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Krüppel-like Factor 5 (KLF5) is a zinc-finger transcription factor associated with cell cycle progression and cell survival. KLF5 plays a key role in mammalian intestinal epithelium development and maintenance, expressed at high levels in basal proliferating cells and low levels in terminally differentiated cells. Considering Barrett's esophagus (BE) and esophageal adenocarcinoma's (EAC) histopathological similarities to intestinal epithelium, we sought to determine KLF5’s role in BE and EAC, as well as KLF5’s possible connection to the sonic hedgehog (SHH) pathway which is highly active in BE and EAC development. Low levels of KLF5 mRNA were found in BE cell lines and tissue– similar to what has been reported in differentiated intestinal epithelium. In contrast, higher KLF5 levels were observed in EAC cells and tissues. KLF5 knockdown in EAC cells caused significant decreases in cell migration, proliferation, and EAC-associated gene expression. Moreover, KLF5 knockdown led to decreased SHH signaling. These results suggest that KLF5 is connected to the SHH pathway in BE and EAC and may represent a potential drug target in EAC; further studies are now indicated to verify these findings and elucidate underlying mechanisms involved.

Objective The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. Background The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. Methods The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. Results BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. Conclusion Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Pneumocephalus is commonly seen on imaging in the setting of craniofacial trauma, skull base tumours, intracranial infection and after neurological intervention. Spontaneous pneumocephalus in the absence of these conditions is exceedingly rare, with only approximately 30 cases reported in the literature to date. Spontaneous otogenic pneumocephalus (SOP) is believed to occur as a result of anomalous communication between the intracranial space and a hyper-pneumatised temporal bone, with either positive extra-to-intracranial pressure or negative intracranial pressure gradient. These anomalous communicating channels may only become clinically apparent when triggered by episodes of acute increase in middle ear pressure during coughing, sneezing, Valsalva manoeuvre or significant change in atmospheric pressure. Patients may exhibit a wide range of neurological symptoms and the aim of treatment is to reduce the risk of complications such as infection and intracranial hypertension. Both conservative and neurosurgical approaches have been described. We report a case of SOP in which the patient was conservatively managed, and spontaneous resolution of pneumocephalus was documented on serial computed tomography (CT) scans. This unique case clearly demonstrates the natural history and temporal evolution of SOP without surgical intervention. This knowledge may potentially obviate the need for surgery, thus reducing morbidity and mortality in patients who are poor surgical candidates.

AimThe presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated.MethodsMutational analyses of the CEBPA gene of 137 AML bone marrow or peripheral blood retrospective specimens were performed by the amplification of the CEBPA gene using the Expand Long Range dNTPack and the amplicons processed by CCS and NGS. CEBPA-specific libraries were then constructed using the Nextera XT V.2 kit. All FASTQ files were then processed with the MiSeq Reporter V.2.6.2.3 using the PCR Amplicon workflow via the customised CEBPA-specific manifest file. The variant calling format files were analysed using the Illumina Variant Studio V.2.2.ResultsA coverage per base of 3631X to 28184X was achieved. 22 samples (16.1%) were found to contain CEBPA mutations, with variant allele frequencies (VAF) ranging from 3.8% to 58.2%. Taking CCS as the ‘gold standard’, sensitivity and specificity of 97% and 97% was achieved. For the transactivation domain 2 polymorphism (c.584_589dupACCCGC/p.His195_Pro196dup), the CEBNX achieved 100% sensitivity and 100% specificity relative to CCS.ConclusionsOur laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of CEBPA. Therefore, our assay is suitable for deployment in the clinical laboratory.

Aims In recent years, genomic technologies have enabled the identification of mutations in acute myeloid leukaemia (AML). DNMT3A is a recurrently mutated epigenetic modifier gene in AML. To date, the prognostic significance of DNMT3A mutations has not been studied in a Southeast Asian AML population. We sought to investigate the clinical implications of DNMT3A mutations in a Southeast Asian cohort of AML patients. Methods DNMT3A mutations were identified using a targeted next-generation sequencing panel in 157 AML patients. We studied the molecular and clinical features of patients with DNMT3A mutations and assessed the prognostic impact of DNMT3A mutations. Results DNMT3A mutations were found in 33 of 157 (21.0%) AML patients. 114 patients were included for statistical analysis. Pretreatment data revealed that patients with DNMT3A mutations were older (≥60 years old), had a higher white blood cell count at diagnosis, had more adverse cytogenetic risk profiles and were more often associated with NPM1 mutations compared with patients with wild-type DNMT3A . Survival analysis showed that DNMT3A mutations were associated with poorer clinical outcomes. This was especially when associated with NPM1 and FLT3-ITD mutations (AML NPM1/FLT3/DNMT3A ), which are common. The AML NPM1/FLT3/DNMT3A subtype was an independent predictor for poorer overall survival (OS). Other independent risk factors for poorer OS include advanced age at diagnosis and adverse cytogenetic risk stratification. Conclusions DNMT3A mutations are associated with an unfavourable clinical outcome in our Southeast Asian AML patient cohort. In particular, AML NPM1/FLT3/DNMT3A patients had the poorest prognosis.

Experimental study. The objective of this study was to establish a non-invasive model to produce pressure ulcers of varying severity in animals with spinal cord injury (SCI). The study was conducted at the Johns Hopkins Hospital in Baltimore, Maryland, USA. A mid-thoracic (T7–T9) left hemisection was performed on Sprague-Dawley rats. At 7 days post SCI, rats received varying degrees of pressure on the left posterior thigh region. Laser Doppler Flowmetry was used to record blood flow. Animals were killed 12 days after SCI. A cardiac puncture was performed for blood chemistry, and full-thickness tissue was harvested for histology. Doppler blood flow after SCI prior to pressure application was 237.808±16.175 PFUs at day 7. Following pressure application, there was a statistically significant decrease in blood flow in all pressure-applied groups in comparison with controls with a mean perfusion of 118.361±18.223 (P

Acute stroke care systems in Southeast Asian countries are at various stages of development, with disparate treatment availability and practice in terms of intravenous thrombolysis and endovascular therapy. With the advent of successful endovascular therapy stroke trials over the past decade, the pressure to revise and advance acute stroke management has greatly intensified. Southeast Asian patients exhibit unique stroke features, such as increased susceptibility to intracranial atherosclerosis and higher prevalence of intracranial haemorrhage, likely secondary to modified vascular risk factors from differing dietary and lifestyle habits. Accordingly, the practice of acute endovascular stroke interventions needs to take into account these considerations. Acute stroke care systems in Southeast Asia also face a unique challenge of huge stroke burden against a background of ageing population, differing political landscape and healthcare systems in these countries. Building on existing published data, further complemented by multi-national interaction and collaboration over the past few years, the current state of acute stroke care systems with existing endovascular therapy services in Southeast Asian countries are consolidated and analysed in this review. The challenges facing acute stroke care strategies in this region are discussed.

See related articles at [[www.cmaj.ca/lookup/doi/10.1503/cmaj.171076][2]][2] and [[www.cmaj.ca/lookup/doi/10.1503/cmaj.170371][3]][3] KEY POINTS Against a background of growing concern that rates of cesarean delivery in Canada and globally are rising, trial of labour after previous cesarean

Background Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. Case presentation The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations (RUNX1, GATA2, DNMT3A, BCORL1, BCOR, 2 PHF6 and CDKN2A) were identified in the pre-induction sample, of which 5 remained (RUNX1, DNMT3A, BCORL1, BCOR and 1 out of 2 PHF6) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1-mutated state. On the contrary, the 5 mutations that persisted post-treatment, including the germline RUNX1 mutation, were likely to be part of the preleukemic clone. Conclusion Further studies are necessary to assess the prevalence of these preleukemic and secondary mutations in the larger FPD/AML patient cohort and establish their prognostic significance. Given the molecular heterogeneity of FPD/AML and other AML subtypes, a better understanding of mutational classes and their involvement in AML pathogenesis can improve risk stratification of patients for more effective and targeted therapy.

Germline CEBPA -mutant acute myeloid leukaemia (AML) is uncommon. There have been reports of single kindreds and small series1–6 since its initial description in 2004;7 and the most comprehensive characterisation comprising 10 CEBPA -mutant families was recently published.8 We herein describe another patient with AML with a germline CEBPA mutation encountered in our haematology practice. The patient was a 33-year-old Vietnamese man who presented with a 2-week history of easy bruising and generalised fatigue. Initial investigations revealed haemoglobin of 11.6 g/dL, white cell count of 3.98×109/L and platelet count of 35×109/L. His marrow was moderately hypercellular with increased pleomorphic blasts, consistent with AML subtype M2 (figure 1). Of note, occasional pelgeroid neutrophils were observed. Flow cytometry was performed using a panel of markers, which included CD7, CD15, CD34, CD36, CD45, CD56, CD71, CD117 and HLA-DR. This revealed the presence of CD34-positive myeloblasts (comprising 5.8% of total cells), as well as dys-coordinated granulopoiesis (with partial CD56 expression) and markedly dysplastic erythropoiesis (with partial CD36/CD71 expression), overall suggestive of clonal evolution from a myelodysplastic syndrome (MDS). Cytogenetic analysis revealed a 46,XY,del(9)(q13q22) karyotype. CEBPA mutational analysis by Sanger sequencing revealed two mutations: c.134dupC and c.937_938ins33 (figure 2). Figure 1 Bone marrow aspirate (May–Grunwald–Giemsa stain) showing myeloblasts and dysplastic neutrophils. Figure 2 Capillary electropherogram of the germline (A, c.134dupC) and somatic (B, c.937_938ins (33 bp)) CEBPA mutations. He was started on induction chemotherapy, and flow cytometric analysis of the bone marrow postinduction revealed 0.8% minimal residual disease (MRD). Unexpectedly, CEBPA mutational analysis revealed persistence of the c.134dupC mutation, raising the suspicion of a germline CEBPA mutation. This was subsequently confirmed by CEBPA mutational …

SummaryTumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis in mouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing antimetastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytosine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoter of EMT and breast cancer stemness toward metastasis.

Aims: We sought to determine whether KLF5 was involved, at either phenotypic or functional levels, in the genesis and progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC), and whether this involvement was related to sonic hedgehog (SHH) signaling. In human adults, KLF5 is expressed in the less differentiated cells of the intestinal crypts, where its activity promotes cell proliferation, contributes to intestinal epithelial homeostasis, and is necessary for villus formation. KLF5 has been found to be highly active in a number of other cancer types, including gastric cancer; moreover, the known role of KLF5 in intestinal epithelial differentiation may contribute to metaplasia and dysplasia in BE and EAC development. We also sought to ascertain whether KLF5 activity was connected to the sonic hedgehog (SHH) pathway, since this pathway is already known to be highly active in BE and EAC. Methods and results: Quantitative reverse transcriptase PCR (qRT-PCR) performed on RNA obtained from BE cell lines (GhTRT and QhTRT) and EAC cell lines (SKGT4 and OE33) revealed that KLF5 is underexpressed in BE cell lines and overexpressed in EAC cell lines relative to normal primary squamous esophageal epithelial cells (HEEpic). Moreover, qRT-PCR performed on clinical BE and EAC tissue specimens showed the same pattern, with downregulation in BE tissues and upregulation in EAC tissues (relative to matched normal esophageal control tissues). Next, we determined KLF5 functional effects in EAC in vitro. The EAC cell line SKGT4 was transfected with KLF5 siRNA, and WST-1 cell proliferation assays revealed significantly lower cell proliferation at 72 hours (p Conclusion: These tissue phenotypic and in vitro functional data identify KLF5 as a potential oncogene in esophageal adenocarcinogenesis. KLF5 is a potential therapeutic and/or chemopreventive target that can be evaluated in future studies of EAC or BE, such as with metformin, which is known to inhibit KLF5 activity. We also identify a potential new connection between KLF5 and SHH pathway activity in the development of EAC. This potential connection deserves further exploration in other malignancies, as well as in physiologic functions such as human development or adult stem cell maintenance. Citation Format: Christopher K. Ng, Yulan Cheng, John M. Abraham, Stephen J. Meltzer. Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3077.

e13552Background: Up-regulated progenitor cell proliferation induced by abnormal activation of the hedgehog (HH) signaling pathway contributes to carcinogenesis in the esophagus and HH is considere...

Background: According to the World Health Organization, as of 2014 9% of the world’s adult population is affected by diabetes. Uncontrolled diabetes is a pro-inflammatory process that increases generation of reactive oxygen species (ROS). Methods: The production of ROS leads to a chronic increase in oxidative stress which results in an increased susceptibility to infections. Individuals with type 2 diabetes mellitus (T2DM) are highly susceptible to Mycobacterium tuberculosis (M. tb) infection. Previous research has demonstrated that glutathione (GSH) plays an important role in the control of M. tb infection. Recent studies have demonstrated that phagocytosis of M.tb is diminished in patients with T2DM. Phagocytosis in macrophages is thought to be mediated in part by complement protein 3b (C3b)-complement protein receptor 3b (C3R) interactions. Since C3b production is not diminished in patients with T2DM we propose that C3R production is reduced and is the cause for impaired macrophage phagocytosis as well as IL-12 and IFN- γsignaling. Conclusion: This study utilizes a quantitative PCR (qPCR), demonstrating decreased transcription of C3R mRNA in patients with T2DM as compared to non-diabetics.

AimsPCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in an off-the-shelf NGS panel (TruSight Myeloid Sequencing Panel) for myeloid neoplasms and attempted to patch the low read depth for one of the affected genes, CEBPA.MethodsWe performed targeted NGS of 158 acute myeloid leukaemia samples and analysed the amplicon read depths across 568 amplicons to identify lower-performance amplicons. We also correlated the amplicon read depths with the template GC content. Finally, we attempted to patch the low read depth for CEBPA using a parallel library preparation (Nextera XT) workflow.ResultsWe identified 16 lower-performance amplicons affecting nine genes, including CEBPA. There was a slight negative correlation between the amplicon read depths and template GC content. Addition of the separate CEBPA library generated a minimum read depth per base across the CEBPA gene ranging from 268x to 758x across eight samples.ConclusionsThe identification of lower-performance amplicons will be informative to laboratories intending to use this panel. We have also demonstrated proof-of-concept that different libraries (TruSight Myeloid and Nextera XT) can be combined and sequenced on the same flow cell to generate additional reads for CEBPA.

Gastric cancer diagnostics has traditionally been histomorphological and primarily the domain of surgical pathologists. Although there is an increasing usage of molecular and genomic techniques for clinical diagnostics, there is an emerging field of personalised drug sensitivity testing. In this review, we describe the various personalised drug sensitivity testing platforms and discuss the challenges facing clinical adoption of these assays for gastric cancer.

Case of cerebrofacial arteriovenous metameric syndrome (CAMS) in a 9-year-old boy is described with arteriovenous malformation simultaneously involving the brain and face, with characteristic CAMS type 1 and 2 involvement. This patient demonstrates the wide spectrum of clinical manifestations of CAMS, and in this particular case, the patient exhibits features of hypopituitarism—an association that was not previously described in the literature to our knowledge. Awareness of the underlying embryological abnormality and recognition of resultant clinical and radiological presentations are paramount for diagnosis and treatment.

Congenital subcostal hernias are a very rare type of flank hernia. They are typically asymptomatic but are more difficult to fix as the child grows, warranting early repair. They are typically found in the setting of multiple congenital anomalies. We report a congenital subcostal hernia associated with Lumbo-Costovertebral Syndrome, which is already known to be associated with congenital lumbar hernias. We will report the background and outcome of primary repair in such a hernia.