Download Catalogue of Mutations

THE HIERARCHY OF
SOMATIC MUTATIONS
IN FOLLICULAR LYMPHOMA
Michael R. Green, Andrew Gentles, Ramesh Nair, Jonathan Irish,
Ron Levy, Ash Alizadeh.
Follicular Lymphoma (FL)
Follicular lymphoma histology
FL flow cytometry
black stain = T Cells (CD3)
3X
B Cells
(follicular structures)
Lymphoma
B cell receptor
Ig light chain (k)
T Cells
(infiltrating tumor)
Tumor-infiltrating T cells (CD3)
10X
Lymphoma
B cell receptor
Ig light chain (k)
BCL2
CD20
Follicular Lymphoma (FL)

Clonally rearranged immunoglobulin

Characterized by t(14;18)(q32;q21) translocation

Incurable using conventional therapy
◦ Good candidate for molecularly-targeted therapies
 Frequent mutation of MLL2 histone methyltransferase
 Recurrent mutation of CREBBP histone acetyltransferase
DLBCL
FL
MZL
PTCL
CLL/SLL
MCL
PMBL
A.
Adapted from WHO 2008
B.
Solal-Celigny et al. Blood 2004;104:1258
Genetic “constants”
Histone Modification by MLL2 and CREBBP
CREBBP/EP300
MLL2/3
INACTIVATING MUTATION
INACTIVATING MUTATION
HAT
MLL
ING
K27
K4
Targeted Therapy: Hitting the Achilles Heel of Cancer
7 July 2012
8 July 2012
9 July 2012
The Theory of “Personalized Oncology”
MacConaill and Garraway J. Clin. Oncol. 2010;28:5219
Roychowdhury et al. Sci. Transl. Med. 2011;3:111
The Reality of “Personalized Oncology”
Peter C. Nowell (1976)
Science.194(4260):23-8.
Mutation 1
Mutation 2
Mutation 3
Catalogue of Mutations
Mutation 1
Mutation 2
Mutation 3
RELAPSE
DRUG
The Reality of “Personalized Oncology”
Mutation 1
Mutation 2
Mutation 3
Catalogue of Mutations
Mutation 1
Mutation 2
RELAPSE
Mutation 3
DRUG
Premise, Aim and Approach

Premise: Early genetic events are likely to be clonally
dominant and represent good targets for mutation-directed
therapy

Aim: To identify the hierarchy of genetic events in FL

Approach: Identify clonally dominant mutations
◦ Consistently represented between intratumoral subpopulations
◦ Maintained from diagnosis to relapse
Experimental approach
FACS
T-cells CD20int CD20hi
DNA Extraction
Whole Exome
Sequencing
Sanger Validation
t(14;18) qPCR Tumor
Purity Measurement
IgHV cloning/sequencing
Genetic “Constants”
Exome Sequencing Methodology




Constructed libraries from 3ug of DNA
Captured exome with with Nimblegen SeqCap (v2)
Indexed with Illumina barcodes
4-plexed samples on a single HiSeq 2000 lane (2x101bp)
Mutation Calling
Called somatic nucleotide variants (SNVs) with stringent
implementation GATK:
◦ GATK score of ≥250 in B-cells
◦ GATK score of <50 in T-cells
 Filtered silent mutations and those in dbSNP/1000genomes
 Only considered cSNVs with:
◦ ≥20X coverage in both T-cells and B-cells
◦ <5% variant allele frequency (VAF) in T-cells
◦ ≥5% VAF in B-cells

 96% validation rate
Exome Sequencing and Mutation Detection

In 10 tumors from 8 cases, identified 877 coding SNVs in 572
unique genes
◦ 95% of genes mutated in only 1/8 cases
100.0%
75.0%
50.0%
25.0%
0.0%
Majority of Mutations in FL are Subclonal
Assessing sub-population skew

Interrogated minor allele frequencies of 232
germ-line coding SNPs/patient (1856 total)

Some noise around VAFs of heterozygous SNPs
◦ By definition, variation in germline SNPs are
false-positives*
◦ Set thresholds to obtain confident calls

At 16% VAF deviation, 85 false-positives
◦ 4.58% error
At 33% VAF deviation, 18 false-positives
◦ 0.97% error


*Possibility of LOH  over-estimating error
Mutation Frequencies in Tumor Subpopulations
More mutations, more clonal divergence
Illustrative Case of Diagnosis/Relapse Comparison
CASE 128





A 40 year old woman with enlarged lymph nodes and fevers found to have advanced follicular
lymphoma
Diagnosis (1996)
◦ Histology: FL grade 1
◦ Stage: 4B
◦ Time to first treatment = 362 days
◦ First treatment = CVP (1997) achieved Complete Remission (CR)
◦ Second treatment = Id-vac (1998)
Relapse (1999)
◦ Histology: FL grade 1
◦ Treatment: Fludarabine + Cyclophosphamide, CR
Second relapse in 2003, treated
Patient alive as of Feb 2013
Interrogation of FL Relapses: Case 128
Genetic Evolution of Case 128
Conclusions

The majority of mutations in FL are not recurrent and are subclonal.

MLL2 and TNFRSF14
◦ Skewed distribution in tumor cell subpopulations
◦ Lost between diagnosis and relapse in LP-J128

Subclonal
=
Late event
CREBBP
◦ Equally represented in tumor cell subpopulations
◦ Maintained between diagnosis and relapse
Clonally dominant
=
Early event
Conclusions
Acknowledgments
Prof. Ron Levy
Prof. Ash Alizadeh
Prof. Jonathan Irish
Dr. June Myklebust
Dr. Itai Kela
Shingo Kihira
Dr. Chih Long Liu
Prof. Sylvia Plevritis
Dr. Andrew Gentles
Dr. Ramesh Nair
Prof. Hanlee Ji
Dr. Eric Hopmans