Mutation Analysis (MutSig v2.0 and MutSigCV v0.9 merged result)
Lung Squamous Cell Carcinoma (Primary solid tumor)
16 April 2014  |  analyses__2014_04_16
Maintainer Information
Citation Information
doi:10.7908/C1V69H83
Maintained by Dan DiCara (Broad Institute)
Cite as Broad Institute TCGA Genome Data Analysis Center (2014): Mutation Analysis (MutSig v2.0 and MutSigCV v0.9 merged result). Broad Institute of MIT and Harvard. doi:10.7908/C1V69H83
Overview
Introduction

This report serves to describe the mutational landscape and properties of a given individual set, as well as rank genes and genesets according to mutational significance. MutSig v2.0 and MutSigCV v0.9 merged result was used to generate the results found in this report.

  • Working with individual set: LUSC-TP

  • Number of patients in set: 178

Input

The input for this pipeline is a set of individuals with the following files associated for each:

  1. An annotated .maf file describing the mutations called for the respective individual, and their properties.

  2. A .wig file that contains information about the coverage of the sample.

Summary

  • MAF used for this analysis:LUSC-TP.final_analysis_set.maf

  • Blacklist used for this analysis: pancan_mutation_blacklist.v14.hg19.txt

  • Significantly mutated genes (q ≤ 0.1): 14

  • Mutations seen in COSMIC: 352

  • Significantly mutated genes in COSMIC territory: 11

  • Significantly mutated genesets: 17

Mutation Preprocessing

  • Read 178 MAFs of type "Broad"

  • Total number of mutations in input MAFs: 65305

  • After removing 2 mutations outside chr1-24: 65303

  • After removing 595 blacklisted mutations: 64708

  • After removing 583 noncoding mutations: 64125

Mutation Filtering

  • Number of mutations before filtering: 64125

  • After removing 343 mutations outside gene set: 63782

  • After removing 47 mutations outside category set: 63735

  • After removing 2 "impossible" mutations in

  • gene-patient-category bins of zero coverage: 63343

Results
Breakdown of Mutations by Type

Table 1.  Get Full Table Table representing breakdown of mutations by type.

type count
Frame_Shift_Del 510
Frame_Shift_Ins 115
In_Frame_Del 44
In_Frame_Ins 3
Missense_Mutation 42416
Nonsense_Mutation 3678
Nonstop_Mutation 55
Silent 15702
Splice_Site 1191
Translation_Start_Site 21
Total 63735
Breakdown of Mutation Rates by Category Type

Table 2.  Get Full Table A breakdown of mutation rates per category discovered for this individual set.

category n N rate rate_per_mb relative_rate exp_ns_s_ratio
Tp*C->mut 12963 692977947 0.000019 19 2 3.3
(A/C/G)p*C->(A/T) 17392 1964339776 8.9e-06 8.9 0.96 2.6
(A/C/G)p*C->G 3301 1964339776 1.7e-06 1.7 0.18 4.9
A->mut 8779 2561387954 3.4e-06 3.4 0.37 3.9
indel+null 5552 5218705677 1.1e-06 1.1 0.12 NaN
double_null 45 5218705677 8.6e-09 0.0086 0.00094 NaN
Total 48032 5218705677 9.2e-06 9.2 1 3.5
Target Coverage for Each Individual

The x axis represents the samples. The y axis represents the exons, one row per exon, and they are sorted by average coverage across samples. For exons with exactly the same average coverage, they are sorted next by the %GC of the exon. (The secondary sort is especially useful for the zero-coverage exons at the bottom). If the figure is unpopulated, then full coverage is assumed (e.g. MutSig CV doesn't use WIGs and assumes full coverage).

Figure 1. 

Distribution of Mutation Counts, Coverage, and Mutation Rates Across Samples

Figure 2.  Patients counts and rates file used to generate this plot: LUSC-TP.patients.counts_and_rates.txt

Lego Plots

The mutation spectrum is depicted in the lego plots below in which the 96 possible mutation types are subdivided into six large blocks, color-coded to reflect the base substitution type. Each large block is further subdivided into the 16 possible pairs of 5' and 3' neighbors, as listed in the 4x4 trinucleotide context legend. The height of each block corresponds to the mutation frequency for that kind of mutation (counts of mutations normalized by the base coverage in a given bin). The shape of the spectrum is a signature for dominant mutational mechanisms in different tumor types.

Figure 3.  Get High-res Image SNV Mutation rate lego plot for entire set. Each bin is normalized by base coverage for that bin. Colors represent the six SNV types on the upper right. The three-base context for each mutation is labeled in the 4x4 legend on the lower right. The fractional breakdown of SNV counts is shown in the pie chart on the upper left. If this figure is blank, not enough information was provided in the MAF to generate it.

Figure 4.  Get High-res Image SNV Mutation rate lego plots for 4 slices of mutation allele fraction (0<=AF<0.1, 0.1<=AF<0.25, 0.25<=AF<0.5, & 0.5<=AF) . The color code and three-base context legends are the same as the previous figure. If this figure is blank, not enough information was provided in the MAF to generate it.

CoMut Plot

Figure 5.  Get High-res Image The matrix in the center of the figure represents individual mutations in patient samples, color-coded by type of mutation, for the significantly mutated genes. The rate of synonymous and non-synonymous mutations is displayed at the top of the matrix. The barplot on the left of the matrix shows the number of mutations in each gene. The percentages represent the fraction of tumors with at least one mutation in the specified gene. The barplot to the right of the matrix displays the q-values for the most significantly mutated genes. The purple boxplots below the matrix (only displayed if required columns are present in the provided MAF) represent the distributions of allelic fractions observed in each sample. The plot at the bottom represents the base substitution distribution of individual samples, using the same categories that were used to calculate significance.

Significantly Mutated Genes

Column Descriptions:

  • N = number of sequenced bases in this gene across the individual set

  • n = number of (nonsilent) mutations in this gene across the individual set

  • npat = number of patients (individuals) with at least one nonsilent mutation

  • nsite = number of unique sites having a non-silent mutation

  • nsil = number of silent mutations in this gene across the individual set

  • n1 = number of nonsilent mutations of type: Tp*C->mut

  • n2 = number of nonsilent mutations of type: (A/C/G)p*C->(A/T)

  • n3 = number of nonsilent mutations of type: (A/C/G)p*C->G

  • n4 = number of nonsilent mutations of type: A->mut

  • n5 = number of nonsilent mutations of type: indel+null

  • n6 = number of nonsilent mutations of type: double_null

  • p_cons = p-value for enrichment of mutations at evolutionarily most-conserved sites in gene

  • p_joint = p-value for clustering + conservation

  • p = p-value (overall)

  • q = q-value, False Discovery Rate (Benjamini-Hochberg procedure)

Table 3.  Get Full Table A Ranked List of Significantly Mutated Genes. Number of significant genes found: 14. Number of genes displayed: 35. Click on a gene name to display its stick figure depicting the distribution of mutations and mutation types across the chosen gene (this feature may not be available for all significant genes).

rank gene description N n npat nsite nsil n1 n2 n3 n4 n5 n6 p_clust p_cons p_joint p_cv p q
1 CDKN2A cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 134387 26 26 23 1 5 5 2 2 12 0 0.0065 2.4e-06 0 0 0 0
2 TP53 tumor protein p53 218677 147 141 98 7 9 42 17 32 47 0 0 0 0 4.7e-15 0 0
3 NFE2L2 nuclear factor (erythroid-derived 2)-like 2 318204 28 27 15 0 19 4 0 4 1 0 0 0 0 4.7e-10 0 0
4 PIK3CA phosphoinositide-3-kinase, catalytic, alpha polypeptide 583734 29 27 16 1 20 4 0 5 0 0 8e-05 0.0016 3.4e-06 6.2e-07 5.9e-11 2.7e-07
5 PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 214719 16 14 15 0 4 1 0 3 8 0 0.044 0.46 0.075 6.4e-11 1.3e-10 4.7e-07
6 KEAP1 kelch-like ECH-associated protein 1 312991 24 22 21 0 9 9 2 2 2 0 0.036 0.18 0.047 7.1e-10 8.4e-10 2.5e-06
7 MLL2 myeloid/lymphoid or mixed-lineage leukemia 2 2584402 41 35 41 5 4 12 4 3 18 0 0.26 0.32 0.38 2.1e-08 1.6e-07 0.0004
8 CSMD3 CUB and Sushi multiple domains 3 2028957 120 81 118 21 35 40 7 17 17 4 0.24 0.21 0.26 3.2e-08 1.6e-07 0.0004
9 RB1 retinoblastoma 1 (including osteosarcoma) 466255 12 12 12 0 1 2 0 1 8 0 0.14 0.69 0.28 1.5e-06 6.4e-06 0.013
10 HS6ST1 heparan sulfate 6-O-sulfotransferase 1 150811 5 5 5 0 1 2 0 0 2 0 0.00077 0.029 0.00089 0.0019 0.000024 0.044
11 FBXW7 F-box and WD repeat domain containing 7 442830 10 10 8 3 1 3 3 0 3 0 0.0052 0.15 0.0084 0.00029 0.000034 0.055
12 ZNF567 zinc finger protein 567 331196 4 3 4 3 2 0 0 1 1 0 0.000091 0.000044 6.6e-06 0.53 0.000047 0.071
13 ELTD1 EGF, latrophilin and seven transmembrane domain containing 1 365327 18 18 18 2 4 3 1 4 6 0 0.5 0.8 0.67 5.4e-06 0.000049 0.071
14 ASCL4 achaete-scute complex homolog 4 (Drosophila) 49103 6 6 6 0 0 2 1 1 2 0 0.34 0.22 0.34 0.000015 7e-05 0.09
15 OR2G6 olfactory receptor, family 2, subfamily G, member 6 169883 17 16 17 4 3 6 1 7 0 0 0.12 0.093 0.074 0.000089 0.000085 0.1
16 HLA-A major histocompatibility complex, class I, A 193690 6 6 6 0 1 0 0 0 4 1 0.09 0.0063 0.014 0.00053 0.000096 0.11
17 NOTCH1 Notch homolog 1, translocation-associated (Drosophila) 1040004 18 14 18 3 1 7 0 3 7 0 0.68 0.00035 0.005 0.002 0.00013 0.13
18 LRRC4C leucine rich repeat containing 4C 342685 19 17 19 1 5 8 1 4 1 0 0.073 0.19 0.063 0.00039 0.00028 0.28
19 ZEB2 zinc finger E-box binding homeobox 2 654993 15 15 15 2 2 9 0 3 1 0 0.000056 0.31 0.00011 0.23 0.00029 0.28
20 TMEM90A transmembrane protein 90A 128556 4 4 4 0 1 1 0 1 1 0 0.0015 0.5 0.002 0.013 0.00029 0.28
21 SLC6A5 solute carrier family 6 (neurotransmitter transporter, glycine), member 5 399899 7 6 7 3 0 3 2 1 1 0 0.000079 0.001 3e-05 1 0.00034 0.29
22 LIN37 lin-37 homolog (C. elegans) 94565 3 3 3 0 1 0 0 1 1 0 0.004 0.052 0.0021 0.016 0.00039 0.32
23 COL6A6 collagen, type VI, alpha 6 1183502 21 19 21 5 6 9 1 4 1 0 0.000046 0.069 0.000045 0.97 0.00049 0.38
24 CBLN4 cerebellin 4 precursor 101248 6 5 6 1 1 4 0 1 0 0 0.0015 0.16 0.0016 0.035 0.00058 0.44
25 LEPROT leptin receptor overlapping transcript 70124 4 4 4 0 0 1 0 1 2 0 0.053 0.64 0.06 0.00094 0.00061 0.44
26 ASB5 ankyrin repeat and SOCS box-containing 5 180540 9 9 9 0 3 1 3 2 0 0 0.013 0.0042 0.0014 0.047 0.0007 0.48
27 COL11A1 collagen, type XI, alpha 1 1000865 45 34 45 7 11 17 6 2 9 0 0.17 0.83 0.28 0.00025 0.00075 0.5
28 SNAI1 snail homolog 1 (Drosophila) 141546 4 4 4 0 2 1 0 1 0 0 0.0012 0.77 0.0022 0.038 0.00086 0.55
29 ZNF80 zinc finger protein 80 146952 7 7 7 1 1 2 1 1 2 0 0.037 0.58 0.065 0.0014 0.00091 0.56
30 ZBBX zinc finger, B-box domain containing 437517 17 17 17 1 4 4 1 5 3 0 0.025 0.68 0.05 0.0019 0.00096 0.58
31 CLSTN2 calsyntenin 2 497245 16 15 16 2 3 5 1 2 5 0 0.9 0.0045 0.019 0.0055 0.0011 0.61
32 PI16 peptidase inhibitor 16 229472 7 7 7 1 2 4 0 0 1 0 0.068 0.049 0.024 0.0045 0.0011 0.61
33 OR4D5 olfactory receptor, family 4, subfamily D, member 5 171040 9 9 9 3 1 4 1 2 1 0 0.00072 0.21 0.0017 0.068 0.0012 0.64
34 NUCB2 nucleobindin 2 230309 7 6 7 0 3 2 0 1 1 0 0.0011 0.61 0.0022 0.057 0.0013 0.67
35 SPOPL speckle-type POZ protein-like 216329 6 6 6 1 4 1 0 0 1 0 0.0038 0.18 0.0041 0.037 0.0015 0.76
CDKN2A

Figure S1.  This figure depicts the distribution of mutations and mutation types across the CDKN2A significant gene.

TP53

Figure S2.  This figure depicts the distribution of mutations and mutation types across the TP53 significant gene.

NFE2L2

Figure S3.  This figure depicts the distribution of mutations and mutation types across the NFE2L2 significant gene.

PIK3CA

Figure S4.  This figure depicts the distribution of mutations and mutation types across the PIK3CA significant gene.

PTEN

Figure S5.  This figure depicts the distribution of mutations and mutation types across the PTEN significant gene.

KEAP1

Figure S6.  This figure depicts the distribution of mutations and mutation types across the KEAP1 significant gene.

MLL2

Figure S7.  This figure depicts the distribution of mutations and mutation types across the MLL2 significant gene.

CSMD3

Figure S8.  This figure depicts the distribution of mutations and mutation types across the CSMD3 significant gene.

RB1

Figure S9.  This figure depicts the distribution of mutations and mutation types across the RB1 significant gene.

HS6ST1

Figure S10.  This figure depicts the distribution of mutations and mutation types across the HS6ST1 significant gene.

FBXW7

Figure S11.  This figure depicts the distribution of mutations and mutation types across the FBXW7 significant gene.

ZNF567

Figure S12.  This figure depicts the distribution of mutations and mutation types across the ZNF567 significant gene.

ELTD1

Figure S13.  This figure depicts the distribution of mutations and mutation types across the ELTD1 significant gene.

ASCL4

Figure S14.  This figure depicts the distribution of mutations and mutation types across the ASCL4 significant gene.

COSMIC analyses

In this analysis, COSMIC is used as a filter to increase power by restricting the territory of each gene. Cosmic version: v48.

Table 4.  Get Full Table Significantly mutated genes (COSMIC territory only). To access the database please go to: COSMIC. Number of significant genes found: 11. Number of genes displayed: 10

rank gene description n cos n_cos N_cos cos_ev p q
1 PIK3CA phosphoinositide-3-kinase, catalytic, alpha polypeptide 29 220 24 39160 9572 1.3e-12 2.6e-09
2 CDKN2A cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) 26 332 25 59096 559 1.7e-12 2.6e-09
3 TP53 tumor protein p53 147 356 144 63368 25865 1.7e-12 2.6e-09
4 PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 16 767 16 136526 579 2.5e-12 2.8e-09
5 HRAS v-Ha-ras Harvey rat sarcoma viral oncogene homolog 5 19 5 3382 1133 2.4e-10 2.1e-07
6 RB1 retinoblastoma 1 (including osteosarcoma) 12 267 7 47526 17 4.1e-07 0.00031
7 FBXW7 F-box and WD repeat domain containing 7 10 91 5 16198 217 5.4e-07 0.00035
8 HEPACAM2 HEPACAM family member 2 4 1 2 178 2 1.3e-06 0.00075
9 NF1 neurofibromin 1 (neurofibromatosis, von Recklinghausen disease, Watson disease) 22 285 6 50730 21 9.7e-06 0.0049
10 BRAF v-raf murine sarcoma viral oncogene homolog B1 8 89 4 15842 77 0.000017 0.0076

Note:

n - number of (nonsilent) mutations in this gene across the individual set.

cos = number of unique mutated sites in this gene in COSMIC

n_cos = overlap between n and cos.

N_cos = number of individuals times cos.

cos_ev = total evidence: number of reports in COSMIC for mutations seen in this gene.

p = p-value for seeing the observed amount of overlap in this gene)

q = q-value, False Discovery Rate (Benjamini-Hochberg procedure)

Geneset Analyses

Table 5.  Get Full Table A Ranked List of Significantly Mutated Genesets. (Source: MSigDB GSEA Cannonical Pathway Set).Number of significant genesets found: 17. Number of genesets displayed: 10

rank geneset description genes N_genes mut_tally N n npat nsite nsil n1 n2 n3 n4 n5 n6 p_ns_s p q
1 G1PATHWAY CDK4/6-cyclin D and CDK2-cyclin E phosphorylate Rb, which allows the transcription of genes needed for the G1/S cell cycle transition. ABL1, ATM, ATR, CCNA1, CCND1, CCNE1, CDC2, CDC25A, CDK2, CDK4, CDK6, CDKN1A, CDKN1B, CDKN2A, CDKN2B, DHFR, E2F1, GSK3B, HDAC1, MADH3, MADH4, RB1, SKP2, TFDP1, TGFB1, TGFB2, TGFB3, TP53 25 ABL1(3), ATM(8), ATR(13), CCNA1(1), CCND1(1), CCNE1(3), CDKN1A(2), CDKN2A(26), CDKN2B(1), E2F1(2), HDAC1(2), RB1(12), SKP2(2), TFDP1(3), TGFB1(1), TGFB2(3), TP53(147) 7994705 230 156 178 17 29 69 22 40 70 0 2.6e-09 <1.00e-15 <2.05e-13
2 P53PATHWAY p53 induces cell cycle arrest or apoptosis under conditions of DNA damage. APAF1, ATM, BAX, BCL2, CCND1, CCNE1, CDK2, CDK4, CDKN1A, E2F1, GADD45A, MDM2, PCNA, RB1, TIMP3, TP53 16 APAF1(4), ATM(8), BAX(1), CCND1(1), CCNE1(3), CDKN1A(2), E2F1(2), GADD45A(1), MDM2(2), RB1(12), TP53(147) 4817072 183 149 134 8 17 53 18 38 57 0 4.3e-10 <1.00e-15 <2.05e-13
3 RNAPATHWAY dsRNA-activated protein kinase phosphorylates elF2a, which generally inhibits translation, and activates NF-kB to provoke inflammation. CHUK, DNAJC3, EIF2S1, EIF2S2, MAP3K14, NFKB1, NFKBIA, PRKR, RELA, TP53 9 CHUK(2), EIF2S2(1), MAP3K14(1), NFKB1(2), RELA(3), TP53(147) 2611540 156 141 107 10 11 43 18 34 50 0 1.7e-07 <1.00e-15 <2.05e-13
4 TERTPATHWAY hTERC, the RNA subunit of telomerase, and hTERT, the catalytic protein subunit, are required for telomerase activity and are overexpressed in many cancers. HDAC1, MAX, MYC, SP1, SP3, TP53, WT1, ZNF42 7 HDAC1(2), MYC(1), SP1(2), SP3(1), TP53(147), WT1(4) 1863398 157 146 108 12 10 45 19 36 47 0 8.6e-07 1.67e-15 2.19e-13
5 SA_G1_AND_S_PHASES Cdk2, 4, and 6 bind cyclin D in G1, while cdk2/cyclin E promotes the G1/S transition. ARF1, ARF3, CCND1, CDK2, CDK4, CDKN1A, CDKN1B, CDKN2A, CFL1, E2F1, E2F2, MDM2, NXT1, PRB1, TP53 15 ARF1(1), CCND1(1), CDKN1A(2), CDKN2A(26), CFL1(2), E2F1(2), MDM2(2), NXT1(1), PRB1(5), TP53(147) 2143952 189 149 137 12 18 56 19 37 59 0 2.5e-10 1.78e-15 2.19e-13
6 PMLPATHWAY Ring-shaped PML nuclear bodies regulate transcription and are required co-activators in p53- and DAXX-mediated apoptosis. CREBBP, DAXX, HRAS, PAX3, PML, PRAM-1, RARA, RB1, SIRT1, SP100, TNF, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF6, TP53, UBL1 13 CREBBP(17), DAXX(3), HRAS(5), PAX3(4), RARA(1), RB1(12), SIRT1(1), SP100(3), TNF(1), TNFRSF1A(2), TNFRSF1B(1), TP53(147) 5020302 197 147 147 17 17 59 21 42 58 0 7.7e-08 2.44e-15 2.51e-13
7 PLK3PATHWAY Active Plk3 phosphorylates CDC25c, blocking the G2/M transition, and phosphorylates p53 to induce apoptosis. ATM, ATR, CDC25C, CHEK1, CHEK2, CNK, TP53, YWHAH 7 ATM(8), ATR(13), CDC25C(1), CHEK1(5), CHEK2(3), TP53(147) 4260894 177 146 128 10 18 52 20 37 50 0 2.2e-07 3.11e-15 2.74e-13
8 RBPATHWAY The ATM protein kinase recognizes DNA damage and blocks cell cycle progression by phosphorylating chk1 and p53, which normally inhibits Rb to allow G1/S transitions. ATM, CDC2, CDC25A, CDC25B, CDC25C, CDK2, CDK4, CHEK1, MYT1, RB1, TP53, WEE1, YWHAH 12 ATM(8), CDC25B(2), CDC25C(1), CHEK1(5), MYT1(4), RB1(12), TP53(147), WEE1(4) 4677103 183 146 134 10 18 53 19 36 57 0 9.3e-09 3.89e-15 2.94e-13
9 TIDPATHWAY On ligand binding, interferon gamma receptors stimulate JAK2 kinase to phosphorylate STAT transcription factors, which promote expression of interferon responsive genes. DNAJA3, HSPA1A, IFNG, IFNGR1, IFNGR2, IKBKB, JAK2, LIN7A, NFKB1, NFKBIA, RB1, RELA, TIP-1, TNF, TNFRSF1A, TNFRSF1B, TP53, USH1C, WT1 18 IFNG(1), IFNGR1(4), IFNGR2(1), IKBKB(2), JAK2(4), LIN7A(1), NFKB1(2), RB1(12), RELA(3), TNF(1), TNFRSF1A(2), TNFRSF1B(1), TP53(147), USH1C(7), WT1(4) 4825619 192 144 143 16 19 51 21 43 58 0 1.1e-07 4.66e-15 2.94e-13
10 ARFPATHWAY Cyclin-dependent kinase inhibitor 2A is a tumor suppressor that induces G1 arrest and can activate the p53 pathway, leading to G2/M arrest. ABL1, CDKN2A, E2F1, MDM2, MYC, PIK3CA, PIK3R1, POLR1A, POLR1B, POLR1C, POLR1D, RAC1, RB1, TBX2, TP53, TWIST1 16 ABL1(3), CDKN2A(26), E2F1(2), MDM2(2), MYC(1), PIK3CA(29), PIK3R1(2), POLR1A(3), POLR1B(2), POLR1D(1), RAC1(1), RB1(12), TBX2(3), TP53(147), TWIST1(1) 5361306 235 155 170 19 43 60 19 44 69 0 1.7e-09 4.77e-15 2.94e-13
Methods & Data
Methods

In brief, we tabulate the number of mutations and the number of covered bases for each gene. The counts are broken down by mutation context category: four context categories that are discovered by MutSig, and one for indel and 'null' mutations, which include indels, nonsense mutations, splice-site mutations, and non-stop (read-through) mutations. For each gene, we calculate the probability of seeing the observed constellation of mutations, i.e. the product P1 x P2 x ... x Pm, or a more extreme one, given the background mutation rates calculated across the dataset. [1]

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] TCGA, Integrated genomic analyses of ovarian carcinoma, Nature 474:609 - 615 (2011)
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