Factors Associated with Intention to Donate Hematopoietic Stem Cells among Blood Donors.

INTRODUCTION: Past studies pay little attention to the intention to donate hematopoietic stem cells (HSC) among blood donors. This study investigated the level of and the influence of socio-demographic characteristics, knowledge, attitude, subjective norm and self-efficacy on the intention to donate HSC among blood donors. METHODS: This cross-sectional study recruited blood donors at selected public hospitals in the Malaysian State of Sarawak in 2019. A structured questionnaire was developed based on the review of relevant literature. It gathered information on socio-demographic characteristics, knowledge, attitude, subjective norm and self-efficacy on the intention to donate HSC. Variables with a p value <0.200 in bivariate analysis were included in the variable selection for regression modeling to examine their associations with the intention to donate HSC. RESULTS: A total of 569 blood donors participated (94.5% response rate). Overall, 87.1% reported a positive intention to donate HSC. In the regression model, the factor with the greatest association with intention to donate HSC was subjective norms about HSC donation (ß = 0.35, 95% CI 0.27-0.42), followed by attitude about regulations of HSC donation (ß= 0.21, 95% CI 0.13-0.35), self-efficacy on HSC donation (ß = 0.15, 95% CI 0.09-0.32), attitude about the potential side effects of HSC donation (ß = 0.14, 95% CI 0.02-0.10) and highest education level (ß = 0.10, 95% CI 0.03-0.44). CONCLUSIONS: The findings can be used to formulate a better strategy in promoting HSC donation among blood donors in the region.

Mesenchymal Stem Cell Expressing TRAIL as Targeted Therapy against Sensitised Tumour.

Tapping into the ability of engineered mesenchymal stem cells (MSCs) to mobilise into the tumour has expanded the scope of cancer treatment. Engineered MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) could serve as a platform for an efficient and targeted form of therapy. However, the presence of cancer stem cells (CSCs) that are resistant to TRAIL and apoptosis may represent a challenge for effective treatment. Nonetheless, with the discovery of small molecular inhibitors that could target CSCs and tumour signalling pathways, a higher efficacy of MSC-TRAIL mediated tumour inhibition can be achieved. This might pave the way for a more effective form of combined therapy, which leads to a better treatment outcome. In this review, we first discuss the tumour-homing capacity of MSCs, its effect in tumour tropism, the different approach behind genetically-engineered MSCs, and the efficacy and safety of each agent delivered by these MSCs. Then, we focus on how sensitisation of CSCs and tumours using small molecular inhibitors can increase the effect of these cells to either TRAIL or MSC-TRAIL mediated inhibition. In the conclusion, we address a few questions and safety concerns regarding the utilization of engineered MSCs for future treatment in patients.

Circulating cytokines and small molecules follow distinct expression patterns in acute myeloid leukaemia.

BACKGROUND: Although aberrant expression of cytokines and small molecules (analytes) is well documented in acute myeloid leukaemia (AML), their co-expression patterns are not yet identified. In addition, plasma baselines for some analytes that are biomarkers for other cancers have not been previously reported in AML. METHODS: We used multiplex array technology to simultaneously detect and quantify 32 plasma analyte (22 reported analytes and 10 novel analytes) levels in 38 patients. RESULTS: In our study, 16 analytes are found to be significantly deregulated (13 higher, 3 lower, Mann-Whitney U-test, P-value <0.005), where 5 of them have never been reported before in AML. We predicted a seven-analyte-containing multiplex panel for diagnosis of AML and, among them, MIF could be a possible therapeutic target. In addition, we observed that circulating analytes show five co-expression signatures. CONCLUSIONS: Circulating analyte expression in AML significantly differs from normal, and follow distinct expression patterns.

Human non-small cell lung cancer expresses putative cancer stem cell markers and exhibits the transcriptomic profile of multipotent cells.

BACKGROUND: Despite significant advances in staging and therapies, lung cancer remains a major cause of cancer-related lethality due to its high incidence and recurrence. Clearly, a novel approach is required to develop new therapies to treat this devastating disease. Recent evidence indicates that tumours contain a small population of cells known as cancer stem cells (CSCs) that are responsible for tumour maintenance, spreading and resistant to chemotherapy. The genetic composition of CSCs so far is not fully understood, but manipulation of the specific genes that maintain their integrity would be beneficial for developing strategies to combat cancer. Therefore, the goal of this study isto identify the transcriptomic composition and biological functions of CSCs from non-small cell lung cancer (NSCLC). METHODS: We isolated putative lung CSCs from lung adenocarcinoma cells (A549 and H2170) and normal stem cells from normal bronchial epithelial cells (PHBEC) on the basis of positive expression of stem cell surface markers (CD166, CD44, and EpCAM) using fluorescence-activated cell sorting. The isolated cells were then characterised for their self-renewal characteristics, differentiation capabilities, expression of stem cell transcription factor and in vivo tumouregenicity. The transcriptomic profiles of putative lung CSCs then were obtained using microarray analysis. Significantly regulated genes (p < 0.05, fold change (FC) > 2.0) in putative CSCs were identified and further analysed for their biological functions using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). RESULTS: The putative lung CSCs phenotypes of CD166(+)/CD44(+) and CD166(+)/EpCAM(+) showed multipotent characteristics of stem cells, including the ability to differentiate into adipogenic and osteogenic cells, self-renewal, and expression of stem cell transcription factors such as Sox2 and Oct3/4. Moreover, the cells also shows the in vivo tumouregenicity characteristic when transplanted into nude mice. Microarray and bioinformatics data analyses revealed that the putative lung CSCs have molecular signatures of both normal and cancer stem cells and that the most prominent biological functions are associated with angiogenesis, migration, pro-apoptosis and anti-apoptosis, osteoblast differentiation, mesenchymal cell differentiation, and mesenchyme development. Additionally, self-renewal pathways such as the Wnt and hedgehog signalling pathways, cancer pathways, and extracellular matrix (ECM)-receptor interaction pathways are significantly associated with the putative lung CSCs. CONCLUSION: This study revealed that isolated lung CSCs exhibit the characteristics of multipotent stem cells and that their genetic composition might be valuable for future gene and stem cells therapy for lung cancer.

Nucleofection optimization and in vitro anti-tumourigenic effect of TRAIL-expressing human adipose-derived mesenchymal stromal cells.

BACKGROUND: Tumour homing capacity of engineered human adipose-derived mesenchymal stromal cells (ADMSCs) expressing anti-tumour agents might be the key for a much safer and yet efficient targeted tumour therapy. However, ADMSCs exhibit resistant to most gene transfection techniques and the use of highly efficient viral vectors has several disadvantages primarily concerning safety risk. Here, we optimized the use of highly efficient and safe nucleofection-based transfection using plasmid encoded for TNF-Related Apoptosis Inducing Ligand (TRAIL) into ADMSCs and investigated the potential anti-tumourigenic of TRAIL-expressing ADMSCs (ADMSCs-TRAIL) on selected cancer models in vitro. METHODS: Different concentration of TRAIL-encoded plasmid and ADMSCs were nucleofected and the percentage of fluorescence cells were analyzed to determine the optimal condition. TRAIL protein and mRNA were validated in nucloeofected ADMSCs using ELISA and RT-PCR respectively. Evaluation of TRAIL specific death receptors were performed on both tumours (A549/lung tumour, LN18/glioblastoma and HepG2/hepatocellular carcinoma) and haematological malignant lines (REH/acute lymphocytic leukaemia, K562/chronic myelogenous leukaemia and KMS-28BM/multiple myeloma) using flow cytometry. ADMSCs-TRAIL was subsequently assessed for anti-tumourigenic properties using both proliferation assay (MTS assay) and apoptosis assay (Annexin-V / Propidium Iodide staining). RESULTS: Nucleofection showed increased total plasmid concentration (2 µg to 8 µg) resulted in significantly higher reporter expression (11.33% to 39.7%) with slight reduction on cells viability (~10%). ADMSCs-TRAIL significantly inhibited ~50% of cell proliferation in LN18, signifying sensitivity of the cell to ADMSCs-TRAIL mediated inhibition. Inhibition of both tumour and malignant lines proliferation by ADMSCs-TRAIL conditioned medium noticed in HepG2, A549 and REH respectively, whereas K562 and KMS-28BM malignant lines exhibit resistant to ADMSCs-TRAIL mediated inhibition. Moreover, we found that native ADMSCs alone were capable of inducing apoptosis in both LN18 and HepG2 tumour lines, despite substantial increased on the percentage of apoptosis by ADMSCs-TRAIL. CONCLUSION: ADMSCs-TRAIL selectively inhibit cancer model and markedly induces apoptosis. Through investigation of the specific TRAIL death receptors expression, we saw that the receptors expression did influence the sensitivity of some but not all cancer lines to TRAIL-mediated inhibition. This study provides further insight into the anti-tumourigenic potential of ADMSCs-TRAIL on different cancer models.

Higher propensity of Wharton's jelly derived mesenchymal stromal cells towards neuronal lineage in comparison to those derived from adipose and bone marrow.

Mesenchymal stromal cells (MSCs) derived from different tissue sources are capable of differentiating into neural and glial cell types. However, the efficiency of differentiation varies between MSCs derived from different tissues. We compared the efficiency of neural progenitor population generation between adipose (AD), bone marrow (BM) and Wharton's jelly (WJ) derived MSCs. MSCs isolated from the three sources were induced to form primary neurospheres using epidermal growth factor (20 ng/mL) and bFGF (20 ng/mL). The self-renewal potential of the primary neurospheres was assessed by secondary neurosphere assay. Primary neurospheres were differentiated to neuronal lineage on fibronectin-coated dishes. The neurospheres and the resulting differentiated cells were characterized by immunocytochemistry and the RT-PCR analyses. We have also investigated the secretome profile of neuronal-related growth factors using Ray biotech cytokine array. The results show that MSCs from the three sources can be induced to generate neurospheres and they expressed neural progenitor markers nestin, Sox2 and Pax6 transcription factors. When differentiated on fibronectin coated dishes in mitogen free culture conditions, the primary spheres from all three sources were able to generate neuron/glial - like cells which expressed Nfl, Map2 and GFAP with varied efficiency. Self-renewal potential of these progenitors was determined by secondary sphere formation. WJ- and BM-derived neurospheres were able to self-renew, while AD derived progenitors failed to do so. Comparison of the secretome profile suggested that WJ derived MSCs secrete more neurotrophic factors. The data suggest that human WJ derived MSCs can be induced to make neural progenitors with higher efficiency compared to BM and AD derived MSCs.

Detection of ß-globin Gene Mutations Among ß-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System-Polymerase Chain Reaction.

BACKGROUND: ß-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of ß-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. METHODS: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 ß-globin gene mutations, were designed and employed to investigate ß-thalassaemia patients and carriers. RESULTS: Out of 169 carriers tested with the MARMS, Cd 41/42 (-TTCT), Cd 26 (A-G) HbE, IVS 1-1 (G-T), and IVS 1-5 (G-C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A-G) HbE, Cd 41/42 (-TTCT), IVS 1-1 (G-T), and IVS 1-5 (G-C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (-TTCT) and IVS 2-654 (C-T) were most common among the Chinese (79.1%). CONCLUSION: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of ß-thalassaemia among Malaysians at the Institute for Medical Research (IMR).

Terminal microdeletion of chromosome 18 in a Malaysian boy characterized with few features of typical 18q- deletion syndrome: a case report.

BACKGROUND: The 18q- deletion syndrome is a rare congenital chromosomal disorder caused by a partial deletion of the long arm of chromosome 18. The diagnosis of a patient with this syndrome relies on the family medical history, physical examination, developmental assessment, and cytogenetic findings. However, the phenotype of patients with 18q- deletion syndrome can be highly variable, ranging from almost normal to severe malformations and intellectual disability, and normal cytogenetic findings are common, thus complicating the diagnosis. Interestingly, only few characteristic features of typical 18q- deletion syndrome were found in the patient, despite sharing the same critical region. To our knowledge, this is the first report of a Malaysian individual with 18q- terminal microdeletion diagnosed with microarray-based technology. CASE PRESENTATION: Here we report a 16-year-old Malaysian Chinese boy, a product of a non-consanguineous marriage, who presented with intellectual disability, facial dysmorphism, high arched palate, congenital talipes equinovarus (clubfoot), congenital scoliosis, congenital heart defect, and behavioral problems. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XY G-banded karyotype. Array-based comparative genomic hybridization was performed using a commercially available 244 K 60-mer oligonucleotide microarray slide according to the manufacturer's protocol. This platform allows genome-wide survey and molecular profiling of genomic aberrations with an average resolution of about 10 kB. In addition, multiplex ligation-dependent probe amplification analysis was carried out using SALSA MLPA kit P320 Telomere-13 to confirm the array-based comparative genomic hybridization finding. Array-based comparative genomic hybridization analysis revealed a 7.3 MB terminal deletion involving chromosome band 18q22.3-qter. This finding was confirmed by multiplex ligation-dependent probe amplification, where a deletion of ten probes mapping to the 18q22.3-q23 region was detected, and further multiplex ligation-dependent probe amplification analysis on his parents showed the deletion to be de novo. CONCLUSION: The findings from this study expand the phenotypic spectrum of the 18q- deletion syndrome by presenting a variation of typical 18q- deletion syndrome features to the literature. In addition, this case report demonstrated the ability of the molecular karyotyping method, such as array-based comparative genomic hybridization, to assist in the diagnosis of cases with a highly variable phenotype and variable aberrations, such as 18q- deletion syndrome.

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation.

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo-, chondro-, and adipo-lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy.

Impact of passing mesenchymal stem cells through smaller bore size needles for subsequent use in patients for clinical or cosmetic indications.

BACKGROUND: Numerous preclinical and clinical studies have investigated the regenerative potential and the trophic support of mesenchymal stem cells (MSCs) following their injection into a target organ. Clinicians favor the use of smallest bore needles possible for delivering MSCs into vascular organs like heart, liver and spleen. There has been a concern that small needle bore sizes may be detrimental to the health of these cells and reduce the survival and plasticity of MSCs. METHODS: In this report, we aimed to investigate the smallest possible bore size needle which would support the safe delivery of MSCs into various tissues for different clinical or cosmetic applications. To accomplish this we injected cells via needle sizes 24, 25 and 26 G attached to 1 ml syringe in the laboratory and collected the cells aseptically. Control cells were ejected via 1 ml syringe without any needle. Thereafter, the needle ejected cells were cultured and characterized for their morphology, attachment, viability, phenotypic expression, differentiation potential, cryopreservation and in vivo migration abilities. In the second phase of the study, cells were injected via 26 G needle attached to 1 ml syringe for 10 times. RESULTS: Similar phenotypic and functional characteristics were observed between ejected and control group of cells. MSCs maintained their cellular and functional properties after single and multiple injections. CONCLUSIONS: This study proves that 26 G bore size needles can be safely used to inject MSCs for clinical/therapeutics purposes.

Ex vivo expanded SSEA-4+ human limbal stromal cells are multipotent and do not express other embryonic stem cell markers.

PURPOSE: The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However, little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated. METHODS: Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition, expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2), tumour protein p63 (p63), paired box 6 (Pax6), cytokeratin 3 (AE5), cytokeratin 10, and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes, osteocytes, and chondrocytes. RESULTS: Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2, p63, Pax6, AE-5, and keratocan sulfate. After passaged, a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60, Tra-1-81, and transcription factors like octamer-binding transcription factor 4 (Oct4), SRY(sex determining region Y)-box 2 (Sox2), and Nanog. Early passaged cells when induced were able to differentiate into adipocytes, osteocytes and chondrocytes. CONCLUSIONS: The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However, despite the expression of SSEA-4, these cells did not express any other markers of ESC. Therefore, we conclude that the cells did not show properties of ESC.

Comparison of chemokine and receptor gene expression between Wharton's jelly and bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: Because of their multilineage differentiation capacity, immunomodulatory role and homing ability, mesenchymal stromal cells (MSC) are emerging as a new therapeutic strategy for treating a variety of disorders. Although bone marrow (BM) is the best characterized source of MSC, Wharton's jelly (WJ) of the umbilical cord holds great promise as an alternative. As delivery direct to the site of injury is not always feasible, efficient homing of MSC to the site of injury is critical for inducing tissue repair and regeneration. MSC express a wide variety of growth factors, chemokines and receptors that are important for cell migration, homing and re-establishment of blood supply for recovery of damaged tissues. METHODS: Detailed chemokine and receptor gene expression profiles of WJ MSC were established, and subsequently compared with those of BM-derived MSC using a polymerase chain reaction (PCR) array. Secretion of growth factors was analyzed and evaluated using culture supernatant from WJ and BM MSC. RESULTS: Our results revealed a differential expression pattern of the chemokines and their receptors between WJ- and BM-derived MSC. Several Glutamic acid-Leucine-Arginine; ELR-positive CXC chemokine genes and secretion of growth factors, which promote angiogenesis, were found to be up-regulated in WJ MSC. CONCLUSIONS: To understand better the localization and mechanism of tissue repair by transplanted WJ MSC, we attempted chemokine and their receptor transcription profiling, followed by analysis of growth factors secreted by WJ MSC, and compared them against those of BM MSC. The data suggest that MSC from different sources can be explored for distinct therapeutic roles.

Cell therapy in critical limb ischemia: current developments and future progress.

Critical limb ischemia (CLI) is a syndrome manifested by ischemic rest pain, non-healing ulcers and tissue loss. CLI patients are at very high risk of amputation and experience poor physical function, leading to severe morbidity and mortality. The fundamental goal for CLI treatment is to relieve ischemic rest pain, heal ulcers, prevent limb loss and improve the quality of life, thereby extending the survival of the patient. Surgical or endovascular revascularization aimed at increasing blood flow is currently available for limb salvage in CLI. However, up to 30% of CLI patients are not suitable for such interventions because of high operative risk or unfavorable vascular anatomy. Therefore exploring new and more effective strategies for revascularization of ischemic limbs is imperative for the establishment of a viable therapeutic alternative. With the emergence of new approaches, this review describes up-to-date progress and developments in cell-based therapy as a novel and promising alternative for CLI treatment. Preliminary clinical data have established the safety, feasibility and efficacy of stem cells, and numerous studies are underway to consolidate this evidence further. However, significant hurdles remain to be addressed before this research can be responsibly translated to the bedside. In particular, we need better understanding of the behavior of cells post-transplantation and to learn how to control their survival and migration proliferation/differentiation in the hostile pathologic environment. Future research should focus on methods of isolation, optimal dosage, appropriate cell type, route of administration, role of tissue-derived factors and supportive endogenous stimulation.

Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

Comprehensive analysis of mutations and clonal evolution patterns in a cohort of patients with cytogenetically normal acute myeloid leukemia.

BACKGROUND: Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability, clonal selection and evolution of leukemic clones during treatment. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to contribute with the investigation of the mutational landscape of CN-AML and clonal evolution during AML treatment. RESULT: A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs being more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that is frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two distinct clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations. CONCLUSIONS: The findings of this study suggest that the relapse-initiating clones may pre-exist prior to therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse.

Construction of a microRNA-mRNA Regulatory Network in De Novo Cytogenetically Normal Acute Myeloid Leukemia Patients.

Background: The association between dysregulated microRNAs (miRNAs) and acute myeloid leukemia (AML) is well known. However, our understanding of the regulatory role of miRNAs in the cytogenetically normal AML (CN-AML) subtype pathway is still poor. The current study integrated miRNA and mRNA profiles to explore novel miRNA-mRNA interactions that affect the regulatory patterns of de novo CN-AML. Methods: We utilized a multiplexed nanoString nCounter platform to profile both miRNAs and mRNAs using similar sets of patient samples (n = 24). Correlations were assessed, and an miRNA-mRNA network was constructed. The underlying biological functions of the mRNAs were predicted by gene enrichment. Finally, the interacting pairs were assessed using TargetScan and microT-CDS. We identified 637 significant negative correlations (false discovery rate <0.05). Results: Network analysis revealed a cluster of 12 miRNAs representing the majority of mRNA targets. Within the cluster, five miRNAs (miR-495-3p, miR-185-5p, let-7i-5p, miR-409-3p, and miR-127-3p) were posited to play a pivotal role in the regulation of CN-AML, as they are associated with the negative regulation of myeloid leukocyte differentiation, negative regulation of myeloid cell differentiation, and positive regulation of hematopoiesis. Conclusion: Three novel interactions in CN-AML were predicted as let-7i-5p:HOXA9, miR-495-3p:PIK3R1, and miR-495-3p:CDK6 may be responsible for regulating myeloid cell differentiation in CN-AML.

Expression and mutational analysis of GATA3 in Malaysian breast carcinomas.

The GATA3 gene is a potential tumour marker and putative tumour suppressor gene in breast cancer. Its expression is associated with better prognosis and disease free survival in breast cancer patients. We aimed to evaluate GATA3 transcriptome expression and mutation in breast carcinomas and correlate its expression with oestrogen receptor (ER), progesterone receptor (PR), lymph node (LN) status, tumour grade and c-erbB-2 expression. Twenty-two breast infiltrating ductal carcinomas and paired normal tissues were used in Branch DNA assay to detect GATA3 mRNA expression. Normalized data for GATA3 mRNA expression were grouped according to the ER, PR and LN status, tumour grade and c-erbB-2 expression of the tumours. Statistical significance was tested using t-test and ANOVA at 95% confidence interval level. Mutational analysis of GATA3 was performed by direct sequencing of the coding regions of GATA3 mRNA. Our findings showed that GATA3 gene were over-expressed and under-expressed by > 2 fold change in 12 and 4 tested samples, respectively. Eighty per cent of ER positive breast carcinomas were GATA3 positive. There was a statistically significant correlation between GATA3 expression and ER at 95% confidence interval level between the study groups. On the contrary, GATA3 expression was not statistically significant with PR, LN, tumour grade and c-erbB-2 expression in our study. In addition, we observed that there was no mutation in mRNA coding region in 16 breast carcinomas that showed GATA3 differential gene expression. Our preliminary results suggested that GATA3 is linked to the ER. This scenario suggests that GATA3 may play a crucial role in oestrogen receptor positive breast cancer patients. Whether GATA3 expression is involved in regulating tumour cell growth in oestrogen responsive breast cancer is a key question that remains to be answered.

Inherited t(9;22) as the cause of DiGeorge syndrome: a case report.

DiGeorge syndrome is associated with microdeletion of chromosome 22q11.2. Most cases occur sporadically although vertical transmission has been documented. We report a rare case of DiGeorge syndrome in an 8-year-old girl. Blood sample of the patient was cultured and harvested following standard procedure. All of the 20 cells analysed showed a karyotype of 45, XX, -22, t (9;22) (p23; q11.2). Cytogenetic investigation done on the patient's mother revealed that she was the carrier for the translocation. Her karyotype was 46, XX, t (9;22) (p23; q11.2). Fluorescence in situ hybridisation (FISH) analysis using TUPLE1 and N25 (Vysis, USA) probes showed deletion of the 22q11.2 region in the patient, confirming the diagnosis of DiGeorge syndrome. FISH analysis showed no deletion of the region in the mother.

Targeting of CD133+ Cancer Stem Cells by Mesenchymal Stem Cell Expressing TRAIL Reveals a Prospective Role of Apoptotic Gene Regulation in Non-Small Cell Lung Cancer.

Mesenchymal stem cells (MSCs) are emerging as vehicles for anti-tumor cytotherapy; however, investigation on its efficacy to target a specific cancer stem cell (CSC) population in non-small cell lung cancer (NSCLC) is lacking. Using assays to evaluate cell proliferation, apoptosis, and gene expression, we investigated the efficacy of MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) to target and destroy CD133+ (prominin-1 positive) NSCLC-derived CSCs. Characterization of TRAIL death receptor 5 (DR5) revealed that it was highly expressed in the CD133+ CSCs of both H460 and H2170 cell lines. The human MSC-TRAIL generated in the study maintained its multipotent characteristics, and caused significant tumor cell inhibition in NSCLC-derived CSCs in a co-culture. The MSC-TRAIL induced an increase in annexin V expression, an indicator of apoptosis in H460 and H2170 derived CD133+ CSCs. Through investigation of mitochondria membrane potential, we found that MSC-TRAIL was capable of inducing intrinsic apoptosis to the CSCs. Using pathway-specific gene expression profiling, we uncovered candidate genes such as NFKB1, BAG3, MCL1, GADD45A, and HRK in CD133+ CSCs, which, if targeted, might increase the sensitivity of NSCLC to MSC-TRAIL-mediated inhibition. As such, our findings add credibility to the utilization of MSC-TRAIL for the treatment of NSCLC through targeting of CD133+ CSCs.

Anti-Proliferative Activity of Triterpenes Isolated from Clinicanthus nutans on Hep-G2 Liver Cancer Cells

Problem statement: Clinicanthus nutans has been used by Malaysian since long time ago. It is used to treat many diseases including cancer. Many studies carried out on its crude extract but no clear report on the specific secondary metabolites responsible for its nature in treating selected diseases. Objective: This study aims to confirm the practice carried out by many people on the usage of Clinicanthus nutans in treating cancer. Methods: C. nutans leaves were extracted by methanol. Thin layer chromatography was used to identify the suitable solvent for fractions separation. The fractions were then separated at larger volume using gravity column chromatography. Each fraction was tested on its anti-proliferative activity on Hep-G2 liver cancer cells by MTT assay. The phytochemical screening was carried out to identify the bioactive compound based on qualitative analysis. Results: The fraction 2 (F2) of C. nutans showed the lowest IC50 value of 1.73 µg/ml against Hep-G2 cancer cells, and it is identified as triterpenes. Conclusion: The fraction F2 identified as triterpenes isolated from C. nutans has potential as an anti-proliferative agent against liver cancer.