Leukaemia represents a group of heterogeneous diseases that are conventionally sub-classified according to morphological, histochemical, immunological, and cytogenetic criteria. Gene expression profiling has been increasingly applied to paediatric and adult leukaemia and will provide important information concerning prognosis, therapy related toxicity, and the likelihood to develop secondary malignancies. Furthermore, microarray data may be valuable for the design of risk-adapted individual therapeutic strategies.
In clinical practice, bone marrow (BM) is the most important tissue type for diagnosis and follow-up monitoring. In western countries, leukaemia patients are almost always enrolled in large multi-centre studies. These require collection of BM samples at the participating clinical centres and transport to central diagnostic laboratories, which perform specialized analyses. Shipping of BM samples does not seem to hamper DNA- and protein-based diagnostics. In contrast, extreme care needs to be taken to preserve gene response patterns in clinical samples.
In our previous work (NGFN1), we have investigated the impact of time delays between sample aspiration and RNA extraction on gene expression profiles (see 1). We have shown that even short-term storage of BM has dramatic effects on mRNA expression of individual transcripts. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses and may mask information in gene response patterns that allow precise tumour classification and the assessment of molecular risk factors for therapy resistance and relapse. Therefore, for diagnostic purposes, reliable standard operating procedures (SOPs) were defined for the proper storage, shipping and pre-analytical handling of BM aspirates, as well as for the extraction of intact RNA.
Our investigations now focus on the detailed analysis of paediatric T-lineage acute lymphoblastic leukaemia (T-ALL). T-ALLs comprise approximately 12 % of all paediatric leukaemias and are characterised by a 5 year event-free survival rate of approximately 60 %. In contrast to the more common B-cell ALL, outcome of patients with relapse is poor, showing an average 20% survival rate. Rare cases of T-lymphoblastic leukaemia (T-ALL) (< 1 %) harbour chromosomal translocations t(7;9) that involve NOTCH1, a gene encoding a single-pass, heterodimeric transmembrane receptor. NOTCH1 is essential for the commitment of pluripotent progenitors to the T-cell fate and for the subsequent assembly of pre T-cell receptor complexes in immature thymocytes (see 2). The NOTCH1 heterodimer consists of non-covalently associated extracellular (NEC) and transmembrane (NTM) subunits. A heterodimerization domain (HD) is responsible for stable subunit association. The HD comprises a 103 amino acid region of the NEC (HD-N) and a 65 amino acid region of the NTM (HD-C) domains. The NEC subunit contains 36 iterated EGF-like repeats that bind DSL (Delta-Serrate-Lag1) ligands, 3 Notch/Lin-12 repeats and HD-N. The NTM subunit contains HD-C, a RAM (RBP-J# (recombination-signal-sequence-binding protein for J# genes) associated molecule) domain, seven ankyrin repeats, a proline-rich and glutamine-rich transcriptional activation domain (TAD), and a PEST (rich in the amino acids proline (P), glutamic acid (E), serine (S), and threonine (T)) domain, where the intracellular receptor fragment is proteolytically inactivated (see 3).
Recently, Weng et al. demonstrated activating NOTCH1 mutations in more than 50 % of paediatric T-ALLs (see 4). Those mutations involve the heterodimerization domain, the C-terminal PEST domain, or both, which result in aberrant up-regulation of NOTCH1-dependent signal transduction. Mutations of HD-N and HD-C enhance production of the active intracellular NOTCH1 (ICN1) by destabilizing the non-covalent intersubunit association. Mutations of the PEST domain increase NOTCH1 signalling by enhancing the stability of ICN1.
Project Status
We systematically analysed DNA samples for the impact of activating NOTCH1 mutations on treatment response and outcome in 157 T-ALLs of the ongoing paediatric ALL-BFM 2000 study. Our own data, generated with an independent study population, confirm the already published observation: more than half of the T-ALLs analysed carry mutations in the NOTCH1 gene. In 82 patients (52.2%), activating NOTCH1 mutations were identified either in the heterodimerization (n = 55), in the PEST (n = 13), or in both domains (n = 14), respectively. We extended previous observations by comparing the mutation status and treatment response and outcome, respectively. The results reveal a surprising positive correlation of activating NOTCH1 mutations and a favourable course of disease. The presence of NOTCH1 mutations was significantly correlated with a good prednisone response (P = 0.001) and favourable kinetics of minimal residual disease (MRD) (P = 0.001) during the course of therapy, which was independent from gender, age and white blood cell count at the time of diagnosis. Considering the favourable effect of NOTCH1 mutations on response to treatment it was interesting to analyse the effect on long term prognosis. Patients with NOTCH1 mutations showed a significantly better relapse-free survival compared to those without mutations. These findings indicate that NOTCH1 mutated T-ALLs are more sensitive to the ALL-BFM 2000 treatment strategy than T-ALLs without NOTCH1 mutations.
Outlook
Based upon our quality criteria and SOPs studies are in progress to analyse global gene expression of paediatric T-ALLs with and without mutated NOTCH1. The mRNA gene expression data will be correlated with clinical parameters, in particular with the kinetics of minimal residual disease (MRD) during the course of therapy. It is expected that a correlation of mRNA gene expression with the kinetics of MRD will yield insight into tumour biology and treatment response. Particular focus will be set on the definition of indicator genes discriminating T-ALLs with and without mutated NOTCH1, to assess therapy responses, and to identify potential drug targets for improved treatment strategies for paediatric T-ALLs,. We further aim to identify additional target genes of the NOTCH1 signalling pathway.
Lit.: 1. Breit S et al. Impact of pre-analytical handling on bone marrow mRNA gene expression. Br J Haematol. 2004 Jul; 126(2):231-43. 2. Radtke F et al. Notch signaling in T- and B-cell development. Curr Opin Immunol 2004;16(2):174-9. 3. Pear WS and Aster JC. T cell acute lymphoblastic leukemia/lymphoma: a human cancer commonly associated with aberrant NOTCH1 signaling. Curr Opin Hematol 2004;11(6):426-33. 4. Weng AP et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 2004;306(5694):269-71.
