1,721,100 research outputs found
Promoter methylation of tumor suppressor genes and microRNAs engaged in TP53 network in acute promyelocytic leukemia
No full textAcute 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_versionMedicineMasterMaster of Philosoph
Standardized minimal residual disease detection by next-generation sequencing in multiple myeloma
No full textBone marrow (BM) based minimal residual disease (MRD) is one of the most powerful prognostic factors in multiple myeloma (MM). Therefore, standardization and easy operation of MRD testing is crucial. Regarding MRD detection by next- generation sequencing (NGS), the majority of data came from LymphoSIGHT platform which is now called clonoSEQ, an expensive customer service provided by a single company in the USA. However, the reported sensitivity of 10-6 of LymphoSIGHT platform by serial dilution experiment in selected patients was not experimentally documented and guaranteed in each & every MRD sample. On the other hand, LymphoTrack, provided by Invivoscribe provides customer with kits comprising multiplex primers compatible with Illumina MiSeq.
Therefore, the first aim of this study was to devise a standardized NGS-based MRD detection approach with a uniform sensitivity. In a pilot experiment of 7 samples, the sensitivity of 10-5 of LymphoTrack-MiSeq platform was achieved by using a standardized protocol of triplicates of one μg DNA input and one million sequencing reads per replicate. Two plasmids cloned from immunoglobulin rearrangements were spiked into MRD sample DNA in parallel with MRD measurement, one for marking sensitivity of 10-5 and the other for MRD
normalization. This platform was further simplified and validated in 19 samples. The plasmid spike-in controls were replaced by genomic DNA from myeloma cells to make the procedure more convenient and accurate. Sensitivity of 10-5 was consistently reached. Reproducibility of MRD detection was confirmed by the small inter-run variation tested in 3 samples. Compared with allele-specific oligonucleotide real-time quantitative-PCR, this approach does not require any patient-specific primer or probe, thus, much less labor- and time-intensive.
Utility of this standardized NGS-based MRD approach in prognostication was evaluated in fifty MM patients who achieved very good partial response or better post autologous stem cell (ASCT) transplant. MRD was detected in 40/50 (80%) of these patients. MRD at the threshold 10-4 was prognostic of both progression-free survival and overall survival. A larger study is warranted to evaluate the prognostic impact of MRD using the cut-off of 10-5.
Furthermore, in four patients, change of MRD from undetectable to positivity was observed in one patient after ASCT despite persistent clinical complete response. More cases are needed to thoroughly evaluate the dynamic change in MRD post-ASCT and the optimal time point for assessing MRD post-ASCT.
Finally, efficacy of a 3-weekly daratumumab-based regimen was evaluated by MRD assessment in 10 relapsed and 3 newly diagnosed MM patients. MRD negativity was achieved in 7/10 (70%) relapsed patients and 2/3 (67%) newly diagnosed patients.
In conclusion, we standardized an NGS-based MRD approach with a sensitivity of 10-5 which is sufficiently sensitive and feasible for MRD measurements. MRD post-ASCT was a prognostic factor of survivals. 3-weekly
daratumumab in combination with proteasome inhibitor and/or immunomodulatory agent are effective and rendered MRD-negativity in the majority of relapsed MM.published_or_final_versionMedicineDoctoralDoctor of Philosoph
DNA methylation of microRNA and long non-coding RNA in multiple myeloma
No full textMultiple myeloma is one form of hematological malignancy characterized by the accumulation and patchy infiltration of the bone marrow by neoplastic plasma cells. It is often preceded by an entirely asymptomatic state, monoclonal gammopathy of undetermined significance (MGUS), that progresses into symptomatic myeloma at the rate of 1% per year. Currently, multiple myeloma remains an incurable disease. Therefore, further insight into the pathogenesis of disease is imperative.
Non-coding RNAs (ncRNAs) are RNA molecules with no protein-coding capacity. Functional ncRNAs include microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). miRNAs contain 19~25 nucleotides and will target and suppress the expression of their target genes by complementary binding to the 3’-UTR of the target mRNAs. lncRNAs are novel class of RNA molecules containing more than 200 nucleotides, which regulate gene expression at both transcriptional and post transcriptional levels by binding to transcription factors, genomic DNAs and mRNAs. Oncogenic or tumor suppressive roles of various miRNAs and lncRNAs have been proved in multiple cancer types, including myeloma.
DNA methylation refers to the addition of a methyl group to carbon five position of the cytosine ring in a CpG dinucleotide. For regulation of ncRNAs, promoter DNA methylation at the CpG island is one of the most well-established mechanisms. Indeed, methylation has been implicated in regulation of numerous miRNAs and lncRNAs in myeloma.
The thesis aimed to investigate the role of promoter DNA methylation in the regulation of candidate ncRNAs in myeloma. Moreover, the prognostic impact of DNA methylation and tumor suppressive function of ncRNAs were studied.
Promoter DNA methylation of miRNAs (miR-340-5p, miR-342-3p and miR-28-5p) and lncRNAs (BM742401 and KIAA0495) were studied in myeloma. Epigenetic silencing of these ncRNAs by tumor-specific methylation were confirmed in human myeloma cell lines (HMCLs). For miR-28-5p, we proved for the first time to our knowledge that it is epigenetically regulated by promoter of its host gene. For miR-342-3p and miR-340-5p, comparable methylation frequencies were detected in primary bone marrows at MGUS and active myeloma at diagnosis and relapse, hence possibly an early event in myeloma pathogenesis. Moreover, methylation of miR-340-5p in diagnostic myeloma was correlated with significantly shorter overall survival (OS), which may serve as an adverse prognosis biomarker. Furthermore, the tumor suppressive roles of miR-340-5p were demonstrated by restoration of miR-340-5p in HMCL, leading to decreased cell proliferation via suppressing MAPK signaling and increased apoptosis via directly targeting X-linked inhibitor of apoptosis protein (XIAP). Besides, lncRNAs, including KIAA0495 and BM742401, are novel targets of DNA methylation in myeloma. In particular, methylation of BM742401 was detected at consecutive stages of myeloma pathogenesis, hence likely an early event, and it was correlated with significantly shorter OS. However, methylation of KIAA0495, as well as miR-28-5p, were rarely detected in primary samples, suggesting methylation of these ncRNAs in HMCLs were acquired during in vitro cell culture.
In conclusion, methylation of miR-340-5p, miR-342-3p and BM742401 are implicated in pathogenesis of myeloma, and methylation of miR-340-5p and BM742401 in prognosis of myeloma.published_or_final_versionMedicineDoctoralDoctor of Philosoph
DNA methylation of microRNA and long non-coding RNA in mantle cell lymphoma
No full textMantle cell lymphoma (MCL), arising from malignant transformation of CD5+ naïve B-cell population in the mantle zone of lymph node, is a rare but aggressive subtype of B-cell non-Hodgkin’s lymphoma (NHL), accounting for 4-6% of all NHL patients. Genetically, MCL is characterized by the presence of t(11;14)(q13;q32) translocation, thereby cyclin D1 overexpression and consequently accelerated G1/S cell cycle transition.
microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two important classes of non-coding RNAs. Mature miRNAs, measuring about 22 nucleotides, downregulate target genes via sequence-specific binding to 3’-UTR of the target genes, resulting in degradation of mRNA or translational block. LncRNAs, generally > 200 nucleotides, may regulate gene expressions by multiple mechanisms operating at epigenetic, transcriptional or post-transcriptional levels. Dysregulation of miRNAs and lncRNAs has been shown in a variety of tumors, hence important in carcinogenesis.
DNA methylation is an epigenetic modification by which a methyl group (-CH3) is added to the C5 position of cytosine in a CpG dinucleotide. Cancer cells are characterized by global DNA hypomethylation and gene-specific promoter DNA hypermethylation. Moreover, promoter DNA hypermethylation emerges to be an important mechanism mediating silencing of both protein-coding and non-coding tumor suppressor genes. However, apart from intergenic tumor suppressor miRNAs, the effect of DNA methylation-mediated silencing of intronic miRNAs and lncRNAs on lymphomagenesis remains largely unexplored.
Herein, it was hypothesized that reversible silencing of intronic miRNAs and lncRNAs was mediated by promoter DNA methylation in MCL and other subtypes of NHL. By a candidate gene approach, promoter methylation of intronic miRNAs (miR-342-3p and miR-1250-5p) and lncRNAs (NKILA and HOTTIP) were investigated. Results showed that both the promoter CpG islands of host genes of both intronic miR-342-3p and miR-1250-5p, and that of lncRNAs (NKILA and HOTTIP) were methylated in a tumor-specific manner. Moreover, intronic miR-342-3p and miR-1250-5p were reversibly silenced and co-regulated by promoter DNA methylation of their host genes, EVL and AATK respectively. Furthermore, in NHL cells, restoration of miR-342-3p or miR-1250-5p resulted in increased cell death and decreased cellular proliferation, hence inherent tumor suppressor functions. Mechanistically, miR-342-3p was shown to inhibit pro-survival autophagy via directly targeting MAP1LC3B, precursor of LC3-II that is a biomarker of autophagy. Moreover, as miR-342-3p was known to target DNMT1, promoter demethylation led to re-expression of a hypermethylated and silenced tumor suppressor, E-cadherin. On the other hand, miR-1250-5p was demonstrated to inhibit NHL cell proliferation by targeting MAPK1 hence repression of MAPK/ERK signaling, and SDF-1-dependent migration by targeting WDR1.
LncRNAs, NKILA and HOTTIP, were reversibly silenced via promoter DNA methylation in NHL cells. Moreover, knockdown of NKILA led to decreased cell death and enhanced cellular proliferation, consistent with a tumor suppressive function in NHL cells.
In primary samples, HOTTIP methylation was frequently detected in MCL. Conversely, miR-342-3p, miR-1250-5p, or NKILA were preferentially methylated in B-NHL other than MCL, implicating an epigenetic heterogeneity among diverse NHL subtypes.
In conclusion, intronic miR-342-3p and miR-1250-5p, as well as lncRNAs HOTTIP and NKILA were regulated by reversible methylation-mediated silencing in MCL and other subtypes of NHL, which implicated in lymphomagenesis.published_or_final_versionMedicineDoctoralDoctor of Philosoph
A staged approach with vincristine, adriamycin and dexamethasone followed by bortezomib, thalidomide and dexamethasone before autologous hematopoietic stem cell transplantation in the treatment of newly diagnosed multiple myeloma
No full textThe 2010 Health Research Symposium, Hong Kong, 11 September 2010
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