Does monitoring of chimerism

predict relapse and allow

pre-emptive immunotherapy?

David Grimwade

Dept. of Medical & Molecular Genetics,

King’s College London School of Medicine, UK

NO!

Investigation of chimerism to predict relapse

post-transplant & guide immunotherapy in AML

Huisman et al. Bone Marrow Transplant 2007; 39: 285-91. Rettinger et al. Blood 2011; 118: 5681-8

CC – Complete Chimerism; MC – Mixed Chimerism;

d-MC – decreasing MC; i-MC – increasing MC

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Schmid et al. J Clin Oncol 2007; 25: 4938-35.

Disease relapse remains a major cause of failure

following allogeneic transplant, with poorer

response to DLI with higher tumour burden

Are chimerism assays fit for purpose to predict relapse?

Issues of standardisation & sensitivity

Exploring the use of over-expressed genes

to track treatment response in AML

Compared to level of expression in

normal PB & BM:

WT1 is sufficiently over-expressed

to discriminate 2-log reduction in

transcripts in 46% and 13% of AML,

according to follow-up sample source

Cilloni et al, J Clin Oncol 2009, 27: 5195-201

204 control samples

620 AML samples

Wilms Tumour gene (WT1) Control samples: n=204

AML samples: n=620

Development of standardised RT-qPCR

assays for detection of WT1 transcripts

3 assays eliminated:

•Cross-reactivity with

genomic DNA (n=1)

•Low sensitivity (n=2)

3 assays eliminated:

•Reduced efficiency & sensitivity (n=3)

Best-performing WT1

assay selected:

•Designated ELN WT1 assay

Phase IPhase I

9 WT1 assays evaluated:

•Cell line dilutions

•Genomic DNA

Phase 2Phase 2

6 WT1 assays evaluated:

•Plasmid standards

Phase 3Phase 3

3 WT1 assays evaluated:

•Cell line dilutions

•Plasmid standards

Phase 4Phase 4

Evaluation of ELN assay

•Normal PB/BM/PBSCs (n=204)

•Diagnostic AML samples (n=620)

•Follow-up samples from 145 pts

Cilloni et al, J Clin Oncol 2009, 27: 5195-201

Pozzi et al, Br J Haematol 2013: 160: 503-9

Investigation of optimised ELN WT1 assay to predict

outcome & guide therapy post-allogeneic transplant in AML

Association between WT1 level post-transplant & relapse Survival following DLI in patients with higher WT1 levels

BM samples analysed with ELN WT1 assay

>180 copies WT1 / 104 ABL used as definition of MRD positivity

Are chimerism assays sophisticated

enough to reliably predict disease relapse?

Development of standardised leukaemia-specific

RT-qPCR assays to track treatment response & guide therapy

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PML-RARA

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TEL-AML1

p210 CML

p210 ALL

p190 ALL

MLL-AF4

E2A-PBX1

Gabert et al, Leukemia 2003; 17: 2318

Europe Against Cancer

Sequential monitoring for leukaemia-

specific transcripts to predict relapse

NPM1 mutant AML

•246 patients

•1441 samples

Adam Ivey, Neesa Bhudia,

Mandy Gilkes, Rosemary Gale

NCRI AML17 trial

Use of MRD monitoring to direct pre-

emptive azacitidine therapy post-SCT

Sockel et al. Haematologica 2011; 96: 1568-70.

Standardised JAK2-V617F qPCR assay improves

prediction of relapse following allogeneic SCT

•JAK2-V617F detected:

•Median of 22 weeks (range 6–85 weeks) before relapse

•Median of 86 days (45-595 days) before loss of full donor chimaerism

•Early intervention with DLI associated with better outcome (Kröger et al, Blood 2009)

Jovanovic et al. Leukemia 2013.

Ley TJ & colleagues: New Engl J Med 1st May, 2013

• NGS of 200 AML cases

• (50 whole genome, 150 exome)

• Average of 13 gene mutations/AML

• 23 genes significantly mutated

• >200 genes mutated in ≥2 cases

• >1000 genes mutated overall

Potential of next generation sequencing

approaches to track treatment response

Thol et al. Genes Chromosomes Cancer 2012.

Diagnosis

Remission

Relapse

Detection of DNMT3A mutation by next generation sequencing

FLT3-ITD

NPM1 mut

DNMT3A mut

Ivey A, Simpson MA, Burnett AK & Grimwade D, unpubl.

•Relapses can occur despite full donor chimerism

•Durable remissions in patients with mixed chimerism

How reliable are chimerism assays

to predict relapse?

Time to upgrade?

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