Detection of minimal residual disease in acute lymphoblastic leukemia

textabstractAcute lymphoblastic leukemia (ALL) represents the most frequent malignancy in childhood. Last decades brought enormous progress in ALL treatment and in the understanding of ALL biology (see Chapter 1.1 ), but still 20 to 30% of children suffer from relapse and many of them will ultimately die of disease progression. The currently used cytomorphological (microscopic) techniques can only detect 1 to 5% of malignant cells, which is not sufficiently sensitive for identification of patients who are prone to relapse and who might be rescued by treatment intensification. During the past 15 years several approaches have been developed for detection of much lower numbers of malignant cells, i.e. for detection of minimal residual disease (MRD) in various hematopoietic malignancies (see Chapter 1.2). Monitoring of MRD with sensitivities of 1 Q-4 to 1 o-6 (i.e. one malignant cell within the background of 104 to 106 normal cells) has significantly higher prognostic value than conventional cytomorphological techniques and other clinical parameters at diagnosis and is therefore currently implemented into clinical practice in several hematopoietic malignancies, including ALL. In childhood ALL, detection of MRD most frequently relies on patient-specific immunoglobulin (lg) and T-cell receptor (TCR) gene rearrangements as molecular markers for PCR studies. The junctional regions of rearranged lg and TCR genes are unique "fingerprint-like" sequences, which are assumed to be different in each lymphoid cell and thus also in each lymphoid malignancy. They can be easily identified and characterized for instance by using heteroduplex PCR analysis (see Chapter 2.2) and direct sequencing. This thesis aimed at detailed evaluation of lg and TCR gene rearrangements in ALL with regard to the following aspects: -characterization of lg/TCR gene rearrangements patterns in precursor-BALL and T-ALL; - immunobiological differences between malignant and normal lymphoid cells; -stability of clonal lg/TCR gene rearrangements at relapse of ALL; -applicability of lg/TCR gene rearrangements as PCR targets for detection of MRD. Virtually all precursor-B-ALL (96%) have rearranged lg heavy chain (/GH) genes. In most cases (80-90%) this concerns complete VH-DH-JH rearrangements on at least one allele. Incomplete DH-JH rearrangements could be identified in 22% of patients, being the sole /GH gene rearrangements in only 5% of patients (see Chapter 2.3). Most precursor-B-ALL contain lg kappa (/GK) light chain gene rearrangements (30%) or deletions (50%); 20% of precursor-B-ALL cases even have lg lambda (IGL) gene rearrangements. Deletions in the IGK genes are predominantly mediated via the IGK deleting element (Kde) sequence. Such Kde rearrangements occur in 50% of precursor-B-ALL case