in partnership with UKMF Spring Day13 th March 2013 Intra-clonal heterogeneity is a critical early event in the preclinical stages of multiple myeloma 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

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 MM Number 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 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

*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 Workflow 6 PatientBone marrow aspiration CD138+ MACS separation Plasma cell DNA isolation Whole Exome/ Genome Sequencing (WES/WGS*) GRCh37 alignment Stampy/BWA GATK R packages Purity > 95% plasma cells Whole Exome Sequencing as in Walker et al., Blood 2012 Patient cohort - 4 MGUS - 4 HR-SMM* * MM - 2 PCL

Project aims 7 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. 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 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* * 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 MM Number 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 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 11 Changes in sub-clonal composition over time (from HR-SMM to MM) AB 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 AB C Walker B, Wardell CP, Melchor L et al., Submitted

Acquired changes in the progression 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. 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.

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

Next generation sequencing 1 16 PatientAspirate CD138 selection Plasma cells Exome Capture Sonicate DNA Ligate 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 Forward read Reverse read