Division of Molecular Pathology The Institute of Cancer Research (ICR) Sutton, UK

1. Intra-clonal heterogeneity is a critical early event in the preclinical stages of multiple myelomaLorenzo Melchor Division of Molecular Pathology The Institute of Cancer Research (ICR) Sutton, UK • UKMF Spring Day 13th March 2013

2. 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

3. Intra-tumour heterogeneity Breast Cancer - H&E staining Breast Cancer - ER staining Homogeneous Tumours No Intra-Tumour Heterogeneity Heterogeneous Tumours Intra-Tumour Heterogeneity Plasma Cells in Multiple Myeloma Courtesy of Dr Tacchetti

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, high-risk (HR) SMM, MM, & PCL. • 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 cohort • 4 MGUS • 4 HR-SMM* • 22 + 4* MM • 2 PCL Purity > 95% plasma cells Whole Exome Sequencing as in Walker et al., Blood 2012 *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 Whole Exome/ Genome Sequencing (WES/WGS*) Patient Bone marrow aspiration CD138+ MACS separation Plasma cell DNA isolation GRCh37 alignment Stampy/BWA GATK R packages

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, high-risk (HR) SMM, MM, & PCL. • 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

8. 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

9. Intra-clonal heterogeneity Walker B, Wardell CP, Melchor L et al., Submitted Clonal heterogeneity is present in all disease states

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, high-risk (HR) SMM, MM, & PCL. • 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

11. A A B B C C Paired patient samples & treatment effect Walker B, Wardell CP, Melchor L et al., Submitted Walker B, Wardell CP, Melchor L et al., Submitted Changes in sub-clonal composition over time (from HR-SMM to MM) Chemotherapy in a HR-SMM patient who evolves to MM results in a reduction in clonal complexity

12. 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

13. Acquired changes in the progression 2 • No significant copy number aberrations (by FISH or WGS) between both HR-SMM and MM samples. • Chromosomal rearrangements or translocations: • Common, unique to HR-SMM or unique to MM. • t(13;21) disrupting BRCA2 in patient 2. • Several complex translocations involving UNC5D in patient 5. Walker B, Wardell CP, Melchor L et al., Submitted

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

15. Acknowledgements ICR Sutton Gareth Morgan Brian Walker Chris Wardell Faith Davies Annamaria Brioli Martin Kaiser David Johnson Fabio Mirabella David González Illumina Sean Humphray Lisa Murray Mark Ross David Bentley University Hospital of Salamanca Lucia López-Corral Norma C Gutiérrez Ramón García-Sanz Jesús San Miguel Funding ICR Chelsea IwankaKozarewa Chris Lord Alan Ashworth

16. Next generation sequencing 1 CD138 selection Patient Aspirate Plasma cells Exome Capture Ligate adaptors Sonicate DNA

17. Next generation sequencing 2

18. 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

19. Next generation sequencing 4 Forward read 200 bp (unsequenced) 75 bp 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