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

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”

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%

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

CREBBP◦ Equally represented in tumor cell subpopulations◦ Maintained between diagnosis and relapse

Subclonal=

Late event

Clonally dominant=

Early event

Conclusions

Acknowledgments

Prof. Ron LevyProf. Jonathan IrishDr. June MyklebustDr. Itai Kela

Prof. Ash AlizadehShingo KihiraDr. Chih Long Liu

Prof. Sylvia PlevritisDr. Andrew GentlesDr. Ramesh Nair

Prof. Hanlee JiDr. Eric Hopmans