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Benzoporphyrin derivative and the photodynamic extracorporeal treatment of leukemia

University of British Columbia

The main question that was addressed in this thesis was whether benzoporphyrin derivative (BPD), a potent photosensitizer, could be used to photodynamically purge residual leukemic cells from bone marrow prior to autologous bone marrow transplantation (ABMT). The ultimate aim was to design a new purging regimen that would selectively eliminate leukemic stem cells while sparing normal stem cells and thereby, provide for better engraftment and a greater log reduction in leukemic cells resulting in prolonged disease free survival or cure. Five systems were used to test this hypothesis. 1) Fluorescence activated cell sorting (FACS) analysis was used to analyze and sort normal and leukemic cells incubated with BPD, which emitted a characteristic red fluorescent signal (Chapter 2). These studies showed that BPD-uptake by leulcemic cells was significantly greater than by normal cells. Differences in BPD fluorescence between normal and leukemic cells facilitated sorting of leukemic from normal cells via FACS thus, providing a novel diagnostic method for assessing leukemic cell burden within bone marrow and possibly a new adjunct to purging regimens based on the use of small volumes of CD34+ cells that could be further purged of leukemic cells via FACS based on BPD fluorescence differences between normal and leukemic cells. 2) Clonogenic and hemopoietic progenitor cell (HPC) assays were used to assess normal or leukemic peripheral blood (PBL) or bone marrow progenitor cell survival subsequent to treatment with BPD and light (Chapters 3 and 4). Clonogenic assays revealed that approximately four logs of leukemic cells derived from the leukemic cell lines EM-2 and K562 could be eliminated at concentrations that eliminated less than one log of normal peripheral blood (PBL) progenitors. Similarly, chronic myelogenous leukemic (CML) PBL progenitors were markedly inhibited by treatment with 10 ng BPD/ml and light while, more than 70% of normal PBL progenitors survived treatment with this dose. Hence, photodynamic purging may be an effective means of eliminating leukemic progenitors from peripheral blood stem cell autogralts while maintaining the capacity of normal PBL stem cells to support engraftment in patients (Chapter 3). Similarly, a therapeutic window existed at 10 ng BPD/ml and 10.8 J/cm 2 of light between normal bone marrow (NBM) and CML bone marrow (CML BM) progenitors. 3) A primary long term marrow culture (LTMC) system demonstrated that CML primitive progenitors were substantially reduced at 10 ng BPD/ml while the majority of normal primitive bone marrow progenitors survived and normal stromal layer development was not impeded (Chapter 5). Two stage LTMC studies revealed that normal hemopoiesis persisted for 8 weeks subsequent to BPD and light treatment of model remission marrows. 4) A reverse polymerase chain reaction (PCR) system was used to detect surviving Philadelphia chromosome positive (Ph’+) cells in the supernatant of LTMCs and in plucked colonies derived from LTMC’s and HPC assays, respectively (Chapters 5). PCR analysis of model remission marrows composed of NBM and 1 % EM-2 leukemic cells revealed that all leukemic cells (four logs) were eliminated by 25 ng BPD/ml and light. Mixing experiments with NBM and 10% CML BM revealed that no Ph+ colonies persisted in 10 ng BPD/ml and light treated samples (Chapter 4). Also, two stage LTMC’s established from NBM and 10% CML BM, treated with 10 ng BPD/ml and light, and PCR analysis revealed that no Ph’ + cells survived this dose (Chapter 5). 5) A murine hemopoietic reconstitution model was established and demonstrated a four log reduction in murine leukemic (L12l0) cells at concentrations of BPD and light that spared normal marrow reconstituting stem cells in 50:50 mixtures of splenocytes and L12l0 cells (Chapter 6).

Thesis/Dissertation

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2008-12-16

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http://hdl.handle.net/2429/2993

Microbiology and Immunology

University of British Columbia

UBCV

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