Outcomes of adolescents with acute lymphoblastic leukaemia.

INTRODUCTION: Compared with young children who have acute lymphoblastic leukaemia (ALL), adolescents with ALL have unfavourable disease profiles and worse survival. However, limited data are available regarding the characteristics and outcomes of adolescents with ALL who underwent treatment in clinical trials. The aim of this study was to investigate the causes of treatment failure in adolescents with ALL. METHODS: We retrospectively analysed the outcomes of 711 children with ALL, aged 1-18 years, who were enrolled in five clinical trials of paediatric ALL treatment between 1993 and 2015. RESULTS: Among the 711 children with ALL, 530 were young children (1-9 years at diagnosis) and 181 were adolescents (including 136 younger adolescents [10-14 years] and 45 older adolescents [15-18 years]). Compared with young children who had ALL, adolescents with ALL were less likely to have favourable genetic features and more likely to demonstrate poor early response to treatment. The 10-year overall survival and event-free survival rates were significantly lower among adolescents than among young children (77.9% vs 87.6%, P=0.0003; 69.7% vs 76.5%, P=0.0117). There were no significant differences in the 10-year cumulative incidence of relapse, but the 10-year cumulative incidence of treatment-related death (TRD) was significantly greater among adolescents (7.2%) than among young children (2.3%; P=0.002). Multivariable analysis showed that both younger and older adolescents (vs young children) had worse survival and greater incidence of TRD. CONCLUSION: Adolescents with ALL had worse survival because they experienced a greater incidence of TRD. There is a need to investigate optimal treatment adjustments and novel targeted agents to achieve better survival rates (without excessive toxicity) among adolescents with ALL.

Incidence and mortality of nasopharyngeal carcinoma: interim analysis of a cluster randomized controlled screening trial (PRO-NPC-001) in southern China.

BACKGROUND: Previous mass screening studies have shown that IgA antibodies against Epstein-Barr Virus (EBV) can facilitate early detection of nasopharyngeal carcinoma (NPC), but the impact of EBV-antibody screening for NPC-specific mortality remains unknown. PATIENTS AND METHODS: A prospective, cluster randomized, controlled trial for NPC screening (PRO-NPC-001) was conducted in 3 selected towns of Zhongshan City and 13 selected towns of Sihui City in southern China beginning in 2008. Serum samples of the screening group were tested for two previously selected anti-EBV antibodies. Subjects with serological medium risk were subsequently retested annually for 3 years, and those with serological high risk were referred to otorhinolaryngologists for diagnostic check-up. An interim analysis was carried out to evaluate the primary end points of the NPC-specific mortality and the early diagnostic rate, and the secondary end point of the NPC incidence, through linkage with the database of Zhongshan City. RESULTS: Among 70 296 total subjects, 29 413 screened participants (41.8% of the total subjects) in the screening group and 50 636 in the control group, 153 (43.3 per 100 000 person-year), 62 (55.3 per 100 000 person-year) and 99 (33.1 per 100 000 person-year) NPC cases were identified. The early diagnostic rates of NPC were significantly higher in the participants (79.0%, P < 0.0001) and the screening group (45.9%, P < 0.0001) compared with the control group (20.6%). Although no differences were found between NPC-specific mortality of the screening group and the control group [relative risk (RR)= 0.82, 95% confidence interval (CI) 0.37-1.79], lower NPC-specific mortality was noticed among participants from the screening group versus the control group (RR = 0.22, 95% CI 0.09-0.49). CONCLUSION: IgA antibodies against EBV can identify high-risk population and was effective in screening for early asymptomatic NPC. Although the mortality reduction was not significant in the primary end point, we noted encouraging evidence of a mortality reduction in screening participants in this interim analysis. CLINICAL TRIAL NUMBER: NCT00941538.

Improved functional assessment of osteoarthritic knee joint after chondrogenically induced cell treatment.

OBJECTIVES: Our previous studies on osteoarthritis (OA) revealed positive outcome after chondrogenically induced cells treatment. Presently, the functional improvements of these treated OA knee joints were quantified followed by evaluation of the mechanical properties of the engineered cartilages. METHODS: Baseline electromyogram (EMGs) were conducted at week 0 (pre-OA), on the locomotory muscles of nine un-castrated male sheep (Siamese long tail cross) divided into controls, adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs), before OA inductions. Subsequent recordings were performed at week 7 and week 31 which were post-OA and post-treatments. Afterwards, the compression tests of the regenerated cartilage were performed. RESULTS: Post-treatment EMG analysis revealed that the control sheep retained significant reductions in amplitudes at the right medial gluteus, vastus lateralis and bicep femoris, whereas BMSCs and ADSCs samples had no further significant reductions (P < 0.05). Grossly and histologically, the treated knee joints demonstrated the presence of regenerated neo cartilages evidenced by the fluorescence of PKH26 tracker. Based on the International Cartilage Repair Society scores (ICRS), they had significantly lower grades than the controls (P < 0.05). The compression moduli of the native cartilages and the engineered cartilages differed significantly at the tibia plateau, patella femoral groove and the patella; whereas at the medial femoral condyle, they had similar moduli of 0.69 MPa and 0.40-0.64 MPa respectively. Their compression strengths at all four regions were within ±10 MPa. CONCLUSION: The tissue engineered cartilages provided evidence of functional recoveries associated to the structural regenerations, and their mechanical properties were comparable with the native cartilage.

Association between HLA-B*58:01 allele and severe cutaneous adverse reactions with allopurinol in Han Chinese in Hong Kong.

BACKGROUND: Allopurinol has been reported as a common cause of severe cutaneous adverse reactions (SCARs). Recent studies in various populations suggest that HLA-B*58:01 is a strong genetic marker for allopurinol-induced SCAR, especially in populations with a high frequency of HLA-B*58:01. OBJECTIVES: To confirm the association link between HLA-B*58:01 and hypersensitivity reactions attributed to allopurinol use in Han Chinese patients in Hong Kong. METHODS: We performed a case-control study to investigate whether the HLA-B*58:01 allele predisposes to allopurinol-induced SCAR in Han Chinese patients in Hong Kong. The HLA-B*58:01 genotyping was performed in 20 patients with allopurinol-induced SCAR or erythema multiforme major (EMM; n = 1) and in 30 patients tolerant to allopurinol. RESULTS: All of the 19 patients with allopurinol-induced SCAR examined but not the patient with EMM carried HLA-B*58:01 whereas only four (13%) of the control patients had this allele. The positive rate of the HLA-B*58:01 was significantly higher in the cases than in the allopurinol-tolerant control group [odds ratio (OR) 123·5, 95% confidence interval (CI) 12·8-1195·1; P < 1 × 10(-4) ] and was even higher after removal of the patient with EMM (OR 229·7, 95% CI 11·7-4520·4). The sensitivity and specificity of the HLA-B*58:01 allele for prediction of allopurinol-induced SCAR were 100% and 86·7%, respectively. CONCLUSIONS: This study confirmed the strong association between the HLA-B*58:01 and allopurinol-induced SCAR in Hong Kong Han Chinese patients. A screening test for the HLA-B*58:01 allele should effectively reduce the risk for allopurinol-induced SCAR in Chinese populations.

Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are widely used in the clinic because they involve fewer ethical issues and safety concerns compared to other stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). MSCs derived from umbilical cord Wharton's jelly (WJ-MSCs) have excellent proliferative potential and a faster growth rate and can retain their multipotency for more passages in vitro compared to adult MSCs from bone marrow or adipose tissue. WJ-MSCs are used clinically for repairing tissue injuries of the spinal cord, liver and heart with the aim of regenerating tissue. On the other hand, WJ-MSCs are also used clinically to ameliorate immune-mediated diseases based on their ability to modulate immune responses. In the field of tissue engineering, WJ-MSCs capable of differentiating into multiple cell lineages have been used to produce a variety of engineered tissues in vitro that can then be transplanted in vivo. This review discusses the characteristics of WJ-MSCs, the differences between WJ-MSCs and adult MSCs, clinical studies involving WJ-MSCs and future perspectives of WJ-MSC research and clinical applications. To summarize, WJ-MSCs have shown promise in treating a variety of diseases clinically. However, most clinical trials/studies reported thus far are relatively smaller in scale. The collected evidence is insufficient to support the routine use of WJ-MSC therapy in the clinic. Thus, rigorous clinical trials are needed in the future to obtain more information on WJ-MSC therapy safety and efficacy.

Correlation of donor age and telomerase activity with in vitro cell growth and replicative potential for dermal fibroblasts and keratinocytes.

Previous studies suggested telomerase activity as a determinant of cell replicative capacity by delaying cell senescence. This study aimed to evaluate the feasibility of adopting telomerase activity as a selection criterion for in vitro expanded skin cells before autologous transplantation. Fibroblasts and keratinoctyes were derived from the same consenting patients aged 9-69 years, and cultured separately in serum-supplemented and serum-free media, respectively. Telomerase activity of fresh and cultured cells were measured and correlated with cell growth rate, donor age and passage number. The results showed that telomerase activity and cell growth were independent of donor age for both cell types. Telomerase was expressed in freshly digested epidermis and dermis and continued expressing in vitro. Keratinocytes consistently showed 3-12 folds greater telomerase activity than fibroblast both in vivo and in vitro. Conversely, growth rate for fibroblast exceeded that of keratinocyte. Telomerase activity decreased markedly at Passage 6 for keratinocytes and ceased by Passage 3 for fibroblasts. The decrease or cessation of telomerase activity coincided with senescence for keratinocyte but not for fibroblast, implying a telomerase-regulated cell senescence for the former and hence a predictor of replicative capacity for this cell type. Relative telomerase activity for fibroblasts from the younger age group was significantly higher than that from the older age group; 69.7% higher for fresh isolates and 31.1% higher at P0 (p<0.05). No detectable telomerase activity was to be found at later subcultures for both age groups. Similarly for keratinocytes, telomerase activity in the younger age group was significantly higher (p<0.05) compared to that in the older age group; 507.7% at P0, 36.8% at P3 and the difference was no longer significant at P6. In conclusion, the study provided evidence that telomerase sustained the proliferation of keratinocytes but not fibroblasts. Telomerase activity is an important criterion for continued survival and replication of keratinocytes, hence its positive detection before transplantation is desirable. Inferring from our results, the use of keratinocytes from Passage 3 or lesser for construction of skin substitute or cell-based therapy is recommended owing to their sustained telomerase expression.

Narcolepsy in Southern Chinese patients: clinical characteristics, HLA typing and seasonality of birth.

OBJECTIVE: To report clinical characteristics, human leukocyte antigen (HLA) typing and seasonality of birth of a series of 54 Southern Chinese patients suffering from narcolepsy. METHODS: All subjects underwent detailed medical and psychiatric interviews and a standardised nocturnal polysomnogram followed by a daytime Multiple Sleep Latency Test. Each subject also completed a set of sleep questionnaires. HLA typing was performed in 91% of subjects. RESULTS: A total of 78% and 22% of patients were diagnosed with suffering from cataplectic and non-cataplectic narcolepsy, respectively. The majority (n = 47, 87%) of patients were referred to our sleep clinic for excessive daytime sleepiness (EDS). The cataplectic narcolepsy differed from non-cataplectic narcolepsy by having more rapid eye movement (REM)-related clinical symptoms (more sleep paralysis and sleep-related hallucination) and sleep disturbances (shorter REM latency), as well as tighter association with HLA DQB1*0602. A bi-modal peak pattern was observed at 11 and 39 years old. A similar bi-modal pattern also occurred for EDS and cataplexy. Excess winter births were observed for this series of patients. 81% of patients with cataplectic narcolepsy were DQB1*0602-positive. There were no differences between early- and late-onset cases in the association with positive DQB1*0602 (71.4% vs 60%). Narcolepsy had prominent pernicious effects on various social, academic, family and mental aspects in our patients. CONCLUSIONS: In our Southern Chinese narcolepsy series, bi-modal peak pattern of age of onset, excess winter birth and tight association of HLA DQB1*0602 with cataplectic narcolepsy were found.

Long-term evaluation of osteoarthritis sheep knee, treated with TGF-ß3 and BMP-6 induced multipotent stem cells.

BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography. RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance. CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-ß3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.

Aortic dissection in an accident and emergency department in Hong Kong.

OBJECTIVES: To compare the clinical characteristics of aortic dissection in Hong Kong with the International Registry of Aortic Dissection, and to evaluate the sensitivity of the Accident and Emergency Department in diagnosing aortic dissection and its role in aortic dissection management. DESIGN: Retrospective observational study. SETTING: Regional public hospital, Hong Kong. PATIENTS: Newly diagnosed aortic dissection patients attending the Accident and Emergency Department from 2002 to 2005 inclusive. MAIN OUTCOME MEASURES: Correct diagnosis in Accident and Emergency Department, tertiary unit transfer, and in-patient mortality. RESULTS: Twenty-eight patients were found to have aortic dissection, with an estimated annual incidence of 2.1 per 100,000 inhabitants. The sensitivity of Accident and Emergency Department in diagnosing aortic dissection was 54%; 11% of the patients were diagnosed at postmortem examination. Compared to the International Registry of Aortic Dissection, the patients in this Tseung Kwan O Hospital study had less abrupt and less severe pain, less chest pain, and a lower proportion were operated on. Higher mortality was associated with age 70 years or older (odds ratio=6.4), female gender (21.0), known hypertension (3.8), systolic blood pressure below 100 mm Hg (6.0), aortic dissection not diagnosed in the Accident and Emergency Department (3.2), and the patient not reaching tertiary unit (33.8). The hourly cumulative mortality rate was 1.32%. The group of aortic dissections diagnosed in the Accident and Emergency Department had 55.1% more transfer to tertiary unit (95% confidence interval, 14.4-79.1%; P=0.006), 84.5 hours less transfer time (95.3-263.6 hours; P=0.232), and 27.2% lower mortality (12.6-58.6%; P=0.246). The yield rate of contrast computed tomography of thorax was 43%. CONCLUSIONS: Diagnosing aortic dissection in the Accident and Emergency Department enabled optimal disposition and lower in-patient mortality.

Specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in SARS patients.

BACKGROUND: Severe acute respiratory syndrome (SARS) is an infectious disease which was caused by a novel coronavirus (SARS-CoV). SARS has caused an outbreak in the world during 2003 and 2004, with 8098 individuals being infected and a death toll of 774 in 28 regions around the world. Specific humoral responses to viral infection remain unclear. OBJECTIVE: To analyse the antigenicity of the SARS-CoV genome and identify potential antigenic epitopes in the structural proteins. METHODS: Potential antigenic epitopes were identified in the structural proteins (nucleocapsid, membrane, spike, and small envelope proteins) and hypothetical proteins (SARS3a, 3b, 6, 7a, and 9b) that are specific for SARS-CoV. A peptide chip platform was created and the profiles of antibodies to these epitopes were investigated in 59 different SARS patients' sera obtained 6-103 days after the onset of the illness. Serial sera from five additional patients were also studied. RESULTS: Epitopes at the N-terminus of the membrane protein and the C-terminus of nucleocapsid protein elicited strong antibody responses. Epitopes on the spike protein were only moderately immunogenic but the effects were persistent. Antibodies were also detected for some putative proteins, noticeably the C-termini of SARS3a and SARS6. CONCLUSIONS: Important epitopes of the SARS-CoV genome that may serve as potential markers for the viral infection are identified. These specific antigenic sites may also be important for vaccine development against this new fatal infectious disease.

Improved outcome of acute lymphoblastic leukaemia treated by delayed intensification in Hong Kong children: HKALL97 study.

OBJECTIVE: To study the outcome of children with acute lymphoblastic leukaemia who were treated using a protocol including one or two delayed intensifications. DESIGN: Prospective single-arm multicentre study. SETTING: Five designated children cancer units of the Hospital Authority of Hong Kong. PATIENTS: Children aged between 1 and 17.9 years with newly diagnosed acute lymphoblastic leukaemia seen from November 1997 to December 2002. INTERVENTION: Chemotherapy was modified from a German Berlin-Frankfurt-Muenster 95 (BFM95) protocol that included a delayed intensification similar to the induction phase repeated 5 months after diagnosis. High-risk patients were given double delayed intensification. MAIN OUTCOME MEASURES: Overall survival and event-free survival of the whole group and the three risk groups (standard-, intermediate-, and high-risk groups), and comparison with historical controls. RESULTS: A total of 171 patients were recruited with a median age at diagnosis of 5.57 years (range, 1.15-17.85 years). The induction remission rate was 95.3% and non-leukaemia mortality during remission was 2.3%. At 4 years, the relapse rate of this (HKALL97) study was significantly lower than that of the HKALL93 study (15.7 vs 37.3%; P<0.001). The 4-year overall survival of HKALL97 and HKALL93 studies were 86.5% and 81.8%, respectively (P=0.51). The 4-year event-free survival for HKALL97 and HKALL93 studies were 79% and 65%, respectively (P=0.007). Nonetheless the difference of event-free survival was most remarkable in the intermediate-risk group: 75.6% and 53.1% for HKALL97 and HKALL93 studies, respectively (P=0.06). CONCLUSION: A more intensive delayed consolidation phase improved the outcome for children with acute lymphoblastic leukaemia by reducing relapses at 4 years. The early treatment complications were manageable and non-leukaemia mortality during remission remained low.

Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction.

We have developed a quantitative method for methylation analysis of the p16 gene based on real-time methylation-specific PCR (MSP). Real-time MSP is sensitive enough to detect down to 10 genome equivalents of the methylated p16 sequence. Application of real-time MSP to DNA from tumor-derived cell lines revealed complete concordance with conventional MSP analysis. Quantitative data generated by real-time MSP were expressed as the methylation index, which was defined as the percentage of bisulfite-converted DNA that consisted of methylated target sequences. The methylation index was shown to be inversely correlated with p16 gene transcription during demethylation treatment of cell lines with 5-aza-2'-deoxycytidine. The application of real-time MSP to bone marrow aspirates from patients with multiple myeloma revealed complete concordance with conventional MSP analysis. Real-time quantitative MSP may have applications in elucidating diverse biological processes involving DNA methylation and may become a valuable diagnostic tool for detecting tumor-associated epigenetic changes in cancer patients.

Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients.

We have studied the feasibility of detecting tumor-associated aberrant p16 methylation in the circulation of patients with hepatocellular carcinoma (HCC). We extracted DNA from the tumor tissues and peripheral blood plasma or serum of 22 HCC patients. p16 methylation was found in 73% (16 of 22) of HCC tissues using methylation-specific PCR. Among the 16 cases with aberrant methylation in the tumor tissues, similar changes were also detected in the plasma/serum samples of 81% (13 of 16) of the cases. No methylated p16 sequences were detected in the peripheral plasma/serum of the six HCC cases without these changes in the tumor, in 38 patients with chronic hepatitis/cirrhosis, or in 10 healthy control subjects. These results suggest that circulating liver tumor DNA may be detected using tumor-associated DNA methylation changes. Because methylation abnormalities have been found in many other genes and tumor types, this approach may have implications for the noninvasive detection of a wide variety of cancers.

Alterations in the structure of the EBV nuclear antigen, EBNA1, in epithelial cell tumours.

The EBV nuclear antigen, EBNA1, is the only viral protein consistently expressed in all virus-infected cells. It is required in trans for viral replication, maintenance of EBV extrachromosomal episomes, and transcriptional transactivation in latently-infected B-cells. It binds RNA suggestive of a regulatory role in post-transcriptional events and in transgenic mice, it is tumorigenic. In RNase protection studies relating to the EBV-associated tumour, nasopharyngeal carcinoma (NPC), we show that a C-terminal EBNA1 RNA probe from the prototype B95-8 marmoset strain can protect its own mRNA from enzymatic digestion, but does not fully protect EBNA1 mRNA from NPC cells. This finding is consistent with changes in the coding region for the antigen. We thus determined the sequences of EBNA1 genes derived from an NPC xenograft and numerous patient biopsies and identified a number of mutations in the gene in these human cells, relative to B95-8. Many of the nucleotide changes would lead to non-conservative amino acid alterations in apparently functionally significant regions of the protein. We show that although some of the mutations lie in regions designated as critical to DNA binding, they have negligible effect on this property of EBNA1. The basic regions in EBNA1 that may bind to RNA, at least in vitro, are exempt from mutation. Thus, unless the alterations are 'silent', which for such a critical viral function seems unlikely, they may relate to as yet unmapped viral activities, such as a role in tumorigenesis and the ability of EBNA1 to evade the cellular immune system, or be associated with the ability of the antigen to regulate gene transcription.

Study on the surface polypeptides isolated from human lymphoblastoid ce-ls by ionic shock.

Ionic shock treatment in the presence of 10% glycerol is an efficient and selective method for extracting cell surface components from Raji cells and effecting the solubilization of up to 22 polypeptides. The majority of these shock-released polypeptides are accessible to lactoperoxidase radioiodination. Sera from rabbits immunized against these soluble extracts are reactive against Raji cell surface as indicated by indirect membrane immunofluorescence and agglutination assays.

Frequent death-associated protein kinase promoter hypermethylation in multiple myeloma.

UNLABELLED: Death-associated protein (DAP) kinase is a novel gene regulating apoptosis induced by IFN-gamma. In B-cell malignancies, loss of DAP kinase expression is commonly associated with promoter hypermethylation. These characteristics of DAP kinase may be of particular relevance in multiple myeloma (MM), a B-lineage malignancy in which prolonged survival capacity of the malignant plasma cells may be critical in the induction and maintenance of tumor cells. PURPOSE: The involvement and potential role of DAP kinase in MM pathogenesis was examined. EXPERIMENTAL DESIGN: In this investigation, methylation-specific PCR was conducted on primary MM and MM cell lines. Methylation status findings were correlated with clinical parameters. RESULTS: We first demonstrated frequent DAP kinase hypermethylation in 24 of 36 primary MMs (20 of 26 at diagnosis and 4 of 10 with relapse/residual MM after treatment), 1 of 2 patients with monoclonal gammopathy of undetermined significance, and 1 of 3 MM cell lines studied. The high frequency of DAP kinase hypermethylation was similarly observed in MM of different stages, immunoglobulin isotypes, and histological grades, with or without plasmacytomas. Although not statistically significant, the overall survival of patients with DAP kinase methylation was notably shortened among 23 MM patients followed prospectively (P = 0.38 by Kaplan-Meier method and log-rank test). This preliminary finding suggests prognostic implications of DAP kinase in MM that may deserve further investigation. CONCLUSIONS: Our data suggest an important role for DAP kinase in MM tumorigenesis.