Identification and functional characterisation of molecular risk factors in acute leukemias

Renate Kirschner1, Michaela Heide1, Peter Rhein1, Leonid Karawajew1, Matthias Nees2, Stephen Breit2, Andreas Kulozik2, Christian Hagemeier1, Wolf-Dieter Ludwig1, Rainer Spang3, Karl Seeger1

1 Charité, Humboldt-Universität Berlin, 2 Universitäts-Kinderklinik Heidelberg, 3 Max-Planck-Institut für molekulare Genetik Berlin,

Initial ALL(ALL-BFM trial)

Relapsed ALL(ALL-REZ BFM trial)

Retrospective Study:

Clinical Outcome Event-free Survival ( 3 years)

1. Relapse Event-free Survival ( 3 years)

2. Relapse Non-Response

Classification

Prospective Study:

Therapy ResponsePrednisone Response1

Minimal Residual Disease (MRD)2

Minimal Residual Disease (MRD)3

Non-Response

Good Poor PositiveNegative PositiveNegative

1 Dördelmann et al. 1999. Blood 94: 1209-1217; 2 van Dongen et al. 1989. Lancet 352: 1731-17383 Eckert et al. 2001. Lancet 358: 1239-1241

Leukemic Cell Sample

1. Pre-enrichment by magnetic cell sorting (MACS™, Miltenyi Biotec)

2. Further isolation by flow sorting(FACS Vantage™, Becton Dickinson)

RNA-Extraction

Linear amplification of cRNAby in vitro transcription

Real time PCR(Candidate Genes)

Gene expression profiling

Isolation of Minor Subpopulations from Heterogeneous Leukemic Samples

Optimising Gene Expression Profiling

• Evaluation and cross-validation of generated data sets with published ALL expression profiles

• RNA Preparation

In order to perform retrospective studies, we isolated RNA from cryopreserved mononuclear cells

(magenta). In a significant number of samples loss of sufficient RNA quality and quantity was observed.

RNA quality was also evaluated on a Bioanalyzer™ and via hybridisation of Affymetrix Test3Arrays™

showing a high degree of consistency. For prospective studies we therefore routinely prepare RNA directly

from incoming bone marrow biopsies with an optimized yet straight forward protocol (light blue). This

should also minimize changes of expression profiles due to cryopreservation. Retrospective studies can only

be performed on a limited number of samples (<25%).

Bone marrow biopsiessent from clinical centers

Lab of the study group:

Isolation of mononuclear cells

RNA Preparation

0-2 days

RNA Preparation

Cryopreservation

18 S

28 S

18 S

28 S

Childhood acute lymphoblastic leukemia (ALL) occurs with an incidence of approximately 600 patients per year in Germany. In general, up to

75% of children can be cured permanently by chemotherapy. ALL relapses (approximately 100 cases per year) are more resistant to treatment with

a cure rate of less than 50%. Therefore, novel approaches in terms of diagnosis and therapy are particularly needful for this group. In order to

identify novel prognostic factors and to unravel molecular mechanisms underlying clinical outcome, we aim to generate gene expression profiles of

initial and relapsed ALL of the Berlin-Frankfurt Münster (ALL-BFM and ALL-REZ BFM) study group by Affymetrix DNA microarray

technology.

In the second part of the project, we focus on distinct, clinically relevant

subpopulations from initially heterogeneous leukemic cell samples. We are

especially interested on minor subpopulations of immature, progenitor-like

leukemic cells as well as on residual leukemic cell populations which have

escaped initial treatment and become more resistant to therapy. In order to

approach this issue experimentally, procedures for identification and

purification of rare leukemic blast cells based on flow cytometric analysis and

flow sorting are under development.

Bone marrow samples for gene

expression profiling are selected from

patients who have been treated

according to the protocols of the

ALL-BFM and ALL-REZ BFM study

groups for intial and relapsed acute

lymphoblastic leukemia, respectively.

For a retrospective study patients are

classified by clinical outcome,

whereas for a prognostic study proven

risk factors as for example ‘minimal

residual disease’ - MRD will be used

to divide patients into subgroups.

MRD sensitively measures the

amount of leukemic cells that are still

present at certain time points during

therapy.

Randomly selected, high quality RNA

cRNA

U95Av2 GeneChip™

U133A GeneChip™

norm

aizi

ng

prof

iles.

Published data sets*

eval

uatio

n

Own data sets

facilitates and improves

Databanks were created containing published genes found to be deregulated in large scale ALL profiling

studies based on Affymetrix U95Av2 GeneChips™ (eg *Yeoh et al. 2002. Cancer Cell 1: 133-143).

Ongoing co-hybridisation of 5 to 10 Probes to U95Av2 and U133A GeneChips™ will allow normalization

of expression profiles for comparing data sets created with “old” and “new” Affymetrix GeneChips.

Databanks and bioinformatic filtering tools can then be used to identify and select against unwanted

signatures in smaller test populations that would otherwise escape recognition. This part therefore aims at

both, utilizing and cross-validating existing data from different laboratories.