Identification of Novel Molecular Markers for the Follow-up of Minimal Residual Disease in Hematooncological Disorders

Abstract 4905

Modern molecular diagnostic techniques allow the detection of minimal residual disease (MRD) in hematological malignancies with high sensitivity, allowing patient-specific assays of MRD levels over the course of treatment. One challenge for these techniques is finding appropriate MRD marker targets. For instance, PCR-based MRD analysis requires unique sequence-specific information for the malignant clone of interest. In many acute leukemias, such detailed information is lacking, either because no abnormality is detected (e.g. in 40–50% of adult Acute myeloid leukemia-AML), or the resolution of cytogenetic methods is too low to precisely define newly-found abnormalities. We have used multicolor-FISH (mFISH) to perform karyotyping of 141 patients. Using this approach we have identified chromosomal abnormalities in about 35% of analyzed cases. The aberrations found involved the recurrent aberrations t(15;17), t(8;21), inv(16), t(9;22),−5q, −7, +8 but also an array of unique abnormalities, such as der(15)t(5;15)(q15;q24), t(6;12)(p22;q13), and der(17)t(12;17)(q14;pter), which may serve as possible targets for molecular MRD follow-up.

To further characterize such targets, we seek to bridge the gap from cytogenetic resolution down to higher-resolution molecular techniques. For this, we first employ the mBAND technique, followed by multicolor FISH using DNA probes with known cytogenetic location. These probes are drawn from the Human Minimal Genomic Clone Set (Version 1.0, Source Bioscience LifeSciences) of over 25,000 bacterial artificial chromosome (BAC) clones with human DNA inserts. These are tiling clones covering almost 100% of the genome, and therefore allow us to develop FISH probes to regions containing almost any conceivable target breakpoint. Starting with breakpoint information from mFISH/mBAND, we perform consecutive multi-color/multi-BAC hybridizations around the region of interest, looking for breakpoint-spanning probes. Using this system, we are able to move from a resolution of around 2 Mbp (the limit of mBAND) to the resolution of individual BAC clones (average 150 kbp) in three hybridizations.

To achieve further resolution, long-range PCR products are designed within the region defined by breakpoint-spanning BACs, with labeling and FISH-mapping of these probes. At present, we are starting to perform these hybridizations on stretched DNA (fiber-FISH or combed DNA), where one micrometer of a stretched DNA molecule represents about 2000 base pairs, allowing visualization of probe distance and spacing, and even higher-resolution breakpoint analysis. Chromosome microdissection followed by high-throughput sequencing (Roche GS Junior) is also being pursued to identify novel breakpoints by rapidly sequencing large chromosomal regions on both sides of a translocation. The final goal is to map genomic abnormalities to a resolution amenable to long-range PCR, yielding specific targets for MRD detection, and allowing clone-specific Real-Time PCR assays for sensitive and specific monitoring of MRD in hematooncological patients.