published_or_final_version / Pathology / 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

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Class of 2014 Abstract / Specific Aims: Evaluate serum posaconazole concentrations following dose adjustment in response to subtherapeutic serum concentrations. Determine optimal dose adjustment schema and identify toxicity with doses above 600 mg daily (e.g.: 200 mg per os three times daily). Methods: The health records were reviewed for 29 patients ≥ 18 years with acute myeloid leukemia over a four-year period. Participants initially received posaconazole 200 mg per os three times daily as prophylaxis and required at least one dose adjustment secondary to a subtherapeutic posaconazole serum concentration. Patients were stratified by posaconazole dosing following dose adjustment (A=200mg QID, B=300mg TID, C=400 mg TID, D=400 QID). Main Results: There was a statistically significant increase in posaconazole serum concentration in each group compared to baseline serum concentration, aside from group C (group A and B P<0.001, group C P=0.236, and group D P=0.0076). The majority of participants in 3 of the 4 groups reached therapeutic serum concentration (A=0.87, B=0.76, D=0.80) whereas group C had a serum posaconazole concentration on average below therapeutic range (0.51). There was no significant difference between the four groups in regards to renal function (p=0.35) or hepatic function (AST p=0.676, ALT p=0.877, total bilirubin p=0.097). Conclusion: A dose increase led to an increase in posaconazole serum concentration except for the dosing regimen of 400 mg three times daily. However, the study is limited by a small patient population, an unequal number of patients in each group, and potentially by poor absorption of posaconazole suspension. Further research is required to expand on these findings.

Acute Myeloid Leukemia (AML) is a type of cancer that affects the process of hematopoiesis. In individuals affected with AML, normal blood cells do not develop into red blood cells, white blood cells, and platelets, leading to symptoms such as anemia, neutropenia, and thrombocytopenia. The prognosis of AML is affected by multiple factors including: the genetic make-up of the leukemic cells, age of the affected individual, and underlying blood disorders such as myelodysplastic syndrome (MDS). MDS affects the development of stem cells into red blood cells, white blood cells, and platelets. Due to their clinical heterogeneity, AML and MDS continue to be a challenge that should be investigated in the community hospital setting. Remission rates between patients diagnosed with de-novo AML and patients diagnosed with AML with MDS were compared in a community hospital setting following induction therapy using a retrospective study design. Length of stay between patients diagnosed with de-novo AML and patients diagnosed with AML with MDS was compared during induction therapy. The association of age at diagnosis and number of chromosomal abnormalities to remission status was evaluated in each disease group. The association of blood transfusion requirements and neutropenic fever to length of stay was evaluated in each disease group. There were no statistically significant differences found between disease groups with respect to remission rates and length of stay. There were no statistically significant associations found between blood transfusion requirements and neutropenic fever in each disease group. There was an association found between age at diagnosis and remission status in patients diagnosed with AML with MDS. This indicates that older patients with AML with MDS are less likely to benefit from therapy and achieve complete remission. It is important to consider the small sample size, rare nature of the disease, and other variables that could have contributed to trends seen in the study population. The impact of predictors such as growth factor use and incidence of fungal infections should be investigated in future studies with AML patients. Considering these factors will allow for the development of targeted therapies and mechanisms against drug resistance for affected individuals.

Acute myeloid leukemia (AML) is a subset of leukemia that is characterized by the clonal expansion of cytogenetically and molecularly abnormal myeloid blasts. These blasts are highly proliferative accumulating in bone marrow and blood which leads to severe infections, anemia, and bone marrow failure. The poor prognosis of AML patients caused by the low tolerance to intensive chemotherapy has encouraged the pursuit of alternative therapeutic approaches. Differentiation therapy which involves the use of agents that can release the differentiation block in these leukemic blasts has emerged as a promising therapeutic approach. The use of All-trans retinoic acid (ATRA) represents a successful example of such an approach, nonetheless its efficacy is restricted to one subtype of AML. Efforts have been focused on finding differentiation agents which are effective for the other more common AML subtypes. Anti-CD44 targeted antibodies that activate the CD44 cell surface antigen are a promising candidate. Previous studies have shown that anti-CD44 treatment has been able to release the differentiation block in AML1 through AML5 subtypes. The exact mechanism by which anti-CD44 treatment is able to induce its effects has not been fully elucidated. Recent studies highlight the role that epigenetic mechanisms play during haematopoiesis and leukemogenesis and therefore, in this work we investigated the epigenetic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44-induced differentiation results in an extensive change of histone modification levels. We found that inhibiting enzymes responsible for the H3K9ac, H3K4me, H3K9me, and H3K27me modifications, attenuated the anti-proliferative and differentiation promoting effects of antic-CD44 treatment. Taken together, these data highlight the promising potential of using anti-CD44 as a therapeutic agent across multiple subtypes in AML