Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiv, 144 p.; also includes graphics (some col.). Includes abstract and vita. Co-advisors: Lawrence E. Mathes and Maria H. Neff, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 122-144).

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 61-64).

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

Acute myeloid leukemia (AML) is a heterogeneous disease both clinically and biologically. Approximately 55% of AML harbour karyotypic changes, and one of the most common chromosomal aberrations is the t(8;21)(q22;q22), which leads to the AML1-ETO fusion protein. Previous studies have found that this fusion protein recruits the N-CoR/mSin3A/HDAC complex, thereby acts as a transcriptional repressor. Recently, DNA methylation array studies have shown that DNA methylation patterns can stratify AML cases into different subgroups, and some of these correspond to certain chromosomal abnormalities, such as the t(8;21). These findings suggest a possible link between the fusion transcript AML1-ETO and epigenetic modifications. Additionally, c-kit mutations have emerged as an important disease modifier in the t(8;21) AML and are correlated with poor overall survival and event free survival in patients with t(8;21) AML. We therefore sought to investigate whether there are different DNA methylation patterns in t(8;21) AML with or without c-kit mutations. In our series, 52.2% of the t(8;21) AMLs harbored c-kit mutations, which were correlated with poor event free survival. We next performed pyrosequencing on a selected panel of genes and pinpointed the THBS4 and PAWR genes as hypermethylated in their promoter CpG islands in 86.4% and 59.1% of the t(8;21) AML patients, respectively. These data suggest that THBS4 and PAWR may be important in the pathogenesis of t(8;21) AML. / published_or_final_version / Pathology / Master / Master of Philosophy

Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts in circulation and/or bone marrow. Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene occurs in about 30% of AML and is associated with an inferior prognosis. Tyrosine kinase domain (TKD) mutations occur in about 5% with uncertain prognostic significance. Intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However these approaches have reached a deadlock with a cure rate of 30-40%. Targeting FLT3 in AML with multi-tyrosine-kinase inhibitors has been evaluated in Phase II/III clinical trials. Despite an initial clearance of myeloblasts, the leukemia invariably progresses despite continuous treatment. The mechanisms of drug resistance and leukemia progression, hence the effective therapeutic strategies are currently unknown, limiting its clinical application. These issues were addressed in the present study. In the first part, 13 patients with chemo-refractory or relapsed FLT3-ITD+ AML received sorafenib 200-400 mg twice daily of whom 12 patients achieved clearance or near clearance of bone marrow blasts after a median of 27 days (range 21-84 days). There was evidence of myeloid differentiation of the leukemia blasts at remission. Leukemia progression occurred in 9 patients after a median of 72 days (range 54-287 days) and in 4 out of 6 patients it was dominated by clones carrying double FLT3-ITD and -TKD mutations. Microarray studies comparing myeloblasts before sorafenib treatment (sorafenib naïve) and at subsequent progression (sorafenib resistant) demonstrated up-regulation of 64 genes including ALDH1A1, JAK3 and TESC whose functions were unknown in AML. Transplantation of sorafenib naïve and resistant myeloblasts into NOD/SCID mice recapitulated their clinical behavior when the animals were treated with sorafenib. Both ITD and TKD mutations at D835 were identified in leukemia initiating cells (LICs) from sorafenib naïve samples. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations. In the second part, the gene encoding tescalcin (TESC), that was up-regulated at sorafenib resistance and was known to activate a sodium/hydrogen exchange (NHE1), was evaluated to examine its link with TKI resistance. TESC was highly expressed in FLT3-ITD+ AML cell lines MOLM-13 and MV4-11 and its knock-down by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene)amiloride (HMA). Induction of sorafenib resistance in MOLM-13 cell line (MOLM-13-RE) significantly increased its sensitivity to HMA. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD+ AML cells significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. Normal CD34+ cells engraftment was not affected. HMA treatment significantly enhanced suppression of FLT3 signaling by sorafenib even in sorafenib resistant cell lines. These observations provided novel information about the pathogenetic role of TESC-NHE1-pHi in sorafenib resistance in AML. In conclusion, the information derived from the present study has provided mechanistic insights to the emergence of drug resistance during sorafenib treatment and important guide for future therapeutic strategies targeting FLT3-ITD+ AML. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Acute promyelocytic leukemia (APL) is one of the subtypes of acute myeloid leukemia carrying t(15;17), and constitutes 10 to 15% of adult AMLs. One of the mechanisms of gene inactivation is hypermethylation of promoter-associated CpG islands. Cancers are characterized by global hypomethylation with locus-specific hypermethylation and hence silencing of tumor suppressor genes. Apart from tumor suppressor genes, microRNA, a class of non-coding RNA measuring 19-25 nucleotides, with tumor suppressive function is also found to be inactivated by DNA methylation in hematological malignancies. microRNAs repress target gene translation and hence expression by binding to 3'-untranslated region of corresponding mRNA. Because TP53 mutation is frequently involved in solid cancer carcinogenesis but is rarely found in APL, TP53 network may be dysregulated through epigenetic inactivation of tumor suppressor gene/miRNAs engaged in TP53 tumor suppressor network. This thesis aimed to study DNA methylation of tumor suppressor genes and miRNAs engaged in TP53 tumor suppressor network in APL. Overall survival (OS) and event free survival (EFS) of patients with or without candidate gene/miRNA hypermethylation were compared to examine their prognostic significances. Promoter methylation of DAPK1, p14ARF, miR-34a, miR-34b/c and miR-605 were studied in 10 normal bone marrow samples, NB4 cell line and 60 APL primary samples at diagnosis by methylation-specific PCR (MSP). DAPK1, miR-34a, miR-34b/c and miR-605 were completely unmethylated in normal bone marrow samples but completely methylated in NB4. Treatment of NB4 by 5'-Aza-2'-deoxyctidine (5-azadC) resulted in promoter demethylation together with re-expression of DAPK1, miR-34a, miR-34b/c and miR-605. Promoter methylation of DAPK1, p14ARF, miR-34a were absent while miR-34b/c and miR-605 methylation were detected in 43% and 10% APL samples respectively. However, methylation of miR-34b/c and miR-605 bore no prognostic significance. Overexpression of miR-34b in NB4 resulted in inhibition of proliferation. In short, methylation of DAPK1, miR-34a, miR-34b/c and miR-605 is associated with gene/miRNAs silencing. miR-34b/c is frequently methylated whereas miR-605 is methylated in small number of APL patients. miR-34b/c is a tumor suppressive miRNA in APL. Methylation of miR-34b/c may contribute to APL leukemogenesis. / published_or_final_version / Medicine / Master / Master of Philosophy

Mixed lineage leukemia (MLL) gene undergoes chromosomal translocation with over 60 different fusion partner genes in human leukemias. The resultant MLL-fusion oncoproteins are profoundly implicated in leukemias with poor prognosis. Epigenetic dysregulations have been frequently reported in MLL-rearranged leukemogenesis. Our study aims to investigate the correlations between epigenetic alterations, including both histone modification and DNA methylation, and gene dysregulation in MLL-rearranged leukemia. My study focused on MLL-EEN fusion protein, which causes an onset of acute myeloid leukemia (AML). A novel Mll-Een expressing cell line, VLA33, was derived from the bone marrow of Mll-Een knockin mouse with AML phenotype. The cells were mainly myeloblast cells, possessing clonogenic ability and showed upregulation of Hoxa cluster genes. Previous study demonstrated that the protein arginine methyltransferase 1 (PRMT1) plays a significant role in MLL-EEN leukemogenesis through conferring H4R3 asymmetric dimethylation (H4R3me2a) mark on HoxA9 locus. Consistently, our ChIP analysis demonstrated enrichment of H4R3me2a at the Hoxa promoters while knockdown of Prmt1 attenuated the expression of Hoxa genes and reduced in vitro clonogenic potential of VLA33 cells. CD41, Runx1 and Tgm2 genes, which showed elevated expression in VLA33 cells, were identified as potential target genes of Mll-Een/Prmt1 complex. However, enrichment of active H4R3me2a was only observed at Runx1 promoters, but not at the regulatory regions of CD41 and Tgm2. Inhibition of Prmt1 by inhibitor AMI-1 reduced Runx1 and CD41 expression. Although Prmt1 knockdown reduced the enrichment of H4R3me2a at Runx1 promoter, it did not suppress the expression of Runx1. These data suggest the involvement of other regulatory mechanism and Prmt1 is not the sole factor causing gene dysregulation. CD41 is a marker of murine definitive hematopoietic progenitors. Interestingly, the CD41+ VLA33 cells demonstrated a trend of enhanced self-renewal ability in colony-forming assay as compared with CD41-/low cells. The CD41 expression was positively correlated with Mll-Een and Prmt1 expression. In addition, CD41+ cells expressed higher level of Hoxa9, Bmi-1, Runx1, Tal-1 and Lmo2 genes that are associated with HSC activities, suggesting reactivation of stem-cell regulatory program in CD41+ leukemia cells, which confer as leukemia stem cell population. The association between DNA methylation and MLL-EEN leukemogenesis was also investigated. The results demonstrated the establishment of stem cell Hox code, which was correlated with DNA hypomethylation status at Hox gene promoters in Mll-Een leukemia cells. Besides, Hox activation through DNA hypomethylation was independent of Prmt1-mediated histone modification, but was found associated with reduction of Bmi-1 binding at Hox loci. In conclusion, my study identified novel dysregulated genes in Mll-Een leukemogenesis. My findings provide insight into the reactivation of stem-cell program in leukemia cells through epigenetic dysregulation, which furthers our understanding of MLL-rearranged leukemogenesis. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Background: Chronic myeloid leukaemia (CML) is a haematological malignant disease involving haematopoietic stem cells. It is caused by a known reciprocal chromosomal t(9;22)(q34;q11) translocation, or known as Philadelphia chromosome. The translocation results in the formation of a chimeric BCR-ABL fusion gene. In the most recent guidelines published by NCCN and European LeukemiaNet in 2013, tyrosine kinase inhibitors (TKI) specifically inhibiting the Bcr-Abl tyrosine kinase, are the first-line therapy for patients with chronic phase CML. Imatinib is the oldest among the 3 TKI, and is the most commonly prescribed. Despite its proven therapeutic role in CML, imatinib is a drug of extreme high cost. Estimated annual drug cost is HKD$223,380for a standard 400mg adult daily dose. Therefore, this study aims to survey on the prescribing pattern of imatinib in CML patients, its funding status, response; and estimate its economic burden on the Hong Kong population. Methodology: This is a retrospective patient chart review study. All patients who were diagnosed with CML from 2003 to 2012 and were managed in QMH or QEH were reviewed. Electronic records were retrieved to see whether imatinib was started as first-line treatment within 6 months of diagnosis. The reasons for not initiating imatinib were also investigated. Patients’ response to imatinib, and funding source for the drug, were documented. Annual drug cost of imatinib was estimated from all CML patients who attended all Hospital Authority institutions in 2012 who were prescribed with the drug. Results: Total 153 patients from the 2 institutions were reviewed. One hundred twenty four (81%) of them started imatinib as first-line therapy within 6 months of diagnosis. Nine patients started second generation TKI as first-line. Among those who did not start TKI, the most common reasons are patient preference (3.9%) and financial difficulties (3.3%). Twelve paediatric patients are identified, and all but one of them started imatinib. Seventy one% patients on imatinib experienced side effects. Most frequently reported adverse reactions are thrombocytopenia, oedema and neutropenia. Twenty eight% switched to second generation TKI due to suboptimal response or intoleranceto imatinib. During their course of treatment, 46.3% patients on imatinib require social subsidy from Samaritan Fund. From the dispensing records, the average drug cost per patient per year is HK$113,902. The estimated annual cost burden on the whole Hong Kong population is HK$43,425,878. Conclusion: The prescribing rate of imatinib in chronic phase CML patients in Hong Kong is comparable to overseas prescribing rate. The drug has become a significant financial burden to patients’ family and the society as a whole. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences