in partnership with

UKMF Spring Day 13th March 2013

Intra-clonal heterogeneity is a critical early Intra-clonal heterogeneity is a critical early event in the preclinical stages of event in the preclinical stages of

multiple myelomamultiple myeloma

Lorenzo Melchor

Division of Molecular Pathology

The Institute of Cancer Research (ICR)

Sutton, UK

Walker B, Wardell CP, Melchor L et al., Submitted

The evolution of plasma cell disorders 2

• Different clinical stages.

• Different initiating carcinogenic events followed by secondary abnormalities.

• Evolution from asymptomatic to symptomatic stages.

• Different locations or tumour environments.

Intra-tumour heterogeneity 3

Breast Cancer - ER staining

Breast Cancer - H&E staining

Courtesy of Dr Tacchetti

Plasma Cells in Multiple Myeloma

Homogeneous Tumours

No Intra-Tumour Heterogeneity

Heterogeneous Tumours

Intra-Tumour Heterogeneity

Extracted from Yates & Campbell, Nature Reviews Genetics 2012

Darwinian tumour evolution 4

Greaves & Malley, Nature, 2012

• FISH & NGS analyses have described tumour intra-clonal heterogeneity.

• Tumours seem to follow a branching or Darwinian evolution model.

• Many human cancers, including multiple myeloma (Walker et al., Blood 2012).

• Implications in:

• Targeted therapies

• Mechanisms of treatment-resistance

Project aims 5

1. To study the genetic make-up of the stages of MMNumber and type of mutations on each clinical stage including MGUS, high-risk (HR) SMM, MM, & PCL.

2. To investigate the genetic relationship of the transition from HR-SMM to MMAnalyse the transition HR-SMM to MM in paired patient samples, to identify likely driver events and/or clonal evolution patterns.

Walker B, Wardell CP, Melchor L et al., Submitted

*Whole Genome Sequencing~100 ng DNA

120 bp paired-end reads on a GAIIx (Illumina)

Median depth of 44x99% of the genome covered at > 1x and 96% >20x coverage

Workflow 6

Patient Bone marrow

aspiration

CD138+ MACS

separation

Plasma cell DNA isolation

Whole Exome/ Genome

Sequencing (WES/WGS*)

GRCh37 alignment Stampy/BWAGATKR packages

Purity > 95% plasma cells

Whole Exome Sequencingas in

Walker et al., Blood 2012

Patient cohort

- 4 MGUS

- 4 HR-SMM*

- 22 + 4* MM

- 2 PCL

Project aims 7

1. To study the genetic make-up of the stages of MMNumber and type of mutations on each clinical stage including MGUS, high-risk (HR) SMM, MM, & PCL.

2. To investigate the genetic relationship of the transition from HR-SMM to MMAnalyse the transition HR-SMM to MM in paired patient samples, to identify likely driver events and/or clonal evolution patterns.

Walker B, Wardell CP, Melchor L et al., Submitted

Results: The genetic make-up of MM 8

Median number of non-synonymous variants (NS-SNVs) increases with disease progression from MGUS to PCL

Walker B, Wardell CP, Melchor L et al., Submitted

Patient cohort

- 4 MGUS

- 4 HR-SMM*

- 22 + 4* MM

- 2 PCL

Intra-clonal heterogeneity 9

Clonal heterogeneity is present in all disease states

Walker B, Wardell CP, Melchor L et al., Submitted

Project aims 10

1. To study the genetic make-up of the stages of MMNumber and type of mutations on each clinical stage including MGUS, high-risk (HR) SMM, MM, & PCL.

2. To investigate the genetic relationship of the transition from HR-SMM to MMAnalyse the transition HR-SMM to MM in paired patient samples, to identify likely driver events and/or clonal evolution patterns.

Walker B, Wardell CP, Melchor L et al., Submitted

Paired patient samples & treatment effect 11

Changes in sub-clonal composition over time (from HR-SMM to MM)

A B C

Walker B, Wardell CP, Melchor L et al., Submitted

Chemotherapy in a HR-SMM patient who evolves to MM results in a reduction in clonal complexity

A B C

Walker B, Wardell CP, Melchor L et al., Submitted

Acquired changes in the progression 1 12

• 19 mutations gained and 36 mutations lost per month.

• 433 mutations gained per sample in the transition. Few within coding regions, and only one NS-SNV.

• Mutation c.G92A/p.W31* in RUNX2.

Walker B, Wardell CP, Melchor L et al., Submitted

Acquired changes in the progression 2 13

• No significant copy number aberrations (by FISH or WGS) between both HR-SMM and MM samples.

• Chromosomal rearrangements or translocations: o Common, unique to HR-SMM or unique to MM.o t(13;21) disrupting BRCA2 in patient 2.o Several complex translocations involving UNC5D in patient 5.

Walker B, Wardell CP, Melchor L et al., Submitted

Conclusions 14

1. The multistep progression from a normal plasma cell to one with leukemic properties is characterised by an increasing number of NS mutations.

MGUS >> HR-SMM > MM >> PCL

2. Sub-clonal heterogeneity is shown in all stages of disease from MGUS to PCL, suggesting clonal competition and Darwinian evolution through progression.

3. The transformation of HR-SMM to MM is not the result of the outgrowth of a single clone but of a number of sub-clones already present in the HR-SMM stage. A process more complex than paediatric ALL (Anderson et al. Nature 2011).

4. What is the driving force?• Few additional coding mutations (RUNX2) or translocations (BRCA2, UNC5D…)• Epigenetic or microenvironment changes may explain the transformation.

• HR-SMM is not a distinct disease entity but is rather a transition state between MGUS and MM where the sub-clonal estructure is evolving.

University Hospital of Salamanca

Lucia López-CorralNorma C Gutiérrez

Ramón García-SanzJesús San Miguel

Acknowledgements 15

ICR Sutton

Gareth MorganBrian WalkerChris WardellFaith Davies

Annamaria BrioliMartin Kaiser

David JohnsonFabio Mirabella

David González

ICR Chelsea

Iwanka KozarewaChris Lord

Alan Ashworth

Illumina

Sean HumphrayLisa MurrayMark Ross

David Bentley

Funding

Next generation sequencing 1 16

Patient AspirateCD138

selectionPlasma cells

Exome Capture

Sonicate DNALigate

adaptors

Next generation sequencing 2 17

Next generation sequencing 3 18

• Sequence the first set of adaptors• Repeat for the second set of adaptors• Result: an equal number of Forward and Reverse short reads,

separated by a known distance

Next generation sequencing 4 19

NGS short read and assembly represents a challenging stage

• Tens of millions of short read pairs• An enormous, complex jigsaw puzzle• Two options

A) De novo assembly

B) Alignment to a reference genome

200 bp (unsequenced)75 bp 75 bp

Forward read

Reverse read