Exome sequencing analysis of the mutational spectrum in carcinogen and genetic models of Kras-driven lung cancer

Peter Westcott, Kyle Halliwill, Minh To, David Quigley, Reyno Delrosario, Erik Fredlund, David Adams1, and Allan Balmain

UCSF Helen Diller Family Comprehensive Cancer Center, 1450 3rd Street, San Francisco.

1 Wellcome Trust Sanger Centre, Cambridge, England.

Why sequence tumors from mice?

Timing of initiation collection

Initiating gene(s), carcinogen(s)

Can distinguish mutations involved in initiation from progression

Control!

Specific goals of this study

Part of the MMHCC TCGA Pilot Project

What is the effect of the causative carcinogen on mutation spectrum?

Characterize the utility of sequencing mouse tumors:

Clean genetic induction (GEM) vs. carcinogen induction?

What mutations arise after Kras initiation?

Exome sequencing

Urethane MNU KrasLA2 (GEM)

44 lung tumorsfrom 17 mice

26 lung tumorsfrom 7 mice

13 lung tumorsfrom 4 mice

Kras+/- (FVB/Ola)

Kras+/- Kras+/+

KrasLA2 (FVB/Ola)

Control tail DNA: 2 Kras+/+ tails

Spontaneous lung tumors

Exome sequencing

Have a confident list of somatic variants

Have aligned reads to mouse genome, called against multiple controls and performed extensive QC (Kyle Hallilwill)

Illumina paired-end sequencing (Wellcome Trust Sanger Centre)

Exome sequencing

Carcinogen models of Kras-driven lung cancer

~90% of lung tumors harbor Kras mutations.

Urethane (ethyl carbamate)

Adenosine and cytidine DNA adducts lead to mispairing:

Kras Q61L (CAACTA), Q61R (CAACGA).

A TReplication

Mispairing

Carcinogen models of Kras-driven lung cancer

MNU (methyl-nitroso urea)

~90% of lung tumors harbor Kras mutations

Guanosine DNA adducts lead to GA transitions

Kras G12D (GGTGAT)

Genome-wide spectrum of these carcinogen mutations not known

GG G

AReplication

Mispairing

Mutation spectrum

Urethane

MNU

LA2

Light shade = Kras+/-

Mutation spectrum

Slight bias for mutationsat G/C nucleotide

Strong bias for mutationsat G nucleotide with flanking G or A

Strong bias for mutationsat A/T nucleotide

Mutation spectrumAv

erag

e co

unts

per

tum

or

Purine bias at 5’ flanking base

5’ A 5’ G

Mutation spectrum

Are non-carcinogen mutations separable?

Aver

age

coun

ts p

er tu

mor

For the most part

670

80

60

40

20

0 NCG->T Other G->A A->T A->G A->C G->C G->T

UrethaneMNULA2

ARE CARCINOGEN MUTATIONS RELEVANT?

Other driver mutations?

Analysis complicated:

High mutation rates: MNU – 21.2/Mb Urethane – 6.4/Mb LA2 – 1.9/Mb Correlation between gene length and mutations

Start with variants within Vogelstein’s 2013 list of drivers:

Selected only consequential mutations at highly conserved sites in expressed genes

Other driver mutations?

GENE EXON_LENGTH NONSYN_MUTMll2 19827 16Sf3b1 6191 5Crebbp 7507 4Asxl1 6674 3Pdgfra 6553 3Met 6652 3Cic 6099 3Atm 11964 3Arid1b 11325 3Alk 5918 3Gnas 3717 2Notch2 10506 2Arid1a 8175 2Fgfr3 4222 2Hnf1a 3186 2Flt3 3656 2Brca2 10540 2Akt1 2640 2Rb1 4625 2

None of these mutations occur in LA2 tumors

Slight enrichment for longer genes

Modest increase in NS mutation ratio

One S367 to F – required for autophosph. and activity

Subclonal Myc T58P?

Conclusions

Clear recapitulation of expected carcinogen mutations

Mutation Spectrum

GEM shows few mutations

Mutations highly specific and distinguishable

Driver Mutations

Kras

Interesting candidates in carcinogen-induced tumors

Future work

InDel analysis.

Optimize list of potential driver mutations (relevant sites?).

Validate top 1000 interesting variants by Sequenom (Wellcome Trust Sanger Centre).

Array CGH (copy number analysis). Inverse correlation of point mutational burden and copy number changes?

Acknowledgments

$: NSF

Kyle HalliwillMinh ToDavid QuigleyReyno Del RosarioErik Fredlund

ALLAN BALMAIN

DAVID ADAMS (WELLCOME TRUST SANGER CENTRE)

$: NIH Training Grant T32 GM007175

$: MMHCC

Supplemental (Kyle’s Pipeline)

• Capture using Agilent mouse whole exome kit

• Sequenced on illumina HiSeq

– Paired end, 75 bp each, average read span of 180 bp

• Converted back to FASTQ, then followed QC pipeline (next slide)

Supplemental (Kyle’s Pipeline)

Align to Mm10 with BWAAlign to Mm10 with BWA

Mark duplicates and fix mate information with picard

Mark duplicates and fix mate information with picard

Base recalibration and realignment with GATK

Base recalibration and realignment with GATK

Alignment and coverage information with picard

Alignment and coverage information with picard

Variant calling with MuTectVariant calling with MuTect

Filter for depth and previously observed variants with vcftools

Filter for depth and previously observed variants with vcftools

QC and Variant Calling Strategy

Supplemental (Kyle’s Pipeline)

Sample .bam

Sample .bam

Control1 .bamControl1 .bam

Control2 .bamControl2 .bam

IntersectVariant List1 .vcfVariant

List1 .vcf

Variant List2 .vcfVariant

List2 .vcf

Variant Calling via MuTect

Candidate Variant List

.vcf

Candidate Variant List

.vcf

Candidate Variants

Candidate Variants

Filter, Annotate

Variant Calling Details