6.0

7

Primary)Tumour)

Pre?neoplas'c)lesion)

Metasta'c)Deposits)

Metasta'c)Deposits)

e.g.)K?Ras)p.G12D,)p53)p.R273H,)p16)(fs))

Targeted)therapy)

Targeted)therapy)

Circula'ng)Tumour))Cells)

6.0

8

Prokyma(

Ion Torrent MSRT-PCR

Identify mutations for subsequent monitoring using <1K sequences with gene specific primers

Wash

Purify & Concentrate CTCs

5 CTCs outnumbered by

~ 50 billion other cells

Monitor CTC levels

And tailor patient specific therapy

CTC Capture Sample Processing

Deep Sequence Monitor

5 CTCs outnumbered by

< 1,000 other cells

Blood

Target mutation identified

Cancer cells monitored

•  Can)iden'fy)single)SNP)from)20,000)wild)type)sequences.)))

•  BUT)need)to)avoid)PCR)error)being)“read”)as)a)muta'on.)

Ion)Torrent)

6.0

9

ATTAGCCTACTCTATATCTCTGGGCTCGATYCGATCCTAGCTCGATCGATATC)

ATTAGCCTACTCTATATCTCTGGGTTCGATYCGATCCTAGCTCGATCGATATC)

Limi'ng)Dilu'on)Next)Genera'on)Sequencing)

Limi'ng)Dilu'on)Next)Genera'on)Sequencing)

6.0

10

Limi'ng)Dilu'on)Next)Genera'on)Sequencing)

3)sequences)with)10%)muta'on)

10)sequences)with)<)1%)PCR)error)

6.0

11

" Prokyma((i4i)(

Acknowledgements(" Pancreas(Research(Group(

" University(of(Heidelberg("   Andrea(Bauer(("   Jörg(D.(Hoheisel("   Markus(Büchler(

"   Eithne(Costello("   John(Neoptolemos("   Paula(Ghaneh("   Robert(SuMon("   Chris(Halloran("   Dan(Palmer("   Victoria(Shaw("   Karen(Aughton("   Li(Yan("   Lucy(Oldfield("   Claire(Jenkinson("   Vicki(EllioM(

" Cold(Spring(Harbor(Laboratories("   David(Tuveson(

"   Brian(Lane("   KaTe(Bullock("   Liz(Garner(("   Sara(Harrison("   Nils(Elander("   James(Nicholson(("   Fran(Atherton("   Thomas(Hanna("   Robert(Ferguson("   Wafa(AbuXAlainin("   Kulbir(Mann("   Sumit(Nandi(

The(paTents(and(volunteers(

" University(College(London("   John(F.(Timms("   Stephane(Camuzeaux("   Usha(Menon("   Stephen(Pereira((

"   Damian(Bond("   John(Hunt("   Chris(Stanley("   Nick(Bryan("   Tom(Hanna(