in partnership with
Intra-clonal heterogeneity is a critical early
event in the preclinical stages of
multiple myeloma
Lorenzo Melchor
Division of Molecular Pathology
The Institute of Cancer Research (ICR)
Sutton, UK
UKMF Spring Day
13th March 2013
The evolution of plasma cell disorders
• Different clinical stages.
• Different initiating carcinogenic events followed by secondary abnormalities.
• Evolution from asymptomatic to symptomatic stages.
• Different locations or tumour environments.
Walker B, Wardell CP, Melchor L et al., Submitted
2
3
Intra-tumour heterogeneity
Homogeneous Tumours
No Intra-Tumour
Heterogeneity
Plasma Cells in Multiple Myeloma
Courtesy of Dr Tacchetti
Heterogeneous Tumours
Intra-Tumour
Heterogeneity
Breast Cancer - H&E staining
Breast Cancer - ER staining
4
Darwinian tumour evolution
• 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
Extracted from Yates & Campbell, Nature Reviews Genetics 2012
Greaves & Malley, Nature, 2012
5
Project aims
1.
To study the genetic make-up of the stages of MM
Number and type of mutations on each clinical stage including MGUS, highrisk (HR) SMM, MM, & PCL.
2.
To investigate the genetic relationship of the transition from HR-SMM
to MM
Analyse 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
6
Workflow
Patient
Bone
marrow
aspiration
Patient cohort
- 4 MGUS
CD138+
MACS
separation
Purity > 95%
plasma cells
Plasma
cell DNA
isolation
Whole Exome/
Genome
Sequencing
(WES/WGS*)
GRCh37 alignment
Stampy/BWA
GATK
R packages
Whole Exome Sequencing
as in
Walker et al., Blood 2012
- 4 HR-SMM*
- 22 + 4* MM
- 2 PCL
*Whole Genome Sequencing
~100 ng DNA
120 bp paired-end reads on a
GAIIx (Illumina)
Median depth of 44x
99% of the genome covered at
> 1x and 96% >20x coverage
7
Project aims
1.
To study the genetic make-up of the stages of MM
Number and type of mutations on each clinical stage including MGUS, highrisk (HR) SMM, MM, & PCL.
2.
To investigate the genetic relationship of the transition from HR-SMM
to MM
Analyse 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
Patient cohort
- 4 MGUS
- 4 HR-SMM*
- 22 + 4* MM
- 2 PCL
Walker B, Wardell CP, Melchor L et al., Submitted
Median number of non-synonymous variants (NS-SNVs) increases with disease
progression from MGUS to PCL
8
Intra-clonal heterogeneity
Walker B, Wardell CP, Melchor L et al., Submitted
Clonal heterogeneity is present in all disease states
9
10
Project aims
1.
To study the genetic make-up of the stages of MM
Number and type of mutations on each clinical stage including MGUS, highrisk (HR) SMM, MM, & PCL.
2.
To investigate the genetic relationship of the transition from HR-SMM
to MM
Analyse 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
A
B
C
Walker B, Wardell CP, Melchor L et al., Submitted
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
11
Acquired changes in the progression 1
• 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
12
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.
5. 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.
15
Acknowledgements
ICR Sutton
Illumina
Gareth Morgan
Brian Walker
Chris Wardell
Faith Davies
Annamaria Brioli
Martin Kaiser
David Johnson
Fabio Mirabella
David González
Sean Humphray
Lisa Murray
Mark Ross
David Bentley
ICR Chelsea
Iwanka Kozarewa
Chris Lord
Alan Ashworth
University Hospital of
Salamanca
Lucia López-Corral
Norma C Gutiérrez
Ramón García-Sanz
Jesús San Miguel
Funding
16
Next generation sequencing 1
Patient
Sonicate DNA
Aspirate
CD138
selection
Exome
Capture
Plasma cells
Ligate
adaptors
Next generation sequencing 2
17
Next generation sequencing 3
•
•
•
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
18
19
Next generation sequencing 4
Forward read
75 bp
200 bp (unsequenced)
75 bp
Reverse read
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