Grimwade, David James; (1998) The molecular biology of acute promyelocytic leukaemia and its response to therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Acute promyelocytic leukaemia (APL) is characterised by the t(15;17) leading the formation of PML-RARα and RARα-PML fusion genes. This rearrangement predicts a favourable differentiation response to all-transretinoic acid (ATRA), which confers improved survival when combined with chemotherapy. Therefore, establishing the presence of the t(15;17) at the molecular or cytogenetic level is critical for optimal management of patients with this disease. This thesis evaluated RT-PCR and PML-immunofluorescence techniques as a means of identifying underlying PML/RARα rearrangements in cases of suspected APL entered into the MRC ATRA trial. These approaches revealed that the classic t(15;17) is not identified cytogenetically in 15% cases with PML/RARα rearrangements. Further characterisation of such cases showed that PML-RARα was the sole fusion gene formed as a result of cytogenetically cryptic rearrangements, thereby supporting its product as the key oncogenic fusion protein. Immunofluorescence studies demonstrated a close correlation between expression of PML-RARα and delocalisation of PML from nuclear body structures; whereas a normal PML staining pattern was found in APL with a PLZF/RARα rearrangement. This showed that delocalisation of PML from nuclear bodies is not a prerequisite for the pathogenesis of APL, or indeed a final common pathway to leukaemogenesis in cases with translocations involving fusion partners other than PML. In addition, nested RT-PCR was also used to define targets for minimal residual disease monitoring, which was shown to provide an independent prognostic variable. Another aspect of this study was the investigation of mechanisms underlying expression of the T cell marker CD2 in APL. DNase I hypersensitivity assays established an identical pattern of hypersensitive sites in the region flanking the CD2 locus in APL samples and myeloid cell lines irrespective of CD2 status, to that detected in T cells. This suggests that regulatory regions of CD2 may be accessible to DNase I during normal myelopoiesis, and that the chromatin configuration and the CD2 expression levels associated with APL blasts could reflect the nature of the haemopoietic progenitors targeted by the PML/RARα rearrangement.

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