t he h ierarchy of s omatic m utations in f ollicular l ymphoma michael r. green, andrew gentles,...
TRANSCRIPT
THE HIERARCHY OF SOMATIC MUTATIONS
IN FOLLICULAR LYMPHOMA
Michael R. Green, Andrew Gentles, Ramesh Nair, Jonathan Irish, Ron Levy, Ash Alizadeh.
Follicular Lymphoma (FL)
B Cells(follicular structures)
T Cells(infiltrating tumor)
LymphomaB cell receptor
Ig light chain (k)
BCL2
Tumor-infiltrating T cells (CD3)
LymphomaB cell receptor
Ig light chain (k)
3X
Follicular lymphoma histologyblack stain = T Cells (CD3)
10X
CD20
FL flow cytometry
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
DLBCLFLMZLPTCLCLL/SLLMCLPMBL
A. Adapted from WHO 2008
B. Solal-Celigny et al. Blood 2004;104:1258
Genetic “constants”
Histone Modification by MLL2 and CREBBP
K4K27
H3H4
ING
MLL
MLL2/3INACTIVATING MUTATION
HAT
CREBBP/EP300INACTIVATING MUTATION
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”
Mutation 1
Mutation 2
Mutation 3
Peter C. Nowell (1976)Science.194(4260):23-8.
DRUG
Mutation 1
Mutation 2
Mutation 3
Catalogue of Mutations
RELAPSE
The Reality of “Personalized Oncology”
Mutation 1
Mutation 2
Mutation 3
DRUGMutation 1
Mutation 2
Mutation 3
Catalogue of Mutations
RELAPSE
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
Sanger Validation
t(14;18) qPCR Tumor Purity Measurement
Whole Exome Sequencing
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
CREBBP
MUC4MLL2
CEP112
NBPF14
AUTS2
BAZ2B
BCL2
BRWD3
C4orf49
CALR
CCAR1CTS
S
DIRAS3
ENTP
D4FA
T2 GNE
KIR2DL3
MATN2
MUC16NEB
OR2M3PEX
14
POTEG
ROS1
SLC9A6
TNFR
SF14
USP6
0.0%
25.0%
50.0%
75.0%
100.0%
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
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
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
CREBBP◦ Equally represented in tumor cell subpopulations◦ Maintained between diagnosis and relapse
Subclonal=
Late event
Clonally dominant=
Early event