related to 'HISTOLOGICAL.TYPE'.

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

HISTOLOGICAL.TYPE Labels N
  OTHER SPECIFY 9
  THYROID PAPILLARY CARCINOMA - CLASSICAL/USUAL 333
  THYROID PAPILLARY CARCINOMA - FOLLICULAR (>= 99% FOLLICULAR PATTERNED) 99
  THYROID PAPILLARY CARCINOMA - TALL CELL (>= 50% TALL CELL FEATURES) 35
     
  Significant markers N = 172
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
HSA-MIR-21 5.137e-42 2.64e-39
HSA-MIR-146B 1.959e-33 1e-30
HSA-MIR-7-2 6.167e-25 3.16e-22
HSA-MIR-204 1.519e-22 7.76e-20
HSA-MIR-345 9.154e-22 4.67e-19
HSA-MIR-1179 2.157e-21 1.1e-18
HSA-MIR-31 2.542e-21 1.29e-18
HSA-MIR-375 3.447e-21 1.75e-18
HSA-MIR-511-2 1.974e-20 9.99e-18
HSA-MIR-148B 5.22e-20 2.64e-17

Figure S8.  Get High-res Image As an example, this figure shows the association of HSA-MIR-21 to 'HISTOLOGICAL.TYPE'. P value = 5.14e-42 with ANOVA analysis.

Clinical variable #9: 'RADIATIONS.RADIATION.REGIMENINDICATION'

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

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

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 14
  YES 462
     
  Significant markers N = 10
  Higher in YES 7
  Higher in NO 3
List of 10 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

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

T(pos if higher in 'YES') ttestP Q AUC
HSA-MIR-888 9.56 1.547e-10 7.74e-08 0.8816
HSA-MIR-374A -8.76 2.583e-07 0.000129 0.9179
HSA-MIR-660 7.96 8.931e-07 0.000445 0.8865
HSA-MIR-324 6.52 9.308e-06 0.00463 0.8434
HSA-MIR-20B 6.41 1.245e-05 0.00617 0.8429
HSA-MIR-3130-1 -5.57 3.227e-05 0.016 0.7455
HSA-MIR-1976 5.63 4.266e-05 0.0211 0.7942
HSA-MIR-3909 -5.21 8.922e-05 0.044 0.7452
HSA-MIR-338 5.33 9.117e-05 0.0449 0.7975
HSA-MIR-532 5.36 9.274e-05 0.0455 0.8304

Figure S9.  Get High-res Image As an example, this figure shows the association of HSA-MIR-888 to 'RADIATIONS.RADIATION.REGIMENINDICATION'. P value = 1.55e-10 with T-test analysis.

Clinical variable #10: 'RADIATIONEXPOSURE'

No gene related to 'RADIATIONEXPOSURE'.

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

RADIATIONEXPOSURE Labels N
  NO 401
  YES 17
     
  Significant markers N = 0
Clinical variable #11: 'EXTRATHYROIDAL.EXTENSION'

71 genes related to 'EXTRATHYROIDAL.EXTENSION'.

Table S20.  Basic characteristics of clinical feature: 'EXTRATHYROIDAL.EXTENSION'

EXTRATHYROIDAL.EXTENSION Labels N
  MINIMAL (T3) 123
  MODERATE/ADVANCED (T4A) 15
  NONE 322
  VERY ADVANCED (T4B) 1
     
  Significant markers N = 71
List of top 10 genes differentially expressed by 'EXTRATHYROIDAL.EXTENSION'

Table S21.  Get Full Table List of top 10 genes differentially expressed by 'EXTRATHYROIDAL.EXTENSION'

ANOVA_P Q
HSA-MIR-30A 1.734e-09 8.91e-07
HSA-MIR-363 5.518e-09 2.83e-06
HSA-MIR-148B 8.699e-09 4.45e-06
HSA-MIR-21 1.209e-08 6.18e-06
HSA-MIR-126 1.574e-08 8.03e-06
HSA-MIR-758 1.596e-08 8.12e-06
HSA-MIR-1180 2.248e-08 1.14e-05
HSA-MIR-411 2.414e-08 1.22e-05
HSA-MIR-376C 3.084e-08 1.56e-05
HSA-MIR-1179 3.706e-08 1.87e-05

Figure S10.  Get High-res Image As an example, this figure shows the association of HSA-MIR-30A to 'EXTRATHYROIDAL.EXTENSION'. P value = 1.73e-09 with ANOVA analysis.

Clinical variable #12: 'COMPLETENESS.OF.RESECTION'

No gene related to 'COMPLETENESS.OF.RESECTION'.

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

COMPLETENESS.OF.RESECTION Labels N
  R0 370
  R1 46
  R2 3
  RX 28
     
  Significant markers N = 0
Clinical variable #13: 'NUMBER.OF.LYMPH.NODES'

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

Table S23.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 3.54 (6.2)
  Significant markers N = 56
  pos. correlated 35
  neg. correlated 21
List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

Table S24.  Get Full Table List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-7-2 -0.3201 3.455e-10 1.78e-07
HSA-MIR-21 0.2807 2.708e-08 1.39e-05
HSA-MIR-1179 -0.29 3.306e-08 1.69e-05
HSA-MIR-511-2 0.2785 3.663e-08 1.87e-05
HSA-MIR-382 0.2794 7.679e-08 3.92e-05
HSA-MIR-31 0.271 9.039e-08 4.6e-05
HSA-MIR-874 -0.2669 1.322e-07 6.72e-05
HSA-MIR-30E -0.265 1.636e-07 8.3e-05
HSA-MIR-134 0.2653 1.711e-07 8.66e-05
HSA-MIR-127 0.2608 2.598e-07 0.000131

Figure S11.  Get High-res Image As an example, this figure shows the association of HSA-MIR-7-2 to 'NUMBER.OF.LYMPH.NODES'. P value = 3.45e-10 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #14: 'MULTIFOCALITY'

One gene related to 'MULTIFOCALITY'.

Table S25.  Basic characteristics of clinical feature: 'MULTIFOCALITY'

MULTIFOCALITY Labels N
  MULTIFOCAL 214
  UNIFOCAL 252
     
  Significant markers N = 1
  Higher in UNIFOCAL 0
  Higher in MULTIFOCAL 1
List of one gene differentially expressed by 'MULTIFOCALITY'

Table S26.  Get Full Table List of one gene differentially expressed by 'MULTIFOCALITY'

T(pos if higher in 'UNIFOCAL') ttestP Q AUC
HSA-MIR-132 -4.16 3.908e-05 0.0201 0.6115

Figure S12.  Get High-res Image As an example, this figure shows the association of HSA-MIR-132 to 'MULTIFOCALITY'. P value = 3.91e-05 with T-test analysis.

Clinical variable #15: 'TUMOR.SIZE'

3 genes related to 'TUMOR.SIZE'.

Table S27.  Basic characteristics of clinical feature: 'TUMOR.SIZE'

TUMOR.SIZE Mean (SD) 2.94 (1.6)
  Significant markers N = 3
  pos. correlated 1
  neg. correlated 2
List of 3 genes significantly correlated to 'TUMOR.SIZE' by Spearman correlation test

Table S28.  Get Full Table List of 3 genes significantly correlated to 'TUMOR.SIZE' by Spearman correlation test

SpearmanCorr corrP Q
HSA-MIR-9-2 -0.2051 5.61e-05 0.0288
HSA-MIR-183 0.2042 6.097e-05 0.0313
HSA-MIR-9-1 -0.2004 8.39e-05 0.043

Figure S13.  Get High-res Image As an example, this figure shows the association of HSA-MIR-9-2 to 'TUMOR.SIZE'. P value = 5.61e-05 with Spearman correlation analysis. The straight line presents the best linear regression.

Methods & Data
Input
  • Expresson data file = THCA-TP.miRseq_RPKM_log2.txt

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

  • Number of patients = 476

  • Number of genes = 514

  • Number of clinical features = 15

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.

Download Results

In addition to the links below, the full results of the analysis summarized in this report can also be downloaded programmatically using firehose_get, or interactively from either the Broad GDAC website or TCGA Data Coordination Center Portal.

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)