Spatial profiling of the microenvironment reveals low intratumoral heterogeneity and STK11-associated immune evasion in therapy-naïve lung adenocarcinomas.

OBJECTIVE: Intratumoral heterogeneity was found to be a significant factor causing resistance to lung cancer therapies, including immune checkpoint blockade. Lesser is known about spatial heterogeneity of the tumor microenvironment (TME) and its association with genetic properties of the tumor, which is of particular interest in the therapy-naïve setting. MATERIALS AND METHODS: We performed multi-region sampling (2-4 samples per tumor; total of 55 samples) from a cohort of 19 untreated stage IA-IIIB lung adenocarcinomas (n = 11 KRAS mutant, n = 1 ERBB2 mutant, n = 7 KRAS wildtype). For each sample the expression of 770 immunooncology-related genes was analyzed using the nCounter platform, while the mutational status was determined by hybrid capture-based next-generation sequencing (NGS) using a large panel covering more than 500 genes. RESULTS: Global unsupervised analyses revealed clustering of the samples into two groups corresponding to a 'hot' or 'cold' immunologic tumor contexture based on the abundance of immune cell infiltrates. All analyzed specific immune cell signatures (ICsig) showed a significantly higher intertumoral than intratumoral heterogeneity (p < 0.02), as most of the analyzed cases (14/19) showed a very homogenous spatial immune cell profile. PD-L1 exhibited a significantly higher intertumoral than intratumoral heterogeneity (p = 1.03e-13). We found a specific association with 'cold' TME for STK11 (11/14, p < 0.07), but not KRAS, TP53, LRP1B, MTOR, U2AF1 co-mutations, and validated this finding using The Cancer Genome Atlas (TCGA) data. CONCLUSION: Early-stage lung adenocarcinomas show considerable intertumoral, but limited intratumoral heterogeneity, which is clinically highly relevant as assessment before neoadjuvant treatment is based on small biopsies. STK11 mutations are specifically associated with a 'cold' TME, which could affect the efficacy of perioperative immunotherapy.

Accuracy of Gastrointestinal Ultrasound and Calprotectin in the Assessment of Inflammation and its Location in Patients with an Ileoanal Pouch.

BACKGROUND AND AIMS: In symptomatic patients with ileoanal pouches, pouchoscopy is needed for accurate diagnosis but is invasive. We aimed to assess the utility of non-invasive gastrointestinal ultrasound and faecal calprotectin in ileoanal pouch patients. METHODS: Patients with an ileoanal pouch were consecutively enrolled in this cross-sectional study from clinics in Victoria, Australia. The pouchitis disease activity index was used as a reference standard. Video-recorded pouchoscopies were reviewed by three gastroenterologists. Pouch, pre-pouch, and cuff biopsies were reviewed by a single pathologist. Ultrasound was performed by a single gastroenterologist transabdominally and transperineally. Faecal calprotectin was measured from morning stool samples. All examiners were blinded to patients' clinical history. RESULTS: A total of 44 participants had a pouchoscopy, of whom 43 had a faecal calprotectin test and 42 had an ultrasound; 17 had pouchitis, 15 had pre-pouch ileitis, and 16 had cuffitis. Pouch wall thickness of <3 mm was 88% sensitive in excluding pouchitis, and pouch wall thickness of ≥4 mm was 87% specific in diagnosing pouchitis. Transabdominal ultrasound had good utility [area under the curve: 0.78] in diagnosing moderate-severe pre-pouch ileitis. Transperineal ultrasound had good utility for the diagnosis of pouchitis [area under the curve: 0.79]. Faecal calprotectin differentiated inflammatory from non-inflammatory pouch disorders, such as irritable pouch syndrome, with an area under the curve of 0.90. Faecal calprotectin <100 µg/g ruled out inflammatory pouch disorders with a sensitivity of 94%. CONCLUSIONS: Faecal calprotectin and ultrasound are accurate and complementary tests to diagnose and localise inflammation of the ileoanal pouch. Prospective studies are needed to validate proposed sonographic indices and calprotectin levels.

The Reliability and Accuracy of Endoscopic Items and Scores Used in the Assessment of the Ileoanal Pouch and Cuff.

BACKGROUND AND AIMS: Currently used endoscopic items for the assessment of pouchitis and cuffitis have deficiencies in reliability and validation. We assessed the reliability and accuracy of new endoscopic items for pouchitis and of the Ulcerative Colitis Endoscopic Index of Severity [UCEIS] for cuffitis. METHODS: Three new endoscopic items were assessed and included in the Monash pouchitis endoscopic subscore: bleeding [absent/contact/spontaneous]; erosions [absent/<10/≥10]; and ulceration [absent/<10%/≥10%]. Three raters evaluated 44 pouchoscopy videos in duplicates, in random order. Intra- and inter-rater reliability of all endoscopic items and UCEIS were assessed. Clinical and histological pouchitis disease activity index [PDAI] subscores were also assessed and faecal calprotectin was measured. RESULTS: All three Monash endoscopic items had substantial intra-rater reliability with intraclass correlation coefficients [ICCs] >0.61 [95% CI >0.61], compared with only ulcers from the currently used PDAI endoscopic subscore, but inter-rater reliability was only substantial for ulceration and no better than those of the currently used endoscopic items. The Monash endoscopic subscore had a strong positive correlation with the reference standard global endoscopic lesion severity r = 0.80 [95% CI 0.80-0.80] and the reference standard PDAI endoscopic subscore r = 0.70 [95% CI 0.67-0.73], which was higher than the correlation observed for the currently used PDAI endoscopic subscore. The UCEIS had substantial intra-rater reliability, but only fair inter-rater reliability and poor diagnostic performance for cuffitis. CONCLUSIONS: The Monash endoscopic items, and endoscopic subscore they generate, have enhanced overall performance compared with the currently used PDAI items and subscore. Further validation and responsiveness to change in disease state are indicated.

Regional registration of whole slide image stacks containing major histological artifacts.

BACKGROUND: High resolution 2D whole slide imaging provides rich information about the tissue structure. This information can be a lot richer if these 2D images can be stacked into a 3D tissue volume. A 3D analysis, however, requires accurate reconstruction of the tissue volume from the 2D image stack. This task is not trivial due to the distortions such as tissue tearing, folding and missing at each slide. Performing registration for the whole tissue slices may be adversely affected by distorted tissue regions. Consequently, regional registration is found to be more effective. In this paper, we propose a new approach to an accurate and robust registration of regions of interest for whole slide images. We introduce the idea of multi-scale attention for registration. RESULTS: Using mean similarity index as the metric, the proposed algorithm (mean ± SD [Formula: see text]) followed by a fine registration algorithm ([Formula: see text]) outperformed the state-of-the-art linear whole tissue registration algorithm ([Formula: see text]) and the regional version of this algorithm ([Formula: see text]). The proposed algorithm also outperforms the state-of-the-art nonlinear registration algorithm (original: [Formula: see text], regional: [Formula: see text]) for whole slide images and a recently proposed patch-based registration algorithm (patch size 256: [Formula: see text] , patch size 512: [Formula: see text]) for medical images. CONCLUSION: Using multi-scale attention mechanism leads to a more robust and accurate solution to the problem of regional registration of whole slide images corrupted in some parts by major histological artifacts in the imaged tissue.

Coordinated responses of academic medical centres to pandemics: Sustaining medical education during COVID-19.

Background: The Corona Virus Disease-19 (COVID-19) has been declared a pandemic by the World Health Organization (WHO). We state the consolidated and systematic approach for academic medical centres in response to the evolving pandemic outbreaks for sustaining medical education.Discussion: Academic medical centres need to establish a 'COVID-19 response team' in order to make time-sensitive decisions while managing pandemic threats. Major themes of medical education management include leveraging on remote or decentralised modes of medical education delivery, maintaining the integrity of formative and summative assessments while restructuring patient-contact components, and developing action plans for maintenance of essential activities based on pandemic risk alert levels. These core principles must be applied seamlessly across the various fraternities of academic centres: undergraduate education, residency training, continuous professional development and research. Key decisions from the pandemic response teams that help to minimise major disruptions in medical education and to control disease transmissions include: minimising inter-cluster cross contaminations and plans for segregation within and among cohorts; reshuffling academic calendars; postponing or restructuring assessments.Conclusions: While minimising the transmission of the pandemic outbreak within the healthcare establishments is paramount, medical education and research activities cannot come to a standstill each time there is a threat of one.

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis.

BACKGROUND: With the accelerated development of next-generation sequencing (NGS), identified variants, and targeted therapies, clinicians who confront the complicated and multifarious genetic information may not effectively incorporate NGS-based circulating tumor DNA (ctDNA) analysis into routine patient care. Consequently, standardized ctDNA testing reports are of vital importance. In an effort to guarantee high-quality reporting performance, we conducted an investigation of the current detection and reporting practices for NGS-based ctDNA analysis. MATERIALS AND METHODS: A set of simulated ctDNA samples with known variants at known allelic frequencies and a corresponding case scenario were distributed to 66 genetic testing laboratories for ctDNA analysis. Written reports were collected to evaluate the detection accuracy, reporting integrity, and information sufficiency using 21 predefined criteria. RESULTS: Current reporting practices for NGS-based ctDNA analysis were found to be far from satisfactory, especially regarding testing interpretation and methodological details. Only 42.4% of laboratories reported the results in complete concordance with the expected results. Moreover, 74.2% of reports only listed aberrations with direct and well-known treatment consequences for the tumor type in question. Genetic aberrations for which experimental agents and/or drug access programs are available may thus be overlooked. Furthermore, methodological details for the interpretation of results were missing from the majority of reports (87.9%). CONCLUSION: This proof-of-principle study suggests that the capacity for accurate identification of variants, rational interpretation of genotypes, comprehensive recommendation of potential medications, and detailed description of methodologies need to be further improved before ctDNA analysis can be formally implemented in the clinic. IMPLICATIONS FOR PRACTICE: Accurate, comprehensive, and standardized clinical sequencing reports can help to translate complex genetic information into patient-centered clinical decisions, thereby shepherding precision oncology into daily practice. However, standards, guidelines, and quality requirements for clinical reports of next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) analysis are currently absent. By using a set of simulated clinical ctDNA samples and a corresponding case scenario, current practices were evaluated to identify deficiencies in clinical sequencing reports of ctDNA analysis. The recommendations provided here may serve as a roadmap for the improved implementation of NGS-based ctDNA analysis in the clinic.

External Quality Assurance of Current Technology for the Testing of Cancer-Associated Circulating Free DNA Variants.

Liquid biopsy testing is rapidly emerging as a diagnostic means of identifying circulating free DNA (cfDNA) disease-associated variants. However, the reporting of cfDNA variants remains inconsistent due in part to the application of multiple testing pipelines which raise uncertainty about current cfDNA detection efficiency. External quality assurance (EQA) programs are required to monitor, evaluate and help improve laboratory performance for cfDNA variant detection and in clinical interpretation. This study therefore evaluated the performance of diagnostic laboratories currently performing cfDNA testing in China, Australia and New Zealand. A total of 89 laboratories participated in this EQA program. Reference testing material comprised of cfDNA manufactured to contain six different genotypes in four different genes (EGFR, KRAS, BRAF, NRAS). The predicted genotypic variant allelic frequencies ranged between 0.5% - 2.5%. Proficiency testing used a z-score on the laboratory consensus allelic frequency data to compare laboratory performance for the detection of the different genotypes. Allelic frequency genotyping data were received from 88 of the 89 laboratories. Next generation sequencing and digital PCR testing platforms were primarily used by participants in this pilot EQA. The average consensus data for each cfDNA genotype identified allelic frequencies ranging between 0.39% - 4.4%. Z-score proficiency testing found that >92% of clinical laboratories were concordant for detecting the cfDNA variants. The data from this pilot study suggest that current cfDNA testing platforms can detect cfDNA allelic frequency variants from 0.39% and above with high levels of confidence. In addition, these data highlight the importance of laboratories enrolling on EQA programs so that proficiency in cfDNA diagnostic testing can be determined and potential sources of error identified and addressed.

Perioperative pregabalin does not reduce opioid requirements in total knee arthroplasty.

PURPOSE: The purpose of this prospective, randomized, double-blinded, placebo-controlled study was to determine if pregabalin, when given perioperatively in addition to patient-controlled analgesia morphine, paracetamol and etoricoxib, is effective in reducing morphine requirements and moderating pain scores after primary total knee arthroplasty. We hypothesize that there would be no difference in postoperative opioid requirements, postoperative pain scores, and functional scores with the use of perioperative pregabalin. METHODS: Eighty-seven patients who underwent primary total knee arthroplasty were randomised and allocated to two groups. One group received capsules containing pregabalin 75 mg, and the other a placebo-one capsule before surgery and one capsule once per night up till postoperative day 2. Multimodal analgesia provided for all patients in this study included femoral nerve block, intravenous patient-controlled analgesia (morphine), paracetamol and etoricoxib. The primary outcome of patient's pain control was based on the measurement of cumulative morphine consumption during the first 72 h postoperatively. RESULTS: Pregabalin did not reduce the cumulative or effective morphine consumption at 48 h and 72 h post-operation. There were also no significant differences noted in pain scores at 48 h and 72 h after surgery, functional range of motion of the operated knee at 72 h post-op, or outcomes recorded on the Knee Society Score (KSS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and 36-Item Short Form Survey (SF-36) questionnaires at 3 and 6 months post-op. None of the patients demonstrated common adverse reactions to pregabalin. CONCLUSION: This study showed no reduction in postoperative opioid requirements, or improvement in early postoperative pain scores or functional outcomes at 6 months, with perioperative use of pregabalin. Orthopaedic surgeons may consider this when selecting an analgesic regimen for their patients. LEVEL OF EVIDENCE: II.

High rates of variation in HLA-DQ2/DQ8 testing for coeliac disease: results from an RCPAQAP pilot program.

AIM: Coeliac disease(CD) is a highly prevalent, gluten-dependent, autoimmune enteropathy. While the diagnosis is based on serological and histological criteria, genotyping of the human leucocyte antigens (HLA) DQ2 and DQ8 has been shown to have substantial clinical utility, especially in excluding the diagnosis in patients who do not carry either antigen. As a result, HLA genotyping is now being performed by more laboratories and has recently become one of the most frequently requested genetic tests in Australia. To date, there has been little scrutiny on the accuracy and reporting of results by laboratories new to HLA typing. In response to clinician feedback that identified potentially clinically significant discrepancies in HLA typing results, the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) undertook a pilot study to assess laboratory performance in the detection of HLA-DQ2/DQ8 and their associated HLA-DQA1 and HLA-DQB1 alleles. METHODS: DNA was extracted from 5 patients and sent to 10 laboratories for external quality assurance (EQA) testing. Laboratories were assessed for reporting in genotyping, interpretation and methodology. RESULTS: Our findings showed that at least 80% of laboratories underperform with respect to recommended guidelines for HLA typing and reporting for CD, with 40% of laboratories failing to provide any clinical interpretation or full genotyping data. This suboptimal level of reporting may lead to ambiguities for downstream clinical interpretation that may compromise patient management. CONCLUSIONS: These findings highlight the importance of adherence to standardised guidelines for optimal performance and reporting of HLA results and substantiate the need for EQA and proficiency testing for laboratories providing this service.

RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

Involvement of vacuolar H(+)-ATPase in killing of human melanoma cells by the sphingosine kinase analogue FTY720.

Targeting the sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) signalling axis is emerging as a promising strategy in the treatment of cancer. However, the effect of such an approach on survival of human melanoma cells remains less understood. Here, we show that the sphingosine analogue FTY720 that functionally antagonises S1PRs kills human melanoma cells through a mechanism involving the vacuolar H(+) -ATPase activity. Moreover, we demonstrate that FTY720-triggered cell death is characterized by features of necrosis and is not dependent on receptor-interacting protein kinase 1 or lysosome cathepsins, nor was it associated with the activation of protein phosphatase 2A. Instead, it is mediated by increased production of reactive oxygen species and is antagonized by activation of autophagy. Collectively, these results suggest that FTY720 and its analogues are promising candidates for further development as new therapeutic agents in the treatment of melanoma.

Oncogenic activation of MEK/ERK primes melanoma cells for adaptation to endoplasmic reticulum stress.

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAF(V600E) or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAF(V600E) into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma.

Obstructive jaundice due to intraductal tumour thrombus in recurrent hepatocellular carcinoma: what is the optimal therapeutic approach?

Icteric Hepatocellular Carcinoma (HCC) is known to cause intraluminal biliary obstruction by one of three mechanisms: hemobilia from the tumour, migration of tumor debris, or continuous growth along the biliary tree. It is however a very rare presentation of HCC and an important differential diagnosis in the approach to obstructive jaundice. We report a case of a recurrent intraductal hepatocellular carcinoma. The patient initially underwent surgical resection of segment five HCC nine months ago with clear margins. The patient now presents with obstructive jaundice and imaging showed a right intraductal tumour involving the confluence, left and common hepatic ducts. He underwent a right hepatectomy and bile duct tumour thrombectomy despite the apparent absence of a parenchymal tumour. Histological examination showed a 2 mm focus of parenchymal tumour with extension of the tumour into the bile duct. In this case report, we reviewed the literature and describe the different surgical approaches to intraductal hepatocellular carcinomas and discuss the pathological aspects of these bile duct tumour thrombus. We report the favourable outcome of surgical resection for intraductal hepatocellular carcinoma and emphasize that intraductal HCC is not a late stage of disease and adequate surgical resection can still provide a reasonable disease free survival.

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress.

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.

PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma.

Inositol polyphosphate 5-phosphatases can terminate downstream signalling of phosphatidylinositol-3 kinase; however, their biological role in the pathogenesis of cancer is controversial. Here we report that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase, has a tumour suppressive role in melanoma. Although it is commonly downregulated in melanoma, overexpression of phosphatidylinositol 4,5-bisphosphate 5-phosphatase blocks Akt activation, inhibits proliferation and undermines survival of melanoma cells in vitro, and retards melanoma growth in a xenograft model. In contrast, knockdown of phosphatidylinositol 4,5-bisphosphate 5-phosphatase results in increased proliferation and anchorage-independent growth of melanocytes. Although DNA copy number loss is responsible for downregulation of phosphatidylinositol 4,5-bisphosphate 5-phosphatase in a proportion of melanomas, histone hypoacetylation mediated by histone deacetylases HDAC2 and HDAC3 through binding to the transcription factor Sp1 at the PIB5PA gene promoter appears to be another commonly involved mechanism. Collectively, these results establish the tumour suppressive role of phosphatidylinositol 4,5-bisphosphate 5-phosphatase and reveal mechanisms involved in its downregulation in melanoma.

The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells.

Emerging evidence has pointed to biological roles of melanoma-associated antigens (MAGEs) in cancer development, progression and resistance to treatment. However, the mechanisms involved remain to be fully elucidated. In this report, we show that one of the MAGE proteins, MAGE-D2, suppresses the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 2 (TRAIL-R2) and plays an important role in protecting melanoma cells from apoptosis induced by TRAIL. MAGE-D2 was commonly expressed at increased levels in melanoma cells compared with melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis. This was associated with enhanced formation of TRAIL death-inducing signaling complex and up-regulation of TRAIL-R2, and was blocked by a recombinant TRAIL-R2/Fc chimeric protein or siRNA knockdown of TRAIL-R2. Regulation of TRAIL-R2 by MAGE-D2 appeared to be mediated by p53, in that knockdown MAGE-D2 did not up-regulate TRAIL-R2 in p53-null or mutant p53 melanoma cells. In addition, inhibition of MAGE-D2 did not result in up-regulation of TRAIL-R2 in wild-type p53 cell lines with p53 inhibited by short hairpin RNA. Indeed, knockdown of MAGE-D2 led to up-regulation of p53 due to a transcriptional increase. The regulatory effect of MAGE-D2 on TRAIL-R2 expression and TRAIL-induced apoptosis was recapitulated in studies on fresh melanoma isolates. Taken together, these results identify the expression of MAGE-D2 as an important mechanism that inhibit TRAIL-induced apoptosis and suggest that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma.

Contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis due to upregulation of TRAIL-R2 and Mcl-1 in human melanoma cells.

Wild-type p53 is commonly expressed in melanoma but does not appear to be effective in the induction of apoptosis. One explanation is that p53 is targeted for degradation by the E3 ligase MDM2. However, we found in this study that blockade of the interaction of p53 and MDM2 by the MDM2 antagonist nutlin-3 in melanoma cells did not induce apoptosis, even though it upregulated p53 and its proapoptotic targets. Nevertheless, nutlin-3 enhanced TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unexpectedly, nutlin-3 upregulated Mcl-1, which attenuated apoptotic signaling triggered by TRAIL, and inhibited apoptosis induced by the microtubule-targeting drug docetaxel. The increase in Mcl-1 was related to a p53-independent transcriptional mechanism, but stabilization of the Mcl-1 protein played a dominant role, as nutlin-3 upregulated the Mcl-1 protein to a much greater extent than the Mcl-1 mRNA, and this was associated with prolonged half-life time and reduced ubiquitination of the protein. Knockdown of p53 blocked the upregulation of the Mcl-1 protein, indicating that p53 plays a critical role in the stabilization of Mcl-1. The contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis were confirmed in fresh melanoma isolates. Collectively, these results show that nutlin-3 may be a useful agent in combination with TRAIL and, importantly, uncover a novel regulatory effect of p53 on the expression of Mcl-1 in melanoma cells on treatment with nutlin-3, which may antagonize the therapeutic efficacy of other chemotherapeutic drugs in addition to docetaxel in melanoma.

Pain prevalence in Singapore.

UNLABELLED: The prevalence of chronic pain is well described in various parts of the world; primarily in Western societies such as Europe, America and Australia. Little is known of the prevalence of chronic pain within Asia or Southeast Asia. In view of the cultural and genetic variation in pain causation, manifestation and reporting, the findings of previous studies cannot be translated to Asian countries. Prevalence studies needed to be carried out to quantify the magnitude and impact of chronic pain within Asian countries to properly allocate precious health funds to deal with this important healthcare issue. We report the findings of the prevalence study within one Asian country: Singapore. OBJECTIVE: To determine the prevalence and impact of chronic pain in adult Singaporeans. MATERIALS AND METHODS: Two sets of questionnaires were designed. The first, a screening questionnaire, to identify the prevalence of chronic pain, and should there be chronic pain; the second, a detailed questionnaire was administered, to characterise the features and the impact of pain. A cross-sectional sampling of Singapore adults were achieved using a computer-based multi-step random sampling of listed telephones numbers. The questionnaires were administered via telephone by a trained interviewer with the aid of a computer-assisted telephone interview system. RESULTS: A total of 4141 screening and 400 detailed questionnaires were completed. The prevalence of chronic pain, defined as pain of at least 3 months' duration over the last 6 months was 8.7% (n = 359). There was a higher prevalence in females (10.9%) and with increasing age. In particular, pain prevalence increased steeply beyond the age of 65 years old. There was a significant impact on work and daily function of those with chronic pain. CONCLUSION: Though the prevalence of chronic pain was marginally lower compared other studies, the impact of pain was just as significant. In a rapidly ageing population such as Singapore, chronic pain is an important emerging healthcare problem which will likely exert increasing toll on the existing social infrastructure within the next 5 to 10 years.